Chemical Properties of Phenolic Compounds
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Chapter title i PHENOLIC COMPOUND BIOCHEMISTRY Contents iii PHENOLIC COMPOUND BIOCHEMISTRY By WILFRED VERMERRIS University of Florida, Gainesville, FL, U.S.A. and RALPH NICHOLSON Purdue University, West Lafayette, IN, U.S.A. iv Contents A C.I.P. Catalogue record for this book is available from the Library of Congress. ISBN-10 1-4020-5163-8 (HB) ISBN-13 978-1-4020-5163-0 (HB) ISBN-10 1-4020-5164-6 (e-book) ISBN-13 978-1-4020-5164-7 (e-book) Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. www.springer.com Printed on acid-free paper All Rights Reserved © 2006 Springer No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Contents v CONTENTS PREFACE xi CHAPTER 1 Families of Phenolic Compounds and Means of Classification 1. Definitions 1 2. Classification 2 3. Classes of phenolic compounds 3 3.1 Simple phenolics 3 3.2 Phenolic acids and aldehydes 4 3.3 Acetophenones and phenylacetic acids 4 3.4 Cinnamic acids 5 3.5 Coumarins 6 3.6 Flavonoids 7 3.6.1 Chalcones 8 3.6.2 Aurones 8 3.6.3 Flavonoids 9 3.6.3.1 Flavanones 10 3.6.3.2 Flavanonols 10 3.6.3.3 Leucoanthocyanidins 11 3.6.3.4 Flavones 12 3.6.3.5 Anthocyanidins and deoxyanthocyandins 12 3.6.3.6 Anthocyanins 15 3.7 Biflavonyls 15 3.8 Benzophenones, xanthones and stilbenes 16 3.9 Benzoquinones, anthraquinones and naphthaquinones 17 3.10 Betacyanins 17 3.11 Lignans 18 3.12 Lignin 20 3.13 Tannins 23 3.13.1 Condensed tannins 24 vi Contents 3.13.2 Gallotannins 25 3.13.3 Ellagitannins 26 3.13.4 Complex tannins 29 3.14 Phlobaphenes 30 4. References 32 CHAPTER 2 Chemical Properties of Phenolic Compounds 1.1 The benzene ring 35 1.2 Planar versus non-planar bonds 38 1.3 The acidic nature of the phenolic hydroxyl group 38 1.3.1 The effects of substituents on acidity 39 1.3.2 Use of acidity in separations 40 1.4 Hydrogen bonding and the phenolic hydroxyl group 40 1.4.1 Intra- and inter-molecular hydrogen bonds 41 1.4.2 Stability of the hydrogen bond ring 41 1.5 Metal complexes 43 1.6 Esterification 45 1.7 Ethers and glycosides 47 1.8 Oxidation of the phenolic hydroxyl group 48 1.8.1 Auto-oxidation of phenolic compounds 48 1.8.2 Enzymatic oxidation of the phenolic hydroxyl group 50 1.8.2.1 E.C. 1.10.3 50 1.8.2.2 E.C. 1.14.18.1 53 1.8.2.3 E.C. 1.11.1. 53 1.9 Reactive oxygen species and antioxidants 58 2. References 61 CHAPTER 3 Biosynthesis of Phenolic Compounds 1. Introduction 63 2. Protein isolation and purification 64 3. Gene cloning strategies 67 3.1 Insertional mutagenesis 70 3.2 Map-based cloning 72 3.3 The candidate-gene approach 72 3.4 QTL mapping 73 4. Isolation and characterization of recombinant proteins 74 5. Carbohydrate catabolism 76 5.1 Glycolysis 77 5.2 The pentose phosphate pathway 77 6. The shikimate pathway 81 Contents vii 7. The general phenylpropanoid pathway 84 8. Biosynthesis of phenolic acids 86 8.1 Salicylic acid 86 8.2 Gallic acid 88 9. Biosynthesis of flavonoids and condensed tannins 90 9.1 Structural genes and enzymes 90 9.2 Regulatory genes 96 10. Monolignol biosynthesis 102 11. Lignan biosynthesis 107 12. Lignin biosynthesis 112 12.1 Genetic control of lignification 112 12.2 Monolignol transport and polymerization 116 12.3 Modification of lignin for agro-industrial applications 120 12.3.1 Pulp and paper industry 121 12.3.2 Forage and silage quality 123 12.3.3 Ethanol production from ligno-cellulosic biomass 124 13. Hydroxycinnamic acid biosynthesis 125 14. Biosynthesis of sinapoyl esters 126 15. Coumarin biosynthesis 129 16. Stilbene biosynthesis 129 17. Biosynthesis of gallotannins and ellagitannins 130 18. References 134 CHAPTER 4 Isolation and Identification of Phenolic Compounds 1. Introduction 151 2. Isolation of phenolic compounds 151 2.1 Total phenolic content: the Folin-Ciocalteu reagent 152 2.2 Determining the content of condensed tannins 154 2.2.1 The butanol-HCl assay 154 2.2.2 The vanillin assay 154 2.2.3 Precipitation of condensed tannins with formaldehyde 155 2.3 Determining the content of gallotannins 155 2.3.1 The potassium iodate assay 155 2.3.2 The rhodanine assay 156 2.4 Determining the content of ellagitannins 157 2.4.1 Nitrous acid oxidation 157 2.4.2 The NaNO2/HCl assay 158 2.5 Determining lignin content 159 2.5.1 Klason lignin 160 2.5.2 Acid detergent lignin 161 2.5.3 Thioglycolic acid lignin 162 2.5.4 Acetylbromide lignin 164 viii Contents 3. Identification and characterization of phenolic compounds 166 3.1 Thin layer chromatography 166 3.2 Liquid chromatography: HPLC and LC-MS 169 3.3 Gas chromatography 170 