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
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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
4.3.1.1 Proton NMR spectral assignment of the different lipid
classes from Bond Elut fractions 193
4.3.1.2 Analysis of fatty acid composition by NMR 202
4.3.2 Analysis of fatty acid composition by GC and GC-MS 205
4.3.3 Determination of phospholipids by ESI-MS electrospray 210
4.4 Discussion 212
5 CHAPTER 5 - ANTIINFLAMMATORY, ANTIMICROBIAL and
ANTIPROTOZOAL ACTIVITIES OF OLIVE SKIN EXTRACTS 218
5.1 Introduction 219
5.2 ANTIINFLAMMATORY activity 223
5.2.1 Materials and methods 223
5.2.2 Inhibition and inflammation 224
5.2.3 Assessment of irritancy of Olive skin extracts alone 224
5.2.4 Results 224
5.3 ANTIMICROBIAL activity 226
5.3.1 Introduction 226
5.3.2 Material and methods 226
5.3.3 Results 227
12 5.4 ANTIPROTOZOAL activity 228
5.4.1 Material and methods 228
5.4.1.1 Preparation of samples for biological testing 228
5.4.1.2 Antiprotozoal activity 229
5.4.1.2.1 Parasites 229
5.4.1.2.2 Plasmodium falciparum test 230
5.4.1.2.3 Trypanosoma brucei trodesiense and Trypanosoma
brucei brucei test 230
5.4.1.2.4 Leishmania donovani promastigotes test 231
5.4.1.2.5 Leishmania donovani amastigote test 231
5.4.1.3 Cytotoxic activity 232
5.4.1.3.1 KB cytotoxicity test 232
5.4.2 Results 233
5.5 Discussion 240
6 CHAPTER 6 - CONCLUSION 243
APPENDIX 1 254
A.1 Membrane lipids 255
A.1.2 Plant membrane: history and functional aspects of plant membrane 255
A.I.3 Membrane lipids: occurrence and distribution of lipids 259
13 APPENDIX 2 261
A.2 Analytical techniques 262
A.2.1 Thin-layer chromatography (TLC) 262
A.2.2 High performance TLC 263
A.2.3 High performance liquid chromatography (HPLC) 263
A.2.4 Gas chromatography (GC) 265
A.2.5 Nuclear magnetic resonances spectroscopy (NMR) 268
A.2.6 Mass spectroscopy (MS) 269
APPENDIX 3 273
Table A.3.1 Biological activities reported for maslinic acid 274
Table A.3.2 Biological activities reported for oleanolic acid 274
REFERENCES 276
14 LIST OF FIGURES
CHAPTER 1
Figure 1.1 Plant storage lipids 46
Figure 1.2 An oleosin protein at the surface of an oil body 47
Figure 1.3 Glycerophospholipids: Basic structure 49
Figure 1.4 The structure of plant glycosylglycerides 52
Figure 1.5 Sphingosine (the sphingenine analogue) 54
Figure 1.6 Chemical structure of benzoic acids 64
Figure 1.7 Chemical structure of hydroxycinnamic acids 65
Figure 1.8 Coumarin 66
Figure 1.9 Magniferin 67
Figure 1.10 Flavone common structure 69
Figure 1.11 Catechin 71
Figure 1.12 Juglone 73
CHAPTER 2
Figure 2.1 TLC Analysis of silica column fractions from Olive-pulp,
Fs, F4, Fs, Fs, F9, Fio, F i3, Fm, F is and Fi6 87
Figure 2.2 TLC Analysis of silica column fractions from Olive-pulp,
Fi, F2, F6, F?, Fii and Fi 2 88
Figure 2.3 HPLC Analysis of silica column fraction from Olive-pulp,
F3 90
15 Figure 2.4 HPLC Analysis of silica column fraction from Olive-pulp,
P4 90
Figure 2.5 HPLC Analysis of silica column fraction from Olive-pulp, p8-10 91
Figure 2.6 HPLC Analysis of silica column fraction from Olive-pulp,
Pl3-15 91
Figure 2.7 HPLC Analysis of silica column fraction from Olive-pulp,
Pi6 92
Figure 2.8 HPLC Analysis of silica column fractions from Olive-pulp 93
Figure 2.9 ID proton NMR spectra of the silica column HPLC fraction A,obtained from Olive pulp pre-separated on silica column 96
Figure 2.10 ID proton NMR spectra of the silica column HPLC fraction B, obtained from Olive pulp pre-separated on silica column 97
Figure 2.11 ID proton NMR spectra of the silica column HPLC fraction C, obtained from Olive pulp pre-separated on silica column 98
Figure 2.12 ID proton NMR spectra of the silica column HPLC fraction D, obtained from Olive pulp pre-separated on silica column 99
Figure 2.13 ID proton NMR spectra of the silica column HPLC fraction E, obtained from Olive pulp pre-separated on silica column 100
Figure 2.14 2D COSY proton NMR spectra of the silica column HPLC fraction A, obtained from Olive pulp pre-separated on silica column 101
Figure 2.15 2D COSY proton NMR spectra of the silica column HPLC fraction B, obtained from Olive pulp pre-separated on silica column 102
16 Figure 2.16 2D COSY proton NMR spectra of the silica column HPLC
fraction C, obtained from Olive pulp pre-separated on silica column 103
Figure 2.17 2D COSY proton NMR spectra of the silica column HPLC
fraction D, obtained from Olive pulp pre-separated on silica column 104
Figure 2.18 2D COSY proton NMR spectra of the silica column HPLC
fraction E, obtained from Olive pulp pre-separated on silica column 105
Figure 2.19 Electrospray mass spectrometry - sample A 108
Figure 2.20 Electrospray mass spectrometry - sample B 109
Figure 2.21 Electrospray mass spectrometry - sample C 109
Figure 2.22 Electrospray mass spectrometry - sample D 110
Figure 2.23 Electrospray mass spectrometry - sample E 110
Figure 2.24 5 ’-deoxy-oleuropein 111
Figure 2.25 Oleuropein 111
Figure 2.26 Oleoside-11-methyl ester 112
Figure 2.27 2-(3 ’-hydroxy 4 ’ -glucopyranosylphenyl)ethanol 112
Figure 2.28 2-(4 ’ -methylphenyl)ethanol 112
Figure 2.29 1,4dimethyl-3 -ethyl-2-naptholglycoside 113
Figure 2.30 Fatty acids detected by GC-MS in the total sample of
Olive pulp 120
TER 3
Figure 3.1 TLC Analysis of Hexane extract from Olive-skin 139
Figure 3.2 ID proton NMR spectra of Hexane extract from Olive
skins 140
17 Figure 3.3 2D COSY NMR proton spectra of Hexane extract from
Olive skins 141
Figure 3.4 TLC Analysis of Chloroform extract from Olive-skin 146
Figure 3.5 ID proton NMR spectra of P-Oleanolic acid extracted from Olive skins 148
Figure 3.6 ^^C NMR spectra of p-Oleanolic acid extracted from
Olive skins 149
Figure 3.7 2D COSY NMR of P-Oleanolic acid extracted from
Olive skins 150
Figure 3.8 P-Oleanolic acid 150
Figure 3.9 FAB-MS spectra of p-Oleanolic acid extracted from
Olive skins 151
Figure 3.10 ID proton NMR spectra of ô-Maslinic acid extracted from Olive skins 152
Figure 3.11 *^C NMR spectra of ô-Maslinic acid extracted from
Olive skins 153
Figure 3.12 2D COSY NMR spectra of ô-Maslinic acid extracted from Olive skins 154
Figure 3.13 ô-Maslinic acid 154
Figure 3.14 FAB-MS spectra of ô-Maslinic acid extracted from
Olive skins 155
Figure 3.15 TLC Analysis of Methanol extract from Olive-skin 157
Figure 3.16 FCPLC Analysis of Methanol extract from Olive-skin 158
18 Figure 3.17 ID proton NMR spectra of ODS column HPLC
fraction M l, obtained from Methanol extract of Olive skins 161
Figure 3.18 ID proton NMR spectra of ODS column HPLC
fraction M2, obtained from Methanol extract of Olive skins 162
Figure 3.19 ID proton NMR spectra of ODS column HPLC
fraction M3, obtained from Methanol extract of Olive skins 163
Figure 3.20 ID proton NMR spectra of ODS column HPLC
fraction M4, obtained from Methanol extract of Olive skins 164
Figure 3.21 HPLC Analysis of Water extract from Olive skins 166
Figure 3.22 ID proton NMR spectra of ODS column HPLC
fraction W l, obtained from Water extract of Olive skins 169
Figure 3.23 ID proton NMR spectra of ODS column HPLC
fraction W2, obtained from Water extract of Olive skins 170
Figure 3.24 ID proton NMR spectra of ODS column HPLC
fraction W3, obtained from Water extract of Olive skins 171
Figure 3.25 ID proton NMR spectra of ODS column HPLC
fraction W4, obtained from Methanol extract of Olive skins 172
Figure 3.26 3-(glucopyranosyl)-cyanidol 179
Figure 3.27 12-01eanene-3,28-diol 180
Figure 3.28 4,14-Dimethyl-9,19-cycloergost-23-en-3-ol 180
Figure 3.29 4’-methyl 8-acetyl 4-(glucopyranosyl)-pinoresinol 181
CHAPTER 4
Figure 4.1 ID proton NMR spectra of Bond Elut fraction 1
19 (neutral lipids) extracted from whole Olive 194
Figure 4.2 ID proton NMR spectra of Bond Elut fraction 3
(non-acidic phospholipids) extracted from whole Olive 199
Figure 4.3 2D J-resolved NMR spectra of Bond Elut fraction 3
(non-acidic phospholipids) extracted from whole Olive 200
Figure 4.4 2D V-resolved NMR spectra of Bond Elut fraction 4
(acidic phospholipids) extracted from whole Olive 201
Figure 4.5 ID proton NMR spectra of Bond Elut fraction 2
(free fatty acid) extracted from whole Olive 204
CHAPTER 5
Figure 5.1 In vitro activity of Olive skin chloroform extract on
P. falciparum YA 236
Figure 5.2 In vitro activity of Olive skin chloroform extract on
P. falciparum 3T>7 237
Figure 5.3 In vitro activity of Olive skin water extract on
Leishmania donovani 238
Figure 5.4 In vitro activity of Olive skin chloroform extract on
KB cells 239
Figure 5.5 Percentage of inhibition of inflammation (erythema)
produced by Olive skin extracts 241
20 CHAPTER 6
Figure 6.1 Compounds identified in the pulp of Olive fruit 246
Figure 6.2 Compounds identified in the skins of Olive finit 248
APPENDIX 1
Figure A. 1.1 Membrane structure 258
APPENDIX 2
Figure A.2.1 Schematic of the APCI source showing the principle
components and pressure regions 272
21 LIST OF TABLES
CHAPTER 1
Table 1.1 Mineral constituents present in the olive pulp of a
Greek cultivar 32
Table 1.2 Major classes of fmit phenolic compounds in olives 34
Table 1.3 Distribution of fatty acids in olive 38
Table 1.4 Constituents reported in Olea europaea 39
Table 1.5 Phospholipids (head group) 50
Table 1.6 Structures of major fatty acids 57
Table 1.7 The presance of common fatty acids in plants 60
Table 1.8 The major classes of phenolics in fhiits 62
CHAPTER 2
Table 2.1 Biological activities reported in Olea europaea 76
Table 2.2 Fractions of Olive pulp from column chromatography 80
Table 2.3 Molecular weights of HPLC fractions from Olive pulp 108
Table 2.4 Fatty acids detected by GC-MS in the Fi fraction 114
Table 2.5 Fatty acids detected by GC-MS in the saponified Fi
fraction 114
Table 2.6 Fatty acids detected by GC-MS in the Fz fraction 115
Table 2.7 Fatty acids detected by GC-MS in the saponified Fz
fraction 115
Table 2.8 Fatty acids detected by GC-MS in the Fô fraction 116
22 Table 2.9 Fatty acids detected by GC-MS in the saponified Fô
fraction 116
Table 2.10 Fatty acids detected by GC-MS in the F? fraction 117
Table 2.11 Fatty acids detected by GC-MS in the saponified F?
fraction 117
Table 2.12 Fatty acids detected by GC-MS in the Fii fraction 118
Table 2.13 Fatty acids detected by GC-MS in the saponified Fii
fraction 118
Table 2.14 Fatty acids detected by GC-MS in the Fn fraction 119
Table 2.15 Fatty acids detected by GC-MS in the saponified Fn
fraction 119
Table 2.16 Fatty acids found in Olive fruit-pulp 121
CHAPTERS
Table 3.1 Compounds already isolated from some fruit skins 128
Table 3.2 Extraction of Olive skins 131
Table 3.3 HPLC Conditions for the analysis of methanol and
water olive fruit-skin extracts 133
Table 3.4 Fatty acids detected by GC-MS in the non-saponified
sample of Hexane extract obtained from Olive skins 143
Table 3.5 Fatty acids detected by GC-MS in the saponified
sample of Hexane extract obtained from Olive skins 144
Table 3.5a Fatty acids detected by GC-MS in the saponified
sample of Hexane extract obtained from Olive skins 145
23 Table 3.6 Methanol extract HPLC fractions from Olive skins -
Molecular weights 160
Table 3.7 Water extract HPLC fractions from Olive skins -
Molecular weights 173
Table 3.8 Fatty acids found in the hexane extract from Olive
skins 175
Table 3.9 Fatty acids found in Olive skin and not in olive pulp 177
CHAPTER 4
Table 4.1 Assigments of ^H NMR resonances in phospholipids 198
Table 4.2 Assigments of H NMR resonances in fatty acids 203
Table 4.3 GC analysis of the saponified Bond Elut fraction 1
(non polar lipids) obtained from whole Olive 205
Table 4.4 GC analysis of the non-saponified Bond Elut fraction 2
(non-esterified fatty acid) obtained from whole Olive 206
Table 4.5 GC analysis of the saponified Bond Elut fraction 3
(non-acidic phospholipids) obtained from whole Olive 206
Table 4.6 GC-MS analysis of the non-saponified total lipids
obtained from whole Olive 207
Table 4.7 GC-MS analysis of the saponified total lipids
obtained from whole Olive 208
Table 4.8 GC-MS analysis of the saponified Bond Elut fraction 4
(acidic phospholipids) obtained from whole Olive 209
24 Table 4.9 Phospholipid masses monitored by MS 210
Table 4.10 Fatty acids found in the total lipids from Olive fruit
byGC 213
Table 4.11 Fatty acids present in the total lipids from olive fhiit
by GC-MS 214
Table 4.12 Phospholipids detected in olives by ESI-MS 216
CHAPTER 5
Table 5.1 Biochemical effects of bioflavonoids 220
Table 5.2 Percentage of inhibition of inflammation
(erythema) produced by Olive skins extracts 225
Table 5.3 Diameters of zones of inhibition (mm)
produced by test compounds 228
Table 5.4 Antiprotozoal activities of Olive skin extracts
on P. falciparum 3D7 and K1 234
Table 5.5 Antiprotozoal activities of Olive skin extracts on
T.b. rhodesiense and T.b. brucei 234
Table 5.6 Antiprotozoal activities of Olive skin extracts on
L. donovani 235
Table 5.7 Cytotoxic activities of Olive skin extracts on
KB cells 235
Table 5.8 In vitro activity of Olive skin chloroform extract
on P. falciparum K1 236
25 Table 5.9 In vitro activity of Olive skin chloroform extract
on P. falciparum 3D7 2 3 7
Table 5.10 In vitro activity of Olive skin water extract
on Leishmania donovani 238
Table 5.11 In vitro activity of Olive skin chloroform extract
on KB cells 239
CHAPTER 6
Table 6.1 Fatty acid detected in Olive skins only 249
Table 6.2 Fatty acids found in Olive fruit 250
Table 6.3 Phospholipids detected in olives by ESI-MS 251
Table 6.4 Fatty acids from phospholipids obtained from Olive
fhiit 252
APPENDIX 3
Table A.3.1 Biological activities reported for maslinic acid 274
Table A.3.2 Biological activities reported for oleanolic acid 274
26 LIST OF ABBREVIATIONS
(in alphabetical orders)
ID and 2D One- and two-dimensional
^H-NMR Proton nuclear magnetic resonance spectroscopy
^^C-NMR Carbon nuclear magnetic resonance spectroscopy
APCI Atmosferic pressure chemical ionization
CHOL Cholesterol
CAR Cardiolipin
CDCb Fully deuterated chloroform; D: deuterium
CDsOD Fully deuterated methanol; D: deuterium
COSY Homonuclear correlation spectroscopy
DG Diacylglycerol
ESI-MS Electrospray-ionization mass spectrometry
FID Free inducation decay
HPLC High performance liquid chromatography
MD Molecular dynamics
MG Monoacylglycerol
MS Mass spectrometry
NMR Nuclear magnetic resonance spetroscopy
NOE Nuclear overhauser enhancement
NOESY Nuclear overhauser enchancment spectroscopy
PA Phosphatidic acid
27 PC Phosphatidylethanolamine
PG Phosphatidylglycerol
PI Phosphatidylinositol ppm Part per million
PS Phosphatidylserine
PUPA Polyunsaturated fatty acid rpm revolutions per minute
SPH Sphingolipid tl time delay in first dimension t2 time delay in second dimension
TFE Triflouroethanol
TO Triacylglycerol
TLC Thin layer chromatography
TMS T etramethylsilane v/v Volume per volume ratio
28 CHAPTER 1
INTRODUCTION
29 C h a p t e r 1 Introduction
1.1 OLIVES
1.1.1 General description
There are approximately 2500 known varieties of olives of which the International Olive
Oil Council (lOOC) classifies 250 as commercial cultivations.
The increasing health consciousness of today's cosmopolitan society explains the rising consumption of olive oil around the world and hence the rapid growth of the olive industry. The beneficial health properties of olive oil have been known for centuries.
Olives and olive oil are part of Mediterranean diet and culture. There is a decreased incidence of cardiovascular disease in these areas which are the lowest in the Western
Hemisphere. These effects have been attributed to the high content of oleic acid in olive oil, which serves to slow the penetration of fatty acids into arterial walls. In addition to oleic acid, the antioxidant properties of tocopherols and phenolic compounds also make very important contributions to the qualities of the olive fhiit. The metabolic pathways of these compounds are particularly complex, and they can have multiple alternative metabolic fates. The interest in plant derived phenol compounds, in the last few decades, has centred around their role in the prevention and treatment of diseases; their biological function and activity as well as relating them to the mechanisms of anticarcinogenicity.
Because of its hardiness, long life and its adaptivnes to areas where it may be the only cultivation possible, types of tree, and different cultivation techniques have evolved.
Although the olive tree is frequently found in isolation in the wild, almost all olive oil production comes fi*om homogeneous olive groves.
The botanical progenitor of the olive is thought to be oleaster, Olea sylvestris, a wild type of olive with a small skinny fruit, still grows around the Mediterranean sea and is occasionally used as grafting stock. The cultivated tree probably evolved around the
Eastern Mediterranean and gradually spread to Southern Europe and North Africa. The
3 0 C h a p t e r 1 Introduction
Spanish missionaries brought the olive tree to California at about 1850, and immigrants
from the Mediterranean introduced it to South America at about the same time.
The olive tree thrives in deep, drained soils, clear dry atmospheres, and moderate temperatures (never below -9 ^C), if fruit is to be produced. Olive fruit production often
follows a 2 year cycle, with a good crop one year followed by a medium or poor crop the following year. Such a pattern creates serious problems for the production industry.
The olive fruit or olive is a drupe, oval in shape, and consists of two main parts, the pericarp and the enclosed seed or kernel. The pericarp consists of epicarp, or skin, mesocarp, or pulp, and the endocarp, or stone (pit) which contains the seed. The pulp is
66-85%; the pit (including the seed) 13-30%, and the skin 1.5-3.5% of the fruit weight.
The size of the olive fruit is affected by generic and environmental factors. At maturity, the weight of a single fhiit may vary from 2 to 12 g, although it can reach 20 g. Over the centuries, many olive cultivates have been developed differing in size, colour and the chemical composition of the fhiit. The olive cultivates best suited for oil production have medium size fruits which may contain 15-40% by weight of oil when ripe, while
olives used for pickling have as little as 8% or less of oil. During the growth period of the olive fhiit many changes are observed. Along with the increase in weight of the
fruit, the oil content increases continually, the sugar content reduces, the fibre content drops sharply initially then gradually afterwards, with the protein content remaining low throughout the development of the fruit, as does the ash content.
Commercially, the mesocarp (pulp) is the most important part of the olive fruit. At maturity, water and oil account for 85-90% of the weight of the pulp. The main sugar in the pulp is glucose, accompanied by fructose. Sucrose, mannose, and galactose have
also been detected in certain species of the finit. Small quantities of organic acids have been reported in olive pulp: citric, malic, oxalic, malonic, fumaric, tartaric, lactic, acetic
and tricarballylic acids. The mineral analysis of olive pulp from a Greek cultivar shows
31 C h a p t e r 1 Introduction that the potassium content is over two-thirds of the total mineral content of the olive pulp.
Tablel.l Mineral constituents present in the olive pulp of a Greek cultivar
Mineral Content (ppm)
Calcium 386 Chlorine 683 Iron 14 Magnesium 192 Manganese 2 Phosphorous 188 Potassium 3642 Sodium 48 Zinc 7
1.1.2 Hydrocarbons, Pigments, Sterols, Tocoferols in olives
Olive pulp also contains small quantities of proteins and other nitrogenous compounds, pectins, salts, pigments and flavour compounds. Gas chromatography in combination with mass spectrometry has made possible the identification of over 70 compounds which contribute to the flavour of the olive and in particular to olive oil. They can be categorised [Fedeli, 1977] into aliphatic and aromatic hydrocarbons, aliphatic and terpenic alcohols, aldehydes, ketones, ethers, esters and thiophene derivatives.
Of particular interest are the green pigments chlorophyll a and chlorophyll b, as well as the brown pheophytina and pheophytin b. Small quantities of these pigments (1 to 10 ppm) are carried into extracted oil. In the presence of light the chlorophylls and pheophytins exert a perooxidation effect on lipids, while in the dark they act as antioxidants [Interese et al., 1971]. Olive oil containing green pigments must be
3 2 C h a p t e r 1 Introduction protected from light for the oxidation to be delayed. The type and amount of pigment in olive tissue depend fundamentally on the species, variety, state of ripeness, development, and cultivation conditions. A gradual decrease can be seen in the fhiit pigment content with time. Both chlorophylls and caretonoids decrease with seasonal changes, almost disappearing at the moment of maturity, giving way to anthocyanic components [Vazquez-Roncero et al, 1976]. Chlorophyll a is the major component at all times, followed by chlorophyll b. Carotenoids are minor components, with p- carotene as the principal carotene.
Hydrocarbons are also present in olives; squalene, a biochemical precursor of sterols, is the major hydrocarbon component of both olives and olive oil. Polycyclic aromatic hydrocarbons have been identified, such as phenanthrene, pyrene, and fluoranthrene.
Both even- and odd-carbon numbered n-paraffins, ranging from Cii to C 30, have also been found, together with the provitamin A and p-carotene in very small quantities 0.3-
3.6 mg/kg.
Three sterols are present in the Italian olive species, with p-sitosterol accounting for about 96% of the sterol mixture; campesterol representing 3% and stigmasterol making up the remaining 1% of the total sterols. In Greek olive oil, a fourth sterol, anemasterol
[Boskou & Morton, 1975] was identified and quantified at 8% of the total sterol content.
The tocopherols have a special significance because of their vitamin E activity. Olives contains a-tocopherol in quantities from 12 to 150 ppm [Vitagliano, 1960; Boatella,
1975] but investigators do not agree on the presence of the other tocopherols
(p, y and 5). It has also been shown that olive seed lipids are richer in tocopherols than olive oil [Karitsakis & Markakis, 1987].
3 3 C h a p t e r 1 Introduction
1.1.3 Phenolic compounds in olives
A variety of phenolic compounds have been found in olive pulp. Phenolic compounds present in olives are conventionally characterised as “polyphenols”, even though not all are polyhydroxy derivatives, but the definition is based on the metabolic origin of substances derived from the shikimate pathway and phenylpropanois metabolism. Plant phenols have been classified into 15 major groupings [Macheix et al., 1990] distinguished by the number of constitutive carbon atoms in conjunction with the
structure of the basic phenolic skeleton. The range of known phenolic compounds is great but only the benzoic acids, cinnamic acids, flavonoids and iridoids are of major significance in olives.
