Flavonoid và các polyphenol khác - Báo chí học thuật _ Flavonoids and Other Polyphenols-Academic Press (2001)

Flavonoid và các polyphenol khác - Báo chí học thuật

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UNIVERSITY OF CALIFORNIA BERKELEY CALIFORNIA

Editorial Advisory Board

Gary Beecher Enrique Cadenas Josiane Cillard Fulvio Ursini Myron Gross Barry Halliwell William Pryor Catherine Rice-Evans Helmut Sies

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

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C o n t r i b u t o r s to V o l u m e 3 3 5

Article numbers are in parentheses following the names of contributors

Affiliations listed are current

VALl~RIE ABALEA (27), Laboratoire de Bio-

logie Cellulaire et Vdg~tale, UFR des Sci-

ences Pharmaceutiques et Biologiques,

Universite de Rennes I, Rennes Cedex

35043, France

GARY E ADAMSON (4), Mars, Inc., Hack-

ettstown, New Jersey 07840

MARK ANGELINI (13), Department of Lab-

oratory Medicine and Pathobiology, Uni-

versity of Toronto, Toronto, Ontario M5G

1L5, Canada

MICHAEL AVIRAM (21), Lipid Research

Laboratory, Rambam Medical Center,

Haifa 31096, Israel

STEPHEN J BLOOR (1), Industrial Research

Limited, Lower Hutt, New Zealand

GINO BONTEMPELLI (30), Department of

Chemical Sciences and Technology, Uni-

versity of Udine School of Food Science,

Udine 1-33100, Italy

WOLF BORS (15), Institutfiir Strahlenbiolo-

gie, GSF Forschungszentrum f~r Umwelt

und Gesundheit, Neuherberg D-85764,

Germany

PRATIMA BOSE (9), Department of Chem-

istry, University of Scranton, Scranton,

Pennsylvania 18510

R A F F A E L L A C A N A L I ( 3 5 ) , National Institute

for Food and Nutrition Research, Rome I-

00178, Italy

VERONIQUE CHEYNIER (7), INRA, UMR

Sciences pour l'Oenologie, Montpellier

34060, France

JOSIANE CILLARD (27), Laboratoire de Bio-

logie Cellulaire et V~g~tale, UFR des Sci-

ences Pharmaceutiques et Biologiques,

Universite de Rennes L Rennes Cedex

35043, France

PIERRE CILLARD (27), Laboratoire de Bi- ologie Cellulaire et Vdg~tale, UFR des Sciences Pharmaceutiques et Biologi- ques, Universite de Rennes L Rennes Cedex 35043, France

OLIVIER DANGLES (28), Universitd d'Avignon, UMR A408, Avignon 84000, France

ELEFTHERIOS P DIAMANDIS (13), Depart- ment of Laboratory Medicine and Patho- biology, University of Toronto, Toronto, Ontario M5G 1L5, Canada

JENNIFER DONOVAN (10), Unit~ des Mal- adies M~taboliques et Micronutriments, INRA Theix, St Genks-Champanelle

63122, France

JEAN-FRAN(~OIS DR1LLEAU (5), Laboratoire

de Recherches Cidricoles, Biotransfor- mation des Fruits et Lggumes, INRA, Le Rheu F-35650, France

CLAIRE DUFOUR (28), Securitg et Qualitd des Produits d'origine Vgg~tale, INRA- Site Agroparc, Avignon 84914, France

JOHN D FOLTS (33), Department of Medicine, Cardiovascular Medicine Sec- tion, University of Wisconsin Medical School, Madison, Wisconsin 53792

EDWIN N FRANKEL (22), Department of Food Science and Technology, University

of California, Davis, California 95616-

8598

KIYOSHI FUKUHARA (18), Division of Organic Chemistry, National Institute of Health Sciences, Tokyo 158-8501, Japan

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x CONTRIBUTORS TO VOLUME 335

Food Science and Microbiology, Division

of Human Nutrition, University of Milan,

Milan 20133, Italy

of Laboratory Medicine and Pathobiol-

ogy, University of Toronto, Toronto, On-

tario M5G 1L5, Canada

ogy and Laboratory Medicine, Mount

Sinai Hospital, Toronto, Ontario M5G

1X5, Canada

QIONG GUO (19, 24, 29), Department of

Molecular and Cell Biology, University of

California, Berkeley, California 94720-

3200

Recherches Cidricoles, Biotransforma-

tion des Fruits et Ldgumes, INRA, Le Rheu

F-35650, France

Biochemistry, National University of Sin-

gapore, 119260, Singapore

Hackettstown, New Jersey 07840

Pharmacy, King's College London, Lon-

don SE1 8WA, United Kingdom

PETER C H HOLLMAN (8), State Institute

for Quality Control of Agricultural Prod-

ucts, RIKILT, Wageningen 6708 PD, The

Netherlands

CARL L KEEN (31), Department of Nutri-

tion, University of California, Davis, Cal-

ifornia 95616

Pharmacy, King's College London, Lon-

don SE1 8WA, United Kingdom

National Institute of Health Sciences,

Tokyo 158-8501, Japan

ganic Chemistry, National Institute of

Health Sciences, Tokyo 158-8501, Japan

gize, Inc., The University of Western On- tario Research Park, London N6G 4X8, Ontario, Canada

pour l'Oenologie, Montpellier 34060, France

Jo~O LARANJINHA (25), Laboratory of Bio- chemistry, Faculty of Pharmacy, and Center for Neurosciences, University of Coimbra, Coimbra 3000, Portugal

SHERYL A LAZARUS (4, 31), Mars, Inc., Hackettstown, New Jersey 07840

ZU D LIU (17), Department of Phar- macy, King's College London, London SE1 8WA, United Kingdom

chemistry, National University of Singa- pore, 119260, Singapore

Maladies Mdtaboliques et Micronutri- ments, 1NRA de Clermont-Ferrand/Theix,

St Genks-Champanelle 63122, France

Recherches Cidricoles, Biotransforma- tion des Fruits et Ldgumes, INRA, Le Rheu F-35650, France

PIERLUIGI MAURI (3), Institute of Ad- vanced Biomedical Technologies, Na- tional Council of Research, Segrate, Mi- lan 20090, Italy

Biotechnology, Technical University of Denmark, Lyngby DK-2800, Denmark

CHRISTA MICHEL (15), Institutfiir Strahlen- biologie, GSF Forschungszentrum fiir Umwelt und Gesundheit, Neuherberg D-

85764, Germany

PAUL E MILBURY (2), Antioxidants Re- search Laboratory, USDA Human Nutri- tion Research Center on Aging at Tufts University, Boston, Massachusetts 02111

HADI MOINI (29), Department of Molecular and Cell Biology, University of Califor- nia, Berkeley, California 94720-3200

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CONTRIBUTORS TO VOLUME 335 xi

CHRISTINE MORAND (10, 28), Unitd des

Maladies Mdtaboliques et Micronutri-

merits, INRA de Clermont-FerrandlTheix,

St Genbs-Champanelle 63122, France

ISABELLE MOREL (27), Laboratoire de

Biologie Cellulaire et Vdgdtale, UFR

des Sciences Pharmaceutiques et Bi-

ologiques, Universite de Rennes I, Rennes

Cedex 35043, France

MICHEL MOUTOUNET (7), INRA, UMR

Sciences pour l'Oenologie, Montpellier

34060, France

GUANGJUN NIL (20), Laboratory of Visual

Information Processing, Department of

Molecular and Cell Biophysics, Institute

of Biophysics, Academia Sinica, Beifing

100101, Peoples Republic of China

ETSUO NIKI (14), Research Center for Ad-

vanced Science and Technology, Univer-

sity of Tokyo, Tokyo 153-8904, Japan

NORIKO NOGUCHI (14), Research Center for

Advanced Science and Technology, Uni-

versity of Tokyo, Tokyo 153-8904, Japan

LESTER PACKER (24, 29), Department of

Molecular and Cell Biology, University of

California, Berkeley, California 94720-

3200

ANANTH SEKHER PANNALA (23), Wolfson

Centre for Age Related Diseases, GKT

School of Biomedical Sciences, King's

College London, London SE1 9RT, United

Kingdom

DEBRA A PEARSON (31), Department of

Human Biology, ES 301, University of

Wisconsin, Green Bay, Wisconsin 54311

PIERGIORGIO PIETTA (3, l 1), Institute of

Advanced Biomedical Technologies, Na-

tional Council of Research, Segrate, Mi-

lan 20090, Italy

JOHN PROCH (9), Department of Chemistry,

University of Scranton, Scranton, Penn-

sylvania 18510

IVAN RAPUZZI (30), Department of Chemi-

cal Sciences and Technology, University

of Udine School of Food Science, Udine 1-33100, Italy

CHRISTIAN REMESY (10, 28), Unitd des Maladies Mdtaboliques et Micronutri- merits, INRA Theix, St Genks-Champ- anelle 63122, France

CATHERINE RICE-EVANS (23), Wolfson Centre for Age Related Diseases, GKT School of Biomedical Sciences, King's College London, London SE1 9RT, United Kingdom

GERALD RIMBACH (24, 34, 35), School of Food Biosciences, Hugh Sinclair Human Nutrition Unit, University of Reading, Reading RG6 6AP, United Kingdom

