ISOLATION AND CHARACTERIZATION OF NATURAL PRODUCTS FROM Olea europaea AND SOME BIOLOGICAL ACTIVITIES by Jelena Milosevic A thesis submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Faculty of Medicine) at the SCHOOL OF PHARMACY, UNIVERSITY OF LONDON 2 0 0 0 ProQuest Number: 10104162 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104162 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 To my loving family Olea europaea L. Identification of Olea europaea L. using the Engler scheme: Division: Angiospermae Class: Dicotyledoneae Subclass: Sympetalae Order: Olea les Family: Oleaceae Genus: Olea Species: europaea L. ABSTRACT Throughout the centuries the olive tree has been regarded as a part of tradition, both socially and culturally. The production of oil is the primary objective in processing olives. The residues from olive oil production are used for soil fertilisation and the waste waters for antioxidant and insecticide purposes. The objective of this study is to explore further the therapeutic and biotechnological potential of the olive, both extracts and pure compounds, by studying their pharmaceutical properties. The skins of fruits are their first defence, both physiological and biological, against the microbiological, parasitic and physical aspects of their environment. Olive skins were isolated and sequentially extracted with hexane, chloroform, methanol and water. Each of the extracts was tested for antimicrobial, antiinflammatory and antiparasitic activity, then further separated and the components structurally identified using ID and 2D Nuclear Magnetic Resonance spectroscopy (NMR) and combined High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS). Potentially, within the European Union hundreds of tons of by-products from olive oil production could have biotechnological, chemical and medicinal uses. The triglycerides in olive oil have been thoroughly studied but the phospholipid content and their structures have received little attention. Using high resolution NMR profiling combined with electrospray MS and ion-exchange chromatography the neutral lipids, fatty acids and phospholipid content were determined. Unlike mammalian lipid extracts, extracts from olives were complicated by the co-extraction of lipid like secondary metabolites. It was therefore a complex matter to extract and purify olive phospholipids for biotechnological proposes. The solvent extracts from olive pulp were separated by silica column chromatography, further separated by HPLC and the fractions obtained identified by NMR and MS. In the analysis of the olive pulp phenol glycosides were found. ACKNOWLEDGEMENTS When a PhD thesis is completed, it is often difficult to assess fully the expert assistance and guidance received throughout the process. I would therefore like to express my sincere thanks and gratitude to the following: To Professor W. A. Gibbons for his advice, help and unfailing interest in this research topic. To Dr. Mire Zloh, Mike Cocksedge and Dr. David Ashton, for their helpful discussions and practical advice. To Dr. Maria Comacho Corona for the help and guidance given, in the extraction of the olive skins and the analysis of the chloroform extract. To Aleksandra Bursae for photographing the flowering tree from which I picked the olives and to Branko Lakusic for then identifying it. I would like to thank the School of Hygiene and Tropical Medicine, University of London for the antiprotozoal testing of the olive skin extracts. I especially wish to thank my husband for his constant support and encouragement and for believing in me. I would also like to acknowledge the support of my family and their faith in me. Finally I would like to thank all my fiiends in London, Belgrade and scattered around the world for their constant support and best wishes. TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENT TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS APPENDIX 1 CHAPTER 1 - INTRODUCTION 29 1.1 Olives 30 1.1.1 General description 30 1.1.2 Hydrocarbons, pigments, sterols, tocoferols in olives 32 1.1.3 Phenolic compounds in olives 34 1.1.4 Glycerophospholipids in olives 37 1.1.5 Fatty acids in olives 38 1.2 Lipids General description 43 1.3 Acyl lipids 44 1.3.1 Acyl Glycerol 44 1.3.2 Glycerophospholipids 49 1.3.3 Glycoglycerolipids 51 1.3.4 Glycerosphingolipids 53 1.3.5 Cutin, Suberin and Waxes 54 1.4 Fatty acids 55 1.5 Phenolic compounds 61 1.5.1 General description 61 1.5.2 Hydroxybenzoic acids 64 1.5.3 