Table 1.2 Major classes of finit phenolic compounds in olives
Basic skeleton Class Example
C6-Cl Benzoic acid /7-Hydroxybenzoic acid Vanillic acid Protocatechuic acid C 6-C 3 Hydroxycinnamic acids Caffeic acid C 6-C 3-C 6 Flavonoids Cyanidin Anthocyanins Flavonoid glycoside Rutin Iridoids Oleuropein Ligstroside Lignins Tannins
Metabolic pathways involving phenolic compounds are complex, differ from one tissue to another and respond to environmental stimuli (e.g. altitude, light and temperature).
34 C h a p t e r 1 Introduction
Establishing a biological activity for phenolic compounds is therefore very difficult; however, almost all possess the common biological and chemical properties of antioxidant activity; the ability to scavenge both active oxygen species and electrophiles; the ability to inhibit nitrosation and to chelate metal ions; the potential for autoxidation, and the capability to modulate certain cellular enzyme activities. The minimum inhibitory concentration of several simple and complex phenolic compounds found in olive fhiits has been measured against four pathogenic bacteria [Chowdhury et al., 1996]. Caffeic acid is the most effective agent although oleuropein, the major phenolic constituent of olives also exhibit bactericidal action [Ruiz-Barba et al., 1991].
Phenolic compounds in olives have attracted attention as antioxidants [Le Tutour et al.,
1992; Amiot et al., 1986] with hydrophilic phenols and the oleosidic forms of hydroxytyrosol (3,4-dihydroxyphenylethanol) correlated with the oxidative stability of virgin olive oil. Tyrosol, /7-hydroxyphenylacetic acid, o-coumaric acid, j9-coumaric acid and vanillic acid had very little or no antioxidant activity.
Phenolic compounds give colour and flavour to the fhiit and contributes to the intense bitterness [Amiot et al., 1986; Vaugh et al., 1961]. Bitter phenolics are often glucosides, of structures containing tyrosol and elenolic acid [Duran et al., 1994].
Oxidation products of oleuropein, together with other native phenolics (anthocyanins,
specifically the glycosides of cyanidin and peonidin), are known to be responsible for the characteristic black colour of mature olive finits [Brenes et al., 1995]. The reaction is catalysed by polyphenol oxidases, a large group of enzymes that provide molecular
oxygen during the oxidation of the phenolics. The major contribution to the browning reaction is from oleuropein, mediated by various metal ions, such as iron and manganese.
The possibility of phenolic compounds serving as biological markers of the physiological stages of the fhiit maturation is well known [Macheix et al., 1990;
Cimato et al., 1990].
3 5 C h a p t e r 1 Introduction
The flowering of the olive tree marks the beginning of the fruit development. After six to eight months the olive is at its maximum fhiit weight. The fhiit development can be characterised using the following criteria: a) the appearance of new compounds; b) the
disappearance of certain compounds; c) the occurrence of various characteristic ratios between certain compounds; d) the evolution of the activity of numerous enzymes
leading to the biosynthesis or degradation of the phenolic compounds. For example,
green maturation is characterised by reduction in chlorophyll content, fruit softening and
an increase in oil content. During the fhiit maturation process there is a hydrolysis of
components with “higher molecular weight” with the consequent formation of other molecules. Oleuropein declines with fhiit maturity, and this is accompanied by the
accumulation of two different compounds, demethyloleuropein and elenolic acid
glycoside. Neither of these compounds was present priory to green maturation [Amiot et
aL, 1989]. It is interesting that the oleuropein content and other phenols, such as
flavonoids and verbacoside (a heterosidic ester of caffeic acid and hydroxytyrosol) have
an inverse relationship, but part of the verbacoside and oleuropein molecule are the
same and therefore, it could be that the oleuropein molecule is responsible for the
formation of the verbacoside, since verbacoside cannot be detected in vary young fruits.
The olive mesocarp contains phenolic compounds that are chiefly water soluble.
However, very small quantities of phenolics are carried into the olive oil. The chief
polyphenols of olive oil are tyrosol and hydroxytyrosol, derived from the hydrolysis of
oleuropein [Vazquez et a i, 1976]. Benzoic acid and cinnamic acid, which probably
originate from the degradation of flavonoids, are also present in olive oil [Montendro &
Cantarelli, 1969; Vazquez et al., 1976 ]. It was observed [Gutfinger, 1981] that olive oil
extracted with solvent (chloroform-methanol mixture) was more stable to oxidation than
mechanically extracted oil. He attributed it to the higher polyphenol content of the
solvent-extracted oil, 321-574 ppm compared to 50-159 ppm for the mechanically
extracted oil where the large quantities of water used in the pressing of the olives.
3 6 C h a p t e r 1 Introduction washes out more of the phenolics from the oil. The average phenol value is 120 ppm for
Greek olive oil, [Kiritsakis, 1982].
1.1.4 Glycerophospholipids in olives
Few definitive studies have been made on phospholipids present in olives. Olive oil
contains small amounts of phosphatidylcholine and phosphatidylethanolamine, ranging
from 40 to 135 ppm; larger quantities of phospholipids are present in olive seed oil.
Oleic acid is a dominant fatty acid in these compounds. Glycerophospholipids and their
fatty acid accumulation in newly formed and ripening fruit were investigated [Marzouk
& Cherif, 1981a, 1981b] and it was showen that there was a considerable increase at a
very rapid rate of neutral triacylglycerols from polar glycerophospholipids and oleic
acid [Marzouk et al., 1990]. Most of the research on glycerolipid biosynthesis in oil-rich
tissues has been done with seeds [Stymne & Stobart, 1987]. Although avocado and olive
fhiits have been used in the past we do not have much knowledge of the fruit in
comparison to the seeds. A reason for this is that fhiits at the right stage of development,
when enzymes involved in lipid biosynthesis are most active, are not as readily
available as seeds are. One way around this problem would be to try and use tissue
cultures from such crops. In comparison to the level of research using tissue cultures
from oilseed of rape and palm, other oil-rich crops like olives, have not been studied as
much. Olive callus cultures have proven to be very stable [Williams et al., 1993] and
therefore are suitable as experimental material for biochemical studies all year round.
The data even shows that microsomes from callus cultures are capable of satisfactory rates of glycerophospholipid and triacylglycerol synthesis, in comparison to those from
olive fruits [Rutter et al., 1997].
3 7 C h a p t e r 1 Introduction
1.1.5 Fatty acids in olives
Most of the fatty acids in olives are present as triglycerides (triacylglycerols). The distribution of the acids in the glycerol follows the l,3-random-2-random rule. The major fatty acids are oleic (18:1), linoleic (18:2), palmitic (16:1) and stearic (18:0) with oleic acid present in much higher concentration than the other acids. The analysis of olive samples originating from six countries gave fatty acid distributions as follows:
Table 1.3 Distribution of fatty acids in olive
Fatty acid Content %
Oleic acid (18:1) 56-83 Palmitic acid (16:0) 7.5-20.0 Linoleic (18:2) 3.5-20.0 Stearic acid (18:0) 0.5-5.0 Palmitoleic acid (16:1) 0.3-3.5 Linolenic acid (18:3) 0.0-1.5 Myristic acid (14:0) trace Arachidonic acid (20:0) trace Behenic acid (22:0) trace Lignoceric acid (24:0) trace Heptadecanoic acid (17:0) trace Heptadecenoic acid (17:0) trace
The presence of elaidic (trans-oleic) acid in olives is doubtful, [Tiscomia and Bertini,
1972; Amellotti et a l, 1973]. It is known that delayed harvesting tends to increase the content in unsaturated fatty acids, especially linoleic (18:2), at the expense of palmitic
acid (16:0). The selection of an optimal harvesting date should be properlly assested to
optimise the production of both olive fruit and olive oil.
3 8 C h a p t e r 1 Introduction
Olive oil is almost unique among vegetable oils in that it can be consumed without any refining treatment. A refining process would destroy the sensory attributes, which are responsible for the extraordinary quality of olive oil.
Extensive work has been done on olives, especially Olea europaea, and this is
summarised in Table 1.4.
Table 1.4 Constituents reported in Olea europaea
Compound Part Reference
Hydrocarbon Squalene fhiit, oil Manzi et al, 1998; DeLeonardis et al, 1998;
Volatile compounds Hexanal, oil Aparicio & Morales, 1998 (E)-3-hexanal, (Z)-3-hexenal, (E)-2-Hexenal, Hexyl acetate, (Z)-3-hexenyl acetate, hexan-l-ol, (E)-2- hexen-l-ol
Waxes Alkanes (C23-C33) finit Blanchi et «/., 1992 Saturated and unsaturated alkylesters (C38-C56) Aldehydes (C24-C30) methylphenylesters triacylglycerols Alcohols (C22-C28)
3 9 C h a p t e r 1 Introduction
Lipids Lipids fruit Mosquera & Fernandez, 1985; Williams et al, 1993 Acyl -lipids pollen Katiyar & Bhatia, 1988; Bianchi & Vlahov, 1994 Gly cerolipids ;triacy Igly cerides fruit Rodrigez Rosales et al, 1990; Rutter et al, 1997 Phosphatidylcholine cultures Sanchez et al, 1992; Santinelli et al, 1992; fruit Williams & Harwood, 1994; pulp,kamel Mamedov & Aslanov, 1985; Bianchi et al, 1992 Acyl seed Thakur & Chadha, 1991 Fatty acids (C16-C28) Mamedova & Aslanov, 1991;
Pigments Caretenoids fruit, oil Mosquera & Fernandez, 1985; Golubev et al, 1986; Manzi et al, 1998: Chlorophyll fruit Minguezmosquera & Garridofemandez, 1989
Carbohydrates Fructose, myo-inositol, leaves Tattini et al, 1996 galatose, galactinol, sucrose, raffinose, stachyose Glucose, mannitol tissues Romani et al, 1994 Carbohydrates fruit Guillen et al, 1992
Polyssacharides Homogalacturonans fruit Jimenez et al, 1995; Coimbra et al, 1996; Sanchez hemicellulose A (HA) Romero et al, 1998 hemicellulose B (HB) Alpha-cellulose (CEL) fruit Guillen et al, 1992; Sanchez Romero et al, 1998 Cellulose seed hull Coimbra et al, 1995; Guillen et al, 1992 Glucoroxylans cell walls Jimenes et a/., 1994 pectic and hemicellolosic fract. and uronic acid Polyssacharides pollen Rodriguez Rosales et al, 1990
Proteins Proteins pollen Rodriguez Rosales et al, 1990 fruit Guillen ef a/., 1992
Cell-wall enzymes Enzymes fruit FemandezBolanos etal, 1995 Chlorophyllase fhiit MinguezMosquera et al, 1994
Micronutrients Iron, magnesium, copper, zinc, pollen Alba etal, 1995 boron
4 0 C h a p t e r 1 Introduction
Lignans (+)-l-Acetoxy-pinoresinol -4’- bark Tsukamoto et al., 1984 (3-D glucoside Tsukamoto et al., 1985 (+)-1 - Acetoxy-pinoresinol-4 ’ - P-D-glucoside-4”-0- methylether (+)-1 -Hydroxy-pinoresinol-1 -P- D-glucoside (+)-Fraxiresinol-1 -P-D- glucoside (+)-1 -Hydroxy-pinoresinol-4 ’ - P-D-glucoside (+)-l-Acetoxy-pinoresinol [IS, 2S, 5R, 6S]-l-acetoxy-2,6-bis (4-hy droxy-3-metoxypheny 1)- 3,4-dioxabicyclo[3.3.0]octane (+)-1 -Hydroxy-pinoresinol (+)-1 - Acetoxy-pinoresinol-4 ’ ’ - 0-methyl ether (+)-1 -Hydroxy-pinoresinol-4 ’ ’ - 0-methyl ether (-)-Olivil (+)-Cyclo-olivil______
Phenolic compounds Phenolic glucosides fruit Tsukamoto ef a/., 1984 Abscisic acid inflorescence Martin et al., 1982 Tocopherol leaves Salidroside (tyrosol-glucose), fruit Golubev et al., 1987 nuzhenide (glucose-elenolic seeds Delatorre ei a/., 1993 acid-glucose-tyrosol) 3.4-Dihydroxyphenylethanol derv. p-hydroxyphenylethanol oil Montedoro etal., 1993 dérivâtes Tyrosol. hydroxytyrosol ligstrosid aglycone oil Angerosa e/fl/., 1995 3.4-Dihydropheny Igly col Hydroxyphenylethanol fruit Bianchi & Pozzi, 1994 (tyrosol) fruit Limiroli et al., 1996 4-Hydroxyphenylethanol glucoside, quinol glucoside Methyl phenyl esters leaves Bianchi ef a/., 1993 2-Pheny 1-ethanol-1 -esters Caffeic acid, hydroxytyrosol fruit Romero etal., 1998 Tyrosol, hydroxytyrosol, oil Tsimidou, 1998 Elenolic acid glucoside fruit Esti et al., 1998 Hydroxytyrosol Phenolic compounds fruit Ryan & Robards, 1998
41 C h a p t e r 1 Introduction
Monoterpenes Secoiridoids fruit Gariboldi et al, 1986; Kuwajima et al., 1988; Loscalzo et al., 1993 Secoiridoid glucosides, bark Tsukamoto etal., 1991; Oleuropein, leaf, fruit, Ficarra etal, 1991; Marsilio etal, 1996; Limiroli bud et al., 1996; Demethyloleuropein, Ficarra et al., 1985; Bianco et al., 1993 verbacoside, leaf, fruit Ligstroside, comoside, Delatorre etal., 1993 halleridone nuzhenide-oleoside seeds
Diterpenes Dammarane dérivâtes fruit Biancbini etal, 1987
Triterpenes Oleanolic acid fruit, leaves, Movsumov & Aliev 1985; Bianchi et al, 1992, Maslinic acid fruit, skin Bianchi et al, 1994 Betulinic acid, a, (3-amyrin leaves Movsumov & Aliev 1985; Bianchi et al., 1992, 1994; Uvaol, reythrodiol leaves Bianchi era/., 1993
Steroids (3-Sitosterol leaves Bianchi et al., 1993 Stigmasta-3, 5-diene oil Cecchi, 1998 a-stigmastene, |3-stigmastene a-cholestane, (3-cholestane cholesta-3, 5-diene cholesterol
Coumarins Esculetin,scopoletin, bark Tsukamotoer a/., 1984, 1985 isoscopoletin scoparone
Anthocyanins Cyanidin-3-mtinoside fruit Vlahov, 1992
Flavonoids Luteolin-7-glucoside, fruit, leaves Tomas & Ferreres, 1980; Ficarraet al, 1991 Quercetin-3-rutinoside fruit, leaves Vlahov, 1992; Heimler et al., 1992 (quercetrin) Luteolin leaves Heimler et al., 1992
42 C h a p t e r 1 Introduction
1.2 LIPIDS
No strict definition of the term "lipid" would appear to exist but it is usually used in one of two ways. The wider frame relates to solubility properties where lipids are considered
as substances which are insoluble in water but soluble in certain organic solvents, including hexane, diethyl ether and chloroform or binary mixtures such as chloroform
and methanol or hexane and isopropanol [Zubay, 1993]. In the more narrow sense,
lipids are defined chemically as derivatives of long chain fatty acids and polyisoprenoids, (such as sterols).
Lipids are often divided into three categories, simple lipids, complex lipids and lipid
derivatives. Simple lipids are composed only of esters of long chain fatty acids with
alcohols, such as triacylglycerols and wax esters. Complex lipids have in addition to
fatty acids and glycerol, some non-lipid components in their molecules, such as phosphoric acid or derivatives of amino acids or a carbohydrate.
Lipids are often subclassified as either "neutral" or "polar" lipids; from simple long
chain fatty acids as least polar to phospholipids as most polar.
Another subclassification can be on the basis on their roles in nature where they act as
energy stores, and as the constituents of membranes they can be involved in the control
of metabolism, e.g. as second mesengers or growth regulators. The storage lipids are
almost exclusively triacylglycerols ( with the exception of jojoba wax, esters), and are
found in small oil droplets within the cell of the seed. The phospholipids, glycolipids
and sterols occur in the membranes of chloroplasts, plastids, mitochondria, and other
membrane organelles. In addition to storage and membrane lipids, there are "surface"
lipids, which are highly apolar, such as wax esters with very long fatty acid chains and
43 C h a p t e r 1 Introduction alcohols.
Interest in plant lipids developed in the 1930s, when it was observed that animals fed lipid-ffee diet food, developed serious symptoms called essential fatty acid (EFA) deficiency. It was found that the essential acids were provided by plant lipids containing polyunsaturated fatty acids with double bonds at 6 and 9 carbons from the methyl terminus. These acids cannot be synthesized by mammals but are needed as precursors of eicosanoids to enact vital processes in healthy individuals.
Today interest in lipids has increased with a wide area of use in by industry for detergents, cosmetic manufacture and as a renewable source of fuel.
Certain lipid molecules are prominent in the plant kingdom. Acyl lipids (i.e. those containing fatty acid moitiés) are by far the most important and form the largest class, although compounds like chlorophyll or caretonoid pigments and sterols can in certain tissues be the major components [Harwood & Grittihs, 1992].
1.3 ACYL LIPIDS
1.3.1 Acyl Glycerols
Triacylglycerols (TAGs) of plant origin are an important class of compounds which have a wide range of applications for both technical and nutritional purposes.
The basic structure of triacylglycerols consists of a glycerol backbone to which are esterified three fatty acids. The combination of acyl groups, their degree of unsaturation
and stereo specific distribution, determines the physical properties of triacylglycerols.
44 C h a p t e r 1 Introduction
The number of possible triacylglycerols is great; for example, 27 different molecular species can be formed if any 3 different fatty acids are randomly distributed on the glycerol backbone [Harwood, 1997].
The triacylglycerol can be substituted at the Sn3 position with a head group such as choline, ethanolamine, ect., while the Snl and Sn2 positions remain esterified with fatty acids. These molecules can form complex mixtures in plants since each lipid class contains various combinations of fatty acid chain.
Triacylglycerides provide a reserve of oil which can be readily hydrolysed. The period over which oil deposition occurs, varies from one species to another, for example in cocoa this can be from 7 days to 2 months. In the first stage when cell division occurs there is little accumulation of these triacylglyceride reserves, containing a high number of polyunsaturated fatty acids (acids like linoleic and a-linolenic [Gurr, 1972], are characteristic of phospholipids). In stage 2, the rapid deposition of storage material consist of lipid and carbohydrate and is a time when unusual fatty acids are synthesised
[Gurr, 1972; Lehrian & Keeney, 1980]. Stage 3, represents a maturation period and there is little further synthesis of reserves. The chronological sequence of events implies some degree of regulation and switching on/off triacylglycerol-synthesizing enzymes, perhaps after the bulk of membrane lipids have been generated.
45 C h a p t e r 1 Introduction
0
CH2OCR 1 0 Triacylglycerol (the major stored lipid) CHOCR 2 0
C H o O C R r
0
CH2OCR 1 Sn-1,2-Diacylglycerol 0 (the metabolic precursor of triacylglycerol is a minor component CHOCR: of lipid stores and racemises to its Sn- 1,3-isomers)
CH2OH
Wax ester 0 (formed by estérification of a fatty acid with a fatty alcohol; only CH3(CH2) x COC(CH2) yCH3 important in jojoba oil)
Figure 1.1 Plant storage lipids
The origin of the storage oil is still a matter of some controversy and two theories should be considered [Wanner & Theimer, 1978]. Some workers [Mudd, 1980] consider that the TAG is deposited and stored in the hydrophobic interior of the bilayers endoplasmic reticulum. However, in a number of studies no evidence of any membrane
4 6 C h a p t e r 1 Introduction can be seen in association with oil droplets, so oil is considered to be synthesized as a
"naked droplet". The oil bodies are surrounded by a half-unit membrane of phospholipid in which proteins [Slack et a., 1980], the oleosins, are located. Oleosins are believed to prevent oil bodies from coalescecing and act as an anchor for the lipases involved in the degradation of the oil reserve, when needed. There are three domains of an oleosin protein at the surface of an oil body. A section with an NH 2 terminal, that may help binding of the lipase; a central section proline-rich hydrophobic domain, which is probably immersed in the triacylglycerol of the oil storage droplet; and a third section which is amphipathic with a charged amino acid in the aqueous phase and uncharged side chains on the lipid side [Murphy, 1990].
NH2
WATER COOH LIPID
Figure 1.2 An oleosin protein at the surface of an oil body
Although free fatty acids, diacylglycerols and monoacylglycerols are true metabolites in the biosynthesis of TAG, (as well as phospholipids), they appear only in trace amounts.
The presence of large quantities are an indication of lipolytic activity in immature or mechanically damaged seeds. Lipolytic enzymes can be active at sub-zero temperature -
20 °C preventing diacylglycerol and monoacylglycerol from accumulating.
The desert plant jojoba stores wax esters which are formed by estérification of a fatty
47 C h a p t e r 1 Introduction acid with a fatty alcohol.
In glycosylglycerides and phospholipids, acyl constituents are necessary to provide membrane function and integrity, but in triacylglycerols (storage lipids), they determine properties which classify oils used in technology, foods and medicine.
The degree of fatty acid chain unsaturation, is the key reason why some oils are used extensively in the production of detergents, or are suitable for cooking, while others are essential components of dietary foods. Short saturated fatty acid chains (8-12 carbon atoms) have good surfactant properties and are used in the production of detergents.
This chain length is also very stable and is good for cooking and margarine formulation.
Oils rich in oleic acid (18:1), are good for both cooking and eating because they have high oxidative stability while linoleic acid (18:2) is an essential dietary fatty acid for animals; both derive their stability from being unable to introduce further double bonds into the acyl chain after C9.
Linolenic acid (18:3) can easily undergo oxidation and therefore is not used in food, but it has excellent drying properties and is used in the manufacture of paints and related coatings [Harwood, 1980].
Gamma (y)-Linolenic acid has recently received attention from medical research. There are reports to suggest that this fatty acid is useful in the treatment of a wide range of atropic disorders [Horrobin & Manku, 1983]. This may be related to its role as a metabolic precursor for ecosanoids. y-Linolenic acid is found only in oil seeds of the
Boraginaceae family and in very small quantities, about 30% of the total oil content
[Jamieson & Reid, 1969].
Capric (10:0) and lauric (12:0) acids are important in the detergent and in the cosmetic industries, as well as providing easily absorbed lipids for patients with digestive
48 C h a p t e r 1 Introduction disorders.
Erucic acid (22:1) is used in lubrication, but since it is absent from the Sn-2 position it can be present only at 60-65%.
Today it is possible to improve oil quality by using conventional plant breeding techniques to change the degree of unsaturation or chain length. For more drastic changes in acyl composition of the seed oils, for example introducing non-endogenous fatty acids or large reductions in acyl chain length, gene manipulation can be used. All of these alternatives to achieve the desired acyl composition usually does not have any adverse effect on the plant.
1.3.2 Glycerophospholipids
Phospholipids are the major lipid component of most cell membranes in both plants and animals. In the animal species neural tissue is the exception and contains cerebrosides and gangliosides. In plant tissues the exception is chloroplast, which is enriched in diacylglycolipids. The most commonly occuring phospholipids have structures based on phosphatidic acid.
CH2O.CO.R1 I R2CO.OCH 0 I II CH2O — p — o x
O"
Figure 1.3 Glycerophospholipids: Basic Structure
49 C h a p t e r 1 Introduction
Table 1.5 Phospholipids
X-Group PHOSPHOLIPID ABBREVIATION
-H Phosphatidic acid PA -CH(0H)-CH(0H)-CH2(0H) Phosphatidylglycerol PG -CH2-CH(NH2)-C00H Phosphatidylserine PS -CH2-CH2-N(CH)3 Phosphatidylcholine PC -CH 2-CH 2-NH 2 Phosphatidylethanolamine PE -inositol Phosphatidylinositol PI
The phospholipid content and composition in the membrane can vary in different membranes within the cell or from one cell type to another. Specificity of the phospholipid content and regulation as well as a distribution in the cell membrane is well known, but still not understood. One of the hypotheses is that lipids optimise the
cell for enzymes requiring a specific membrane. The total phospholipid content and
composition is very different in mammalian and plant cells. Mammalian membranes
contain 40-80% of phospholipids, where as plant tissue membranes can have 30-40% of phospholipids. Bacteria and fimgi produce different yields of phospholipid due perhaps to the different requirements of the cell fimctions.