3.4 Methods for the identification of lignin subunit composition 171 3.4.1 The nitrobenzene oxidation 172 3.4.2 Thioacidolysis 173 3.4.3 Derivatiation Followed by Reductive Cleavage 176 3.4.4 Analytical pyrolysis 178 3.4.5 Nuclear magnetic resonance 178 3.4.6 Fourier-transform infrared spectroscopy and near 181 infrared reflectance spectroscopy 4. Visualization of phenolic compounds in planta using 183 histochemical stains 4.1 An overview of histochemical staining protocols 183 4.2 Visualizing plant-pathogen interactions involving phenolics 185 with histochemical stains 5. References 190 CHAPTER 5 Analysis of Phenolic Compounds with Mass Spectrometry 1. The principles of mass spectrometry 197 1.1 Ionization 197 1.2 Mass Analysis 198 1.3 Detectors 199 1.4 Sample introduction 200 2. New developments in mass spectrometry 202 2.1 Fast atom bombardment 202 2.2 Plasma desorption ionization 203 2.3 Electrospray ionization 204 2.4 Matrix-assisted laser desorption ionization 206 3. Quantitation 208 4. References 210 CHAPTER 6 The Role of Phenols in Plant Defense 1. Preformed antimicrobial and insecticidal metabolites 211 1.1 Chlorogenic acid 213 1.2 Phloridzin and phloretin 214 1.3 Cyanogenic glycosides 215 1.4 Tuliposides 216 1.5 Protocatechuic acid 217 Contents ix 1.6 Lignin 217 1.7 C-glycosyl flavones 217 2. Compounds formed in response to pathogen attack 222 2.1 3-Deoxyanthocyanidins 222 2.2 Pisatin 224 2.3 Stilbenes 224 2.4 Salicylic acid 225 2.5 Lignin 227 3. References 230 CHAPTER 7 Phenolic Compounds and their Effects on Human Health 1. Introduction 235 2. Antioxidant properties 237 3. Disease prevention 246 4. Activity against toxins 251 5. References 253 Appendix 257 Index 267 Contents xi PREFACE Phenolic compounds represent a large group of molecules with a variety of functions in plant growth, development, and defense. Phenolic compounds include signaling molecules, pigments and flavors that can attract or repel, as well as compounds that can protect the plant against insects, fungi, bacteria, and viruses. Most phenolic compounds are present as esters or glycosides rather than as free compounds. Tannins and lignin are phenolic polymers. Tannins are used comercially as dyes and astringents, and lignin accounts for structural ridgidity of cells and tissues and is essential to vascular development. From this brief overview it is apparent that phenolic compounds make up a large and fascinating family. Our intention is to provide the reader with an overview of this family of compounds that will show their diversity and provide a basis for continued investigations. The target audience is intended to include phytopathologists, botanists, agronomists, entomologists, and people with a general interest in plant biochemistry. This book will also be a valuable resource when used as a textbook in a course on phenolic compounds, aimed at advanced undergraduate or beginning graduate students in the life sciences. While writing this book we assumed the reader would have basic knowledge of organic chemistry, biochemistry of DNA, RNA, proteins and lipids, and cell physiology. At the end of the chapters we have provided some additional references for further reading, either to obtain background information, or to obtain more details. The focus of this book is centered on structure, nomenclature and occurrence of phenolic compounds (Chapter 1), and their chemical properties (Chapter 2). Chapter 3 describes the biosynthetic pathways leading to the major classes of phenolics. This chapter presents an up-to-date overview of the genetic approaches that have been used to elucidate these pathways. Chapter 4 presents an overview of methods for the isolation and identification of plant phenolic compounds. Given that much of the recent xi xii PContentsreface advances in the identification of phenolic compounds have been made possible through the development of mass spectrometry, we have dedicated an entire chapter to the use of mass spectrometry in the identification of phenolic compounds (Chapter 5). This chapter was written by Dr. Karl V. Wood of the Mass Spectrometry Facility in the Department of Chemistry at Purdue University. We are grateful for his contribution to this book. Chapter 6 highlights the role of phenolic compounds in plant defense. We have included a number of examples, including phytoanticipins, phytoalexins and lignin synthesized in response to pathogen attack, to complement the more chemical nature of the other chapters, and to illustrate the important role phenolic compounds play in plant survival.