CLAUDE SALIOU (34, 35), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-

3200

PHILIPPE SANONER (5), Laboratoire de Recherches Cidricoles, Biotransforma- tion des Fruits et IMgumes, INRA, Le Rheu F-35650, France

HAROLD H SCHMITZ (4, 31), Mars, Inc., Hackettstown, New Jersey 07840

DHANANSAYAN SHANMUGANAYAGAM (33), Department of Medicine, Cardio- vascular Medicine Section, University

of Wisconsin Medical School, Madison, Wisconsin 53792

SHENGRONG SHEN (20), Department of Tea, Zhejiang University, Hangzhou, 310029, China

HONGLIAN SHI (14), Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan

PAOLO SIMONETTI (11), Department of Food Science and Microbiology, Division

of Human Nutrition, University of Milan, Milan 20133, Italy

GEORGE J SOLEAS (12, 13), Quality Assur- ance, Liquor Control Board of Ontario, Toronto, Ontario M5E 1A4, Canada

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xii CONTRIBUTORS TO VOLUME 335

JEREMY P E SPENCER (26), International

Antioxidant Research Centre, King's Col-

lege London, Guy's Campus, London SE1

DIETER STRACK (6), Abteilung Sekudiirstof-

fwechsel, Leibniz-lnstitut far Pflanzen-

biochemie, Halle (Saale) D-06120,

Germany

search Laboratories, Kyowa Hakko Ko-

gyo Co., Ibaraki 305-0841, Japan

FULVIO URSINI (30), Department of Bio-

logical Chemistry, University of Padova

School of Medicine, Padova 1-35121,

Italy

Medicine, University of California,

Davis, California 95616

JACOB VAYA (21), Laboratory for Natu-

ral Medicinal Compounds, Migal-Galilee

Technological Center, Kiriat-Shmona, Israel

JOE A VINSON (9), Department of Chem- istry, University of Scranton, Scranton, Pennsylvania 18510

FABIO VIRGILI (35), National Institute for Food and Nutrition Research, Rome

00178, Italy

Information Processing, Department of Molecular and Cell Biophysics, Institute

of Biophysics, Academia Sinica, Beijing

100101, Peoples Republic of China

Antioxidant Research Centre, King's Col- lege London, Guy's Campus, London SE1 8RT, United Kingdom

WENJUAN XIN (19), Institute of Biophysics, Academia Sinica, Beijing 100101, Peo- ples Republic of China

JOE YAN (12), Quality Assurance, Liquor Control Board of Ontario, Toronto, On- tario, Canada M5E 1A4

BAOLU ZHAO (19, 20), Laboratory of Vi- sual Information Processing, Department

of Molecular and Cell Biophysics, Insti- tute of Biophysics, Academia Sinica, Bei- jing 100101, Peoples Republic of China

KAICUN ZHAO (26), International Antiox- idant Research Centre, King's College London, Guy's Campus, London SE1 8RT,, United Kingdom

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Preface

Reactive oxygen and nitrogen species (ROS) and antioxidants are increasingly playing an important role in oxidative stress and disease Certainly ROS are important in regulating oxidative processes in all biological systems This volume

of Methods in Enzymology on Flavonoids and Other Polyphenols was prepared

in recognition of compelling evidence that these substances important components of our food chain (such as fruits and vegetables and beverages such as tea or red wine) have been reported to have health benefits In pure form or as herbal extracts or plant products they have been reported to have antioxidant and cell regulation activity and to affect gene expression

However, many unsolved problems exist with respect to the action of polyphenols and bioflavonoids in biological systems There is a lack of information on bioavailability, metabolism, biochemical, and molecular biological effects on cell regulation and on effects on health New methodologies described in this volume will aid progress in solving these unanswered questions

In bringing this volume to fruition, credit must be given to the experts on various aspects of research in this field Appreciation is extended to the contributors selected to contribute and to produce this state of the art volume The topics included were chosen on the excellent advice of Drs Gary Beecher, Enrique Ca- denas, Josiane Cillard, Myron Gross, Barry Halliwell, William Pryor, Catherine Rice-Evans, Helmut Sies, and Fulvio Ursini To these colleagues I extend my sincere thanks and appreciation

LESTER PACKER

xiii

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M E T H O D S IN ENZYMOLOGY

VOLUME I Preparation and Assay of Enzymes

Edited by SIDNEY P COLOWICK AND NATHAN O KAPLAN

VOLUME II Preparation and Assay of Enzymes

VOLUME III Preparation and Assay of Substrates

VOLUME IV Special Techniques for the Enzymologist

VOLUME V Preparation and Assay of Enzymes

VOLUME VI Preparation and Assay of Enzymes (Continued)

Preparation and Assay of Substrates

Special Techniques

VOLUME VII Cumulative Subject Index

VOLUME VIII Complex Carbohydrates

Edited by ELIZABETH E NEUFELD AND VICTOR GINSBURG

VOLUME IX Carbohydrate Metabolism

Edited by WILLIS A WOOD

VOLUME X Oxidation and Phosphorylation

VOLUME XI Enzyme Structure

Edited by C H W HIRS

VOLUME XII Nucleic Acids (Parts A and B)

VOLUME XIII Citric Acid Cycle

Edited by J, M LOWENSTEIN

VOLUME XIV Lipids

Edited by J, M LOWENSTEIN

VOLUME XV Steroids and Terpenoids

Edited by RAYMOND B CLAYTON

VOLUME XVI Fast Reactions

Edited by KENNETH KUSTIN

XV

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xvi METHODS 1N ENZYMOLOGY

VOLUME XVII Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR

VOLUME XVIII Vitamins and Coenzymes (Parts A, B, and C)

VOLUME XlX Proteolytic Enzymes

VOLUME XX Nucleic Acids and Protein Synthesis (Part C)

VOLUME XXI Nucleic Acids (Part D)

VOLUME XXII Enzyme Purification and Related Techniques

Edited by WILLIAM B JAKOBY

VOLUME XXIII Photosynthesis (Part A)

Edited by ANTHONY SAN PIETRO

VOLUME XXlW Photosynthesis and Nitrogen Fixation (Part B)

VOLUME XXW Enzyme Structure (Part B)

Edited by C H W HIRS AND SERGE N TIMASHEFF

VOLUME XXVI Enzyme Structure (Part C)

VOLUME XXVII Enzyme Structure (Part D)

VOLUME XXVIII Complex Carbohydrates (Part B)

Edited by VICTOR GINSBURG

VOLUME XXIX Nucleic Acids and Protein Synthesis (Part E)

VOLUME XXX Nucleic Acids and Protein Synthesis (Part F)

VOLUME XXXI Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

VOLUME XXXII Biomembranes (Part B)

VOLUME XXXIII Cumulative Subject Index Volumes I-XXX

Edited by MARTHA G DENNIS AND EDWARD A DENNIS

VOLUME XXXIW Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAM B JAKOBY AND MEIR WlLCHEK

VOLUME XXXV Lipids (Part B)

Edited by JOHN M LOWENSTEIN

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METHODS IN ENZYMOLOGY xvii

VOLUME XXXVI Hormone Action (Part A: Steroid Hormones)

Edited by BERT W O'MALLEY AND JOEL G HARDMAN

VOLUME XXXVII Hormone Action (Part B: Peptide Hormones)

VOLUME XXXVIII Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G HARDMAN AND BERT W O'MALLEY

VOLUME XXXIX Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems)

Edited by JOEL G HARDMAN AND BERT W O'MALLEY

VOLUME XL Hormone Action (Part E: Nuclear Structure and Function)

VOLUME XLI Carbohydrate Metabolism (Part B)

Edited by W A WOOD

VOLUME XLII Carbohydrate Metabolism (Part C)

Edited by W A WOOD

VOLUME XLIII Antibiotics

Edited by JOHN H HASH

VOLUME XLIV Immobilized Enzymes

Edited by KLAUS MOSBACH

VOLUME XLV Proteolytic Enzymes (Part B)

Edited by LASZLO LORAND

VOLUME XLVI Affinity Labeling

Edited by WILLIAM B JAKOBY AND MEIR WILCHEK

VOLUME XLVII Enzyme Structure (Part E)

VOLUME XLVIII Enzyme Structure (Part F)

VOLUME XLIX Enzyme Structure (Part G)

VOLUME L Complex Carbohydrates (Part C)

VOLUME LI Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A HOFFEE AND MARY ELLEN JONES

VOLUME LII Biomembranes (Part C: Biological Oxidations)

VOLUME LIII Biomembranes (Part D: Biological Oxidations)

VOLUME LIV Biomembranes (Part E: Biological Oxidations)

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xviii METHODS IN ENZYMOLOGY

VOLUME LV Biomembranes (Part F: Bioenergetics)

VOLUME LVI Biomembranes (Part G: Bioenergetics)

VOLUME LVII Bioluminescence and Chemiluminescence

Edited by MARLENE A DELUCA

VOLUME LVIII Cell Culture

Edited by WILLIAM B JAKOBY AND IRA PASTAN

VOLUME LIX Nucleic Acids and Protein Synthesis (Part G)

VOLUME LX Nucleic Acids and Protein Synthesis (Part H)

VOLUME 61 Enzyme Structure (Part H)