Phospholipids in the plant cell are primarily constituents of lipid membrane structures.
Phosphatidylcholine, phoaphatidylethanolamine, and phosphatidylglycerol are the major phospholipids in higher plants with phosphatidylcholine the major phospholipid in most plant tissues and membranes. Phosphatidylglycerol is the main and in some cases the
only phospholipid in chloroplast but phosphatidylethanolamine can also be distributed
5 0 C h a p t e r 1 Introduction in chloroplast, certain seeds and in all membranes. Phosphatidylinositol can be found in lesser amounts, but diphosphoinositide and triphosphoinositide have not been detected.
Phosphatidylserine and phosphatidic acid are widely distributed in plants, but in trace amounts. Cardiolipin (diphosphatidylglycerol) is exclusively found in the inner mitochondrial membrane. Phospholipids are only 15% of the total lipids in chloroplasts; however the phospholipid content of plant mitochondria are significantly higher.
Lyso-lipid derivatives have been reported in several tissues; lyso-phosphatidylcholine
(LyPC) is an important glycerophospholipid in the cereal grains, wheat and barley.
They are many different fatty acids in plants, but only a few common fatty acids are major fatty acids in phospholipids. Palmitic, linoleic, and linolenic are the major fatty acids in higher plants; stearic and oleic acids are usually present in smaller amounts.
Chain lengths other than 16 and 18 carbons are rare. The fatty acid composition and distribution of the plants is similar to that in animal tissues, although compositions are very dependent on conditions, such as light and temperature.
1.3.3 Glycoglycerolipids
Glycoglycerolipids are present in large amounts in plant tissues, but in lesser amounts in
animal and microbial tissues.
The most important three glycoglycerolipids are :
- Monogalactosyl-diacylglycerol (MGDG)
- Digalactosyl-diacylglycerol (DGDG)
-Sulphoquinovosyl-dyacylglycerol (SQDG)
51 C h a p t e r 1 Introduction
Common name Structural and chemical name
HgCOH
Monogalactosyl CHz diacylglycerol I (MGDG) " ë - CHOCOR^ I HO CHgOCOR^
l,2-diacyl-[P-D-galactopyranosyl-(l-3)]-5«-glycerol
HgCOH
Digalactosyl HO diacylglycerol OH Ô — CH2 (DGDG) HO J— HO O-CH
CHOCOR OH
1,2-diacyl-[a-D-galactopyranosyl-( 1-6)- P-D-galactopyranosyl-(l-3)]-5«-glycerol
H2C— SO3 Plant sulpholipid (sulphoquinovosyldiacylglycerol (SQDG) O — CH2
A u CHOCOR^
CHgOCOR^
1,2-diacyl-[6-sulpho-a-D-quinovopyranosyl-(l -3)]-5«-glycerol
Figure 1.4 The structure of plant glycosylglycerides
5 2 C h a p t e r 1 Introduction
Diacylgalactosylglycerol (monogalactosyldiglyceride) and diacylgalabiosylglycerol
(digalactosyldiglyceride) occur in very high amounts in plastid membranes, and are probably among the most common lipids found in chloroplast together with a unique sulpho lipid, sulphoquinovosyldiacylglycerol, found in higher plants and algae
[Harwood, 1980; Harwood, 1985].
Chloroplasts as photosynthetic tissue and plastids as a non-photosynthetic tissue, have membranes consisting mainly of glycosylglycerides. Unlike any other membrane in nature, chloroplast membranes contain protein-glyco-lipid structures.
Monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG) carry no charge although their head groups are polar. These lipids are orientated in the membrane according to its bilayer polarity; diacylglycerol which is hydrophobic, orientates itself to the interior of the membrane, while the hydrophilic head group is on the outside surface of the membrane. The plant sulfo lipid (SQDG), carries a full negative charge at physiological pH, and it is likely to be associated with positively charged proteins or water soluble cations.
1.3.4 Glycosphingolipids
This group of lipids contain sphingosine or a related amino alcohol. Cerebrosides, ceramide and phytoglycolipids are of minor importance in plants.
Cerebrosides can contain a variety of bases, but only glucose as their component sugar
[Carter et <2/., 1961; Karlessos & Holm, 1965; Ito & Fujino, 1973]. Ceramide has been found in alfalfa leaves and rice grain. Phytoglycolipids are sphingosine-containing lipids, and they contain inositol as well as other monosaccharide residues [Kaul &
5 3 C h a p t e r 1 Introduction
Lester, 1975; Hitchcock & Nichols, 1971].
3 CH2-OH
2 CH-NHo
HO-CH-CH=CH-CH2(CH2) iiCH3 1 a b c
Figure 1.5 Sphingosine (the sphingenine analogue)
Although phosphoglycerides and glycosylglycerides are the most important membrane
lipids, a large number of minor lipids can also be found. Sterols and sphingolipids may be significant in the plasma membrane, whereas ether-linked lipids may be minor
components in many plant membranes.
1.3.5 Cutins, suberin and waxes
While phospholipids and glycolipids are mainly membrane constituents and
triacylglycerol is a storage lipid, the surface coverings of plants contain lipids of
different compositions. The nature of the suface layer is different for various plant parts;
leaf covering is formed of cutin and wax, whereas roots (and wounds) contain suberin.
These have very long chain components which vary in their proportions, not only from
species to species but also with age. Other lipids that are important in plant surface
coverings contain hydrocarbons, ketones, secondary alcohols, aldehydes, diketones and
5 4 C h a p t e r 1 Introduction di- or polyesters.
All plant surfaces are covered in a protective layer which serves the dual function of preventing water loss and stopping microbial entry into the plant tissues.
Waxes are found in barks, roots and wounds as well as in the seeds of certain plants
(e.g. jojoba) and can be readily extracted using hexane or chloroform, because they usually contain hydrocarbons (alkanes). Odd-chain alkanes, monoesters of long-chain alcohols and fatty acids are present in virtually every plant together with very-long chain alcohols (C22-C34), very-long chain saturated fatty acids (C20-C30), ketones and beta-diketones. Their proportions can vary from a few per cent, to being the major constituents of some waxes.
Cutin is composed of a C16 and Cl 8 monomer. The hydroxy groups of these monomers can form ester links and from there, polyester structures to be formed.
The structure of suberin consists of ©-hydroxy fatty acids, the corresponding
dicarboxylic acids, very long chain acids and alcohols (>C18). There are also phenolic
compounds (the most common is p-coumaric acid), which are the basis of suberin the polymeric structure.
1.4 FATTY ACIDS
The building blocks of acyl lipids are fatty acids. Although the number of fatty acids
detected in plants is about 300 [Hitchcock & Nichols, 1971], only a few are commonly
used as storage or membrane lipids; 89-97% of all known leaf acids consist of only
seven fatty acids. These are both saturated and unsaturated monocarboxylic acids with
5 5 C h a p t e r 1 Introduction an unbranched even-numbered carbon chain [Harwood, 1980; Gustone et al., 1986;
Harwood & Jones, 1989].
The saturated fatty acids are, lauric, myristic, palmitic, and stearic, while the unsaturated fatty acids are oleic, linoleic and linolenic.
The traditional names are inspired by the original source of the oil or fat from which the fatty acid was originally isolated; palmitic acid was named after palm oil, and oleic acid after olive oil. Since the number of acids is large, trivial names have been given to some but many have only systematic names.
The fatty acid chain determines many of the physical properties; lipids that are solid
(fats) at room temperature have saturated fatty acids as a major component or if the lipids are liquid (oils) they have unsaturated fatty acids as a major component. It is believed that membrane acyl chains must be above their gel to liquid transition temperature (Tc) and therefore be fluid, which is important, since plants cannot regulate their own temperature under different environmental conditions.
The position and stereochemistry of double bonds are of considerable importance. Most unsaturated fatty acids in seed lipids have double bonds in the cis- conformation but there are some naturally occurring fatty acids with trans-double bonds. In systematic names, the position of the double bond is defined by numbering the carbon atoms from the carboxyl group end, marking the first unsaturated carbon and adding the cis- or trans- prefix to define the double bond position and conformation.
5 6 C h a p t e r 1 Introduction
Table 1.6 Structures of mai or fattv acids
Common Symbol Structure Systematic name name
Lauric 12:0 CH3(CH2)10COOH Dodecanoic acid Myristic 14:0 CH3(CH2)12C00H Tetradecanoic acid Palmitic 16:0 CH2(CH2)14C00H Hexadecanoic acid Stearic 18:0 CH2(CH2)16C00H Octadecanoic acid Oleic 18:1 CH3(CH2)7CH=CH(CH2)7C00H Octadecanoic acid Linoleic 18:2 CH3(CH2)4CH=CHCH2CH=CH(CH2)7C00H Octadecadienoic acid Linolenic 18:3 CH3(CH2CH=CH)3(CH2)C00H Octadecatrenoic acid
Trans unsaturated acids have melting properties similar to saturated compounds.
The distance between the methyl-end of the carbon chain and the first double bond can be important structurally and from the biological functional view point. The fatty acid composition in plants depends on environmental conditions, particularly light and temperature.
Various membranes in plants contain special lipids and proteins, and each class of these lipids possesses different acyl patterns. Their distribution and combination on the glycerol backbone is important for the properties and function of these lipids. Particular acids with their positions and combinations can influence a lipid’s ability to become a substrate for an enzyme. The gel-liquid thermal transition (Tc) temperature in PC can differ by 4 °C, depending on whether stearic or oleic acid is bound to the glycerol backbone at positions 1 or 2 [Harwood, 1997].
57 C h a p t e r 1 Introduction
Palmitic, linoleic and linolenic acids are the major components in photosynthetic tissues, while oleic acid is a major component of non-photosynthetic tissues. Much less information is available about fatty acid positional distribution in natural triacylglycerols, where unsaturated fatty acids are at positions 1 and 2, while saturated acids are normally at position 3. In phospholipids chain lengths other than 16 and 18, are unusual and their distribution on the glycerol backbone is usually saturated fatty acid chains at position-1 and unsaturated fatty acids at position-2 [Harwood, 1989].
In phosphatidylcholine (PC), linoleic acid is the main component together with palmitic, oleic and linolenic acids, except in PC from non-photosynthetic tissue. Studies with this phospholipid have show that saturated acids are preferred at position 1 [Sastry & Kates,
1964; Heinz & Harwood, 1977; Devor & Mudd, 1971].
Phosphatidylglycerol, the lipid in photosynthetic tissues, contains 15-30% of trans-3- hexadecenoate and trans-3-hexadecanoic acids, esterifred at position 2 [Haverkate &
Van Deenen, 1965]. Phosphatidylglycerol is the acidic lipid characteristic of chloroplast and has a linolenic acid group at position-1. The actual percentage of saturated fatty acids chains varies from 10-65% for different plants. Plants with a higher percentage of saturated chains have poor cold tolerance.
Phosphatidylethanolamine (PE) is similar in this respect to PC [Harwood (b), 1985].
Phosphatidylinositol (PI) contains predominantly the saturated acids, palmitic and stearic acid [Erdahl et al., 1973; Gaillard, 1973; Harwood, 1975; Harwood & Stumpf,
1970].
Phosphatidylserine (PS) has been reported to have a high concentration of very long chain fatty acids [Murata et al., 1984].
In higher plants, thylakoid lipids in leaves have rather characteristic fatty acid patterns.
5 8 C h a p t e r 1 Introduction
Monogalactosyldiacylglycerol contains around 90% of linoleic acid whereas digalactosyldiacylglycerol besides linoleic has, an increased amount of palmitic acid compared to monogalactosyldiacylglycerol. The positional distribution of fatty acids has been analysed following lipase hydrolysis and mass spectrometry of individual molecular species [Auling et al, 1971; Rullkotter et a l, 1975; Tulloch et al, 1973].
One important observation from this work is that, plants which synthesize hexadecatrienoic acid, have this acid localised specifically at the Sn2 position, which is similar to the palmitic acid in DGDG.
SQDG is more similar to phospholipids than galactolipids, when it comes to fatty acid composition. Palmitic, linoleic and linolenic acids are nearly always major components.
In some plants oleic acid is predominant. Positional specifities vary with plant type
[Heinz & Harwood, 1977].
There are strikingly similar fatty acid compositions of leaf tissues from different plants.
This is in contrast with the variability seen in the seeds, where the seeds can contain
90% or so of their fatty acids as a single component.
In membrane lipids, oleic acid is the most commonly occurring mono-unsaturated fatty acid, together with linoleic and a-linolenic acids which are the most commonly occurring poly-unsaturated fatty acids. In membrane lipids (like in leaves) there is a consistency of fatty acid components, while in seeds there can be extensive variations.
This is related to their function; membrane structure and properties which have to be tightly regulated. By contrast storage lipids have to be accessed by degenerative enzymes to be broken down as required.
59 C h a p t e r 1 Introduction
Table 1.7 The presence of common fattv acids in plants
Fatty acid Comments % content % content in leaves in seed oils
Lauric widespread minor trace 70-80% in palm (12:0) component but major in kemal and coconut palm and coconut fhiits Myristic widespread minor trace 80% in coconut (14:0) component Palmitic the main saturated fatty 10-13% 45% in palm seed (16:0) acid of plants oil Stearic wide spread minor 1-4% 34% in cocoa butter (18:0) component, occasionally major as in cocoa butter Oleic the main 3-7% 78% in olive (18:1) monounsaturated fatty acid of alga and plant tissues; also major component of edible oils Linoleic a major component of all 8-15% 75% in safflower (18:2) plant tissues; the most common in edible oils Linolenic the main fatty acid of 53-67% 60% in linseed (18:3) leaves; minor component in seed oils, eccept linseed Arachidonic not usually found in trace trace (20:4) significant amounts in higher plants but major component of mosses, liverworts, marine algae
6 0 C h a p t e r 1 Introduction
1.5 PHENOLIC COMPOUNDS
1.5.1 General description
In a addition to the primary metabolites which are common to all plants, research has been carried out to identify numerous secondary metabolites. Among them, phenolics embrace a considerable range of substances from some which possess an aromatic ring bearing one or more hydroxyl substituents to highly complex polymeric substances such as tannins and lignins. These compounds are present in all plant tissues and frequently form the most abundant secondary metabolites in fruits, where they can reach high concentrations. Phenolics are important constituents of some medicinal plants and in the food industry they are used as colouring agents, flavourings, aromatizers and antioxidants. There are several thousand polyphenols which have been found in plants and they can be grouped in several classes, most of which are found in fruits. Table 1.8.
61 C h a p t e r 1 Introduction
Table 1.8 The major classes of phenolics in fhiits
No of Basic Class Example Fruit carbon skeleton (example) atoms
7 Cô-Ci Hydroxybenzoic acids /^-Hydroxybenzoic Strawberry 9 Cô-Cs Hydroxycinnamic acid Caffeic Apple Coumarins Scopolin Citrus 10 C6-C4 Naphthoquinones Juglone Walnut 13 Cô-Ci-Cô Xanthones Magniferin Mango 14 C6 -C2-C6 Stilbenes Resveratrol Grape 15 Cô-Cs-Cô Flavonoids Quercetin, cyanidin Cherry Isoflavonoids Daidzein French bean n Lignins Stone fruits Tannins Persimmon
Functional aspects of phenolic compounds are of great importance in plants. They are
indispensable elements in anatomical and morphological structures and form an integral part of cell walls, with polymeric compounds such as lignins and suberins. These provide mechanical support, form barriers against microbial invasion, and help plants to
adapt to terrestrial life by building woody stems and conducting cell elements for water transport. Lignins are the second most abundant organic structures on earth. Today there is an increasing interest in phenols as compounds of prime ecological importance for plant survival.
Anthocyanins together with flavones are co-pigments and contribute to flower and fruit colours. This is important for attracting animals and insects to the plant for pollination
and seed dispersal. There is strong evidence that DNA damaging ultraviolet light
62 C h a p t e r 1 Introduction induces the accumulation of UV light-absorbing phenolics which are predominantly placed in the dermal tissues of a plant body [Strack, 1997].
Phenolics can protect plants against predators. Herbivores react sensitively to the phenolic content in plants. Both simple and complex compounds affect plant-bird interactions by interfering with digestion through interaction with the microbial flora of the cecum. Phenol compounds can accumulate as "phytoalexins", as a result of microbial attack which the plant recognises by the detection of parasite derived molecules called elicitors. Phytoalexins are low-molecular weight compounds that might be present in a plant at a low concentration but can rapidly accumulate upon the attack, as induced compounds. In contrast there are preinfectional toxins present as constitutive compounds at high concentration in healthy tissue as free toxins or in conjugated forms from which they are released after the attack. Hydroxycoumarins and hydroxycinnamates are among the major phenolic phytoalexinst and contribute to the disease resistance mechanisms in plants.
Phenolic compounds may influence competition among plants and this phenomenon is called "allelopathy".
The most recent findings concerning the function of phenolic compounds, is that they can act as signal molecules [Strack, 1997]. This consists of an interaction between nitrogen fixing bacteria in certain plant families, for example Fabaceae. Flavonoids from this plants induce nodulation gene transcription by activating regulatory proteins which enables the plant to use atmospheric nitrogen through the action of the bacterial nitrogen-activating enzyme nitrogenaze.
Along with other plant compounds, phenols are of great importance and although poisonous or hallucinogenic are of pharmacological value. Polyphenols are important
6 3 C h a p t e r 1 Introduction in food stuffs, nutrition, and in wines and herbal teas, for their astringent taste. Because of the ability of tannins to complex with proteins they have been used for centuries in the leather making industry. Anthocyanins are of interest and importance in plant colouring and are used in food colouring.
1.5.2 Hydroxybenzoic Acids
Hydroxybenzoic acids (HBA) have a general structure of the C6-C1 type derived directly from benzoic acid. Variations in structure lie in the hydroxylations and méthoxylations of the aromatic ring. From the quantitative point of view, the HBA content is generally low, except in certain fruits of the Rosaceae family and in particular blackberry in which protocatechuic and gallic acid contents may be very high [Mosel &
Herrmann, 1974]. Quantitative and qualitative investigations of HBA in native products can play a role in the organoleptic qualities of the fruit.
HO GOGH
R
Figure 1.6 Chemical structures of benzoic acids
Ri=H Rz=H /7-Hydroxybenzoic acid
R1=0CH3 Rz=H Vanillic acid
Ri=OH R2=0H Gallic acid
64 C h a p t e r 1 Introduction
1.5.3 Hydroxycinnamic acids
Hydroxycinnamates preserve the C6 -C3 structure. Among fruit phenolics hydroxycinnamic acids (HCD), play an important role because of both their abundance and diversity. They are derived from cinnamic acid and are essentially present in four molecular types; />-coumaric, caffeic, ferulic and sinapic acids. The diversity of HCD found in plants and particularly in fruits results from the nature of the bonds in the molecules involved. Conjugating moieties can be anything from carbohydrates, proteins, lipids, amino acids, amines, carboxylic acids, terpenoids, alkaloids, flavonoids, cutins, suberins, lignins or polysaccharides, which can be found in a cell wall.
Conjugation reactions may determine whether the compound is going to become metabolically inactive as a final end product (storage) or an accumulating intermediate for further metabolic pathways. In addition to this, for each of these compounds the presence of a double bond in the lateral chain leads to the possible existence of two isomeric forms cis and trans. Distribution of hydroxycinnamates shows a correlation with the systematic arrangements of plant families.
COOH
Figure 1.7 Chemical structures of hydroxycinnamic acids
Ri=H R2=H p-Coumaric acid
Ri=OH R2=H Caffeic acid
65 C h a p t e r 1 Introduction
Coumarins are also derived from hydroxycinnamic acids. They are lactones from the cyclization and ring closure between the o-hydroxy and carboxy groups. Today, nearly a thousand coumarins are known in nature and although some of these coumarins are present in plants as their glycosides, most of them exist in the free form in the cell.
Considerable study of fruit coumarins has been carried out on the Umbelliferae and
Rutaceae, with emphases on citrus fruits because of their economic importance.
Figure 1.8 Coumarin
1.5.4 Xanthones
Xanthones are compounds with a Cô-Ci-Cô structure that is encounterd in several families of Angiosperms, mainly in the Guttiferae and Gentianaceae. Magniferin is perhaps the best known xanthone, and has been identified in the pulp of three varieties of mango, where it is abundant in the leaves and bark [Saeed et al., 1976]. Magniferin has anti-inflammatory, antihepatotoxic and antiviral properties.
66 C h a p t e r 1 Introduction
0 OH
Figure 1.9 Magniferin
1.5.5 Flavonoids
There are more than 500 different known flavonoids. Various structural classes of the
Ci5 aglycone skeleton are modified by hydroxylation and méthoxylation. Three of the numerous classes of flavonoids which are very widespread in fruits and quantitatively dominant are anthocyanins, flavonols and flavan-3-ols. The other classes flavones, flavanones, flavanonols or dihydroflavonols, chalcones and dihydrochalcones, are less important except in certain special cases, such as Citrus fhiits.
Flavonoids are usually glycosylated and many are acylated with aliphatic and aromatic acids. The flavonoid aglycones are classified according to the oxidation state of the pyran ring. Important groups are isoflavones and phenylpyrens, that are infection- induced phytoalexins which are characteristic constituents in some of the Fabiacece possessing fungicidal and bactericidal activities. It does not necessarily follow that the compounds are present in all parts of the plant, but they are often specifically located in certain organelles. The occurrence and distribution of flavones (Figure 1.10) and flavonols, has been reviewed [Macheix et al., 1990]. In recent years attention has been given to the importance of glycosylation in relation to the functions of flavones and
67 C h a p t e r 1 Introduction flavonols in plants although a significant number of these compounds occur in plants in the free state without sugar attachment. Free aglycones, especially those highly methylated, are likely to occur externally or associated with secretory structures and lipophilic constituents and may be restricted to certain parts of the plant, e.g. the waxy coatings of the leaves. Free flavones and flavonols are potentially toxic to living cells and inhibit many enzymatic activities. Flavonols are flavonoids characterised by an unsaturated 3-C chain with a double bond between C-2 and C-3 and by the presence of a hydroxyl group in the 3-position. Nearly 200 flavonol aglycones have been identified in plants and many of them are methylated derivatives. Approximately 90% of these flavonols are hydroxylated at the 5- and 7- positions in addition to the hydroxyl at position 3.
Flavones are flavonoids characterised by an unsaturated 3-C chain and have a double bond between C-2 and C-3, like flavonols, and differ by the absence of the hydroxyl in the 3-position. What appears to be a simple difference between flavones and flavonols has very important consequences in biogenesis, physiological and pharmacological roles. There are over 100 natural flavones that have been identified in plants and they are widely distributed among the higher plants in the form of aglycones or glycosides.
However, these compounds are not common in fruits and are never predominant.
68 C h a p t e r 1 Introduction
OH
OH O
Figure 1.10 Flavone common structure
Rl=0-Glucosyl R2=0H Luteolin 7-glucoside
1.5.6 Isoflavonoids
Isoflavonoids are biogenetically related to the flavonoids but constitute a distinctly separate class because they contain a rearranged Cis skeleton and may be regarded as derivatives of 3-phenylcoumarin. Structurally the isoflavonoids may be subdivided into several classes according to “oxidation” of the skeleton, and its complexity. Isoflavones are the most abundant of the natural isoflavonoid derivatives.
1.5.7 Lignins
Lignins are important polymeric substances in plants consisting, of heterogeneous phenylpropane polymers (C6-C3)n. Lignins from different biological sources vary in
69 C h a p t e r 1 Introduction composition, depending on the particular monomeric units of which they are composed.
They form important constituents of cell walls within supporting and conducting tissues, which enable vascular plants to develop into large upright structures, like trees.
Lignins are always associated with the wall polysaccharides, cellulose and hemicellulose.
1.5.8 Lignans and neolignans
Lignans are dimers, (Ce-Cs):. At one time it was thought that they were early intermediates in the formation of lignins. Lignans are widely distributed in plants accumulating as soluble components, some of them as glycosides. Some 300 lignans have been isolated and categorised into a number of groups according to their structure.
There is increasing knowledge about their antimicrobial, antifungal and anticancer properties [Strack, 1997]. Lignans are generally defined as phenylpropane dimers, where phenylpropane units are C-C linked mostly tail-to-tail, through their Cs-side chain. Neolignans have phenylpropane units linked head-to tail.
