ISOLATION AND CHARACTERIZATION OF APIGENIN-7-B-GLUCURONIDE FROM SNAPDRAGON (ANTIRRHINUMMAJUSL.) A Thesis Presented to Faculty of Graduate Studies of The University of Guelph by LINDA JEAN VELDHUIS In partial fulfillment of requirements for the degree of Master of Science August 2008 © L. J. 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The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada ABSTRACT ISOLATION AND CHARACTERIZATION OF APIGENIN-7-B-GLUCURONIDE FROM SNAPDRAGON (ANTIRRHINUM MAJUS L.) Linda Jean Veldhuis Advisor: University of Guelph, 2008 Professor JC Hall The greenhouse industry uses hydroponic systems for the production of ornamental plants. Government regulations have forced the growers to reuse hydroponic solutions resulting in potential accumulation of allelopathic compounds that may affect subsequent crops. This study focuses on the identification and characterization of the flavone, apigenin-7-|3-glucuronide, that was found in snapdragon, an important greenhouse crop in Ontario, CA. The allelopathic activity of apigenin-7-p-glucuronide was examined using several types of bioassays, i.e., germination and growth of lemna, bentgrass, lettuce, carrot and snapdragon, and a bacterial growth assay using Pseudomonas aeruginosa. There was biological activity of the purified compound with lettuce and carrot germination at 250 mM. Whereas, the aglycone, apigenin, inhibited bacterial growth at 50, 100 and 250 mM, and stimulated the germination of lettuce at all concentrations except 1 mM and snapdragon seed at 250 mM. Therefore, apigenin-7-p-glucuronide isolated from snapdragon does not pose a potential problem for greenhouse producers. ACKNOWLEDGEMENTS This was a great experience for me. I was able to test a number of my ideas, and hypotheses putting some of my experience to good use as well, learn how to write scientifically. I would like to extend a great thank you to Dr. J. Christopher Hall. He has been my advisor, my mentor and my friend. He was supportive as I worked part-time on this project. I would like to thank the members of my advisory committee, Dr. Gerry Stephenson and Dr. Keith Solomon for their guidance and assistance in the preparation of this thesis. I'd also like to thank Ms. Yongqing Niu and Dr. Mithila Jugulam for their assistance and friendship throughtout my studies and to everyone in the Hall Lab who also provided support and friendship during my studies. I would also like to thank Dr. Clifford Beninger for his assistance in isolating large quantities of the main flavonoid and Dr. Donald Hughes from McMaster University for running samples and his advice on the analysis of the NMR data. Finally I would like to thank my parents and my kids: Misty, Lucy and Pepper for their continual love, support and encouragement. i LIST OF ABBREVIATIONS 4CL - 4-coumaroyl:CoA ligase ALS - advanced life support ap-7-p-gluc - apigenin-7-(3-glucuronide APCI - atmospheric pressure chemical ionisation BAMT - S-adenosyl-L-methione benzoic acid carboxyl methyl transferase BAW - butanol-acetic acid-water CFU - colony forming units CHH - cinnamate-4-hydroxylase CHI - chalcone isomerase CHR - chalcone reductase COSY - correlation spectroscopy DAT - days after treatment DFR - dihydroflavonol-4-reductase DMID - 7,2'-dihydroxy 4'-methylisoflavanol-dehydratase ESI - electrospray ionization F3'5'H - flavonoid 3',5' hydroxylase F3'H - flavonoid-3'-hydroxylase FNS II - flavone synthase II FSI - flavone synthase I H3PO4 - phosphoric acid HCL - hydrochloric acid HMBC - heteronuclear multiple bond correlation ii HPLC - high pressure liquid chromatography HSQC - heteronuclear single quantum coherence I1'H - isoflavon-2'-hydroxylase IFR - isoflavone reductase IFS - isoflavone synthase LCMS - liquid chromatography mass spectrometry LCR - leucoanthocyanidin reductase LDOX - leucoanthocyanidin dioxygenase NADPH - nicotinamide adenine dinucleotide phosphate NMR - nuclear magnetic resonance ODS - octadecyl silane OMT - O-methyltransferase PAL - phenylalanine