VOLUME 62 Vitamins and Coenzymes (Part D)

VOLUME 63 Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhi- bitor Methods)

Edited by DANIEL L PURICH

VOLUME 64 Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems)

VOLUME 65 Nucleic Acids (Part I)

VOLUME 66 Vitamins and Coenzymes (Part E)

Edited by DONALD B MCCORMICK AND LEMUEL D WRIGHT

VOLUME 67 Vitamins and Coenzymes (Part F)

Edited by DONALD B MCCORMICK AND LEMUEL D WRIGHT

VOLUME 68 Recombinant DNA

Edited by RAY WU

VOLUME 69 Photosynthesis and Nitrogen Fixation (Part C)

VOLUME 70 Immunochemical Techniques (Part A)

Edited by HELEN VAN VUNAKIS AND JOHN J LANGONE

VOLUME 71 Lipids (Part C)

VOLUME 72 Lipids (Part D)

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METHODS IN ENZYMOLOGY xix

VOLUME 73 Immunochemical Techniques (Part B)

Edited by JOHN J LANGONE AND HELEN VAN VUNAKIS

VOLUME 74 Immunochemical Techniques (Part C)

VOLUME 75 Cumulative Subject Index Volumes XXXI, XXXII, XXXIV-LX

Edited by EDWARD A DENNIS AND MARTHA G DENNIS

VOLUME 76 Hemoglobins

Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE VOLUME 77 Detoxication and Drug Metabolism

VOLUME 78 Interferons (Part A)

Edited by SIDNEY PESTKA

VOLUME 79 Interferons (Part B)

VOLUME 80 Proteolytic Enzymes (Part C)

Edited by LASZLO LORAND

VOLUME 81 Biomembranes (Part H: Visual Pigments and Purple Membranes, I)

Edited by LESTER PACKER

VOLUME 82 Structural and Contractile Proteins (Part A: Extracellular Matrix)

Edited by LEON W CUNNINGHAM AND DIXIE W FREDERIKSEN

VOLUME 83 Complex Carbohydrates (Part D)

VOLUME 84 Immunochemical Techniques (Part D: Selected Immunoassays)

VOLUME 85 Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton)

Edited by DIXIE W FREDERIKSEN AND LEON W CUNNINGHAM

VOLUME 86 Prostaglandins and Arachidonate Metabolites

Edited by WILLIAM E M LANDS AND WILLIAM L SMITH

VOLUME 87 Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereo- chemistry, and Rate Studies)

VOLUME 88 Biomembranes (Part I: Visual Pigments and Purple Membranes, II)

VOLUME 89 Carbohydrate Metabolism (Part D)

VOLUME 90 Carbohydrate Metabolism (Part E)

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x× METHODS IN ENZYMOLOGY

VOLUME 91 Enzyme Structure (Part I)

VOLUME 92 Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)

VOLUME 93 Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity)

VOLUME 94 Polyamines

Edited by HERBERT TABOR AND CELIA WHITE TABOR

VOLUME 95 Cumulative Subject Index Volumes 61-74, 76-80

Edited by EDWARD A DENNIS AND MARTHA G DENNIS

VOLUME 96 Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

VOLUME 97 Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]

VOLUME 98 Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling)

VOLUME 99 Hormone Action (Part F: Protein Kinases)

Edited by JACKIE D CORBIN AND JOEL G HARDMAN

VOLUME 100 Recombinant DNA (Part B)

Edited by RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE

VOLUME 101 Recombinant DNA (Part C)

Edited by RAY Wu, LAWRENCE GROSSMAN, AND KIVlE MOLDAVE

VOLUME 102 Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins)

Edited by ANTHONY R MEANS AND BERT W O'MALLEY

VOLUME 103 Hormone Action (Part H: Neuroendocrine Peptides)

Edited by P MICHAEL CONN

VOLUME 104 Enzyme Purification and Related Techniques (Part C)

VOLUME 105 Oxygen Radicals in Biological Systems

VOLUME 106 Posttranslational Modifications (Part A)

Edited by FINN WOLD AND KIVIE MOLDAVE

VOLUME 107 Posttranslational Modifications (Part B)

Edited by FINN WOLD AND KIVIE MOLDAVE

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METHODS IN ENZYMOLOGY xxi

VOLUME 108 Immunochemical Techniques (Part G: Separation and Characteri- zation of Lymphoid Cells)

Edited by GIOVANNI DI SABATO, JOHN J LANGONE, AND HELEN VAN VUNAKIS VOLUME 109 Hormone Action (Part I: Peptide Hormones)

Edited by LUTZ BIRNBAUMER AND BERT W O'MALLEY

VOLUME 110 Steroids and Isoprenoids (Part A)

Edited by JOHN H LAW AND HANS C RILLING

VOLUME 111 Steroids and Isoprenoids (Part B)

Edited by JOHN H LAW AND HANS C RILLING

VOLUME 112 Drug and Enzyme Targeting (Part A)

Edited by KENNETH J WIDDER AND RALPH GREEN

VOLUME 113 Glutamate, Glutamine, Glutathione, and Related Compounds

Edited by ALTON MEISTER

VOLUME 114 Diffraction Methods for Biological Macromolecules (Part A)

Edited by HAROLD W WYCKOFF, C n W HIRS, AND SERGE N TIMASHEFF VOLUME 115 Diffraction Methods for Biological Macromolecules (Part B)

Edited by HAROLD W WYCKOFF, C H W HIRS, AND SERGE N TIMASHEFF VOLUME 116 Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions)

Edited by GIOVANNI DI SABATO, JOHN J LANGONE, AND HELEN VAN VUNAKIS VOLUME 117 Enzyme Structure (Part J)

VOLUME 118 Plant Molecular Biology

VOLUME 119 Interferons (Part C)

VOLUME 121 Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)

VOLUME 122 Vitamins and Coenzymes (Part G)

VOLUME 123 Vitamins and Coenzymes (Part H)

VOLUME 124 Hormone Action (Part J: Neuroendocrine Peptides)

VOLUME 125 Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems)

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xxii METHODS IN ENZYMOLOGY

VOLUME 126 Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force)

VOLUME 127 Biomembranes (Part O: Protons and Water: Structure and Transloca- tion)

VOLUME 128 Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology)

Edited by JERE P SEGREST AND JOHN J ALBERS

VOLUME 129 Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism)

Edited by JOHN J ALBERS AND JERE P SEGREST

VOLUME 130 Enzyme Structure (Part K)

VOLUME 131 Enzyme Structure (Part L)

VOLUME 132 Immunochemical Techniques (Part J: Phagocytosis and Cell-Mediated Cytotoxicity)

Edited by GIOVANNI DI SABATO AND JOHANNES EVERSE

VOLUME 133 Bioluminescence and Chemiluminescence (Part B)

VOLUME 134 Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton)

Edited by RICHARD B VALLEE

VOLUME 135 Immobilized Enzymes and Cells (Part B)

VOLUME 136 Immobilized Enzymes and Cells (Part C)

VOLUME 137 Immobilized Enzymes and Cells (Part D)

VOLUME 138 Complex Carbohydrates (Part E)

VOLUME 139 Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins)

Edited by ANTHONY R MEANS AND P MICHAEL CONN

VOLUME 141 Cellular Regulators (Part B: Calcium and Lipids)

Edited by P MICHAEL CONN AND ANTHONY R MEANS

VOLUME 142 Metabolism of Aromatic Amino Acids and Amines

Edited by SEYMOUR KAUFMAN

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METHODS IN ENZYMOLOGY x x i i i

VOLUME 143 Sulfur and Sulfur Amino Acids

Edited by WILLIAM B JAKOBY AND OWEN GRIFFITH

VOLUME 144 Structural and Contractile Proteins (Part D: Extracellular Matrix)

Edited by LEON W CUNNINGHAM

VOLUME 145 Structural and Contractile Proteins (Part E: Extracellular Matrix)

Edited by LEON W CUNNINGHAM

VOLUME 146 Peptide Growth Factors (Part A)

Edited by DAVID BARNES AND DAVID A SIRBASKU

VOLUME 147 Peptide Growth Factors (Part B)

Edited by DAVID BARNES AND DAVID A SIRBASKU

VOLUME 148 Plant Cell Membranes

Edited by LUSTER PACKER AND ROLAND DOUCE

VOLUME 149 Drug and Enzyme Targeting (Part B)

Edited by RALPH GREEN AND KENNETH J WIDDER

VOLUME 150 Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors)

Edited by GIOVANNI DI SABATO

VOLUME 151 Molecular Genetics of Mammalian Cells

Edited by MICHAEL M GOTTESMAN

VOLUME 152 Guide to Molecular Cloning Techniques

Edited by SHELBY L BURGER AND ALAN R KIMMEL

VOLUME 153 Recombinant DNA (Part D)

Edited by RAY Wu AND LAWRENCE GROSSMAN

VOLUME 154 Recombinant DNA (Part E)

VOLUME 155 Recombinant DNA (Part F)

Edited by RAY Wu

VOLUME 156 Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na, K-Pump)

VOLUME 157 Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps)

VOLUME 158 Metalloproteins (Part A)