1.5.9 Tannins
Tarmins are complex substances that occur as a mixtures of polyphenols that are difficult to separate because they do not crystallise. Tannins can be divided into two chemical classes: hydrolysable and non-hydrolysable (condense) tannins. Both tannin groups are usually located in a specific plant part: leaves, fhiits, barks or stems. Tannins possess a number of hydroxyl groups which makes it possible for them to bond
7 0 C h a p t e r 1 Introduction reversibly with other natural macromolecules (e.g.polysaccharides, proteins) and with various metabolites. This bonding can occur naturally during the physiological development of the fruit or during the decompartmentation which occurs when the fruit is crushed for technological processing or after extraction.
Hydrolysable tannins are complex polyphenols which may be degraded under hydrolytic conditions (either acid, alkaline, or hydrolytic enzymes) into smaller fragments, like sugars and some phenolic acids. Hydrolysable tannins are polyesters based on gallic acid (Figure 1.7) and/or hexahydroxydiphenic acid which are united by ester linkages to a central glucose molecule. Two principal types of hydrolysable tannins are gallitannins and ellagitannins.
Unlike hydrolysable tannins, condensed tannins are not readily hydrolysed to simpler molecules and they do not contain a sugar moiety. Non-hydrolysable tannins or condensed tannins are also named proanthocyanidins, because they can give anthocyanidins in the presence of acids. Most of the condensed tannins result from the condesation of two or more flavan-3 ols such as (+) catechin (Figure 1.11) and (-) epicatechin. Together with flavan-3,4-diols (leucocyanidins) they are intermediates in the insoluble biosynthesis of the polymeric molecules, usually red coloured, known as phlobaphenes.
OH
HO, OH
OH OH
Figure 1.11 (+) Catechin
71 C h a p t e r 1 Introduction
Complex tannins are a newly discovered group which are biosynthesised from both a hydrolysable tannin (mostly a C-glycoside ellagitannin) and a condensed tannin. The union occurs through a C-C bond between the C-1 of the glucose unit of the ellagitannin and the C-8 or C-6 of the flavan-3-ol derivative. The monomers are also involved in oligomer formation.
Tannins are of wide occurrence in plants and they exert an inhibitory effect on many enzymes due to protein precipitation which makes them contribute to a protective function in barks and heartwoods.
1.5.10 Quinones
Quinones form an important group of natural compounds because of their reactivity, their involvement in electron transport and their biological and pharmaceutical properties [Leistner, 1981]. The commonly occurring quinones in nature are benzo-, naphtho- and anthraquinones. They derive from different biosynthetic routes but their structures can contain phenolic moieties (e.g. Juglone, found in walnut shells. Figure
1.12). Benzoquinone isoprenoid derivatives such as plastoquinone and ubiquinone play important roles in primary metabolism. Secondary benzoquinones are typical constituents in fungi but are more rare in higher plants where many naphtho- and anthraquinones occur. Naphthoquinones are often responsible for the pigmentation of coloured heartwood and bark. Anthraquinones occur in bacteria, fungi and lichen and in members of higher plant families (Rubiaceae). They have been isolated from leaves, stems and roots but it is rare for antraquinones as well as other quinones to be found in fhiits.
72 C h a p t e r 1 Introduction
o
OH
Figure 1.12 Juglone
7 3 CHAPTER 2
OLIVE FRUIT-PULP ANALYSIS
74 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2 OLIVE FRUIT-PULP ANALYSIS
2.1 Introduction
Natural foods contain many compounds whose nutritional effects are unknown. Some of these naturally-occurring compounds have therapeutical effects and are of interest to the pharmaceutical industry in the treatment of human diseases and can be exploited in healthy diet.
Extensive studies have been made on the chemical composition of the primary and
secondary metabolism of Olea europaea, (table 1.4) with the view to both improving the organoleptic quality of the oil and identifying the substances in this plant that have biological activities (table 2.1). The distribution of components in olive fhiit is greatly
varied in the lipid fractions obtained from skin, pulp, wood shell and seed [Bianchi &
Vlahov, 1994].
Several studies of phenolic compounds present in olives have been carried out in relation to the processing of the fruit. These compounds are important parameters in the
organoleptic quality of the fhiit and as antioxidants in the conservation of the oil. In
contrast to other oils, virgin olive oil is consumed unrefined, and therefore contains polyphenols which are usually removed from other edible oils at various refining stages.
Olive oils are low in tocoferols, so the presence of other phenolic constituents capable
of antioxidant activity is of particular importance. It has been shown [Gutfinger, 1981], that the method of oil extraction from the fhiit affects the amount of polyphenols in the
oils. Solvent extracted oils were richer in polyphenols than oils obtained by pressing, with values of 321-574 pg/g and 44-194 pg/g of oil, respectively. The value of the olive
75 C h a p t e r 2 O live f r u it - p u l p a n a l y sis fruit, with olive oil as its main product, is determined by the acyl composition. Olive fruit contains the essential fatty acids linoleic and linolenic, and it is particularly rich in the monoenoic acid, oleic acid. Understanding lipid metabolism in olive fruit together with knowledge of the variations in phenolic compounds will make it possible to have a better understanding of the relationships that may exist between different substances and fruit physiology.
Table 2.1 Biological activities reported for Olea eurovaea
BIOLOGICAL ACTIVITY SAMPLE REFERENCES
Allergenogenic Allergenes (Ole-E-1, Ole-E-3) Obispo et al., 1993; Vilialba et al., 1994;Lahoz et al., 1985; Batanero et al., 1996; Boluda et al., 1997; Decesare et al., 1993; Rubio et al., 1987; Lauzurica et al., 1988; Blanca et al., 1983
Antioxidative activity Phenolic compounds Letutour & Guedon, 1992; Oleuropein, hydroxytyrosol Saija et al., 1998; Manzi et al., Carotene, alpha-tocopherol, squalene 1998
Free radical-scavenger Oleuropein, hydroxytyrosol Saija et at., 1998
Nitric oxide production Oleuropein Visioli et al., 1998
Antiinflammatory activity Oleuropein Visioli et al., 1998
Insecticide and anti fungal Oleuropein Kubo & Matsumoto, 1985;
Molluscides Olive extract Kubo & Matsumoto, 1984
Anti fungal Oleanolic acid Kubo & Matsumoto, 1984
Effects on excito-conduction and on Oleuropein Occhiuto et al., 1 990 monophasic action potential in anesthesized dogs Vasodilator effect leaf, fruits Zarzuelo et a/.,1991; Rauwald et al., 1994 Phytotoxic effects Catechol, 4-methyl catechol, tyrosol, Capasso et a i, 1994 hydroxytyrosol Hypoglycemic activity leaf Gonzales, 1992
7 6 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Effects on olive fruit-fly Olea-europea volatiles Scarpati et al, 1993
Effects on bactrocera-oleae Polyphenols Capasso et al, 1994 (Diptera, tephritidae) Repellent to Dacus-oleae females olive juice Loscaizo et a l, 1994
Feeding stimulant to the weevil Oleuropein Nakajima et a l, 1995 (Dyscerus perforatus) Antimicrobial activity Long-chain alpha, beta-unsaturated Kubo et al, 1995 aldehydes Effects on rat isolated ileum and trachea leaf extract Fehri et a l, 1995
Angiotensin-converting enzyme (ACE) Secoiridoid Hansen et al, 1996 inhibitor 2-(3,4-dihydroxyphenyl )ethyl -4-formyl - 3-(2-oxoethyl)-4E-hexenoate (oleacein) Toxicity against brine shrimp Oleacein Hansen et al, 1996
Anticomplementary activity Apigenin, apigenin-4’-0- Pieroni et a l, 1996 (inhibition of the classical pathway of rhamnosylglucoside, apigenin-7-0- the complement system) glucoside, luteoline, luteolin-4’-0- glucoside, chrysoeriol, chrysoeriol-7-0- glucoside and quercetin-3-O-rhamnoside Hypertension and diabetes Olea europea Ziyyat et a l, 1997
2.2 Materials and methods
2.2.1 Sample
Black Greek Olives {Olea europaea L.) were obtained from the island of Kim. The
olives were peeled and the pulp separated from the skin and seed.
2.2.2 Reagents/equipment for the analysis of Olive pulp
Chloroform, methanol, hexane and 1.2 dichloroethane, were all of HPLC grade and
obtained from Fisher Scientific, Limited, UK.
77 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Deuterated chloroform ( 99.8% ), perdeuteromethanol ( 99.8% ) and NMR tubes (5mm) were obtained from the Aldrich Chemical Company, Inc., USA.
The estérification reagent, BCb-methanol was ordered from the Supelco, Co., USA.
Aluminium backed Kiesel gel 60 F-254 silica gel plates for thin layer chromatography were purchased from B. D. H. Merck Ltd., UK.
Lipid standards were obtained from the Sigma Chemical Company.
2.2.3 Preparation of extracts from Olive pulp
The olive pulp was removed carefully and air dried in a shadowed room for seven days.
The air dried pulp (209 g) was then frozen in liquid nitrogen and ground to a paste. This was then mixed with 300 ml of chloroform:methanol (2:1) in a glass beaker and
sonicated in an ultrasonic bath at 0 °C for 20 min. The suspension was then filtered through cheese cloth to remove particulate matter and then centrifuged at 2 000 rpm for
10 min (at 4 °C), to separate the aqueous and organic layers. Three sequential
extractions were carried out.
From the first extraction two layers separated out, an upper aqueous layer and a lower
organic layer (250 ml). Only discrete organic fractions were collected. The interface with the water layer were combined with the residual material from the cheese cloth
filtration and re-extracted with a further 300 ml of chloroform : methanol (2:1). From this second extraction two layers again separated out, an upper aqueous layer and a
lower organic layer (250 ml). The third extraction using 300 ml of chloroform : methanol (2:1) again produced two layers, an upper aqueous layer and a lower organic
layer (240 ml).
78 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
The lower layer from each extract (denoted Ei, Ei, Es-respectively) was concentrated by rotary evaporation to a volume of 2 ml.
2.2.4 Chromatographic techniques
2.2.4.1 Column chromatography
Each of the three solvent extracts was separated by column chromatography. A 6 cm x
2.5 cm eluting tube was packed with silica gel 60-100, Davisil grade 63s, and pre conditioned by washing with 50 ml of HPLC grade chloroform and methanol (2:1).
An aliquot of 500 pi of the concentrated extract (Ei) was loaded on to the column which was eluted first with chloroform and then with methanol, and 20 ml fractions were
collected. A yellow band was eluted by the chloroform (80 ml) Fi. The eluting solvent was then changed to methanol and a further 60 ml ¥2, collected. Further elution with
methanol gave three coloured bands, each contained within a 20 ml volume designated
as F 3 , F4 and Fs respectively.
An aliquot of 500 pi from the second extraction (E 2) was processed in a replicate way using the same solvent volumes as in the first extraction to produce a fraction Fô, from the chloroform elution and from the methanol elution Ft, Fs, F9 and Fio. Fs, F9 and Fio
were also coloured and corresponded with Fa, F4 and Fs from the chromatography of the
first extract.
An aliquot of 500 pi from the third extraction (Es) was processed in the same way using
the same solvent volumes to produce a chloroform fraction Fii (corresponding to Fi and
79 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Fô) and methanol fractions F 12, F 13, Fi4, Fis and F 16 . The fractions F 13 - F16 were slightly darker in colours than the corresponding F 3 - Fs and Fs - Fio from the first and second extractions. The fractions collected (Table 2.2) were monitored by TLC, HPLC,
GC-MS and electospray MS.
Table 2.2 Fractions of Olive pulp from column chromatography
Eluting Solvent Extraction E l Extraction E2 Extraction E3
Chloroform Fi Fô Fii
Chloro/Methanol F2 F? F i2 Methanol F3 Fs F13
Methanol F4 F9 F i4 Methanol Fs Fio Fis
Methanol F i6
2.2.4.2 Thin layer chromatography (TLC) on fractions from Olive pulp
Thin layer chromatography was performed using Merck aluminium backed precoated thin layer Kieisel gel 60 F-254 plates. The solvent system was equilibrated before use in glass TLC tanks, lined with filter paper. Samples were applied 2 cm above the bottom edge and separated by 2 cm divisions. Samples were eluted until the solvent front was
18 cm above the origin. All chromatographed plates were initially observed under UV before treatment with a spray reagent. The plates were stained with a mixture of 1%
80 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Vanillin in EtOH and 40% Sulphuric acid in EtOH (1:1), until saturated and heated in an oven at 100 °C for 10 min., before visualisation.
Fractions Fi and F 2; Fô and F?; Fii and F 12 gave no obvious separation when examined in chloroform : methanol (9 : 1) under the conditions used to separate fractions Fs - Fs;
Fs - Fio and Fis - F 16 (Fig. 2.1). Fractions Fi and F 2; Fô and F?; Fii and F 12 were therefore examined in hexane : chloroform (9:1) (Fig. 2.2).
Fractions Fs, ¥9 and Fio which had identical TLC profiles were pooled. Fractions Fis,
F i4 and Fis which also had identical TLC profiles were also pooled.
2.2.4.3 High performance liquid chromatography (HPLC) on fractions from Olive
pulp
HPLC was performed on a Perkin Elmer Series 3 pump unit coupled to an LC 65T oven/variable wavelength UV detector (Perkin Elmer, Beaconsfield UK). Zorbax Silica columns of dimensions 25cm x 0.46cm and 25cm x 0.94cm, particle size 5pm, were eluted isocraticly with 90% 1.2 dichloroethane and 10% methanol.
Fractions Fs, F4, Fs- 10, Fis-is and F 10 were first examined on an analytical column. The chromatograms are shown in Figures 2.3-2.8. All of the chromatograms gave similar profiles, however Fs and F4 showed more early eluting components than Fs-io; Fis-is and F 10. The combined fraction Fis-is was selected for preparative HPLC because of its common profile and the relative intensity of the component peaks. Twenty injections were made and the main component fractions collected and labelled A, B, C, D and E respectively. These fractions were evaporated to dryness and dissolved in CDCls:
CDsOD (2:1) for NMR spectroscopy.
81 C h a p t e r 2 O live fr u it - p u l p a n a l y sis
2.2.5 Spectroscopic methods
2.2.5.1 Nuclear magnetic Resonance Spectroscopy (NMR) of fractions from Olive
pulp
Fractions were evaporated to dryness and dissolved in CDsOD : CDCb (2:1) for NMR
spectroscopy. Samples were bubbled with nitrogen prior to recording the spectrum in order to prevent any possible oxidation. Both and 2D COSY spectra were obtained
from a Bruker AM 500 NMR instrument (Bruker, Germany). The spectra were processed using felix 2.3 [Biosym, 1987, 1993].
A field frequency lock was provided using the deuterium signal in CDsOD.
The spectra were recorded at 25 °C in the Fourier transform mode (FT) and were
acquired using a 3 ps, 45° pulse, a spectral width of 6000 Hz, an acquisition time of
1.36 s, and a relaxation delay of 4 s. The free induction decays (FIDs) were collected with 16 K data points.
Connectivity among protons and hence confirmation of the identity of different
compounds was established with the magnitude COSY pulse sequence [Nagayama, et
al., 1980]. 2D COSY experiments were performed at 298 K with a special width (SW)
of 6024.10 Hz and a relaxation delay of 2 s and were recorded in 2 K (2048) data points,
obtained from 512 FIDs of 48 scans each, with zero-filling in the FI dimension.
Chemical shifts were referenced to the residual methanol peak at 3.31 ppm for the
samples in CDsODiCDCb (2:1).
8 2 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2.2.S.2 Mass Spectrometry (MS) of fractions from Olive pulp
Mass spectrometry measurements were made in both positive and negative electrospray
ionisation modes on a Finnegan Navigator mass spectrometer (Finnegan, Altrincham
UK). The samples were introduced by loop injection from a Waters 2690 pump and injector unit.
Samples A, B, C, D and E were evaporated to dryness and redissolved in methanol (1 ml) to which 50 pi of water had been added. Each sample (10 pi) was injected into the mass spectrometer via the injector and pumping system using acetonitrile : water (50
:50). Spectra were recorded in negative ion mode over the mass range 100 - 1000
Daltons at a cone voltage of 35 V.
To determine the number of exchangeable hydrogens each fraction was evaporated to
dryness and redissolved in CHsOH (1 ml) to which 50 pi of D 2O had been added. The
solvent system on the, pump and injector unit was changed to CHsCN : D 2O (50 : 50).
Spectra were again recorded over the mass range 100 - 1000 Daltons at a cone voltage
of 35 V.
83 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2.2.S.3 Gas Chromatography- Mass Spectrometry (GC-MS) of fractions from Olive
pulp
Analyses were performed on a Hewlett Packard 5 890A gas chromatography interfaced to a VG 12-12F mass spectrometer (VG, Altrincham UK). A Chrompack - CP-Sil 88 fused silica capillary column was used with helium as carrier gas.
Gas chromatography (GC) analysis of fatty acids is most commonly performed after they have been converted to their, methyl ester derivatives. Metcalfe and Schmitz,
[Metcalfe & Schmitz, 1961] showed that BCb/methanol was a suitable reagent for the méthylation of free fatty acids and by 1969 this was established as an official method.
2.2.5.3.1 GC-Method
To establish whether there was any free acids present the samples were examined both saponified and non-saponified. Sodium hydroxide (NaOH) 0.5 M in methanol was used to saponify each sample prior to estérification with BCb-methanol.
Sodium hydroxide solution (0.4 ml) was added to the sample (100 pi) in a reaction vial and the mixture heated until the droplets of fat had disappeared. This would normally take up to 5-10 minutes. Petroleum ether was then added to dissolve the fatty acids, and the whole mixture vortexed, before being heated for 3 min. at 100 °C. The upper layer which consisted of the methyl esters was removed for examination by GC.
84 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2.2.S.3.2 GC-MS conditions for fractions from Olive pulp
Instrument: Carlo Erba HRGC 5300 Mega Series
Column: 50m x 0.22mm ID; Cyanopropyl CP-Sil (non CB)
Temperature and
Programme: 70°C for 5min.; then 8°C/min tol50°C; 150°C for
lOmin.; then 3°C/min to 240°C and 240°C for 20min;
Carrier Gas: Helium (5) 170 kPa
Injection: Split (1:50); lpl@ 280°C
Detector: VG 12-12 F quadropole mass spectrometer
Ionisation mode: Electron Impact (EI)
Scan analysis: 40-600
2.3 Results
2.3.1 Thin layer chromatography (TLC) of fractions from Olive pulp
Thin layer chromatography on silica showed similar profiles for fractions Fs, F9 and Fio
and fractions Fis, Fi4 and Fis as shown in Figure 2.1. Fractions Fs, Fo and Fio indicated the presence of four components (Rf. 0.01-0.22) in each fraction while fractions Fis, Fi4
and Fis indicated the presence of five components (Rf. 0.03-0.25). Fractions Fs and F4
(Rf. 0.02-0.28) showed similar profiles to Fs, Fo and Fio but of different intensities.
Fraction Fio showed only two weak spots (Rf. 0.01-0.15) with retention fronts matching two of the spots from the other fractions analysed, while Fs gave no component spots
85 C h a p t e r 2 O live f r u it - p u l p a n a l y sis under the TLC conditions used, chloroform : methanol (9:1), Figure 2.1. The fractions with identical TLC profiles were pooled for further analysis by HPLC. Fractions Fs, F9 and Fio were therefore pooled as were fractions Fis, F14 and Fis. Fractions Fs, F4, and
Fi6 were also examined by HPLC.
Fractions Fi and F 2; Fô and F?; Fii and F 12 gave no obvious separation when examined by TLC under the conditions used for fractions Fs - Fs; Fs - Fio and Fis Fiô, chloroform : methanol (9 : 1), Figure 2.1. Fractions Fi and F 2; Fô and F?; Fii and F 12 were therefore examined in hexane : chloroform (9:1), Figure 2.2. These fractions showed similar profiles when run in this solvent system and some separation was achieved. In fractions Fi, Fô and Fii five spots were observed, one at the solvent front, three closely spaced of mid retention and one elongated trailing spot at the origin.
Fractions F 2, F? and F 12 had only two spots, one was at the solvent front, the other was elongated and trailing from the origin. Fractions Fi, F 2, Fô, Ft, Fii and F 12 were derivatised for GC and GC-MS analysis.
86 C h a p t e r 2 O live f r u it- p u l p a n a l y s is
F3 F4 F5 F8 F9 F10 F13 F14 F15 F16
Figure 2.1 TLC Analysis of silica column fractions from Olive-pulp, F3, F4, Fs, Fs, F9, Fio, Fi3, Fi4, Fis and F16
TLC: Silica gel
Solvent system: Chloroform: Methano 1 (9:1)
Solvent front: 18 cm
Stain: 1 % Vanillin in EtOH and 40% Sulphuric acid in EtOH (1:1)
(heated at 100"C for 10 min., for visualisation)
87 C h a p t e r 2 O live f r u it- p u l p a n a l y s is
1^
FI F6 F11 F2 F7 F12
Figure 2.2 TLC Analysis of silica column fractions from Olive-pulp, Fi, F2, Fô, F?, Fil and F12
TLC: Silica gel
Solvent system: Hexane; Chloroform (9:1 )
Solvent front: 18 cm
Stain: 1% Vanillin in EtOH and 40% Sulphuric acid in EtOH (1:1)
(heated at 100 °C for 10 min., for visualisation)
88 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2.3.2 High performance liquid chromatography (HPLC) of fractions from Olive
pulp
Analysis of the combined fractions Fs-io by HPLC on a silica column, eluted with 90%
1.2 dichloroethane and 10% methanol showed three major components (Rt. 10.8 min.,
12.3 min. and 13.6 min.) to be present. Figure 2.5. Analysis under the same conditions of fractions Fis-is showed the presence of the same three components labelled A, B and
C plus two additional smaller components labelled D and E (Rt. 21.8 min. and 27.8 min.). Figure 2.6. Fractions Fa and F4 gave chromatograms which contained higher proportions of the early eluting components (Rt. 2.5-7.1 min.)than those observed in fractions Fs-io and Fia-is. In fractions Fa and F4 peaks A, B and E were present. Fraction
F i6 contained only the components B and C.
Separation of the component peaks A, B, C, D and E was undertaken by preparative
HPLC using a semi-preparative silica colunm (25 cm x 0.94 cm) under the same solvent conditions as used with the analytical column. Twenty injections of the combined fractions Fia-is were made and the main component fractions A, B, C, D and E collected from each injection. Fia-is was preferred because of the relative proportions of the main peaks A, B, C, D and E that it contained. A typical preparative scale chromatogram is shown in Figure 2.8. Fractions were collected at the times shown for the components A,
B, C, D and E. Each fraction was concentrated by rotary evaporation prior to being dissolved in CDCb : CDsOD (2:1) for NMR spectroscopy.