PBS - phosphate buffered saline PGHS-1(2) - prostaglandin endoperoxide hydrogen synthase-1 (2) PHC - 2',4',6',3,4-pentahydroxylchalcone ROESY - rotational nuclear Overhauser effect spectroscopy RT - rhamnosyltransferase STS - stilbene synthase TFA - trifluoroacetic acid THC - 2',4',6',4-tetrahydroxychalcone TLC - thin layer chromatography UFGT - UDPG-flavonoid-glycosyl transferase iii UV-VIS - ultraviolet - visible VR - vestitone reductase TABLE OF CONTENTS ACKNOWLEDGEMENTS i LIST OF ABBREVIATIONS iii TABLE OF CONTENTS v LIST OF TABLES vii LIST OF FIGURES viii 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 4 2.1 Allelopathy 4 2.1.1 Definition 4 2.1.2 History 6 2.1.3 Allelochemicals in agriculture 7 2.1.4 Allelochemicals as secondary plant metabolites 9 2.1.5 Testing methods for phytotoxic allelochemicals 11 2.1.5.1 Method 1, Seed Germination 12 2.1.5.2 Method 2, Lemna Bioassay 14 2.1.5.3 Method 3. Root Growth/Radicle Elongation Bioassay 16 2.1.5.4 Method4. Bacterial Bioassay 16 2.2 Phenolic Compounds 17 2.2.1 Introduction 17 2.3 Flavonoids 19 2.3.1 Introduction 19 2.3.2 Flavonoid identification 27 2.3.2.1 Extraction and analysis methods for flavonoids 27 2.3.3 Colour and flavonoid production 28 2.3.4 Biosynthetic pathway for flavonoids 29 2.4 Snapdragon 32 2.4.1 Introduction 32 2.4.2 Phenolic identification 32 2.4.3 Isolation methods of flavonoids from snapdragons 33 2.4.4 Allelopathic compound identification 34 2.5 Hypothesis and Objectives 39 2.6 Possible Applications 39 v 3 METHODOLOGY 40 3.1 Growth of plants 40 3.2 Isolation and purification 40 3.3 Lemna bioassay 43 3.4 Chlorophyll assay 44 3.5 Germination assay 44 3.6 Bacterial assay 45 3.7 Statistical Analysis 46 4 RESULTS 46 4.1 Isolation and identification 46 4.2 Lemna Bioassay 58 4.3 Seed Germination and Growth Assay 59 4.4 Bacterial Activity Assay 69 5 DISCUSSION 69 6 CONCLUSION 73 7 LITERATURE CITED 76 8 APPENDIX 91 8.1 NMR results for the isolated compounds: 91 8.1.1 NMR Proton spectra for apigenin-7-B-glucuronide 91 8.1.2 NMR Carbon spectra for apigenin-7-B-glucuronide 98 8.1.3 NMR Carbon spectra negative ion mode for apigenin-7-B-glucuronide 99 8.1.4 NMR Carbon HMBC spectra for apigenin-7-B-glucuronide 100 8.1.5 NMR Proton-Carbon Gradient HSQC 101 8.2 Mass spectrometric data for the isolated compounds: 102 8.2.1 Apigenin using negative ion mode 102 8.2.2 Apigenin using positive ion mode 103 8.2.3 Apigenin-7-B-glucuronide using negative ion mode 104 8.3 Other materials: 105 8.3.1 TLC plates 105 VI LIST OF TABLES TABLE 1. Structures of seven of the subclasses belonging to the flavonoids 21 TABLE 2. Glycosidic flavonoids in Antirrhinum majus and the flower colour for which they are responsible 38 TABLE 3. 1H Chemical Shifts and coupling constants for apigenin- 7-P-glucuronide in DMSO-d6. 52 TABLE 4. 13C Chemical Shifts for apigenin-7-(3-glucuronide in DMSO-de 57 vii LIST OF FIGURES FIGURE 1. Relationship of plant interference with competition and allelopathy (Fuest, 1983) 6 FIGURE 2. Structure of two typical phenolic acids, cinnamic acid (a) and benzoic acid (b) .10 FIGURE 3. Structure of two typical terpenes, 1,3-cineole(a) and camphor(b) 10 FIGURE 4. Structure of two typical alkaloids, caffeine (a) and a- picolinic acid (b) 11 FIGURE 5. Synthetic pathway of phenolic compounds starting from the amino acid, pyruvate, in the shikimate pathway (Putnam, 1978) 18 FIGURE 6. Basic flavonoid structure 19 FIGURE 7. Chemical structure of a chalcone; e.g. 2',4',6',4- tetrahydroxychalcone (THC) 20 FIGURE 9. Schematic of the flavonoid biosynthesis pathway starting with phenylpropanoid metabolism 30 FIGURE 10. Chemical structure of the two aglycones discovered by Harborne(1963) 35 FIGURE 11. Pathway of flavanone conversion to flavone showing the enzymes involved in the process 36 FIGURE 12. Aurone biosynthesis pathway 37 FIGURE 13. Lemna bioassay of the initial extract from snapdragon flowers 47 FIGURE 14. HPLC trace of the initial methanol fraction from a 50 pi sample volume 48 FIGURE 15.