Edited by JAMES E RIORDAN AND BERT L VALLEE

VOLUME 159 Initiation and Termination of Cyclic Nucleotide Action

Edited by JACKIE D CORBIN AND ROGER A JOHNSON

VOLUME 160 Biomass (Part A: Cellulose and Hemicellulose)

Edited by WILLIS A WOOD AND SCOTT T KELLOGG

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xxiv METHODS IN ENZYMOLOGY

VOLUME 161 Biomass (Part B: Lignin, Pectin, and Chitin)

Edited by WILLIS A WOOD AND SCOTT T KELLOGG

VOLUME 162 Immunochemical Techniques (Part L: Chemotaxis and Inflamma- tion)

VOLUME 163 Immunochemical Techniques (Part M: Chemotaxis and Inflamma- tion)

VOLUME 164 Ribosomes

Edited by HARRY F NOLLER, JR., AND KIVIE MOLDAVE

VOLUME 165 Microbial Toxins: Tools for Enzymology

Edited by SIDNEY HARSHMAN

VOLUME 166 Branched-Chain Amino Acids

Edited by ROBERT HARRIS AND JOHN R SOKATCH

VOLUME 167 Cyanobacteria

Edited by LESTER PACKER AND ALEXANDER N GLAZER

VOLUME 168 Hormone Action (Part K: Neuroendocrine Peptides)

VOLUME 169 Platelets: Receptors, Adhesion, Secretion (Part A)

Edited by JACEK HAWlGER

VOLUME 170 Nucleosomes

Edited by PAUL M WASSARMAN AND ROGER D KORNBERG

VOLUME 171 Biomembranes (Part R: Transport Theory: Cells and Model Membranes)

VOLUME 172 Biomembranes (Part S: Transport: Membrane Isolation and Characterization)

VOLUME 173 Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]

VOLUME 174 Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]

VOLUME 176 Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics)

Edited by NORMAN J OPPENHEIMER AND THOMAS L JAMES

VOLUME 177 Nuclear Magnetic Resonance (Part B: Structure and Mechanism)

Edited by NORMAN J OPPENHEIMER AND THOMAS L JAMES

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METHODS IN ENZYMOLOGY xxv

VOLUME 178 Antibodies, Antigens, and Molecular Mimicry

Edited by JOHN J LANGONE

VOLUME 179 Complex Carbohydrates (Part F)

VOLUME 180 RNA Processing (Part A: General Methods)

Edited by JAMES E DAHLBERG AND JOHN N ABELSON

VOLUME 181 RNA Processing (Part B: Specific Methods)

Edited by JAMES E DAHLBERG AND JOHN N ABELSON

VOLUME 182 Guide to Protein Purification

Edited by MURRAY P DEUTSCHER

VOLUME 183 Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences

Edited by RUSSELL E DOOLITTLE

VOLUME 184 Avidin-Biotin Technology

Edited by MEIR WILCHEK AND EDWARD m BAYER

VOLUME 185 Gene Expression Technology

Edited by DAVID V GOEDDEL

VOLUME 186 Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants)

Edited by LESTER PACKER AND ALEXANDER N GLAZER

VOLUME 187 Arachidonate Related Lipid Mediators

Edited by ROBERT C MURPHY AND FRANK A FITZPATRICK

VOLUME 188 Hydrocarbons and Methylotrophy

Edited by MARY E LIDSTROM

VOLUME 189 Retinoids (Part A: Molecular and Metabolic Aspects)

VOLUME 190 Retinoids (Part B: Cell Differentiation and Clinical Applications)

VOLUME 191 Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells)

VOLUME 192 Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells)

VOLUME 193 Mass Spectrometry

Edited by JAMES A MCCLOSKEY

VOLUME 194 Guide to Yeast Genetics and Molecular Biology

Edited by CHRISTINE GUTHRIE AND GERALD R FINK

VOLUME 195 Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase

Edited by ROGER A JOHNSON AND JACKIE D CORBIN

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xxvi METHODS IN ENZYMOLOGY

VOLUME 196 Molecular Motors and the Cytoskeleton

VOLUME 197 Phospholipases

Edited by EDWARD A DENNIS

VOLUME 198 Peptide Growth Factors (Part C)

Edited by DAVID BARNES, J P MATHER, AND GORDON H SATO

VOLUME 200 Protein Phosphorylation (Part A: Protein Kinases: Assays,

Purification, Antibodies, Functional Analysis, Cloning, and Expression)

VOLUME 201 Protein Phosphorylation (Part B: Analysis of Protein

Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases)

VOLUME 202 Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes)

VOLUME 203 Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs)

VOLUME 204 Bacterial Genetic Systems

Edited by JEFFREY H MILLER

VOLUME 205 Metallobiochemistry (Part B: Metallothionein and Related

Molecules)

Edited by JAMES F RIORDAN AND BERT L VALLEE

VOLUME 206 Cytochrome P450

Edited by MICHAEL R WATERMAN AND ERIC F JOHNSON

VOLUME 207 Ion Channels

Edited by BERNARDO RUDY AND LINDA E IVERSON

VOLUME 208 Protein-DNA Interactions

Edited by ROBERT T SAUER

VOLUME 209 Phospholipid Biosynthesis

Edited by EDWARD A DENNIS AND DENNIS E VANCE

VOLUME 210 Numerical Computer Methods

Edited by LUDWIG BRAND AND MICHAEL L JOHNSON

VOLUME 211 DNA Structures (Part A: Synthesis and Physical Analysis of DNA)

Edited by DAVID M J LILLEY AND JAMES E DAHLBERG

VOLUME 212 DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA)

Edited by DAVID M J LILLEY AND JAMES E DAHLBERG

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METHODS IN ENZYMOLOGY xxvii

VOLUME 213 Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation)

VOLUME 214 Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis)

VOLUME 215 Platelets: Receptors, Adhesion, Secretion (Part B)

Edited by JACEK J HAWIGER

VOLUME 216 Recombinant DNA (Part G)

VOLUME 219 Reconstitution of Intracellular Transport

Edited by JAMES E ROTHMAN

VOLUME 220 Membrane Fusion Techniques (Part A)

Edited by NEJAT DOZGUONES

VOLUME 221 Membrane Fusion Techniques (Part B)

Edited by NEJAT DOZGONES

VOLUME 222 Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors)

Edited by LASZLO LORAND AND KENNETH G MANN

VOLUME 223 Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors)

VOLUME 224 Molecular Evolution: Producing the Biochemical Data

Edited by ELIZABETH ANNE ZIMMER, THOMAS J WHITE, REBECCA L CANN, AND ALLAN C WILSON

VOLUME 225 Guide to Techniques in Mouse Development

Edited by PAUL M WASSARMAN AND MELVIN L DEPAMPHILIS

VOLUME 226 Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins)

Edited by JAMES F RIORDAN AND BERT L VALLEE

VOLUME 227 Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins)

Edited by JAMES E RIORDAN AND BERT L VALLEE

VOLUME 228 Aqueous Two-Phase Systems

Edited by HARRY WALTER AND GOTE JOHANSSON

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xxviii METHODS IN ENZYMOLOGY

VOLUME 230 Guide to Techniques in Glycobiology

Edited by WILLIAM J LENNARZ AND GERALD W HART

VOLUME 231 Hemoglobins (Part B: Biochemical and Analytical Methods)

Edited by JOHANNES EVERSE, KIM D VANDEGRIFF, AND ROBERT M WINSLOW VOLUME 232 Hemoglobins (Part C: Biophysical Methods)

Edited by JOHANNES EVERSE, KIM D VANDEGRIFF, AND ROBERT M WINSLOW VOLUME 233 Oxygen Radicals in Biological Systems (Part C)

VOLUME 234 Oxygen Radicals in Biological Systems (Part D)

VOLUME 235 Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors)

Edited by VIRGINIA L CLARK AND PATRIK M BAVOIL

VOLUME 236 Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells)

Edited by VIRGINIA L CLARK AND PATRIK M BAVOIL

VOLUME 237 Heterotrimeric G Proteins

Edited by RAVI IYENGAR

VOLUME 238 Heterotrimeric G-Protein Effectors

Edited by RAVl IYENGAR

VOLUME 239 Nuclear Magnetic Resonance (Part C)

Edited by THOMAS L JAMES AND NORMAN J OPPENHEIMER

VOLUME 240 Numerical Computer Methods (Part B)

Edited by MICHAEL L JOHNSON AND LUDWIG BRAND

VOLUME 241 Retroviral Proteases

Edited by LAWRENCE C Kuo AND JULES A SHAFER

VOLUME 242 Neoglycoconjugates (Part A)

Edited by Y C LEE AND REIKO T LEE

VOLUME 243 Inorganic Microbial Sulfur Metabolism

Edited by HARRY D PECK, JR., AND JEAN LEGALL

VOLUME 244 Proteolytic Enzymes: Serine and Cysteine Peptidases

Edited by ALAN J BARRETT

VOLUME 245 Extracellular Matrix Components

Edited by E RUOSLAHTI AND E ENGVALL

VOLUME 246 Biochemical Spectroscopy

Edited by KENNETH SAUER

VOLUME 247 Neoglycoconjugates (Part B: Biomedical Applications)