89 C h a p t e r 2 O ljve FRUfi-pyLP a n a l y s i &
LU
O 10 15 2 0 26 6 T IM E (m in )
Figure 2.3 HPLC Analysis of Olive-pulp fraction Fa
10 15 20 ~2^ TIM E (m in )
Figure 2.4 HPLC Analysis of Olive-pulp fraction F4
9 0 C h a p t e r 2 O live f r u it- p u l p a n a l y s is
I
3> 5 s flO flC o s <
-4— » 20 15 10 TIME (m in)
Figure 2.5 HPLC Analysis of Olive-pulp fraction Fs-io
16 TIME (m in)
Figure 2.6 HPLC Analysis of Olive-pulp fractions Fo-is
91 C h a p t e r 2 O live f r u it- p u l p a n a l y s is
I g
IOQ flC
i<
25 20 15 10 TIME (m in)
Figure 2.7 HPLC Analysis of Oiive-pulp fraction Fi6
Figures 2.3-2.7 HPLC Analysis of silica column fractions from Olive-pulp,
F3, F4, Fs-io, F i3-i5 and F i6
Column: Zorbax Silica (5|im) (25 cm X 0.46 cm ID)
Solvent: 90% 1.2 DCE : 10% Methanol (isocratic mode)
Flow: Iml/min
Detector: UV 254 nm
Injection: 5 |il (2 mg/ml)
9 2 C h a p t e r 2 O live f r u it- p u l p a n a l y s is
29 28 27 26 2S 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 S 8 7 6 5 4 3 2 1 TIME (min)
Figure 2.8 HPLC Analysis of Silica Column Fractions from Olive pulp
Column; Zorbax Silica (5p,m)
(25 cmx 0.94 cm ID)
Solvent: 90% 1.2 DCE : 10% Methanol (isocratic mode)
Flow: 2ml/min
Detector: UV 254 nm
Injection: 50 p,l (2 mg/ml)
9 3 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2.3.3 Nuclear magnetic Resonance Spectroscopy (NMR) of fractions from Olive
pulp
Both and 2D COSY spectra were obtained for each of the samples A, B, C, D and E
The NMR spectra (Figures 2.9 and 2.14) of sample A showed the following resonances to be present:
7.02 ppm (d, H-5’; d, H- 7’); 6.96 ppm (d, H-4’; d,H-8’); 4.2 ppm (q, H-2’); 4.1 ppm (q,
H-2’); 3.9 ppm (dd, H-5); 2.8 ppm (t, H-T); 2.7 ppm (dd, H-6); 2.4 ppm (t, H-6); 5.89 ppm (s, H-1); 6.08 ppm (q, H-8); 1.58 ppm (d, CHs-lO);
The NMR spectra (Figures 2.10 and 2.15) of sample B showed the following resonances to be present:
7.02 ppm (d, H- 7’); 6.96 ppm (s, H-4’; d, H-8’); 4.2 ppm (q, H-2’); 4.1 ppm (q, H-2’);
3.9 ppm (dd, H-5); 2.8 ppm (t, H-1’); 2.7 ppm (dd, H-6); 2.4 ppm (t, H-6); 5.89 ppm(s,
H-1); 6.08 ppm (q, H-8); 1.58 ppm (d, CHa-lO);
The NMR spectra (Figure 2.11 and 2.16) of sample C showed the following resonances to be present:
3.9 ppm (dd, H-5); 3.75 ppm (CHsO-); 2.8 ppm (t, H-1’); 2.7 ppm (dd, H-6); 2.4 ppm (t,
H-6); 5.89 ppm (s, H-1); 6.08 ppm (q, H-8); 1.58 ppm (d, CHs-lO);
The NMR spectra (Figure 2.12 and 2.17) of sample D showed the following resonances to be present: a) 3.7 ppm (t, H-1); 2.71 ppm (t, H-2); 6.73 ppm (s, H-2’); 6.64 ppm (d, H-6’); 7.08 ppm (d, H-5’); 3.75 ppm (CHsO-); b) 3.7 ppm (t, H-1); 2.77 ppm (t, H-2); 7.01 ppm (d, H-2’; d, H-6’); 7.13 ppm (d, H-3’; d, H-5’); 2.31 ppm (s, CH3-4’);
94 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
The NMR spectra (Figures 2.13 and 2.18) of sample E showed the following resonances to be present:
7.0 ppm (d, H-1; d, H-4); 6.13 ppm (t, H-2, t, H-3); 3.76 ppm (q, H-1’); 2.05 ppm (t, H-
2’);
In all of the samples examined, because of overlapping, the glucose moiety was assigned from the 2D COSY connectivity at:
4.8 ppm (H-1); 3.34 ppm (H-2); 3.68 ppm (H-3); 3.8 ppm (H-4); 3.33 ppm (H-5); 3.7 ppm (H-6);
95 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
ppm
ppm
ppm
Figure 2.9 ID proton NMR spectra of the silica column HPLC fraction A, obtained from Olive pulp pre-separated on a silica column; 1024 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
96 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
ppm
ppm
ppm
Figure 2.10 ID proton NMR spectra of the silica column HPLC fraction B, obtained from Olive pulp pre-separated on a silica column; 10 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
97 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
ppm
ppm
ppm
Figure 2.11 ID proton NMR spectra of the silica column HPLC fraction C, obtained from Olive pulp pre-separated on a silica column; 10 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
98 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 ppm
2.03.5 ppm
Figure 2.12 ID proton NMR spectra of the silica column HPLC fraction D, obtained from Olive pulp pre-separated on a silica column; 20 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 pip was used as the reference chemical shift.
99 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
ppm
ppm
ppm
Figure 2.13 ID proton NMR spectra of the silica column HPLC fraction E, obtained from Olive pulp pre-separated on a silica column; 10 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
100 C h a pt e r 2 O live fr u it -pu l p analysis
Cp pm?
4 : _ 5 4 _ 0 5. .02.52.0 1.5 1.00.
Figure 2.14 2D COSY proton NMR spectra of the silica column HPLC fraction A, obtained from Olive pulp pre-separated on a silica column; 1024 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
101 C h a p t e r 2 O liv e f r u it- p u lp a n a l y s is
LO o
GO
e . OF’. ^ F . 2^ . 8^ . <48. 0 5 D 1
J LO
LO
O OvI . [=3 R LO CD
CE. 4 2 . a . a D 1
Figure 2.15 2D COSY proton NMR spectra of the silica column HPLC fraction B, obtained from Olive pulp pre-separated on a silica column; 10 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
102 C h a p t e r 2 O l iv e f r u it- p u lp a n a l y s is
LO CD
oo N± oo
e. 4-e. oô’. . 2^0 85". <8. 05. o D 1
Figure 2.16 2D COSY proton NMR spectra of the silica column HPLC fraction C, obtained from Olive pulp pre-separated on a silica column; 10 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
1 0 3 C h a p t e r 2 O liv e f r u it- p u lp a n a l y s is
IT) CD
• 0
CO
O - o Or^ o a 2^ V, 0 ^ = ^ 0 . 0 5 z. 0 Cp p
I
LO OCD
LO
e ro CD
< . 5< . 05. 55. OO. 50 . OI . 51 . OO. 5 Cp p
Figure 2.17 2D COSY proton NMR spectra of the silica column HPLC fraction D, obtained from Olive pulp pre-separated on a silica column; 20 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
1 0 4 C h a p t e r 2 O l iv e f r u it- p u lp a n a l y s is
oLO ~ VO \î-
OO Cl_
o
oo
e . O P '. . e g . 4 - g . 0 5 . g Cp p rr>;>
LO o O
in
P) Ln
XI-
go 5g c OO. 50. OO. 50. Oi . 51 = OO . 5 Cp p
Figure 2.18 2D COSY proton NMR spectra of the silica column HPLC fraction E, obtained from Olive pulp pre-separated on a silica column; 10 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
105 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2.3.4 Mass Spectrometry (MS) of fractions from Olive pulp
The separated HPLC fractions A, B, C, D and E were examined by electrospray mass spectrometry. This technique readily produces a pseudo molecular ion and therefore gives molecular weight information on each fraction.
Injections were made directly from separated fractions into the mass spectrometer via the injector loop and pump unit. The samples were initially examined in methanol solution (1 ml) containing 50 pi of water and were sprayed into the mass spectrometer with CHsCN : H2O (50 : 50) [Lagley, J. et al., 1998].
The spectra obtained for each fraction are shown in Figures 2.19-2.23.
Sample A showed a strong pseudo molecular ion cluster (M-1) at m/z 523, with other minor components observed at m/z 559, 569 and a weak mass ion at m/z 637.
Sample B showed a strong pseudo molecular ion cluster (M-1) at m/z 539, with a minor component observed at m/z 575 together with weaker mass ions at m/z 585 and 653.
Sample C showed a strong pseudo molecular ion (M-1) at m/z 403 together with ions at m/z 539 and 807. Smaller mass ions were observed at m/z 439, 449 and 575.
Sample D showed a strong pseudo molecular ion (M-1) at m/z 315 together with numerous ions at higher mass.
Sample E showed a dominant ion (M-1) at m/z 361. Other strong ions were observed at m/z 315, 353, 403, 441 and 429 together with other weaker more numerous ions which
could have been part of the sample background.
To obtain an indication of the number of exchangeable hydrogens in each compound
deuterium exchange was effected by the evaporation of each sample to dryness under nitrogen and then redissolving it in CH3OH (1 ml) plus 50 pi of D 2O and spraying into the mass spectrometer withCHsCN : D 2O (50:50).
106 C h a p t e r 2 O live f r u it -p u l p a n a l y sis
Sample A now gave a pseudo molecular ion cluster (M-1) displaced to m/z 527 with other minor components at m/z 564 and 574, indicating four exchangeable hydrogens in the major component and five in each of the minor components.
Sample B now gave a pseudo molecular ion cluster (M-1) displaced to m/z 544 indicating five exchangeable hydrogens. Some minor components were observed at higher mass.
Sample C now gave a strong pseudo molecular ion (M-1) at m/z 407 indicating four exchangeable hydrogens. Other ions were observed at m/z 544 and 816.
Sample D now showed a strong pseudo molecular ion (M-1) displaced to m/z 320 indicating five exchangeable hydrogens; together with other ions at higher mass .
Sample E now showed a strong ion (M-1) at m/z 366, indicating five exchangeable hydrogens. Other strong ions were observed at m/z 319, 356, 407, 417 and 434.
107 C h a p t e r 2 O live fr u it - pu l p a nalysis
Table 2.3 Molecular weights of HPLC fractions from Olive pulp
HPLC fraction from Molecular weight of Moleeular weight of
Olive pulp main component minor components
A 524 560 570 638
B 540 576 586 654
C 404 540 808 440 450 576 D 316
E 362 316 354 404 442 430
Figure 2.19 Electrospray mass spectrometry - sample A
108 C h a pt e r 2 O live fr u it - pu l p analysis
Figure 2.20 Electrospray mass spectrometry - sample B
ikw
Figure 2.21 Electrospray mass spectrometry - sample C
1 0 9 C h a p t e r 2 O liv e f r u it- p u lp a n a l y s is
509 3 523* I
290 300 320 340 3«0 360 400 420 *40 460 *80 500 520 540 500 5«0 600 620 640 560 680 7 » 720 / « Î60 fSC 800 520 M3
Figure 2.22 Electrospray mass spectrometry - sample D
Figure 2.23 Electrospray mass spectrometry - sample E
110 C h a p t e r 2 O live f r u it -p u l p a n a l y sis
2.3.5 Assignment of structures from NMR and MS data
The NMR resonances and molecular weight information obtained by mass spectrometry enabled the structures listed to be deduced.
Sample A
MW: 524.19
ooc S COOMe
Figure 2.24 5’-deoxy-oleuropein
Sample B
MW: 540.52
OH
OOC H COOMe
OGlc
Figure 2.25 Oleuropein
111 C h a p t e r 2 O l iv e f r u it - p u l p a n a l y s is
Sample C
MW: 404.37
HOOC COOMe
OGlc
Figure 2.26 Oleoside- 11 -methyl ester
Sample D
MW: 316.31
CH 2 CH 2 OH
k ^ O H OGlc
Figure 2.27 2-(3’-hydroxy 4’-glucopyranosylphenyl)ethanol
Minor component in sample D
MW: 136.09
CH^CH.OH
CH3
Figure 2.28 2-(4’ -methylpheny l)ethanol
112 C h a p t e r 2 O l iv e f r u it - p u l p a n a l y s is
Sample E
MW: 362.17
CH 3 OGlc
CH 2 CH 3 CH
Figure 2.29 1,4 dimethyl-3-ethyl-2-naptholglycoside
2.3.6 Analysis of fatty acid composition by GC-MS in olive pulp
Fractions Fi and F2; Fô and F? and Fii and F12 (Table 2.2) give no obvious separation when examined by TLC in a chloroform : methanol solvent system. They were therefore examined in hexane : chloroform (9:1) (Figure 2.2), where partial separation was obtained. The fractions Fi and F2; Fôand F? and Fii and F12 were further examined by
GC-MS, as saponified and non-saponifred samples. The results are shown in tables 2.4-
2.15 and are summarised in Figure 2.19 as fatty acids detected in non-saponifred and saponified samples in the total sample of olive pulp.
113 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Table 2.4:
Fatty acids detected by GC-MS in the FI fraction (Table 2.2) The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 5.8 Stearic (18:0) 3.4 Oleic (18:1) n-9 66.5 Linoleic (18:2) n-9,12 11.5 Behenic (22:0) 6.8 Lignoceric (24:0) 6.1
Table 2.5:
Fatty acids detected by GC-MS in the saponified FI fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 5.5 Stearic (18:0) 2.5 Oleic (18:1) n-9 73.6 Linoleic (18:2) n-9,12 18.5
114 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Table 2.6:
Fatty acids detected by GC-MS in the F2 fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 10.4 Stearic (18:0) 3.5 Oleic (18:1) n-9 78.9 Linoleic (18:2) n-9,12 7.3
Table 2.7:
Fatty acids detected by GC-MS in the saponified F2 fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 8.8 Palmitoleic (16:1) n-9 1.4 Heptadecanoic (17:0) 0.8 Stearic (18:0) 8.4 Oleic (18:1) n-9 40.5 Linoleic (18:2) n-9,12 38.5 Arachidic (20:0) 1.6
115 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Table 2.8:
Fatty acids detected by GC-MS in the F6 fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 4.55 Oleic (18:1) n-9 82.39 Linoleic (18:2) n-9,12 8.8 Behenic (22:0)______3.79
Table 2.9:
Fatty acids detected by GC-MS in the saponified F6 (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 12.1 Stearic (18:0) 8.5 Oleic (18:1) n-9 63.2 Linoleic (18:2)n-9,12 14.6 Arachidic (20:0)______1.6
116 C h a p t e r 2 O live f r u it -p u l p a n a l y sis
Table 2.10:
Fatty acids detected by GC-MS in the F7 fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2..
Fatty acid Rel. %
Palmitic (16:0) 29 Oleic (18:1) n-9 62.6 Linoleic (18:2) n-9,12 8.4
Table 2.11:
Fatty acids detected by GC-MS in the saponified F7 (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 19.2 Stearic (18:0) 4.0 Oleic (18:1) n-9 66.1 Linoleic (18:2) n-9,12 9.6 Arachidic (20:0)______1.1
117 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Table 2.12:
Fatty acids detected by GC-MS in the FIX fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid Rel. %
Oleic (18:1) n-9 100
Table 2.13:
Fatty acids detected by GC-MS in the saponified FIX (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Myristic (14:0) 12 Palmitic (16:0) 12.8 Palmitoleic (16:1) n-9 6.4 Stearic (18:0) 16 Oleic (18:1) n-9 28.8 Linoleic (18:2) n-9,12 12 Behenic (22:0) 12
118 C h a p t e r 2 O live f r u it -p u l p a n a l y sis
Table 2.14:
Fatty acids detected by GC-MS in the F12 fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 3.7 Oleic (18:1) n-9 34.2 Linoleic (18:2) n-9,12 6.2 Lignoceric (24:0) ______55.9
Table 2.15:
Fatty acids detected by GC-MS in the saponified F12 fraction (Table 2.2). The conditions for GC-MS are described in method 2.2.5.3.2.
Fatty acid______Rel. %
Palmitic (16:0) 10.9 Palmitoleic (16:1) n-9 0.6 Heptadecanoic (17:0) 0.5 Stearic (18:0) 5.0 Oleic (18:1) n-9 64. Linoleic (18:2) n-9,12 18.8 Arachidic (20:0) 0.6
119 C h a p t e r 2 O l w e f r u h -p u l p analysjs
80'
70--^
GO
nMyristic acid (14:0) 50- a Palmitic acid (16:Q) HPatmitoteic acidt10:1) B Heptadecanoic acid (17:0) 40 B Stearic acid (18:0) □ Oleic acid (18:1) □ Unoleic acid (18:2) □ Arachidic acid(20:0) 30- BBehenic acid (22:0)
2 0 -
ID-
Non-saponified Saponified sample sample
Figure 2.30 Fatty acids detected by GC-MS in the total sample of Olive pulp
The relative % o f each component was obtained as described in method 2.2.3. The conditions for GC-MS are described in method 2.2.5.3.2
120 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
2.4 Discussion
The fractions obtained from the silica column chromatography of olive pulp were initially screened by TLC. The chloroform fractions when examined by HPLC showed no significant component peaks under the conditions used. HPLC of the methanol fractions under the same conditions showed a consistent elution profile of three major and two minor component peaks.
The chloroform fractions were further examined by GC-MS. The analysis of these fractions by GC-MS showed the presence of both saturated and unsaturated fatty acids.
The non-saponified material showed the presence of six fatty acids while the saponified samples showed the presence of ten fatty acids. The fatty acids found are shown in
Table 2.16.
Table 2.16 Fatty acids found in Olive fmit-pulp
Non-saponified sample Saponifeid sample Carbon chain Rel. % Carbon chain Rel. % - - (14:0) 2 (16:0) 9.93 (16:0) 11.5
- - (16:1) 1.4
- - (17:0) 0.2 (18:0) 1.31 (18:0) 7.4 (18:1) n-9 79.54 (18:1) n-9 56.03 (18:2) n-9,12 7.45 (18:2) n-9,12 18.66
- - (20:0) 0.8 (22:0) 1.77 (22:0) 2
121 C h a p t e r 2 O live f r u it -p u l p a n a l y sis
All of the fatty acids found to be present in olive pulp are found in olive oil and in similar ratios.
The saponified sample showed an increase in the number of unsaturated fatty acids from two to three and an increase in the amount of linoleic acid [(18:2) n-9,12] present, Table
2.7. The oleic acid content appeared to have fallen when compared to the non-saponified sample. Table 2.6. This may reflect sampling difficulties or incomplete saponification since there are no reports of oxidation of the double bond in oleic acid.
The three major and two minor components in the methanol fractions were separated by preparative HPLC. The fractions obtained were examined by NMR and by -ve ion
Electrospray mass spectrometry and the following structures were deduced.
Sample A
MW: 524.19
3' r ooc COOMe
10
OGlc
5 ’deoxy-oleuropein
122 C h a p t e r 2 O live f r u it - p u l p a n a l y sis
Sample B
MW: 540.52
OH
OOC COOMe
10
OGlc
Oleuropein
Sample C
MW: 404.37
HOOC COOMe
OGlc
Oleoside-11-methyl ester
Sample D
MW: 316.31
CH2CH2OH
k ^ O H OGlc
2-(3 ’-hydroxy 4’-glucopyranosylphenyl)ethanol
123 C h a p t e r 2 O live f r u it -p u l p a n a l y sis
Minor component in sample D
MW:136.09
CH2CH2OH
CH
2-(4 ’ -methylphenyl)ethanol
Sample E
MW: 362.17
CH,
CH 2 CH 3 CH
l,4-dimethyl-3-ethyl-2-naptholglycoside
Compounds A, B, and C share a common template
-OOC S COOMe 10 r OGlc
124 C h a p t e r 2 O live f r u it -p u l p a n a l y sis with A and B and are closely related derivatives of a common structure. Compound B also shares a common molecular substructure with D although this molecule is in fact glycosylated. Compounds of the type A, B and D are of common occurrence in olives
[Inouye et al., 1974; Walter et al., 1973; Capasso et al., 1992]. Compound C could be considered a hydrolysis product of either A or B [Gariboldi et al., 1986].
Compound E represents a novel glycosylated structure. This compound type has not been previously reported.
125 CHAPTER 3
OLIVE FRUIT-SKIN ANALYSIS
126 C h a p t e r 3 O live f r u it - sk in a n a l y sis
3 OLIVE FRUIT-SKIN ANALYSIS
3.1 Introduction
Current therapeutic treatments use not only synthetic drugs but also drugs derived from plants together with chemical modification of these materials. Since the 1970’s the amount spent on plant based pharmaceuticals, their importation, processing and formulation, has steadily increased and now represents an enterprise worth billions of dollars.This large investment demonstrates the interest of the pharmaceutical industry in plant-based drugs and also the considerable research carried out in this field of natural resources.
Fruit plants like many other plants have been studied in the context of medical and pharmaceutical research and have involved the flesh of fruits, the leaves of fruit trees and other parts of fruit plants but little systematic or comprenhensive research has been done on the skins of fruits. Some of the results are summarised in Table 3.1.
Plant materials are now being tested for a wide range of different biological activities.
The skins of fruits are considered to be not only a physical but also a chemical barrier to protect the fhiit against animals and more importantly micro organisms. Because of this role-effect antimicrobial activity can be expected in a large number of naturally occurring compounds. Alkaloids, steroids, terpenes and flavonoids with different biological activities have been discovered and are currently used in various medical therapies and for example the anticancer drug Taxol is derived from Taxus Brevifolia.
If a potent compound could therefore be isolated from olive skin the raw material could then be cheaply purchased as a waste product of the food industry.
127 C h a p t e r 3 O live f r u it -sk in a n a l y sis
Table 3.1 Comüounds already isolated from some fruit skins
Fruit Compounds Reference
Apple Anthocyanin Diener & Naumann, 1981; (4-hydroxy-beta-damascones ; Faragher, 1983; 3-hydroxy-7,8-dihydro-beta-ionone; Brohier & Faragher, 1984 3-oxo-alpha-ionol, 3-hydroxy-beta- ionol ; 3 -hydroxy-7,8 -dihydro-beta- ionol; 3-hydroxy-beta-ionone; 3-hydroxy-5,6-epoxy-beta-ionone; dehydrovomifoliol; vomifoliol) Aroma compounds Krammer et al, 1991 (monoterpene-diols:3,7-imethyloct- l-ene-3,7-diol; (E)-2,6-imethylocta- 2.7-diene-1,6-diol; 2,6-dimethyl- 1.8-octanediol;(2E,6Z)-3,7-imethyl- 2,6-octadiene-l,8-diol) Sesquiterpene (a-famese) Allen et a l, 1995 Cyanidin Lancaster er a/., 1997 Quercetin glycosides Lancaster er a/., 1997 Banana Polyamine (Putrescine) Takeda et a l, 1997 Phenylphenalenones Kamo et a l, 1998 Cycloartane triterpenes Akihisa e/ûf/., 1998 Mango Glycosidically bound aroma Adedeji et a l, 1992 compounds (monoterpene alcohols, aldehydes, acids and esters) Carotenoids Wilberg et al, 1995;
Orange Volatile fatty acids Nishio et al, 1982 Carotenoids Rosenberg et a l, 1983 Pectin Elnawawi & Shehata, 1987 Polymethoxylated flavones Hadjmahammed et n/.,1987; Gaydou et al, 1998 Carotenoids Tzia et al, 1992 Flavonoids Locurto et al, 1992; Manthey & Grohmann, 1996 Hydroxycinnamic acid esters Manthey & Grohmann, 1997 Peach Anthocyanin (cyanin-3-glycoside) Cheng & Crisoto, 1997 Chlorogenic acid, catechin, epicatechin
128 C h a p t e r 3 O l iv e f r u it - s k in a n a l y s is
Pear Phenolic fatty acid esters Whitaker, 1998 Isoflavonoid fatty acid esters Plum Free and glycosidically bound Adedeji et al, 1991 aroma compounds (isobutyl 3 -hydroxybutanoate)
3.2 Material and methods
3.2.1 Sample
Black Greek Olives, (Olea europaea L.) were hand picked from the island of Kim. The olives were peeled and the skin separated from the olive tissue.
3.2.2 Reagents/equipment for the preparation of extracts from Olive skins
The solvents that were used in the extractions and analyses were, chloroform, methanol, hexane and 1.2 dichloroethene, all of HPLC grade and obtained from Fisher Scientific
Limited, U.K.
Deuterated chloroform ( 99.8% ) and perdeuteromethanol ( 99.8% ) together with NMR tubes (5mm) were obtained from the Aldrich Chemical Company, Inc., USA.
The estérification reagent, BC13-methanol, was obtained from Supelco, Co., USA.
Aluminium backed, Kiesel gel 60 F-254 thin layer chromatography silica gel plates were purchased from B. D. H. Merck Ltd., UK.
Lipid standards were obtained from the Sigma Chemical Company.
129 C h a p t e r 3 O live f r u it -sk in a n a l y sis
3.2.3 Preparation of extracts from Olive skins The skin of the olives was removed carefully, air dried in a shadowed room for four days then powdered using a domestic blender. The air-dried and powdered skins (26g) were put into a screw topped glass bottle which was covered with aluminium foil to prevent degradation of the compounds. The olive skins were extracted sequentially with hexane, chloroform, methanol, and water by maceration at room temperature.
The skins were suspended in a volume of solvent for a minimum of 24 hours. The solvent was then removed by filtration through Whatman Nol filter paper and stored at
-80 °C. The filtered leaf paste was scraped back into the screw top bottle and further volumes of solvent added for further periods of time. Each volume was removed by filtration and the leaf paste then allowed to dry overnight before extraction with the next solvent.