Hydroxycinnamic acid 65 1.5.4 Xanthones 66 1.5.5 Flavonoids 67 1.5.6 Isoflavonoids 69 1.5.7 Lignins 69 1.5.8 Lignans and neolignans 70 1.5.9 Tannins 70 1.5.10 Quinones 72 2 CHAPTER 2 - OLIVE FRUIT-PULP ANALYSIS 74 2.1 Introduction 75 2.2 Materials and methods 77 2.2.1 Sample 77 2.2.2 Reagents/equipment for the analysis of Olive pulp 77 2.2.3 Preparation of extracts from Olive pulp 78 2.2.4 Chromatographic techniques 79 2.2.4.1 Column chromatography 79 2.2.4.2 Thin layer chromatography (TLC) of fractions from Olive pulp 80 2.2.4.3 High performance liquid chromatography (HPLC) of fractions from Olive pulp 81 2.2.5 Spectroscopic methods 82 2.2.5.1 Nuclear magnetic resonance spectroscopy (NMR) of fractions from Olive pulp 82 2.2.5.2 Mass spectrometry (MS) of fractions from Olive pulp 83 2.2.5.3 Gas chromatography-Mass spectrometry (GC-MS) of fractions from Olive pulp 84 2.2.5.3.1 GC-Method 84 2.2.5.3.2 GC-MS conditions for fractions from Olive pulp 85 2.3 Results 85 2.3.1 Thin layer chromatography (TLC) of fractions from Olive pulp 85 2.3.2 High performance liquid chromatography (HPLC) of fractions from Olive pulp 89 2.3.3 Nuclear magnetic resonance spectroscopy (NMR) of fractions from Olive pulp 94 2.3.4 Mass spectrometry (MS) of fractions from Olive pulp 106 2.3.5 Assignment of structures from NMR and MS data 111 2.3.6 Analysis of fatty acid composition by GC-MS in olive pulp 113 2.4 Discussion 121 3 CHAPTER 3 - OLIVE FRUIT-SKIN ANALYSIS 126 3.1 Introduction 127 3.2 Materials and methods 129 3.2.1 Sample 129 3.2.2 Reagents/equipment for the preparation of extracts from Olive skins 129 3.2.3 Preparation of extracts from Olive skin 130 3.3 Analytical techniques 131 3.3.1 Thin Layer Chromatography (TLC) of extracts from Olive skins 131 3.3.2 Preparative Thin layer Chromatography (PTLC) of extracts from Olive skins 132 3.3.3 High Performance Chromatography (HPLC) of extracts from Olive skins 133 3.3.4 Nuclear Magnetic resonance Spectroscopy (NMR) of extracts from Olive skins 134 3.3.5 Mass Spectrometry (MS) of extracts from Olive skins 135 3.3.6 Gas Chromatography-Mass Spectrometry (GC-MS) of extracts from Olive skins 136 3.3.6.1 GC-Method of extracts from Olive skins 136 3.3.6.2 Fast GC-MS Analysis conditions for FAMEs of extracts from Olive skins 137 3.4 Results 137 3.4.1 Hexane extract 138 3.4.1.1 Thin layer chromatography (TLC) of the hexane extract from Olive skins 138 3.4.1.2 Nuclear magnetic Resonance spectroscopy (NMR) of hexane extract from Olive skins 138 3.4.1.3 Gas chromatography - Mass spectroscopy (GC-MS) of hexane extract from Olive skins 142 3.4.2 Chloroform extract 146 3.4.2.1 Thin layer chromatography (TLC) 146 3.4.2.2 Preparative thin layer chromatography (PTLC) 147 3.4.2.3 P-Oleanolic (30-hydroxy-12-oleanen-28-oic acid) 148 3.4.2.3.1 ‘HN M R (400M H z,Py-d6) 148 3A2.3.2 '^C NMR (100 MHz, Py - d6) 149 3.4.2.3.3 2D NMR COSY (400 MHz, Py-d6) 150 3.4.2.3.4 Fast atom bombardment - mass spectrometry (FAB-MS) 151 3.4.2.4 p-maslinic acid (2a, 3p-dihydroxy-13(18)-oleanin-28-oic acid) 152 3.4.2.4.1 'H NMR (400 MHz, Py-d6) 152 3.4.2.4.2 "C NMR (100 MHz, Py-d6) 153 3.4.2.4.3 2D NMR COSY (400 MHz, Py-d6) 154 3.4.2.4.4 Fast atom bombardment - mass spectrometry (FAB-MS) 155 3.4.3 Methanol extract 156 3.4.3.1 Thin layer chromatography (TLC) of the methanol extract from Olive skins 156 10 3.4.3.2 High performance liquid chromatography (HPLC) of the methanol extract from Olive skins 156 3.4.3.3 Nuclear magnetic Resonance spectroscopy (NMR) of methanol extract from Olive skins 159 3.4.3.4 Mass spectroscopy of methanol extract from Olive skins 160 3.4.4 Water extract 165 3.4.4.1 High performance liquid chromatography (HPLC) of water extract from Olive skins 165 3.4.4.2 Nuclear magnetic resonance spectroscopy (NMR) of water extract from Olive skins 167 3.4.4.3 Mass spectroscopy of water extract from Olive skins 173 3.5 Discussion 174 4 CHAPTER 4 - PHOSPHOLIPID ANALYSIS 182 4.1 Introduction 183 4.2 Materials and methods 186 4.2.1 Sample 186 4.2.2 Reagents/equipment 187 4.2.3 Extraction of total lipids from olives 187 4.2.4 Separation of total lipids by Bond Elut (NH2-aminopropyl) solid phase separation method 188 4.2.5 Proton NMR of total lipids and Bond Elut fractions 189 4.2.6 Gas chromatography of lipids 190 4.2.6.1 GC-Method 190 11 4.2.6.2 GC-conditions (for Bond Elut fractions) 190 4.2.6.3 Fast GC-MS Analysis conditions for FAMEs 191 4.2.7 Mass Spectrometry 192 4.3 Results 192 4.3.1 Identification and quantification of lipids by NMR 192