Edited by Y C LEE AND REIKO T LEE

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METHODS IN ENZYMOLOGY xxix

VOLUME 248 Proteolytic Enzymes: Aspartic and Metallo Peptidases

Edited by ALAN J BARRETT

VOLUME 249 Enzyme Kinetics and Mechanism (Part D: Developments in Enzyme Dynamics)

Edited by DANIEL L, PURICH

VOLUME 250 Lipid Modifications of Proteins

Edited by PATRICK J CASEY AND JANICE E BUSS

VOLUME 251 Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals)

Edited by LUSTER PACKER

VOLUME 252 Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation)

VOLUME 253 Adhesion of Microbial Pathogens

Edited by RON J DOYLE AND ITZHAK OFEK

VOLUME 254 Oncogene Techniques

Edited by PETER K VOGT AND INDER M VERMA

VOLUME 255 Small GTPases and Their Regulators (Part A: Ras Family)

Edited by W E BALCH, CHANNING J DER, AND ALAN HALL

VOLUME 256 Small GTPases and Their Regulators (Part B: Rho Family)

Edited by W E BALCH, CHANNING J DER, AND ALAN HALL

VOLUME 257 Small GTPases and Their Regulators (Part C: Proteins Involved in Transport)

VOLUME 258 Redox-Active Amino Acids in Biology

Edited by JUDITH P KLINMAN

VOLUME 259 Energetics of Biological Macromolecules

Edited by MICHAEL L JOHNSON AND GARY K ACKERS

VOLUME 260 Mitochondrial Biogenesis and Genetics (Part A)

Edited by GIUSEPPE M ATTARDI AND ANNE CHOMYN

VOLUME 261 Nuclear Magnetic Resonance and Nucleic Acids

Edited by THOMAS L JAMES

VOLUME 262 DNA Replication

Edited by JUDITH L CAMPBELL

VOLUME 263 Plasma Lipoproteins (Part C: Quantitation)

Edited by WILLIAM A BRADLEY, SANDRA H GIANTURCO, AND JURE P SEGREST VOLUME 264 Mitochondrial Biogenesis and Genetics (Part B)

Edited by GIUSEPPE M ATTARDI AND ANNE CHOMYN

VOLUME 265 Cumulative Subject Index Volumes 228, 230-262

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xxx METHODS IN ENZYMOLOGY

VOLUME 266 Computer Methods for Macromolecular Sequence Analysis

Edited by RUSSELL F DOOLITTLE

VOLUME 267 Combinatorial Chemistry

Edited by JOHN N ABELSON

VOLUME 268 Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase)

VOLUME 269 Nitric Oxide (Part B: Physiological and Pathological Processes)

VOLUME 270 High Resolution Separation and Analysis of Biological Macro- molecules (Part A: Fundamentals)

Edited by BARRY L KARGER AND WILLIAM S HANCOCK

VOLUME 271 High Resolution Separation and Analysis of Biological Macro- molecules (Part B: Applications)

Edited by BARRY L KARGER AND WILLIAM S HANCOCK

VOLUME 272 Cytochrome P450 (Part B)

Edited by ERIC F JOHNSON AND MICHAEL R WATERMAN

VOLUME 273 RNA Polymerase and Associated Factors (Part A)

Edited by SANKAR ADHYA

VOLUME 274 RNA Polymerase and Associated Factors (Part B)

Edited by SANKAR ADHYA

VOLUME 275 Viral Polymerases and Related Proteins

Edited by LAWRENCE C KUO, DAVID B OLSEN, AND STEVEN S CARROLL VOLUME 276 Macromolecular Crystallography (Part A)

Edited by CHARLES W CARTER, JR., AND ROBERT M SWEET

VOLUME 277 Macromolecular Crystallography (Part B)

Edited by CHARLES W CARTER, JR., AND ROBERT M SWEET

VOLUME 278 Fluorescence Spectroscopy

Edited by LUDWIG BRAND AND MICHAEL L JOHNSON

VOLUME 279 Vitamins and Coenzymes (Part I)

Edited by DONALD B MCCORMICK, JOHN W SUTI'IE, AND CONRAD WAGNER VOLUME 280 Vitamins and Coenzymes (Part J)

VOLUME 281 Vitamins and Coenzymes (Part K)

VOLUME 282 Vitamins and Coenzymes (Part L)

VOLUME 283 Cell Cycle Control

Edited by WILLIAM G DUNPHY

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METHODS IN ENZYMOLOGY xxxi

VOLUME 284 Lipases (Part A: Biotechnology)

Edited by BYRON RUBIN AND EDWARD A DENNIS

VOLUME 285 Cumulative Subject Index Volumes 263, 264, 266-284, 286-289 VOLUME 286 Lipases (Part B: Enzyme Characterization and Utilization)

Edited by BYRON RUBIN AND EDWARD A DENNIS

VOLUME 287 Chemokines

Edited by RICHARD HORUK

VOLUME 288 Chemokine Receptors

Edited by RICHARD HORUK

VOLUME 289 Solid Phase Peptide Synthesis

Edited by GREGG B FIELDS

VOLUME 290 Molecular Chaperones

VOLUME 291 Caged Compounds

Edited by GERARD MARRIOTT

VOLUME 292 ABC Transporters: Biochemical, Cellular, and Molecular Aspects

Edited by SURESH V AMBUDKAR AND MICHAEL i GOTTESMAN

VOLUME 293 Ion Channels (Part B)

VOLUME 294 Ion Channels (Part C)

VOLUME 295 Energetics of Biological Macromolecules (Part B)

Edited by GARY K ACKERS AND MICHAEL L JOHNSON

VOLUME 296 Neurotransmitter Transporters

Edited by SUSAN G AMARA

VOLUME 297 Photosynthesis: Molecular Biology of Energy Capture

Edited by LEE MCINTOSH

VOLUME 298 Molecular Motors and the Cytoskeleton (Part B)

VOLUME 299 Oxidants and Antioxidants (Part A)

VOLUME 300 Oxidants and Antioxidants (Part B)

VOLUME 301 Nitric Oxide: Biological and Antioxidant Activities (Part C)

VOLUME 302 Green Fluorescent Protein

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xxxii METHODS IN ENZYMOLOGY

VOLUME 303 cDNA Preparation and Display

Edited by SHERMAN M WEISSMAN

VOLUME 304 Chromatin

Edited by PAUL M WASSARMAN AND ALAN P WOLFFE

VOLUME 305 Bioluminescence and Chemiluminescence (Part C)

Edited by THOMAS O BALDWIN AND MIRIAM M ZIEGLER

VOLUME 306 Expression of Recombinant Genes in Eukaryotic Systems

Edited by JOSEPH C GLORIOSO AND MARTIN C SCHMIDT

VOLUME 307 Confocal Microscopy

VOLUME 308 Enzyme Kinetics and Mechanism (Part E: Energetics of Enzyme Catalysis)

Edited by DANIEL L PURICH AND VERN L SCHRAMM

VOLUME 309 Amyloid, Prions, and Other Protein Aggregates

Edited by RONALD WETZEL

VOLUME 310 Biofilms

Edited by RON J DOYLE

VOLUME 311 Sphingolipid Metabolism and Cell Signaling (Part A)

Edited by ALFRED n MERRILL, JR., AND YUSUF A HANNUN

VOLUME 312 Sphingolipid Metabolism and Cell Signaling (Part B)

Edited by ALFRED H MERRILL, JR., AND YUSUF A HANNUN

VOLUME 313 Antisense Technology (Part A: General Methods, Methods of Delivery, and RNA Studies)

Edited by M IAN PHILLIPS

VOLUME 314 Antisense Technology (Part B: Applications)

Edited by M IAN PHILLIPS

VOLUME 315 Vertebrate Phototransduction and the Visual Cycle (Part A)

Edited by KRZYSZTOF PALCZEWSKI

VOLUME 316 Vertebrate Phototransduction and the Visual Cycle (Part B)

Edited by KRZYSZTOF PALCZEWSKI

VOLUME 317 RNA-Ligand Interactions (Part A: Structural Biology Methods)

Edited by DANIEL W CELANDER AND JOHN N ABELSON

VOLUME 318 RNA-Ligand Interactions (Part B: Molecular Biology Methods)

Edited by DANIEL W CELANDER AND JOHN N ABELSON

VOLUME 319 Singlet Oxygen, UV-A, and Ozone

Edited by LESTER PACKER AND HELMUT StEM

VOLUME 320 Cumulative Subject Index Volumes 290-319

VOLUME 321 Numerical Computer Methods (Part C)

Edited by MICHAEL L JOHNSON AND LUDWIG BRAND

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METHODS IN ENZYMOLOGY xxxiii

VOLUME 322 Apoptosis

Edited by JOHN C REED

VOLUME 323 Energetics of Biological Macromolecules (Part C)

Edited by MICHAEL L JOHNSON AND GARY K ACKERS

VOLUME 324 Branched-Chain Amino Acids (Part B)

Edited by ROBERT A HARRIS AND JOHN R SOKATCH

VOLUME 325 Regulators and Effectors of Small GTPases (Part D: Rho Family)

VOLUME 326 Applications of Chimeric Genes and Hybrid Proteins (Part A: Gene Expression and Protein Purification)

Edited by JEREMY THORNER, SCOTT D EMR, AND JOHN N ABELSON

VOLUME 327 Applications of Chimeric Genes and Hybrid Proteins (Part B: Cell Biology and Physiology)