The hexane, chloroform and methanol extracts were evaporated to dryness by rotary evaporator, weighed and then stored at -80 °C. The water extract was first frozen and then freeze dried. The solid material obtained was weighed, then stored at -80 °C.
The extraction solvent volumes, times and weights are shown in Table 3.2 for each
solvent used.
1 3 0 C h a p t e r 3 O live f r u it -sk in a n a l y sis
Table 3.2 Extraction of Olive skins
Solvent Volume Time Total weight (ml) (hrs) (g)
Hexane 700 24 700 24 6.1 700 24 Chloroform 700 65 700 48 10.2 700 48 700 72 Methanol 700 48 700 48 700 72 3.8 800 24 Water 800 24 2.9
3.3 Analytical techniques
3.3.1 Thin Layer Chromatography (TLC) of extracts from Olive skins
Thin Layer Chromatography was performed using Merck aluminium backed thin layer
Kiesel gel 60 F-254 plates. Different solvent systems were used for the individual hexane, chloroform and methanol extracts.
The solvent systems were equilibrated before use in glass TLC tanks, lined with filter paper. Samples were applied 1 cm above the bottom edge and separated by 1 cm
131 C h a p t e r 3 O live fr u it -sk in a n a l y sis divisions. Samples were eluted until the solvent front was 18 cm above the origin. All chromatographed plates were initially observed under UV before treatment with a spray reagent. The plates were stained with a mixture of 1% vanillin in EtOH and 40% sulphuric acid in EtOH (1:1), until saturated and heated in an oven at 100 °C for 10 min., before visualisation.
Each solvent extract from the olive skins was examined under different conditions to achieve an optimum separation of components. Hexane:acetone (9:1), give 8 components from the hexane extract; chloroform:methanol (8:2), give 2 components from the chloroform extract; chloroform : methanol (8:2), give 4 components from the methanol extract;
3.3.2 Preparative Thin layer Chromatography (PTLC) of extracts from Olive
skins
Preparative TLC plates coated with silica gel 6F-254 (MERCK Ltd.) were prepared using a Camag silica gel spreader adjusted to 0.5 mm. To prepare two PTLC plates 20 g of silica gel was dissolved in 40 ml of distilled water and mixed to a suspension. The glass plates were placed in the plate maker and the suspension poured into the metal block, which was then pulled over the plate maker to cover the plates evenly with the suspension. The plates were kept in a rack for 12 hours and activated by heating in an oven at 110 °C for one hour. The sample was chromatographed as five separate loadings on the same plate. After the sample had been run the area of the plate containing the elution of the first loading was isolated and stained with a mixture of 1% vanillin in
EtOH and 40% sulphuric acid in EtOH (1:1) until saturated and then heated with a hair
132 C h a p t e r 3 O live f r u it -sk in a n a l y sis dryer.
The chloroform extract was eluted with chloroform : methanol (9:1) and two purple bands were observed.
3.3.3 High Performance Liquid Chromatography (HPLC) of extracts from Olive
skins
A Perkin Elmer Series 3 pump unit equipped with an LC 65T variable wavelengh UV detector and oven unit was used with a Zorbax RP CIS column. Two diameters of this column were used to fulfil both the analytical and semipreparative roles. The conditions are shown below in Table 3.3.
Table 3.3 HPLC Conditions for the analvsis of methanol and water olive fruit skin extracts
Column Zorbax RPC18
Size 25 cm X 0.46 cm 25 cm X 0.94 cm Solvent 30% Methanol : 70% Water 30% Methanol : 70% Water Flow Iml/min 2ml/min Temp 30 °C 30 °C UV 254 nm 254 nm
The methanol and water extracts were first examined on a Zorbax RP CIS analytical column. Conditions from the analytical column which gave the best separation and peak
1 3 3 C h a p t e r 3 O live f r u it -sk in a n a l y sis intensity, were suitably modified for the preparative HPLC. Twenty five injections were made of both the methanol and water extracts on to the preparative column and the main component fi*actions collected and labelled:
Ml - M4, for the methanol extract run on the ODS column (Figure 3.11),
Wi - W4, for the water extract run on the ODS column (Figure 3.16).
The methanol fractions were evaporated to dryness and dissolved in CDCbiCDaOD
(1:2), while the water fi*actions after evaporation to dryness were dissolved in
CD30D:D20 (1:1), for NMR spectroscopy.
3.3.4 Nuclear Magnetic Resonance Spectroscopy (NMR) of extracts from Olive
skins
Both ’H and 2D COSY spectra were obtained from either a Bruker AM 400 or a Bruker
AM 500 NMR spectrometer and processed using Felix 2.3 [ Biosym, 1987, 1993 ]. ^^C spectra were obtained firom a Bruker AM 100 instrument.
Nitrogen was bubbled through each sample prior to recording the spectrum in order to prevent any possible oxidation.
A field fi*equency lock was provided using the deuterium signal from the solvent.
The spectra were recorded at 25 °C in the Fourier transform mode (FT). The spectrum of each extract was acquired using a 3 ps, 45° pulse, a spectral width of 6000Hz, an acquisition time of 1.36 s, and a relaxation delay of 4 s. The free induction decays
(FIDs) were collected with 16 K data points.
Connectivity among protons and hence conformation of the identity of different lipids was established with the magnitude COSY pulse sequence [Nagayama, et al, 1980]. 2D
134 C h a p t e r 3 O live f r u it -s k in a n a l y sis
COSY experiments were performed at 298 K with a special width (SW) of 6024.10 Hz and a relaxation delay of 2 s. They were recorded in 2K (2048) data points, obtained from 512 FIDs of 48 scans each, with zero-filling in the FI dimension.
The number of carbons in the spectra were obtained using 9ps, 45° pulse, a spectral width of 15625 Hz, an acquisition time of 0.524 s, and a relaxation delay of 0.5s. The free induction decays (FIDs) were collected with 16 K data points.
Chemical shifts were referenced to the residual methanol peak at 3.31 ppm and the TSP peak at 0.00 ppm, for the samples in CDsOD : CDCb (2:1) and CDsOD : D 2O (1:1), respectively.
3.3.5 Mass Spectrometry (MS) of extracts from Olive skins
Mass spectrometry measurements were made in both positive and negative electrospray ionisation modes on a Finnegan Navigator mass spectrometer. The samples were introduced by loop injection from a Waters 2690 pump and injector unit.
A VG ZAB SE mass spectrometer was also used in the positive ion fast atom bombardment ionisation mode to examine the preparative TLC bands obtained from the chloroform extract.
All of the collected fractions from the preparative HPLC were first examined by NMR spectroscopy then evaporated to dryness and redissolved in 30% methanol : 70% water
(1 ml) prior to mass spectrometry examination. Each sample (10 pi) was injected into the mass spectrometer via the auto injector and pumping system using methanol : water
(30 : 70). Spectra were recorded in positive ion mode over the mass range 100 - 1000
Daltons at a cone voltage of 35 V.
1 3 5 C h a p t e r 3 O live f r u it -sk in a n a l y sis
3.3.6 Gas Chromatography - Mass Spectrometry (GC-MS) of extracts from Olive
skins
Analyses were performed on a Hewlett Packard 6890 Series gas chromatograph interfaced to a Hewlett Packard 5973 Series MSD mass spectrometer. A HP-WAX capillary column was used with helium as carrier gas.
Gas chromatography (GC) analysis of fatty acids is most commonly performed after they have been converted to their, methyl esters derivatives.
Metcalfe and Schmitz, [Metcalfe & Scmitz, 1961] showed that BFB/methanol was a suitable reagent for the méthylation of free fatty acids and by 1969 this was established as a standard method.
3.3.6.1 GC-Method of extracts from Olive skins
To be able to establish whether there was any free acids present in the samples they were examined both saponified and non-saponified. Sodium hydroxide (NaOH) 0.5 M in methanol was used to saponify each sample prior to estérification with BCb- methanol.
Sodium hydroxide solution (0.4 ml) was added to the sample (100 pi) in a reaction vial and the mixture heated until the droplets of fat had disappeared. This would normally take up to 5-10 minutes. Petroleum ether was then added to dissolve the fatty acids, and the whole mixture vortexed, before being heated for 3 min.. The upper layer which consisted of the methyl esters was removed for examination by GC.
1 3 6 C h a p t e r 3 O live f r u it -sk in a n a l y sis
3.3.6.2 Fast GC-MS Analysis conditions for FAMEs of extracts from Olive skins
Instrument: HP 6890 Series
Column: 10m x 0.10mm ID x 0.2pm HP-WAX
Temperature and Programme: 60 °C for 0.4 min.; then 16 °C/min to 240 °C
Carrier gas/flow rate: Helium/0.5ml/min
Injection mode: Manual: Split 40:1, splitless
Injection volume: O.SpL split, l.OpL, splitless
Inlet temp: 280 °C
Mass spectrometer: HP 5973 Series MSD
Interface temp: 280 °C
Ion source temp: 230 °C
Quad Temp: 150 °C
Ionisation mode: Electron Impact (El)
Scan analysis: 40-400 amu
3.4 Results
A hexane, chloroform, methanol and water extract from the skins of Olive fruit were sequentially prepared and individually analysed by TLC, HPLC, GC-MS, MS and
NMR. The ^H and 2D COSY NMR spectra for the chloroform fraction were recorded on a Bruker AM 400 spectrometer while the ^^C spectra were recorded on a Bruker AM
100 instrument. All other ^H and 2D COSY spectra for the hexane, methanol and water fractions were measured on a Bruker AM 500 spectrometer.
137 C h a p t e r 3 O live f r u it -sk in a n a l y sis
3.4.1 Hexane extract
3.4.1.1 Thin layer chromatography (TLC) of the Hexane extract from Olive skins
When the hexane extract from olive skins was run in chloroform : hexane (9:1), (Figure
3.1), some separation was achieved. Two spots were observed at the solvent front (Rf:
0.9 and 1), four closely spaced of mid retention time (Rf: 0.52, 0.46, 0.3 and 0.2) and another two closely spaced towards the origin (Rf: 0.1 and 0.04). This sample was further examined by NMR spectroscopy and by GC-MS analysis.
3.4.1.2 Nuclear magnetic resonance spectroscopy (NMR) of hexane extract from
Olive skins
The NMR spectra of the total hexane extract confirmed the presence of both saturated and unsaturated fatty acids (Figure 3.2 and 3.3). Chemical shifts at 0.9 ppm confirmed the presence of CHs- groups, at 1.59 ppm (CH 2)n- groups, at 1.65 ppm -CH 2-CH2-
COOH and at 2.3 ppm -CH 2-COOH. The total number of unsaturated fatty acids was identified at 1.98-2.10 ppm, by calculating the integral area of the CH 2 groups in the sequence. The presence of higher polyunsaturated fatty acid chains was established by the allylic methylene proton signals from within the series of double bonds in the chain, at 2.80 ppm.
1 3 8 C h a p t e r 3 O live f r u it- s k in a n a l y s is
Figure 3.1 TLC Analysis of Hexane Extract from Olive-skin
TLC: Silica gel
Solvent system: Hexane: Acetone (9:1)
Solvent front: 18 cm
Stain: 1 % Vanillin in EtOH and 40% Sulphuric acid in EtOH (1:1)
(heated in the oven at 100 for 10 min., for visualisation)
1 39 C h a p t e r 3 O live f r u it -s k in a n a l y sis
U
8.0 F.Q 6,0 5.0 4.0 3.0 2.0 KO ppm
Figure 3.2 ID proton NMR spectra of Hexane extract from Olive skins 1028 scans were obtained for Fourier transformation and the residual methanol peak at 3.31 ppm was used as the reference chemical shift
Assignments of H NMR resonances in Fatty Acids
Chemical Shift/ppm Functional Group comment
0.87-0.9 - c m 1.59 -CH=CH-CH2-CH2-CH2-C00H 1.65 -CH2-CH2-COOH 1.98-2.10 -CH2-CH=CH-CH2-CH=CH-CH2- total UFA 2.3 -CH2-COOH 2.8 -CH=CH-CH2-(CH=CH-CH2-)n PUFA 5.33 -CH=CH- Vinyl proton
140 C h a p t e r 3 O live fr u it - skin an a ly sis
A
4. 54. 03, 53. 02, 52, 01,51, 00,5 F2 (ppm)
Figure 3.3 2D COSY NMR proton spectra of Hexane extract from Olive skins 2D spectrum consisted of 2K data points obtained from 512 FIDs of 48 scans with zero-filling in the FI dimension
141 C h a p t e r 3 O live f r u it - sk in a n a l y s is
3.4.1.3 Gas Chromatography-Mass Spectrometry (GC-MS) of hexane extract from
Olive skins
The analysis of the hexane extract from Olive skins by GC-MS showed the presence of fifteen components in the non-saponified extract (Table 3.4) and nineteen components in the saponified extract (Tables 3.5, 3.5a). In both non-saponified and saponified samples the most abundant acid is oleic acid C (18:1) n-9, followed by palmitic acid C
(16:0) and linoleic acid C (18:2) n-9,12. The saponified sample from the hexane extract of olive skins was re-examined using a different temperature profile (Table 3.5a), to effectively determine the total number of compounds, in particular the longer carbon chains that are present in this sample.
142 C h a p t e r 3 O live f r u it - sk in a n a l y sis
Table 3.4
Fatty acids detected by GC-MS in the non-saponified sample of Hexane extract obtained from Olive skins, as described in method 3.2.3. The conditions and sample preparation for GC-MS are described in method 3.3.6.
Instrument: Hewlett Packard HP 6890/5973 GC-MS Column: 10m X 0.10mm ID x 0.2um HP-WAX Carrier gas/flow rate: Helium. 0.5ml/min Injection mode/volume: Manual:split 40:1, 0.5pl Oven temperature: 60(04)-16-240 Ionization mode: Electron Impact (El) Scan analysis: 40-400 amu
Carbon chain Detected as Rel.%
Myristic acid (14:0) FAME 0.06 Palmitic acid (16:0) FAME 15.08 Hexadecanoic acid (16:1) n-7 FAME 0.24 Palmitoleic acid (16:1) n-9 FAME 0.53 Palmitic acid (16:0) FAEE 0.14 N-valerylalanine 0.05 Heptadecanoic acid (17:0) FAME 0.34 Oleic acid (18:1) n-9 FAME 59.14 Stearic acid (18:0) FAME 4.43 Octadecenoic acid (18:1) n-8 FAME 0.32 Oleic acid (18:1) n-9 FAEE 0.58 Linoleic acid (18:2) n-9,12 FAME 11.65 Linolenic acid (18:3) n-9,12,15 FAME 0.84 Arachidic acid (20:0) FAME 0.78 Eicosenoic acid (20:1) n-11 FAME 0.49 Behenic acid (22:0) FAME 1.24 Lignoceric acid (24:0) FAME 2.58 Oleic acid (18:1) n-9 FFA 1.49
a) FAME Fatty acid methyl ester b) FADME Fatty acid dimethyl ester c) FAEE Fatty acid ethyl ester d) FF A Free fatty acid
143 C h a p t e r 3 O live f r u it - sk in a n a l y sis
Table 3.5
Fatty acids detected by GC-MS in the saponified sample of Hexane extract obtained from Olive skins, as described in method 3.2.3. The conditions and sample preparation for GC-MS are described in method 3.3.6.
Instrument: Hewlett Packard HP 6890/5973 GC-MS Column: 10m X 0.10mm ID x 0.2um HP-WAX Carrier gas/flow rate: Helium. 0.5 ml/min Injection mode/volume: Manual:split 40:1, 0.5pl Oven temperature: 60(04)-16-240 Ionization mode: Electron Impact (El) Scan analysis: 40-400 amu
Carbon chain Detected as Rel. %
Myristic acid (14:0) FAME 0.05 9-oxo-nonanoic acid (9:0) FAME 0.23 Palmitic acid (16:0) FAME 13.80 Hexadecenoic acid (16:1) n-7 FAME 0.23 Palmitoleic acid (16:1) n-9 FAME 0.58 Heptadecanoic acid (17:0) FAME 0.50 Stearic acid (18:0) FAME 4.48 Oleic acid (18:1) n-9 FAME 61.24 Linoleic acid (18:2) n-9,12 FAME 11.72 Nonadecenoic acid (19:1) n-10 FAME 0.15 Linolenic acid (18:3) n-9,12,15 FAME 0.63 Octadecadienoic acid (18:2) n-10,13 FAME 0.14 Arachidic acid (20:0) FAME 0.77 Eicosenoic acid (20:1) n-11 FAME 0.83 Behenic acid (22:0) FAME 0.60 Palmitic acid (16:0) FFA 0.48 Lignoceric acid (24:0) FAME 0.92 Oleic acid (18:1) n-9 FFA 2.22
a) FAME Fatty acid methyl ester b) FADME Fatty acid dimethyl ester c) FAEE Fatty acid ethyl ester d) FFA Free fatty acid
144 C h a p t e r 3 O live f r u it - sk in a n a l y s is
Table 3.5 a
Fatty acids detected by GC-MS in the saponified sample of Hexane extract obtained from Olive skins, as described in method 3.2.3. The conditions and sample preparation for GC-MS are described in method 3.3.6.
Instrument: Hewlett Packard HP 6890/5973 GC-MS Column: 10m X 0.10mm ID x 0.2um HP-WAX Carrier gas/flow rate: Helium. 0.5 ml/min Injection mode/volume: Manual:split 40:1, O.Spl Oven temperature: 70(l)-38-150(2)-14-240 Ionization mode: Electron Impact (El) Scan analysis: 40-400 amu
Carbon chain Detected as Rel. %
Palmitic acid (16:0) FAME 15.59 Palmitoleic acid (16:1) n-9 FAME 0.61 Heptadecanoic acid (17:0) FAME 0.36 Oleic acid (18:1) n-9 FAME 69.44 Linoleic acid (18:2) n-9,12 FAME 6.88 Linolenic acid (18:3) n-9,12,15 FAME 0.17 Octadecadienoic acid (18:2) n-10,13 FAME 0.55 Arachidic acid (20:0) FAME 0.83 Eicosenoic acid (20:1) n-11 FAME 1.41 Octadecatrienoic acid (18:3) n-6,9,12 FAME 0.15 Behenic acid (22:0) FAME 0.66 Lignoceric acid (24:0) FAME 1.55 Hexacosanoic acid (26:0) FAME 1.50 Octacosanoic acid (28:0) FAME 0.28
a) FAME Fatty acid methyl ester b) FADME Fatty acid dimethyl ester c) FAEE Fatty acid ethyl ester d) FFA Free fatty acid
The temperature programme was modified to accommodate the analysis of the C (26:0) and C (28:0) fatty acids.
145 C h a p t e r 3 O live f r u it-sk in a n a l y s is
3.4.2 Chloroform extract
3.4.2.1 Thin layer chromatography (TLC)
Thin layer chromatography on silica using chloroform : methanol (9:1), gave two predominant components (Rf. values 0.47 and 0.82 respectively). Figure 3.2.
Figure 3.4 TLC Analysis of Chloroform extract from Olive-skins
TLC: Silica gel Solvent system: Chloroform : Methanol (9:1). Solvent front: 18 cm Stain: 1% Vanillin in EtOH and 40% Sulphuric acid in EtOH (1:1) (heated at 100°C for 10 min., for visualisation)
146 C h a p t e r 3 O l iv e f r u it - s k in a n a l y s is
3.4.2.2 Preparative Thin layer chromatography (PTLC)
The chloroform extract (62mg) was separated by PTLC on a 20 cm x 20 cm silica gel plate (6F-254, MERK Ltd.) eluted with chloroform : methanol (9:1). After the PTLC plate was developed, a margin of 2 cm at one side of the plate was sprayed with a mixture of 1% vanillin in EtOH and 40% sulphuric acid in EtOH (1:1), until saturated and then heated with a hair drier. Two purple bands were observed, which were scraped off into separate flasks, suspended in CHCb : MeOH (1:1) and sonicated for 20 minutes.
The silica gel was then removed by filtration and the solutions evaporated to dryness at
35 °C by rotary evaporator. The two compounds obtained were subsequently identified as the triterpenes, p-oleanolic acid [(17.75 mg)(Rf. 0.82)] and ô-maslinic acid [(17.4 mg) (Rf. 0.47)], by NMR spectroscopy and mass spectrometry.
147 C h a p t e r 3 O l iv e f r u it- s k in a n a l y s is
3.4.2.3 P-Oleanolic acid (3p-hydroxy-12-oleanen-28-oic acid)
3.4.2.3.1 NMR (400 MHz, Py-d6):
7CH.1
Py-d6 12 Py-d(. Py-tl6
Figure 3.5 ID proton NMR spectra of P-OIeanoIic acid extracted from Olive skins
5.42 (t, J = 3.3 Hz, H-12), 3.35 (dd, J = 10, 9.96 Hz, H-3), 3.15 (dd, J = 13.73, 3.9 Hz,
H-18), 1.63 (t, J = 8.6, 9.12 Hz, H-9), 1.23 (s, CH3-27), 1.15 (s, CH3-23), 0.99 (s, CH3-
30), 0.94 (s, CH3-24), 0.94 (s, CH3-29), 0.93 (s, CH3-26), 0.89 (s, CH3-25), 0.81 (d, J =
10.92 Hz, H-5).
148 C h a p t e r 3 O liv e f r u it- s k in a n a l y s is
3.4.2.3.2 13C NMR (100 MHz, Py-d6):
7C
TT T 20 0
Figure 3.6 13 C NMR spectra of p-Oleanolic acid extracted from Olive skins
180 (C-28), 145.65 (C-13), 123,40 (C-12), 79.13 (C-3), 56.71 (C-5), 49.02 (C-9),
47.56(C-17), 47.45 (C-19), 43.05 (C-14), 42.78 (C-18), 40.61 (C-8), 40.27 (C-4), 39.91
(C-1), 38.31 (C-10), 35.26 (C-21), 34.39 (C-29), 34.15 (C-7), 34.02 (C-22), 31.90 (C-
20), 29.70 (C-23), 29.19 (C-2), 28.82 (C-15), 27.19 (C-27), 24.86 (C-30), 24.73 (C-11),
24.56 (C-16), 19.73 (C-6), 18.30 (C-26), 17.39 (C-24), 16.57 (C-25).
1 4 9 C h a p t e r 3 O l iv e f r u it- s k in a n a l y s is
3.4.2.3.3 2D NMR COSY (400 MHz, Py-d6):
1
Figure 3.7 2D COSY NMR spectra of p-OleanoIic acid extracted from Olive skins
30 29
Figure 3.8 P-Oieanolic acid
1 5 0 C h a p t e r 3 O liv e f r u it- s k in a n a l y s is
3.4.2.3.4 Fast atom bombardment - mass spectrometry (FAB-MS)
100%, 289 . OEb
90j
80_ 439 7 0 r~
f.O 273
50.
261 - 1 . 3E5 40 -1 . 2T.5
25. 301 39 3
2 0 , : 5 .9E4 15. 353 319 479 L 4 . 4 E4 j}34 10 1 468 ; 3 . 0E4
hî/ I ■ 3K4 ■ OHO 4è0 ào s6o 5io m/ 2
Figure 3.9 FAB -MS spectra of |3-Oleanolic acid extracted from Olive skins
FAB-MS (Matrix MNOBA + Na) ; [M+1 (-Hz)] 455; [M+Na] 479;
151 C h a p t e r 3 O l iv e f r u it- s k in a n a l y s is
3.4.2.4 ô-Maslinic acid (2a, 3p-dihydroxy-12(13)-oleanen-28-oic acid)
3.4.2.4.1 H NMR (400 MHz, Py-d6):
7CH1
Py-d6
12 Py-d6 f\-d6 8 j L T ppm y .1 I I)
Figure 3.10 ID proton NMR spectra of Ô-Maslinic acid extracted from Olive skins
5.46 (brs, H-12), 4.08 (dt, J=16, 2.68 Hz, H-2), 3.38 (d, J = 9.36 Hz, H-3),3.27
(dd,J=12, 4 Hz, H-1 8), 1.8 (m, H-9), 1.26 (s, CH3-27), 1.26 (s, CH3-30), 1.065 (s, CH3-
26), 1.03 (d, J = 1 0 Hz, H-5), 1.0 (s, CH3-24), 1.0,(s, CH3-29), 0.97 (s, CH3-25), 0.95 (s,
CH3-23).
152 C h a p t e r 3 O l iv e f r u it- s k in a n a l y s is
3.4.2.4.2 13CNMR (100 MHz, Py-d6):
7C
COOH
ftrmi n rjr r r rrrirr^ ppm 200 ISO " 120 xo
Figure 3.11 13 C NMR spectra of ô-Maslinic acid extracted from Olive skins
181.14 (C-28), 145.71 (C-13), 123.31 (C-12), 84.62 (C-3), 69.45 (C-2), 56.76 (C-
5),49.02 (C-9), 48.56 (C-19), 47.51 (C-17), 47.32 (C-1), 43.06 (C-14), 42.83 (C- 18),
40.71 (C-8), 40.66 (C-4), 39.93 (C-10), 35.11 (C-21), 34.19 (C-23), 34.07 (C-22), 34.06
(C-7), 31.83 (C-20), 30.21 (C-27), 29.14 (C-15), 27.07 (C-30), 24.79 (C-16), 24.67 (C-
29), 24.55 (C-11), 19.73 (C-6), 18.56 (C-26), 18.34 (C-24), 17.73 (C-25).
153 C h a p t e r 3 O l iv e f r u it- s k in a n a l y s is
3.4.2.43 2D NMR COSY (400 MHz, Py-d6);
Figure 3.12 2D COSY NMR spectra of ô-Maslinic acid extracted from Olive skins
30 29
COOH 28
Figure 3.13 ô-Maslinic acid
154 C h a p t e r 3 O l iv e f r u it-s k in a n a l y s is
3.4.2A.4 Fast atom bombardment - mass spectrometry (FAB-MS)
loot 7 .8E4
£-7 ,4E4
7 . OE-l 6 . 6E4
6.2E4
5.8E4
-5 .OE-1 ,7E4
. 3K4 -3.9E4
_ 3 .5E4
3 . 1E4
-.2.7E4
2 . 3E
11.9E4
1 . 6E4
■1.2E4 L7.9E3 - 3 .9E3
0 . CEO 370 3é0 3^0 4Ô0 4io 420 4 0 510
Figure 3.14 FAB -MS spectra of ô-Maslinic acid extracted from Olive skins
FAB-MS (Matrix MNOBA + Na); [M+1] 473; [M+Na] 495;
155 C h a p t e r 3 O live f r u it -sk in a n a l y s is
3.4.3 M ethanol extract
3.4.3.1 Thin layer chromatography (TLC) of the Methanol extract from Olive skin
The methanol extract from olive skins when run in chloroform : methanol (8:2), gave four spots with a good separation, Figure 3.15. One spot was close to the solvent front
(Rf. 0.9), two closely spaced just below mid retention time (Rf. 0.23 and 0.21), and one elongated trailing spot at the origin (Rf. 0.08). This sample was further examined by
HPLC.
3.4.3.1 High performance liquid chromatography (HPLC) of the Methanol extract
from Olive skins
Analysis of the methanol extract by HPLC was performed on a Zorbax RP C18 (CDS) column eluted with 30% Methanol and 70% Water. Twenty five injections of the extract were made on the semi-preparative CDS column (25cm x 0.94cm) and a typical preparative chromatogram is shown in Figure 3.16. Fractions were collected from each injection at the times shown for components Mi, Mi, Ms and M4.
Each fraction was concentrated by rotary evaporation prior to being dissolved in
CDCbiCDaOD (2:1) for NMR spectroscopy.
156 C h a p t e r 3 O l iv e f r u it- s k in a n a l y s is
Figure 3.15 TLC Analysis of Methanol Extract from Olive-skin
TLC: Silica gel
Solvent system: Chloroform;Methanol (8:2)
Solvent front: 18 cm
Stain: 1% Vanillin in EtOH and 40% Sulphuric acid in EtOH (1:1)
(heated in the oven at 100 ”C for 10 min., for visualisation)
157 C h a p t e r 3 O l iv e f r u it-s k in a n a l y s is
o LU
C/3
M4 M3 M2 M1
13 12 11 10 9 8 7 6 5 4 3 2 1 TIME (min)
Figure 3.16 HPLC Analysis of Methanol Extract from Olive skins
Column: Zorbax CDS (5^im)
(25 cm X 0.94 cm ID)
Solvent: 30% Methanol : 70% Water (isocratic mode)
Flow: 2ml/min - -
Detector: UV 254 nm
Injection: 60 (2mg/ml)
158 C h a p t e r 3 O live fr u it -sk in a n a l y s is
3.4.3.3 Nuclear magnetic reasonances (NMR) of Methanol extract from Olive skins
Fractions Mi, M2, Ms and M4 obtained from the HPLC separation on a Zorbax RP CIS
(CDS) column of the methanol fraction from Olive skins were examined by NMR spectroscopy.
The spectra obtained were complex due in part to the difficulty in obtaining a more effective chromatographic separation. The NMR spectra are shown in Figures 3.7-
3.10, and 3.12-3.15.
Functional groups which can be identified from these spectra are aromatic rings, non conjugated double bonds, anomeric sugar protons as well as other sugar moieties and methine/methylene protons from the steroid resonances. The methyl group signal is very prominent, both for singular methyl groups and the methyl group as a part of an aliphatic chain.
Methanol HPLC fraction Mi (Figure 3.17) contains a dominant component with one or two aromatic rings which can be identified downfield from 7.0 to 7.2 ppm. Non conjugated double bonds (6.27-5.62 ppm) could be part of this molecule. Upfield, there are peaks (2.29-2.04 ppm) which could be from the main molecule but interference from the chemical shifts of fatty acid aliphatic chains and steroidal protons makes it impossible to elucidate the complete structure.
Methanol HPLC fraction M 2 (Figure 3.18) contains structures that are very much the same as in Mi. Peaks that are specific only to this fraction are from the aromatic ring and non-conjugated double bonds, but their intensity is too weak for full characterisation.
Methanol HPLC fraction Ma (Figure 3.19) has an individual peak at 6.04 ppm (quartet) which could be part of the structure x-CH=CH-CH3. Additional upfield peaks in this
1 5 9 C h a p t e r 3 O live f r u it -sk in a n a l y sis fraction suggest the conjugation of the -CH 2 structure with the aromatic part of the molecule. Resonances of the glucose molecule are observed at 5-3 ppm.
Methanol HPLC fraction M4 (Figure3.20) showed a strong comparison with fraction Mi with resonances at 7.0 to 7.2 ppm and in the area 6.3 to 5.5 ppm.
3.4.3.4 Mass Spectrometry of Methanol extract HPLC fractions
Fractions Mi, M2, Ma and M4 from the NMR experiments were evaporated to dryness and redissolved in 30% methanol and 70% water. Each fraction was in turn examined by positive ion Atmospheric Pressure Chemical Ionisation (APCI) mass spectrometry.
Attempts to examine these fractions by Electrospray ionisation and FAB were discounted due to the low mass ions observed inferring fragmentation of the molecular ion species. APCI is a soft ionisation technique, preserving the pseudo molecular ion
(M+1). The molecular weights obtained for each fraction are shown in Table 3.6.
Table 3.6 Methanol extract HPLC fractions from Olive skins - Molecular weights
HPLC fractions from Molecular weights - MW
Methanol extract
"Mi 480 446 442 428 238
M2 477 445 386
M3 592 449 238
M4
1 6 0 C h a p t e r 3 O live fr u it -sk in a n a l y s is
ppm
ppm
2. ,^ 2. 42. 1 1.8 1.5 1.20. so. 60. 3 p p m
Figure 3.17 ID proton NMR spectra of ODS column HPLC fraction Mi, obtained from Methanol extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
161 C h a p t e r 3 O live f r u it -s k in a n a l y sis
6 6 6.0 6 ppm
ppm
ppm
Figure 3.18 ID proton NMR spectra of ODS column HPLC fraction M2, obtained from Methanol extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
162 C h a p t e r 3 O live f r u it - sk in a n a l y s is
8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 ppm
4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 ppm
ppm
Figure 3.19 ID proton NMR spectra of ODS column HPLC fraction Ma, obtained from Methanol extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
1 6 3 C h a p t e r 3 O live f r u it -sk in a n a l y sis
8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 ppm
ppm
IL -A*, T------1------1------1 1 1 1 1 1------2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 ppm
Figure 3.20 ID proton NMR spectra of ODS column HPLC fraction M4, obtained from Methanol extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
164 C h a p t e r 3 O live f r u it -sk in a n a l y sis
3.4.4 Water extract
3.4.4.1 High performance liquid chromatography (HPLC) of the Water extract
from Olive skins
The water extract was examined by HPLC using the semi-preparative Zorbax RP CIS
(ODS) column (25cm x 0.94cm) under the same solvent conditions, 30% methanol :
70% Water, as used for the methanol extract examined by this column. Twenty five injections of the water extract were made and the main component fractions Wi, W2,
Wa and W4, collected from each injection at the times shown. A typical preparative scale chromatogram is shown in Figure 3.21.
Each fraction was concentrated by rotary evaporation prior to being dissolved in
CDsODiDzO (1:1) for NMR spectroscopy.
165 C h a p t e r 3 O liv e f r u it-s k in a n a l y s is
g? 6 5 4 3 2 1 T»ME (mn)
Figure 3.21 HPLC Analysis of Water extract from Olive skins
Column: Zorbax ODS (5p.m)
(25 cm X 0.94 cm ID)
Solvent: 30% Methanol : 70% Water (isocratic mode)
Flow: 2ml/min
Detector: UV 254 nm
Injection: 60 jil (2mgZml)
166 C h a p t e r 3 O live f r u it -sk in a n a l y s is
3.4.4.2 Nuclear magnetic resonances (NMR) of Water extract from Olive skins
The fractions Wi, W 2, W 3 and W4 were obtained from the HPLC separation on a
Zorbax RP CIS (ODS) column of the water fraction from Olive skins. The spectra obtained were complex; the low concentrations preventing 2D NMR measurements from being made. The NMR spectra are shown in Figures 3.22-3.25.
Functional groups which can be identified from the spectra are a single aromatic ring, sugar anomeric protons, other sugar moieties and methine/methylene protons. Signals for methyl groups, both individual and in chains can be distinguished. There are no conjugated or non-conjugated double bonds present in any of the water fractions.
HPLC fraction Wi (Figure 3.22) has an aromatic component at 7.75-7.67 ppm and 7.28-
7.21 ppm. Sugar resonances can be observed from 4.77 to 3.32 ppm, but it is difficult to determine how many sugar molecules could be present because of the steroid protons overlapping in that area. Further resonances observed are 3.6 ppm (triplet) for >CH-OH,
4.0 ppm (quartet) for -CO-CH 2-OH, possibly the side chain of a steroidal ring, and 4.25 ppm (multiplet), possibly a steroid ring proton. Between 2.32 ppm and 0.87 ppm aliphatic chain protons for CHs-, -(CH 2)n- and -CH 2-CH2-COOH were present together with signals for individual -CHs groups (0.92-0.95 ppm, 1.32-1.30 ppm, 2.36 ppm and
2.11 ppm) and methine/methylene protons from steroid rings.
HPLC fraction W 2 (Figure 3.23) has an aromatic part of the molecule which is the same as in WI. Sugar resonances have stronger intensities then in the Wi fraction but the difference lies in the peaks at 2.75 ppm (doublet), 2.50 ppm (doublet) and 2.41 ppm
(multiplet). These peaks occur where some of the protons from steroid moieties can be present, for example at 2.7 ppm.
HPLC fraction W 3 (Figure 3.24) has chemical shifts of lesser intensity and definition
167 C h a p t e r 3 O live f r u it - sk in a n a l y sis than in fraction W 2, and therefore it is more difficult to characterise. The multiplet at
2.41 ppm is not present in fraction Ws.
HPLC fraction W4 (Figure 3.25) has additional chemical shifts in the area of aromatic and conjugated/non-conjugated double bonds, at 6.14 ppm (quartet) and 5.9 ppm
(singlet). These can be from a structure of the type shown: -CH=CH-CO-CH=CH-.
A singlet is observed at 3.75 ppm which could be indicative of a methoxy group. A multiplet at 3.88 ppm suggests a structure such as: -CH 2-CH(OH)-CH2-. In this fraction a strong chemical shift is observed at 0.9 ppm suggesting the methyl group is part of an aliphatic chain. Other signals from aliphatic chain protons are dominant in the spectra.
168 C h a p t e r 3 O live f r u it -sk in a n a l y s is
j J jUl 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 ppm
4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 ppm
2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 ppm
Figure 3.22 ID proton NMR spectra of ODS column HPLC fraction Wi, obtained from Water extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
169 C h a p t e r 3 O live f r u it -sk in a n a l y sis
I I I I I I I 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 ppm
ppm
2.4 2.1 1.8 1.5 1.2 0.9 0.62.7 ppm
Figure 3.23 ID proton NMR spectra of ODS column HPLC fraction W2, obtained from Water extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
1 7 0 C h a p t e r 3 O live f r u it -sk in a n a l y sis
8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 ppm
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
Figure 3.24 ID proton NMR spectra of ODS column HPLC fraction Wa, obtained from Water extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
171 C h a p t e r 3 O live f r u it -sk in a n a l y sis
8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 ppm
4.5 4.0 3.5 ppm
Figure 3.25 ID proton NMR spectra of ODS column HPLC fraction W4, obtained from Water extract of Olive skins; 12 000 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
172 C h a p t e r 3 O live f r u it -sk in a n a l y sis
3.4.4.3 Mass Spectrometry of Water extract from HPLC fractions
Fractions Wi, Wz, W 3 and W4 from the NMR experiments were evaporated to dryness and redissolved in 10% methanol and 90% water. Each fraction was in turn examined by positive ion APCI mass spectrometry. The molecular weight information so obtained is shown in Table 3.7.
Table 3.7 Water extract HPLC fractions from Olive skins-Molecular weights
HPLC fractions from Molecular weights - MW
Water extract wi
W2 480 442 440 426
W3 832 518 477 445 386
W4 386
173 C h a p t e r 3 O live f r u it -sk in a n a l y sis
3.5 Discussion
The olive skins were examined using four solvent extracts, hexane, chloroform, methanol and water.
The distribution of fatty acids found in olive oil has been discussed in Chapters 1 and 2, and comparisons made with the fatty acids found in olive pulp. A comparison with the fatty acids found in olive skins is therefore of interest, particularly since olive skins have not been analysed in detail. The hexane extract from olive skins was therefore analysed by GC-MS and the identified skin components compared with these found in olive pulp and olive oil. The results of this study are summarised in Table 3.8.
174 C h a p t e r 3 O live f r u it -sk in a n a l y sis
Table 3.8 Fatty acids found in the hexane extract from Olive skins
Non-saponified sample Saponified sample Carbon chain Rel % Carbon chain Rel. % (14:0) 0.06 (14:0) 0.05 (16:0) 15.08 (16:0) 13.8 (16:1) n-7 0.24 (16:1) n-7 0.23 (16:1) n-9 0.53 (16:1) n-9 0.58 (17:0) 0.34 (17:0) 0.50 (18:0) 4.43 (18:0) 4.48 (18:1) n-9 59.14 (18:1) n-9 61.24 (18:1) n-8 0.32 - (18:2) n-9,12 11.65 (18:2) n-9,12 11.72 - - (18:2) n-10,13 0.14 (18:3) n-9,12,15 0.84 (18:3) n-9,12,15 0.63 - - (18:3) n-6,9,12 0.15 -- (19:1) n-10 0.15 (20:0) 0.78 (20:0) 0.83 (20:1) n-11 0.49 (20:1) n-11 0.83 (22:0) 1.24 (22:0) 0.6 (24:0) 2.58 (24:0) 1.55 -- (26:0) 1.5 - - (28:0) 0.28 N-valerylalanine 0.05 9-oxo-nonanoic 0.23
1 7 5 C h a p t e r 3 O live f r u it -sk in a n a l y s is
The analysis of the hexane extract from Olive skins showed the presence of 14 different fatty acids in the non-saponified sample. Comparison of the non-saponified sample with the saponified sample showed the presence of five additional acids in the saponified fraction namely:
C (19:1) n-10; C (18:2) n-10,13; C (18:3) n-6,9,12; C (26:0) and C (28:0).
Fatty acids not previously identified in olive oil but found to be present in the non- saponified skin extract were:
C (16:1) n-7; C (18:1) n-8 and C (20:1) n-11.
While in the saponified skin extract the following additional fatty acids were identified:
C (16:1) n-7; C (18:2) n-10,13; C (18:3) n-6,9,12; C (19:1) n-10; C (20:1) n-11; C
(26:0) and C (28:0).
A comparison of the fatty acids present in olive skin with those present in olive pulp would indicate that the olive skin contains a more complex pattern of fatty acids both in the non-saponified and in the saponified samples. This comparison is shown in Table
3.9.
176 C h a p t e r 3 O live f r u it -s k in a n a l y sis
Table 3.9 Fatty acids found only in olive skin and not in olive pulp
Non-saponified sample Saponified sample Carbon chain Rel. % Carbon chain Rel. % (14:0) 0.06 - - (16:1) n-7 0.24 (16:1) n-7 0.23
(16:1) n-9 0.53 - -
(17:0) 0.34 - - (18:1) n-8 0.32 - - - - (18:2) n-10,13 0.14 (18:3) n-9,12,15 0.84 (18:3) n-9,12,15 0.63 - - (18:3) n-6,9,12 0.15 - - (19:1) n-10 0.15 (20:0) 0.78 - - (20:1) n-11 0.49 (20:1) n-11 0.83 (24:0) 2.58 (24:0) 1.55 - - (26:0) 1.5 -- (28:0) 0.28 N-valery alanine 0.05 9-oxo-nonanoic 0.23
The presence of C (26:0) and C (28:0) acids in the saponified sample indicates the presence of these chains in the non-saponified conjugates. This is the first reported
finding of such substituent chains. The presence of 9-oxo-nonanoic acid is a novel
finding and is of interest in understanding both the structure and role of the conjugate molecules present in olive skin.
177 C h a p t e r 3 O live f r u it - sk in a n a l y sis
The N-valerylalanine found in the saponified sample would on saponification be converted to alanine and valeric acid. Valeric acid would not be detected in the present
FAME analysis because of the temperature profile used. In olive skins, the N- valeryalanine indicates the likely presence of simple amino acids conjugated to fatty acids.
The chloroform extract was chromatographed to yield two compounds, the triterpenes,
p-oleanolic acid and 0-maslinic acid.
30 29
25 11 COOH 28
H O ^
24 23
p-oleanoIic acid
30 29
COOH HO 28
H O ^
24 23
0-masllnic acid
178 C h a p t e r 3 O live f r u it -sk in a n a l y sis
Both of these compounds have previously been identified in a study on olives by
[Bianchi et al., 1994].
A comparison of the four extracts from olive skins was carried out by NMR profiling.
This method yielded spectra more complex and difficult to analyse than the more traditional lipid NMR profiling. The methanol and water extracts were subjected to
HPLC to obtain fractions for NMR and MS analysis. The latter gave masses (Tables 3.6,
3.7) for the principal components but contained insufficient material for detailed
conformation by 2D NMR.
The functional groups observed by NMR in the methanol and water fractions together
with the molecular weight information obtained from the APCI mass spectrometry
suggested the presence of the following structures:
MW: 449.39 HO
3
Figure 3.26 3-(glucopyranosyl)-cyanidol
179 C h a p t e r 3 O live f r u it -sk in a n a l y sis
MW: 442.72
CH.OH
H a
Figure 3.27 12-01eanene-3,28-diol
MW: 426.72
Figure 3.28 4,14-Dimethyl-9,19-cycloergost-23-en-3-ol
180 C h a p t e r 3 O live f r u it -sk in a n a l y sis
MW: 592.59
O M e
H —4 o(—O-C-CH3
^ C H 3 O 4' I O M e
Figure 3.29 4' -methyl- 8 -acetyl 4-(glucopyranosyl)-pinoresinol
Further analytical detail on these structures is difficult to obtain due to the apparent instability of the extracts and the complexity of the chromatography. Some individual, although poorly resolved component peaks observed in the water fraction were of the same elution volume as peaks observed in the methanol fraction analysis. This observation was subsequently confirmed by the APCI mass spectrometry. Common components in each fraction would not be unexpected considering the compound types proposed. Oxidation of the separated components is also a possibility in these analyses.
A quinoid structure could easily be generated from the compound shown in Figure 3.26 and these together with the presence of carbohydrate moieties would be susceptible to oxidation. Some of these compounds found in olive skin have been found in previous studies on whole olives where the complete olive fruit was crushed and extracted
[Tsukamoto et ah, 1984, Chapman & Hall, 2000].
181 CHAPTER 4
PHOSPHOLIPID ANALYSIS
182 C h a p t e r 4 P hospholipid a n a l y sis
4 PHOSPHOLIPID ANALYSIS
4.1 Introduction
The lipid content of olives such as Olea europaea L., has been the object of many
investigations not only in relation to plant metabolism but also nutrition and medicine.
Neutral lipids together with the fatty acid content of olives are thus now well established
areas. However, little has been reported on the phospholipid content of olives [Marzouk
& Cherif, 1981a, 1981b]. To understand better the importance of the techniques and
experimental processes used in obtaining comprehensive information on lipid mixtures,
it is necessary to review the steps involved in the analytical procedures. The isolation of
a particular class of lipids is carried out by solvent extraction of the total lipids [Bligh-
Dyer, 1959; Folch, 1957]. The purification and analysis is then carried out by one or a
combination of the following techniques; TLC [Bligh-Dyer, 1959], HPLC [Christie,
1987], GC [Christie, 1989] after derivatisation of the fatty acid components into methyl
esters and GC-MS [Metcalfe & Schmitz, 1969]. Lipid content determination has always
been a time consuming multiple step process, until the advent of one and two
dimensional high resolution proton NMR spectroscopy which was used to obtain
comprehensive information on the total lipid extract without prior chromatographic
separation or any chemical modification [Higgins, 1987]. This technique has been
successfully applied to determine qualitatively and quantitatively the lipids extracted
from cell tissues and body fluids, [Choi et al, 1993; Nicholson et al., 1995] parasites
and organisms [Adosraku et al., 1993, 1996].
As with lipid analysis, conventional approaches to metabolite analysis are laborious and
183 C h a p t e r 4 P hospholipid a n a l y sis often involve elaborate sample preparation with multiple extraction procedures, each limited in compound specificity, resulting in a variable or poor recovery. An alternative approach is to use crude extracts, for which many enzymatic and colorimetric methods are available, but these detection methods are usually not sufficiently specific and are often complicated by interference from other compounds. Equally important, only predetermined metabolites can be analysed in this way while unexpected or unknown components are easily overlooked.
With the advent of high-resolution gas and liquid chromatography many of these problems were solved. However, GC analysis is limited to volatile compounds (or those that can be made volatile by derivatisation) while HPLC is better suited for more polar compounds with conveniently detectable chromophores or functional groups. Recent advances in NMR methodology have overcome most of these limitations, with the exception of very low molecular concentrations.
The profile of a comprehensive range of organic metabolites can be acquired by multinuclear NMR methods, with the previous limitations involving volatility, the presence of a chomophore or polarity being totally overcome. NMR is non-destructive and therefore allows samples to be analysed subsequently by other techniques.
Definitive structural information can be obtained directly from crude extracts with minimal sample preparation. This allows a specific and simultaneous determination of a number of metabolites while maximising the opportunity for identifying important but unexpected or previously unknown metabolites. As such, it is particularly valuable for acquiring metabolite profiles on samples where prior knowledge is limited. Moreover, a substantial reduction in labour and time can be achieved from multiple component analysis. Complex metabolite pools have been assigned by a combination of
184 C h a p t e r 4 P hospholipid a n a l y sis
multidimensional and multinuclear NMR experiments [Jardetski & Roberts, 1981; Fan
et al., 1993; Fan, 1996; Nicholson et al., 1995]. These techniques have been commonly
applied to animal tissues and body fluids, but metabolites from plants and olives in particular, have mainly been investigated by traditional chromatographic methods.
In this study, we report for the first time, the analysis of both intact and purified lipid
extracts from Olea europaea L., using ID and 2D-Jresolved proton NMR experiments.
Different types of phospholipids and neutral lipids were identified from intact lipid
mixtures and quantified from their NMR spectra. Information on the fatty acid
composition and the average level of unsaturation was obtained from the same spectra.