Edited by JEREMY THORNER, SCOTT D EMR, AND JOHN N ABELSON

VOLUME 328 Applications of Chimeric Genes and Hybrid Proteins (Part C: Protein- Protein Interactions and Genomics)

Edited by JEREMY THORNER, ScoTT D EMR, AND JOHN N ABELSON

VOLUME 329 Regulators and Effectors of Small GTPases (Part E: GTPases Involved in Vesicular Traffic)

VOLUME 330 Hyperthermophilic Enzymes (Part A)

Edited by MICHAEL W W ADAMS AND ROBERT M KELLY

VOLUME 331 Hyperthermophilic Enzymes (Part B)

VOLUME 332 Regulators and Effectors of Small GTPases (Part F:

Ras Family I)

VOLUME 333 Regulators and Effectors of Small GTPases (Part G:

Ras Family II)

VOLUME 334 Hyperthermophilic Enzymes (Part C)

VOLUME 335 Flavonoids and Other Polyphenols

VOLUME 336 Microbial Growth in Biofilms (Part A: Developmental and Molecular Biological Aspects) (in preparation)

VOLUME 337 Microbial Growth in Biofilms (Part B: Special Environments and Physicochemical Aspects) (in preparation)

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xxxiv METHODS IN ENZYMOLOGY

VOLUME 338 Nuclear Magnetic Resonance of Biological Macromolecules (Part A) (in preparation)

Edited by THOMAS L JAMES, VOLKER DOTSCH, AND ULI SCHMITZ

VOLUME 339 Nuclear Magnetic Resonance of Biological Macromolecules (Part B) (in preparation)

Edited by THOMAS L JAMES, VOLKER DOTSCH, AND ULI SCHMITZ

VOLUME 340 Drug-Nucleic Acid Interactions (in preparation)

Edited by JONATHAN B CHAIRES AND MICHAEL J WARING

VOLUME 341 Ribonucleases (Part A) (in preparation)

Edited by ALLEN W NICHOLSON

VOLUME 342 Ribonucleases (Part B) (in preparation)

Edited by ALLEN W NICHOLSON

VOLUME 343 G Protein Pathways (Part A: Receptors) (in preparation)

Edited by RAVI IENGAR AND JOHN D HILDEBRANDT

VOLUME 344 G Protein Pathways (Part B: G Proteins and Their Regulators) (in preparation)

Edited by RAVI IYENGAR AND JOHN D HILDEBRANDT

VOLUME 345 G Protein Pathways (Part C: Effector Mechanisms) (in preparation)

Edited by RAVI IYENGAR AND JOHN D HILDEBRANDT

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[ l ] ANALYSIS AND IDENTIFICATION 3

[ 1] Overview of Methods for Analysis

a n d Identification of Flavonoids

By STEPHEN J BLOOR

The flavonoids are a class of plant secondary metabolites derived from the condensation of a cinnamic acid with three malonyl-CoA groups All flavonoids arise from this initial reaction, which is catalyzed by the chalcone synthase enzyme The chalcone is usually converted rapidly into a phenylbenzopyran, and further modification leads to the flavones, isoflavones, flavonols, or anthocyanins (Fig 1) Additional structural elaboration, mainly through glycosylation but also via acylation or alkylation, gives us the huge variety of flavonoid structures seen throughout the plant kingdom

Many books and review articles have been written on the subject of flavonoids, their occurrence, and analysis The series "The Flavonoids: Advances in Research ''1-3 provides the most comprehensive coverage of occurrence and structural variation, whereas more specific texts or articles relate to the analysis of flavonoids 4-7 Although the general methodology used for analysis and identification of flavonoids has much in common with the techniques used for many other groups of natural products, a number of useful techniques have evolved that can provide shortcuts enabling the rapid identification of flavonoid type and substitution pattern Despite the plethora of flavonoid structures presented in the scientific literature, the number of common, basic structural units remains limited; the flavone and flavonol compounds are by far the most common structural types and so are the main focus of this article Another particular advantage the ana- lyst has in flavonoid analysis is the distinctive UV (or UV-VIS) spectra of these

1 j B Harborne and T J Mabry (eds.) "The Flavonoids-Advances in Research." Chapman and Hall, London, 1982

2 j B Harbome (ed.) "The Flavonoids-Advances in Research since 1980." Chapman and Hall, London, 1988

3 j B Harborne (ed.) "The Flavonoids-Advances in Research since 1986." Chapman and Hall, London, 1994

4 K R Markham and S J Bloor in "Flavonoids in Health and Disease" (C A Rice-Evans and

L Packer, eds.), pp 1-33 Dekker, New York, 1998

5 T J Mabry, K R Markham, and M B Thomas "The Systematic Identification of Flavonoids." Springer-Verlag New York, 1970

6 K R Markham "Techniques of Flavonoid Identification." Academic Press, London, 1982

7 p M Dey and J B Harbome (eds.) "'Methods in Plant Biochemistry," Vol 1 Academic Press, London, t989

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4 SOURCES, CHARACTERIZATION, AND METHODS [1]

eg, eriodictyol chalcone

FIG 1 Basic flavonoid structural types

compounds where minor differences in structure are often seen as significant differences in their UV spectra, enabling rapid diagnosis of certain structural features Modern instrumental techniques enable us to gain much information regarding the mass and U V - V I S spectra of individual components in a complex mixture A combination of some more traditional analytical techniques combined with these modern techniques enables at least a partial identification of most flavonoid components without large-scale purification of the individual compounds Although more specific examples of applications are described elsewhere in this volume, this article gives a general overview of the typical procedures used to determine type and quantities of flavonoid compounds

For the purposes of analysis, the flavonoids can be basically classified into three types: flavonoid glycosides, nonpolar flavonoids (aglycones, methylated or alkylated flavonoids), and anthocyanins Each type requires a different analytical technique The proanthocyanidins, 8 especially the oligomeric forms, would con- stitute a fourth class but are not discussed in detail here

8 L J Porter in "The Flavonoids: Advances in Research since 1986" (J B Harborne, ed.), pp 23-53 Chapman and Hall, London, 1994

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[ 1] ANALYSIS AND IDENTIFICATION 5

C h r o m a t o g r a p h i c A n a l y s i s

Thin-Layer Chromatography (TLC) [or Paper Chromatography (PC)]

Historically, paper chromatography has been the preferred method for flavonoid analysis, and relative mobility data are available for a large variety of compounds.9,10 However, not all laboratories are equipped to perform PC, but TLC

on cellulose sheets (e.g., Schleicher and Schuell, Keene, NH, Avicel) is a useful alternative Cellulose TLC is especially useful for quick analyses of materials containing flavonoids, especially flavonoid glycosides The relative mobility and appearance of spots under UV, before and after spraying with various reagents, enable a good approximation of structural type Two solvent systems are routinely used for such preliminary analyses: 15% acetic acid (acetic acid : H20, 15 : 85) and TBA (t-BuOH : acetic acid : H20, 3 : 1 : 1) Rough relative mobilities for various flavonoid types are shown in Table I Typically, plastic-backed sheets are cut to a suitable size, e.g., 5 cm wide × 7 cm high, the samples are applied as small 1- to 2-mm spots, allowed to dry, and the sheets developed in a glass tank (a slide-staining jar is useful for small sheets) The relative mobilities in TBA and 15% acetic acid should give a rough guide as to flavonoid type Sheets are dried and viewed under UV light (366 nm) The significance of the spot color and the behavior on exposure to ammonia vapor are detailed in Table II The sheet is then sprayed with NA reagent (a 1% solution of diphenylboric acid-ethanolamine complex in methanol), dried, and again viewed under UV light Most flavonoids show some color, but most significantly 3', 4'-dihydroxyflavones or flavonols are orange and the 4'-hydroxy equivalents are yellow-green If desired, a fingerprint type of two-dimensional chromatogram can be prepared by applying one sample only on a spot in one corner of the sheet, developing the sheet with TBA in one dimension, and then, after drying, developing in the second dimension with 15% acetic acid Generally, most flavonoids will be separated on two-dimensional TLC and their spot characteristics can be noted Silica TLC is also a useful screening system for flavonoid compounds 11 Ethyl acetate-formic acid-acetic acid-H20 (100 : 11 : 11 : 27) gives a good range of mobilities for flavone and flavonol glycosides (Rf : diglycosides < monoglycosides < aglycones) and, as discussed earlier, most spots can be visualized with the NA reagent Anthocyanins are best analyzed

by TLC using a quite acidic solvent system A useful general eluting solvent for cellulose TLC of these compounds is HC1 :formic acid : H20 (1 : 1:2) 12 The intensely colored spots (violet for trihydroxylated B-ring, red for dihydroxy and orange for mono-) do not require spraying, and generally the R f increases as the

degree of glycosylation increases

9 K R Markham and R D Wilson Phytochem Bull 20, 8 (1988)

10 K R Markham and R D Wilson Phytochern Bull 21, 2 (1989)

I I H Wagner, S Bladt, and E M Zgainski "Plant Drug Analysis." Springer-Verlag, Berlin, 1983

12 O M Andersen and G W Francis J Chromatogr 318, 450 (1985)

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6 SOURCES, CHARACTERIZATION, AND METHODS [ 1 ]

TABLE I Two-DIMENSIONAL PC MOB1LITY a OF VARIOUS FLAVONOID TYPES IN TBA AND 15% ACETIC ACID b

and anthocyanidins

and cinnamic acids

'~ Mobility code (typical Rf range): low, 0 to 33 med, 0.33 to 0.65; high, 0.66 to 1.0; "+" indicates

high end of range Hyphenated categories indicate a range covering both

b Reproduced from K R Markham and S J Bloor, in "Flavonoids in Health and Disease" (C A Rice-Evans and L Packer, eds.), pp 1-33 Dekker, New York, 1998, with permission

H P L C

T h e distinctive U V - V I S spectra o f m o s t flavonoids and the w i d e s p r e a d avail- ability o f h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) s y s t e m s w i t h multi-

w a v e l e n g t h capability, or the ability to r e c o r d o n - l i n e spectra, has m e a n t H P L C is

n o w the m e t h o d o f c h o i c e for f l a v o n o i d a n a l y s i s J 3-15 M o s t often separations are

p e r f o r m e d u s i n g a r e v e r s e d - p h a s e c o l u m n (RP-18) and a g r a d i e n t elution s y s t e m starting w i t h a p r e d o m i n a n t l y a q u e o u s phase and i n t r o d u c i n g an i n c r e a s i n g p r o -

p o r t i o n o f an organic s o l v e n t such as m e t h a n o l or acetonitrile A s m o s t flavonoids are ionizable, s o m e acid is usually added to the m o b i l e phase to control the pH

13 K Vande Casteele, C Van Sumere, and H Geiger J Chromatogr 240, 81 (1982)

14 C Van Sumere, P Fache, K Vande Casteele, L De Cooman, and E Everaert Phytochem Anal 4,

279 (1993)

15 M C Pietrogrande and Y D Kahie J Liq Chrom 17, 3655 (1994)

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TABLE II INTERPRETATION OF TYPICAL FLAVONOID SPOT COLORS ON Two-DIMENSIONAL PC a

Dark purple Yellow or yellow-green shades Flavones (5- and 4'-OH)

Flavonols (3-OR and 4/-OH) Red or orange Chalcones (2/-OH, with free 2- or 4-OH) Little change Flavone C-glycosides (5-OH)

Flavones (5-OH and 4~-OR) Flavones (5-OH and 3,4'-OR) Isoflavones (5-OH) Dihydroflavones (5-OH) Dihydroflavones (5-OH) Biflavonyls (5-OH) Flavonols (3-OH) murones Anthocyanins/anthocyanidins (Pelargonidin-3, 5-OR, yellow fluorescence) Flavones (5-OR) Dihydroflavones Flavonols (3,5-OR) Isoflavones (5-OR) Cinnamic acids and derivatives

Yellow fluorescent Little change

Red or orange Magenta, pink, Blue with time

yellow fluorescence

Blue fluorescence Yellow-green or

blue-green

No change Brighter blue

a Reproduced from K R Markham and S J Bloor, in "Flavonoids in Health and Disease" (C A Rice-Evans and L Packer, eds.), pp 1-33 Dekker, New York, 1998, with permission

b -OR, O-glycoside or O-alkyl

The order of elution from most polar through to least polar means triglycosides (and higher glycosides) are eluted early, along with most anthocyanin glycosides, followed by di- and m o n o g l y c o s i d e s and then acylated or alkylated glycosides and aglycones The requirement for a low p H solvent system for anthocyanins, a high organic modifier content for aglycones, and an extended gradient system to separate the many possible mono- and diglycosidic combinations means no one solvent system will give optimal separation o f all flavonoid types

Three useful gradients solvent systems are outlined All are designed for use with the same RP-18 column [Merck Lichrospher 100 RP-18 endcapped (5 Ixm, 11.9 × 4 cm) or Supersphere (4 ~m)] The first is a general-purpose solvent system with high acid content and relatively fast total analysis time for routine analysis

o f plant extract, including those containing anthocyanin pigments Table III gives

a list o f retention times for a variety o f flavonoid compounds using this system Note the bunching together o f the aglycones at the end o f the chromatographic run The second system is designed for m a x i m u m resolution o f flavone and flavonol

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HPLC RETENTION TIMES FOR Flavonoids a'b

Retention Anthocyanidin glycosides Anthocyanidins Flavonoid aglycones Flavone/flavonol glycosides time (rains.)

Luteolin Y-O-glucoside 19.6 Luteolin 5-O-glucoside 19.6

Luteolin 7-O-glucoside 20.4

Quercetin 3-O-glucoside 21.0 Kaempferol 3-O-glucoside 21.0

Apigenin 7-O-neohesperidoside 27.7 Kaempferol 3-O-rhamnoside 29.2

aReproduced from K R Markham and S J Bloor, in "Flavonoids in Health and Disease" (C A Rice-Evans

and L Packer, eds.), pp 1-33 Dekker, New York, 1998, with permission

9HPLC column and conditions as outlined in text [solvent system (1)]

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[ 11 ANALYSIS AND IDENTIFICATION 9 glycosides A slow increase in acetonitrile content between 0 and 52 min is used, but most glycosides elute between 25 and 40 min The third solvent system is useful for nonpolar flavonoids, such as those found on the leaf surface, and uses a high methanol content

1 Elution (0.8 ml/min) is performed using a solvent system comprising solvents

A (1.5% H3PO4) and B [acetic acid: CH3CN : H3PO4 : H20 (20:24 : 1.5 : 54.5)] mixed using a gradient starting with 80% A, linearly decreasing to 33% A after

30 min, 10% A after 33 min, and 0% A after 39.3 min (column temperature 30°)

2 Solvents A (water adjusted to pH 2.5 with H3PO4) and B (CH3CN) are mixed

in a gradient (0.8 ml/min) starting with 100% A, linearly decreasing to 91% A after

12 min, 87% A after 20 min, 67% A after 40 min and then held at 67% A for 2 min, then a linear decrease to 57% A after 52 rain, and then finally to 0% A at 55 min (column temperature 24°)

3 Elution (1.0 ml/min) is performed using a solvent system comprising 5% formic acid in water (A) and methanol mixed according to a gradient, starting with 65% A, linearly decreasing to 55% A after 10 min, held at 55% A until 20 min, then linearly decreased to 20% A at 55 min, and 5% A at 60 min (column temperature 30°)

The utility of HPLC is best illustrated by an example such as that shown

in Fig 2 A mixture of flower extracts has been used to present a broad spectrum of compound type Detection in the range of 340-360 nm is suitable for flavones and flavonols (e.g., 352 nm for chromatograms in Fig 2) In the crude mixture the series of peaks can be grouped based on analysis of on-line spectra (Fig 3), as luteolin or apigenin flavone glycosides, kaempferol glycosides, an obvious chalcone glycoside, and possibly some aromatically acylated flavonoid glycosides Alkaline hydrolysis (see later) removes any acyl groups, dramatically increasing the relative level of one of the kaempferol glycosides, confirming that the acylated late-eluting compounds are acyl derivatives of compound 3 (Fig 3) Acid hydrolysis (see later) to cleave O-glycosides gives the expected peaks for luteolin and kaempferol, but the peaks assigned to apigenin glycosides are still present, indicating these are flavone C-glycosides This example demonstrates the considerable amount of information that can be gleaned from a few small-scale experiments

Quantification of flavonoids is another forte of HPLC in combination with

UV detection In Fig 2a, a rough estimate of the flavonoid level can be arrived

at by comparing integration data for that chromatogram with that from the injec- tion of a known amount of a readily accessible standard such as rutin (quercetin 3-rutinoside) run under the same conditions A more rigorous quantification will involve the use of several standard compounds; in this case, a flavone glycoside

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t _ L _ ,

15 2 0 25

J 3'0 3~ ,0 4 5 50

a large relative increase in peak 3 and loss of the acylated kaempferol glycoside peaks; and (c) the acid-hydrolyzed mixture showing luteolin and kaempferol [8] Peaks due to apigenin glycosides are still present, showing these are apigenin C-glycosides

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12 SOURCES, CHARACTERIZATION, AND METHODS [ 1 ]

In many cases, some pretreatment of extracts is advisable prior to analysis Chlorophyll can be removed by washing the aqueous alcoholic extract with hexane or diethyl ether, or the source material may be preextracted with such a solvent Excessive amounts of sugars (e.g., in fruit extracts) may be removed by solid-phase extraction, e.g., on a RP SPE column In this case, most of the alcohol in the extract should be evaporated, the sample made up to >90% water, and applied to the preconditioned cartridge The cartridge is washed with water and the flavonoids eluted with alcohol Some extracts have excessive quantities of nonflavonoid phenolic acids, which overwhelm the flavonoids, making analysis difficult These can often be removed by SPE with cellulose The sample is dried, reconstituted in 1% acetic acid in water, and applied to a small cellulose column; washing with 2% acetic acid removes most of the phenolic acids and the flavonoids are recovered by washing the column with alcohol

S t r u c t u r a l A n a l y s i s b y H y d r o l y s i s

The almost limitless range of flavonoid glycosides means that for most analyses the starting point for identification is the recognition of the flavonoid aglycone Acid hydrolysis will cleave most sugars from flavonoid glycosides 6,16 For this type of hydrolysis the flavonoid (0.25-0.5 mg) mixture is dissolved in 300 ixl 2 N HC1 : methanol (1 : 1 v/v) or in trifluoroacetic acid (TFA) : methanol (1 : 1 v/v), sealed in a screw-top polypropylene tube, and heated on a steam bath for 30 rain The sample can then be analyzed directly by TLC or HPLC, and the aglycone iden- tified by comparison with common standard compounds For TLC, better results are obtained if the posthydrolysis mixture is extracted with an equal volume of ethyl acetate or isoamyl alcohol The upper layer is evaporated to dryness, redis- solved in methanol, and analyzed The lower aqueous layer can be used for analysis

of sugars Most common flavonoid O-glycosides undergo hydrolysis under these conditions; however, glycuronides generally require more extreme conditions, e.g.,

2 hr at 100 ° Flavonoid C-glycosides are not cleaved under these conditions, but the sugar can isomerize between the C-6 and the C-8 positions 6

Alkaline hydrolysis is generally only employed if the presence of acyl groups

is suspected To avoid oxidation of the flavonoid, these hydrolyses should be performed in the absence of air A syringe or small sealed polypropylene vial may

be used An equal volume of 2 M NaOH is added to the flavonoid sample solution (generally the sample becomes intensely colored) and left at room temperature for

2 hr The reaction is halted by neutralization with acid (returned to original color) The sample can then be analyzed directly by HPLC or TLC Once again, better analytical results are achieved if the hydrolysis mixture is partially purified, in this case RP SPE is best Comparison of chromatograms of crude and hydrolyzed

16 j B Harborne, Phytochemistry 4, 107 (1965)

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material will show which flavonoids are acylated (see section on HPLC) Aromatic acyl groups, from acid or alkaline hydrolysis, such as p-coumaric or caffeic acid are detected easily by HPLC, whereas the less easily detected aliphatic acids are more suited to TLC or (after derivatization) gas chromatography

Enzymatic hydrolysis is used occasionally with fiavonoids and can provide se- lective cleavage of 3-0- or 7-O-glycosides 6' 13 These hydrolyses can be performed

by dissolving the sample (0.34).5 mg) in water (500 Ixl), adding about 10 mg enzyme (e.g., 13-glucosidase), and standing at room temperature The sample is then analyzed as described earlier

Individual flavonoids are dissolved in methanol (concentration such that the maximum absorbance is between 0.05 and 1.00 AU), and the basic spectrum is measured Most flavonoids show a band in the 210- to 290-nm region (band II) and a second band at 320-380 nm (band I) Compilations of spectral data are available for comparison, s'I7 For anthocyanins, the latter band is in the visible region (490-540 nm)) s

The procedure for shift reagent tests and the resultant shifts for flavones or flavonols 5'6 are as follows

i Two to three drops of 2.5% NaOMe in methanol are added directly to the cuvette containing the methanolic solution of the flavonoid A 45- to 65-nm shift

in band 1 of flavones and flavonols with no loss of intensity indicates a free 4'-OH

A decrease in intensity of this band indicates a substituted 4'-OH If band I reduces

or degrades after several minutes, then either a free 3,4'-OH or three adjacent OHs are likely, and if a new low intensity band appears at 320-335 nm, a free 7-OH is indicated

ii Several milligrams of solid sodium acetate are added to a fresh methanolic solution of the flavonoid A shift of band II to longer wavelength indicates a free 7-OH

17 L Jurd in "The Chemistry of Flavonoid Compounds" (T A Geissman, ed.), pp 107-155 Pergamon Press, Oxford, 1962

t8 E J Francis in "'Anthocyanins as Food Colors" (P Markakis, ed.), pp 181-207 Academic Press, New York, 1982

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iii Several milligrams of solid boric acid are added to the solution from step

ii to diagnose for the presence of ortho di-OH groups Without these groups the spectrum will revert to the original methanol spectrum Movement of band I (12-36 nm) indicates an ortho di-OH group(s)

iv Another methanolic flavonol solution is prepared and tested for response to A1C13 A few drops of 5% A1C13 in methanol solution are added, and a shift of band II by 20 to 40 nm again indicates ortho di-OH group Two to three drops of 20% aqueous HC1 are then added to the methanol/A1C13 flavonoid solution, and a shift o f 35 to 70 nm indicates a free 5- and/or 3-OH

These shift reagents have proven to be extremely useful guides to substitution patterns for a variety of flavonoids Little material is required and the tests are performed directly in the sample cuvette, taking only a few minutes to complete Quantification of flavonoids is often performed by U V - V I S spectroscopy However, the contribution of interfering compounds in a mixture should be ac- counted for Anthocyanins are generally free of interference if the measurements are made at the visible maximum (500-540 nm, measure at pH 1.0) A useful e value is 30,175 for the c o m m o n glycoside, cyanidin 3,5-diglucoside 13 Flavones and flavonol glycosides can be measured at their band I maxima of 340-360 nm and a general ~ value of 14,500 used (i.e., in a 1-cm path length cell, a 1 M solution will give a absorption value of 14,500 AU; specific values for many flavonoids are available)

For many plant extracts the presence of cinnamic acid derivatives are a problem when quantifying flavonoids using this wavelength range of the U V - V I S spectrum Most cinnamic acids can be removed by SPE with cellulose (see earlier)

The overview presented here has only skimmed the surface of this subject There are many steps separating a quick analysis of a mixture and the accurate determination of the structural features of each constituent flavonoid A number of techniques used in flavonoid analysis such as nuclear magnetic resonance, (NMR), electrophoresis, and liquid chromatography-mass spectrometry (LC-MS) have not been discussed and the reader is referred to more detailed reviews (13C NMR, 19 1H N M R 2°, electrophoresis21-24) Some of these methods are also discussed in other articles in this volume that deal with more specific examples of flavonoid analysis

19 K R Markham and V M Chaff in "The Flavonoids: Advances in Research" (J B Harborne and

T J Mabry, eds.), pp 19-134 Chapman and Hall, London, 1982

20 K R Markham and H Geiger in "The Flavonoids: Advances in Research since 1986" (J B Harborne,

ed.), pp 441-497 Chapman and Hall, London, 1993

21 E A Tomas-Barberan Phytochem Anal 6, 177 (1995)

22 p Pietta, P Mauri, A Bruno, and C Gardana Electrophoresis 15, 1326 (1994)

23 T K McGhie and K R Markham Phytochem Anal 5, 121 (1994)

24 K R Markham and T K McGhie Phytochem Anal 7, 300 (1996)

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[2] HPLC ELECTROCHEMICAL DETECTION METHODS 15

[21 Analysis of Complex Mixtures of Flavonoids and Polyphenols by High-Performance Liquid

as well as efficacy studies in human and animal trials

Flavonoids and polyphenols, derived from plant or animal sources, comprise an enormous number, exceeding 4000 individual analytes As a group, flavonoids are plant secondary metabolites based on the structure of a pyran ring flanked by at least two phenyl rings, designated as A and B rings (Fig 1) They vary from one another subtly in the degree of unsaturation, the pattern of hydroxylation or methylation, and type of sugar attached or the degree of polymerization Flavonoids in most abundance are classified as flavonols, anthocyanidins, and proanthocyanidins Considering such numbers and complexity, even the best high-performance liquid chromatography (HPLC) methods cannot resolve and separate all phenolic compounds from complex mixtures and often elaborate preparative procedures are necessary to investigate some selected and unique compounds Further com- plicating analysis is the limited number of authentic or purified standards available for many of the flavonoids Fortunately, the majority of the flavonoids constituent

in significant amounts in the human diet and in "natural" medicinal preparations number far fewer than the total number known to exist

Separation methods have been reviewed I and equally adequate HPLC methods have been described previously 2 The majority of chromatographic methods utilized for the study of these colorful and UV absorptive plant compounds have naturally favored UV/visible detection Photodiode array detectors facilitate col- lection of spectra of unknown flavonoids during chromatographic analysis The absorption spectrum can help identify a compound if it is cleanly resolved and if

I D J D a i g l e a n d E J C o n k e r t o n , J Liquid Chrom 11, 3 0 9 (1988)

2 A L Waterhouse, S E Price, a n d J D M c C o r d , Methods Enzymol 299, 113 (1999)

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of electrochemical array detection

A significant body of literature has accumulated, primarily from in vitro in- vestigations, regarding the antioxidant properties of flavonoids and other plant polyphenolics 3 As the essence of redox chemistry involves electron transfer, it would seem natural that electrochemical detection would rival UV, visible, and fluorescent detection for those compounds that are purported antioxidants Until recently, technical difficulties inherent in the instrumental methods required to op- erate electrochemical detectors made their use, to say the least, inconvenient With improvements over the last decade in both electrochemical detector geometries and electronics, coupled with a requirement for increased sensitivity, as the field moves from analysis of plant composition to bioavailablility and bioactivity studies, the use of electrochemical detectors may offer significant additional advantages when combined with traditional UV/VIS detection in the analysis of plant polyphenols

3 C A Rice-Evans, N J Miller, and George Paganga, Free Radic Biol Med 20, 933 (1996)

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