In order to obtain a more detailed analysis of the sample, GC and GC-MS were used for
fatty acid analysis while MS measurements for phospholipids were made in both
positive and negative electrospray ionisation modes. Electro spray ionizaton mass
spectrometry (ESI-MS) was used in the determination of individual phospholipid
molecular species in our plant extract. ESI-MS is a rapid and sensitive method for
obtaining molecular weight information and complements NMR spectroscopy. Polar
lipids in principal can be analyzed using soft ionization techniques such as field
ionisation [Lehmann & Kessler, 1983b], chemical ionization [Haroldsen & Murphy,
1987; Jungalwala et a l, 1984], or by fast atom bombardment ionisation [Lehmann &
Kessler, 1983a; Chilton & Murphy, 1986]. Although complete structural information
can be obtained for phosphilipids by FAB-MS-MS, electro spray ionization offers
several advantages over this technique, including lower background signals because of
the absence of matrix ions, longer lasting and more stable primary ion currents, ease of
sampling, and compatibility with liquid chromatography. There are relatively few
reports in the literature on the analysis of lipids by ESI-MS. However investigations to
185 C h a p t e r 4 P hospholipid a n a l y sis date include the analysis of a platelet activating factor [Weintraub et al., 1991], and triacylglycerides [Cole & Harrata, 1992].
Plant extracts can be separated by solid phase ion exchange chromatography into four fractions corresponding to a) neutral lipids, b) free fatty acids, c) neutral phospholipids and d) acidic phospholipids [Kates et al, 1988]. NMR and ESI-MS can then be used to confirm the identity of the lipids belonging to the different classes contained within the
four fractions and their respective fatty acid compositions can be confirmed by GC-MS
[Choi et al., 1993].
The study of plant lipids by NMR is new and has some drawbacks, mainly because of
limitations in the maps of membrane lipids in various plant tissues. Although
chloroform and methanol are conventionally used to extract low polarity secondary
metabolites from plants, it was not clear to what extent the plant lipid extract would be
free from these metabolites. With animal tissue this type of complication is minimal.
4.2 Materials and methods
4.2.1 Sample
Black Greek olives (Olea europaea L.) were obtained from the island of Kim.
1 8 6 C h a p t e r 4 P hospholipid a n a l y sis
4.2.2 Reagents/equipment
Chloroform, methanol and hexane, were all of HPLC grade and obtained from Fisher
Scientific Limited, UK.
Potassium chloride (KCI), sodium sulphate (Na2S04), sodium hydroxide (NaOH), ammonium-acetate, ammonia and diethyl-ether, were research grade obtained from
BDH Laboratory Supplies, Pool, UK,
Bond Elut, NH2-aminopropyl columns, size (3cc, 500mg), were obtained from Varian,
CA, USA.
Standards of phospholipids and fatty acid methyl-esters were obtained from the Sigma
Chemical Co., USA.
Deuterated chloroform (CDCb 99.8%), and perdeuteromethanol (CD 3OD 99.8%) and
NMR tubes (5mm) were obtained from the Aldrich Chemical Company, Inc., USA.
The estérification reagent, BCb-methanol was ordered from the Supelco Co., USA.
4.2.3 Extraction of total lipids from whole olives
The olive tissue was first cut out from the fruit and separated from the seed. 100 g of this material was frozen in liquid nitrogen and ground to a paste, which was then mixed with 250 ml of chloroform : methanol (2:1) in a glass beaker and sonicated in an ultrasonic bath at 0 °C for 20min. The suspension was filtered through cheese cloth to remove particulate matter and then centrifuged at 2000 rpm for 10 min (at 4 °C), to separate the aqueous and organic layers.
Lipids which were left in the water layer and the remaining tissue were re-extracted with a further 250 mis of CHCbiCHsOH (2:1), and the lower organic layers obtained
1 8 7 C h a p t e r 4 P hospholipid a n a l y sis from both steps combined. The combined organic extract was washed twice with equal volumes of 0.5 M KCI in 50% HzO/methanol, dried over Na2S04, filtered and concentrated using a rotary evaporator and then under a stream of dry nitrogen. The lipid residue was resuspended in 0.5 ml of chloroform; 0.1 ml was transferred to a 5mm
NMR tube, evaporated down under a stream of dry nitrogen and resuspended in 0.6 ml of CDCbiCDsOD (1:2). The remaining volume was retained for Bond Elut separation.
The tubes were kept at -20 °C while awaiting NMR analysis. [Bligh-Dyer, 1959].
4.2.4 Separation of total lipids by Bond Elut (NHi-aminopropyl) solid phase
separation method
The column was conditioned by washing with 8 ml of HPLC grade hexane priory to the application of 0.2 ml of the lipid extract (from 4.2.3). According to their different polarities, lipids are separated into four individual fractions [Floch, 1957] by passing different eluants through the column in the following order: (1) chloroform (which elutes non-polar lipids and sterols), (2) diethyl ether with 2% acetic acid (which elutes non-esterified fatty acids), (3) methanol (which elutes non-acidic phospholipids) and (4)
0.05 M ammonium acetate in chloroform:methanol (4:1) plus 2% (v/v) of 28 % aqueous ammonium solution (which elutes acidic phospholipids). A total of 6.0 ml of each eluent (2 times 3.0 ml) was used for each elution. All of the eluates were concentrated under a stream of dry nitrogen and the residues then resuspended in 0.6 ml of
CDCl3:CD30D (1:2) and transferred to 5 mm NMR tubes which were kept at -20 °C while awaiting analysis.
188 C h a p t e r 4 P hospholipid a n a l y sis
4.2.5 Proton NMR of total lipids and Bond Elut fractions
Samples were bubbled with nitrogen prior to recording the spectrum in order to prevent any possible oxidation. The NMR spectra were determined using a Bruker AM 500
NMR spectrometer and processed using Felix 2.3 [ Biosym, 1987,1993 ].
The spectra were recorded at 25 °C in the Fourier transform mode (FT). The lipid extract spectra were acquired using a 3 ps, 45° pulse, a spectral width of 6000Hz, an acquisition time of 1.36 s, and relaxation delay of 4s. The free induction decays (FIDs) were collected with 16 K data points.
Connectivity among protons and hence conformation of the identity of different lipids was established with the magnitude COSY pulse sequence [Nagayama et al, 1980]. 2D
COSY experiments were performed at 298 K with a special width (SW) of 6024.10 Hz and a relaxation delay of 2 s. They were recorded in 2K (2048) data points obtained from 512 FIDs of 48 scans each, with zero-filling in the FI dimension.
Two-dimensional V-resolved spectra [Aue et al., 1976] were measured with solvent presaturation at 298 K. Experiments were recorded in 4 K (4096) data points with a spectral width (SW) of 6024.10 Hz. The FI (J-coupling) domain spectral width covered
45 Hz. 128 FIDs of 128 scans were collected.
Chemical shifts were referenced to the residual methanol peak at 3.31 ppm for the samples in CDsODiCDCb (2:1).
4.2.6 Gas chromatography of total lipids and Bond Elut fractions
Gas chromatography (GC) is used extensively in the analysis of the major lipid classes.
Fatty acids are the class of lipid most commonly analysed by GC and carbon numbers
1 8 9 C h a p t e r 4 P hospholipid a n a l y sis can range from C4 to Cso, but the most commonly occurring are those of chain length
Ci4 to C 22.
Gas chromatography (GC) analysis of fatty acids is most commonly performed after they have been converted to their methyl esters derivatives (FAME’s).
Metcalfe and Schmitz, [Metcalfe & Schmitz, 1961] showed that BFs/methanol was a suitable reagent for the méthylation of free fatty acids and by 1969 this was established as a standard method. Subsequently BFs/methanol was replaced by BCb/methanol which provided a more stable reagent preparation.
4.2.6.1 GC-Method
To be able to establish whether there was any free acids present the samples were examined both saponified and non-saponified. Sodium hydroxide (NaOH) 0.5 M in methanol was used to saponify each sample prior to estérification with BCb-methanol.
Sodium hydroxide solution (0.4 ml) was added to the sample (100 pi) in a reaction vial
and the mixture heated until the droplets of fat had disappeared. This normally takes up to 5-10 minutes.
Petroleum ether was then to added to dissolve the fatty acids, and the whole mixture vortexed, before being heated for 3 min.. The upper layer which consisted of the methyl
esters was removed for examination by GC.
1 9 0 C h a p t e r 4 P hospholipid a n a l y s is
4.2.6.1 GC-conditions (for Bond Elut fractions)
Instrument: Carlo Erba HRGC 5300 Mega Series
Column: 50m x 0.22mm ID; Cyanopropyl CP-Sil 88 (non CB) ;
Temperature and Programme: 142°C for Imin.; than 4°C /min to 240°C;
Carrier Gas/flow rate: Helium/0.5 ml/min
Injection: Split (1:50); lpl@ 280°C
Detector: FID (a) 300 "C
4.2.6.3 Fast GC-MS Analysis conditions for FAMEs
Instrument: HP 6890 Series
Column: 10m x 0.10mm ID; 0.2pm HP-WAX
Temperature and Programme: 60 °C for 0.4 min.; then 16 °C/min to 240 °C
Carrier gas/flow rate: Helium/ 0.5 ml/min
Injection mode: Manual: Split 40:1, Splitless
Injection volume: 0.5pL split, l.OpL splitless
Inlet temp: 280 °C
Mass spectrometer: HP 5973 Series MSD
Interface temp: 280 °C
Ion source temp: 230 °C
Quad Temp: 150 °C
Ionisation mode: Electron Impact (El)
Scan analysis: 40-400 amu
191 C h a p t e r 4 P hospholipid a n a l y s is
4.2.7 Mass Spectrometry (MS)
Mass spectrometry measurements on intact phospholipids were made in both positive and negative electrospray ionisation (ESI) modes on a Finnegan Navigator mass spectrometer. The samples were introduced by loop injection from a Waters 2690 pump and injector unit.
4.3 Results
4.3.1 Identification and quantification of lipids by NMR
The identification of chemical shifts was achieved by comparison with spectra of standard lipids from each class of lipid; and by the analysis of two-dimensional peaks in the lipid extracts. The relative concentrations of individual lipids were determined by measuring the relative integrals under their characteristic chemical shifts in their ID and
2D-J resolved spectra.
The total lipid extract showed the presence of only long fatty acid chains and glycerol moieties Snl, Sn2 and Sn3 in the ratios corresponding to triglycerides. As expected the olive fruit proved to be extremely rich in oils, which made it difficult for other classes of lipids to be detected. Bond Elute column chromatography, proved to be quick and efficient in providing further separation into different lipid classes and especially in removing the complications of triglycerides.
192 C h a p t e r 4 P hospholipid a n a l y s is
4.3.1.1 Proton NMR spectral assignment of the different lipid classes from Bond
Elut fractions
Class 1. Mono, di- and triglycerides
Triglyceride (TG) species were identified by their structure specific resonances at
4.32ppm (Snld, Sn3d), 4.15ppm (Snlu, Sn3u) and 5.25 ppm (Sn2), Figure 4.1.
The specific chemical shifts of diglycerides (DO) overlap with TG and other moieties but were readily identified by the multiplet at 5.12ppm corresponding to the Sn2 moiety in DG. The amount of TG was deduced readily from the integral of the multiplet at 4.32ppm, which corresponded to DG Snl and TG Snl,3 downfield, and gave a good approximation to the proportions of DG and TG in the mixture. Accordingly, TG was found in a ratio of 90% to 10% of DG.
Monoglycerides (MG) resonances at 3,5ppm and 4.0ppm overlapped with other features of the spectrum making their determination difficult.
Class 2. Steroidal lipids
The lipid extract spectra (Figure 4.1) was examined also for the possible presence of cholesterol, cholesterol esters and ergosterol. It is possible to identify them from high- field CIS proton resonances at 0.63-0.68ppm with other resonances at 2.22ppm (C4),
3.4ppm (C3), 1.76ppm (C2) for cholesterol only, and 2.25ppm (C4), 4.55ppm (C3),
1.82ppm (C2) for the cholesterol ester only. Specific resonances for ergosterol are at
1.04ppm (C21 methyl), 5.46ppm (C6 proton) and 5.72ppm (C7 proton).
Cholesterol and cholesterol esters were not identified, but ergosterol was present only in
1 9 3 C h a p t e r 4 P hospholipid a n a l y sis trace amounts. However, stero-methyl resonances between 0.5-1.Oppm on the NMR spectrum demonstrates a concentration of sterols in the sample. Literature results
[Bianchi et al., 1993; Cecchi, 1998] suggest that some of the sterols could be a and p stigmastene, a and p cholestane, P-sitosterol and cholesta-3,5-diene. A more detailed sterol analysis could be performed by GC-MS.
5. O 3 . 0 1 . 0 ppm
Figure 4.1 ID proton NMR spectra of Bond Elut fraction 1 (neutral lipids) extracted from whole Olive; 41654 scans were obtained for Fourier transformation. The residual methanol peak at 3.31 ppm was used as the reference chemical shift.
194 C h a p t e r 4 P hospholipid a n a l y s is
Class 3. Ether lipids
In ether glycerophospholipids (Bond Elut fraction 3) a fatty acid chain is attached to the
C-1 oxygen of the glycerol, by an ether linkage. The head groups are usually ethanolamine or choline.
The C-1 alkyl chain can be saturated or unsaturated, and they can be distinguished from each other by specific chemical shifts for saturated ether lipids; these are at 3.44ppm (a) and 1.6ppm (b), in the RC^H 2C% 0C^HzC^H moiety. The absence of a peak at 3.44 ppm or the overlapping of the resonance at 1.6 ppm in both ID and 2D spectra was consistent with the low abundance of this class of lipids.
Similarly unsaturated ether lipids with their chemical shifts at 5.9ppm (a), 4.3ppm (b) and 2.00ppm (c), in the RC^HgÇ^HzÇ^H^Ç^HOCmoiety could not be observed, being not above noise level or overlapped. It was concluded therefore that neither saturated (alkyl-acyl glycerolipids) nor unsaturated (alkenyl-acyl glycerolipids) ether lipids were present at concentrations which could be studied. If present their spectra would be dominated by polar steroid resonances.
Class 4. Sphlngolipids
This class of lipids (Bond Elut fraction 3) contains the base 4-sphinganine or 4- sphingenine (sphingosine) instead of glycerol. The principal compounds of this class of lipids are sphingomyelin and ceramide phosphoethanolamine. There are other sphingolipids, like cerebrosides and gangliosides, with the phospho head group replaced by carbohydrate moieties, but they were not observed in this extract.
Sphingenine lipids can be identified by their specific vinyl proton resonances at 5.7ppm
1 9 5 C h a p t e r 4 P hospholipid a n a l y sis
(multiplet) and 5.4ppm (quartet), in 2D-J resolved spectra. In addition, it is possible to distinguish if these sphingolipids possess a head group of choline or ethanolamine by checking for splitting of the choline resonance at 3.21 ppm. No splitting was observed, therefore, the sphingolipids present in the mixture were identified as being mainly ceramide phosphoethanolamines.
3 CH2-OH
2 CH-NH2
HO-CH-CH=CH-CH2(CH2)iiCH3 1 a b c
sphingosine (the sphingenine analogue)
Class 5. The Diacylglycerophospholipids (DAGP)
This class of lipids (Bond Elut fractions 3 and 4) possesses a glycerol backbone
substituted with acyl chains at the Snl and Sn2 positions, a phosphate diester at the Sn3 position, and can have a variety of head groups. The head groups include serine (for PS- phosphatidylserine), ethanolamine (for PE-phosphatidylethanomamine) in its mono and di-N-methylated forms, choline (for PC-phosphatidylcholine), inisitol (for PI- phosphatidylinositol), hydrogen (for PA-phosphatidic acid) and glycerol (for PG- phosphatidylglycerol), (Figure 1.3, Table 1.5).
Cardiolipin (CAR)-diphosphatidyldyglycerol is also a member of this class.
These lipids can form subclasses, depending on the composition (saturated, unsaturated
or in rare cases branched) of the fatty acid chains, and they can also lose one of the fatty
1 9 6 C h a p t e r 4 P hospholipid a n a l y s is chains, forming lysolipids.
Their structures can be represented by the following formula:
CH2 O.CO.R1 I R 2C O .O C H Q I II CH2 O — P — OX
O-
The one dimensional spectrum and 2D J-resolved spectra from Bond Elut fraction 3 and
4 were obtained at 500 MHz and revealed the presence of many lipids, (Figures 4.3 and
4.4). Although there was considerable overlap of resonances from different lipid components, most of the lipids had structure-specific resonances which enabled their identification and quantification.
The diacylglycerolphospholipids could be identified and quantified by the glycerol backbone protons, Sn2-multiplet (5.21 ppm); both the corresponding magnetically inequivalent Snl,d (4.42ppm) and Snl,u (4.15ppm) and both Sn3 methylene protons
(4.0ppm).
1 9 7 C h a p t e r 4 P hospholipid a n a l y sis
Table 4.1 Assignment of NMR resonances in nhosnholinids
Phospholipid Assignment Chemical Functional group shift/ppm
Diacylglycerolphospho Snl,d 4.42 -CHd-OCO- lipids-glycerol Snl,u 4.15 -CHu-OCO- Backbone Sn2 5.21 -CH-OR- Sn3 4.00 -CH2-0-P03- Phosphatidic acid PA Snl,d 4.46 -CHd-OCO- Cl 3.88 -CH2-CH(OH)-CH2(OH) Phosphatidylglycerol C2 3.58 -CH2-CH(OH)-CH2(OH) PG C3 3.74 -CH2-CH(OH)-CH2(OH) Phosphatidylserine Cl 3.9 -CH2-CH(NH2)-COOH PS C2 4.28 -CH2-CH(NH2)-COOH Cl 4.23 -CH2-CH2-N Phosphatidylcholine C2 3.6 -CH2-CH2-N PC 3.2 -CH2-N(CH3)3 Phosphatidylethanolamine Cl 4.05 -CH2-CH2-NH3 PE C2 3.11 -CH2-CH2-NH3 Cl 3.2 C2 3.4 Phosphatidylinositol C3 3.6 inositol ring proteins PI C4 3.8 C5 4.2 Lysophosphatidylcholine Cl,d 4.08 -CH2-CH2-N LyPC Cl,u 4.15 -CH2-CH2-N Sn2 3.95
Determination of both non-acidic and acidic phospholipids by their head group resonances is possible but the ID spectra of Bond Elut fraction 3 contained to many overlaps from the polar steroid components for this to occur. However, head group resonances were detected in the highly resolved 2D-J spectra. The NMR spectrum of
Bond Elut fraction 3 showed only low concentrations of glycerol containing non-acidic phospholipids. This was confirmed by the low concentration of fatty acid chains in the same spectrum (at resonances 2.0 ppm, 2.2 ppm and 2.8 ppm). Figure 4.2 shows the
1 9 8 C h a pt e r 4 P hospholipid an a ly sis spectra with very small resonances at 4.32 ppm which is the marker resonance for triglyceride moieties of the non-acidic phospholipids. The 4.15 ppm (Snlu/Sn3u) and
5.25 ppm (Sn2) resonances confirm this low concentration with respect to the polar steroids, of non-acidic glycerophospholipids in Bond Elute fraction 3.
Determination of acidic phospholipids proved to be more difficult. Cardiolipin, PS and
PA, if present, were at a concentration below the noise level, but PI and PG appeared to be present in small quantities in both the ID and 2D spectra of Bond Elute fraction 4.
Figure 4.2 ID proton NMR spectra of Bond Elut fraction 3 (non-acidic phospholipids) extracted from whole Olive; 41654 scans were obtained for Fourier transformation. The residual methanol peak at 3.31ppm was used as the reference chemical shift.
199 C h a p t e r 4 P hospholipid a n a ly sis
4.4 4.2 4.0 3.8 3.6 3.4 3.2 F2 (ppm)
Figure 4.3 2D /-resolved NMR spectra of Bond Elut fraction 3 (non-acidic phospholipids) extracted from whole Olive; 41654 scans were obtained for Fourier transformation. The residual methanol peak at 3.31ppm was used as the reference chemical shift
200 C h a p t e r 4 P hospholipid an a ly sis
N Z -o U_
4.2 4. i 4.0 3.9 3 .6 F2 ( ppm
Figure 4.4 2D 7-resolved NMR spectra of Bond Elut fraction 4 (acidic phospholipids) extracted from whole Olive; 41654 scans were obtained for Fourier transformation. The residual methanol peak at 3.31ppm was used as the reference chemical shift
201 C h a p t e r 4 P hospholipid a n a l y sis
4.3.1.2 Analysis of fatty acid composition by NMR
Further insight into the presence of phospholipids can be gained from their fatty acid analysis.
The number of fatty acid chains was determined from the ID spectrum by taking the area of the total CH 3 resonances at 0.87ppm to represent 100% of the fatty acid chains.
The total number of unsaturated fatty acid chains was calculated using the integral area of the CH 2 groups in the sequences -CH 2-CH =C H -(C H 2-C H =C H )-C H 2- at 2,04ppm.
Unsaturated fatty acid (UFA) chains containing only two double bounds were determined from their structure specific proton resonance at 2.74ppm -CH =CH -CH 2-
CH =CH-; [e.g. linoleic acid C (18:2) n-6].
The presence of higher polyunsaturated fatty chains (PUFA) is indicated by the methylene protons within the series of double bonds in the chain [-CH=CH-CH 2-
(CH=CH-CH2-)n] at 2.80ppm.
The diverse PUFAs have diagnostic resonances related to the number and the position of their carbon-carbon double bonds. Arachidonic acid [C ( 20:4) n-5,8,11,14] with the specific structure -CH=CH-CH 2-CH2-CH2-COOH has protons at resonances 1.65ppm,
2.2ppm and 2.4ppm.
The unsaturated fatty acid chains with structure specific R-CH=CH-CH2-CH2-COOH
[e.g. docosahexaenoic C (22:6) n-4,7,10,13,16,19] have isolated methylene group resonances at 2.38ppm.
Other alkyl resonances of importance are at 2.3ppm for the -CH 2-COOH group; and at
1.59ppm for the -CH 2-CH2-COOH group.
The quantitation of monounsaturated fatty acids (MUFA’s) was performed by subtraction of the total PUFA’s from the total UFA’s.
202 C h a p t e r 4 P hospholipid a n a l y sis
The total percentage of imsaturated chains was calculated using the ratio of the peak at
2.04ppm to the integral of all fatty acid chains at 0.87ppm. This confirmed the predominance of unsaturated fatty chains in the olive extract.
An estimation of unsaturation in fatty acid chains is calculated from a ratio of the vinyl proton resonances (5.33ppm) (minus ergosterol resonances) to the methyl proton resonances from fatty acids (0.87ppm).
The majority of free fatty acids in Bond Elut fraction 2 are either saturated or monosaturated which can be determined from the relative areas at 2.0 ppm and 2.7 ppm.
A more detailed analysis of both unsaturated and saturated fatty acid chains was made by GC-MS.
Table 4.2 Assignments of NMR resonances in Fattv Acids
Chemical Shift/ppm Functional Group comment
0.87-0.95 -c m 1.35 -CH=CH-CH2-CH2-CH2-C00H 1.59 -CH 2-CH 2-COOH e.g.arachidonic acid 2.2 -CH=CH-CH2-CH2-CH2-C00H 2.4 -CH=CH-CH2-CH2-CH2-C00H 1.98-2.10 -CH2-CH=CH-CH2-CH=CH-CH2- total UFA 2.3 -CH 2-COOH 2.38 R-CH=CH-CH2-CH2-C00H e.g. docosahexanoic 2.74 -CH=CH-CH2-CH=CH- e.g. linoleic acid 2.8 -C H = C H -C H 2-(C H = C H -C H 2-)n PUFA 5.33 -C H = C H Vinyl proton ______
203 C h a p t e r 4 P hospholipid a n alysis
2. ^ 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 ppm
Figure 4.5 ID proton NMR spectra of Bond Elut fraction 2 (free fatty acids) extracted from Olive; 1028 scans were obtained for Fourier transformation. The residual methanol peak at 3.31ppm was used as the reference chemical shift
204 C h a p t e r 4 P hospholipid a n a l y s is
4.3.2 Analysis of fatty acid composition by GC and GC-MS
The total lipid fraction, together with the component fractions 1,2,3 and 4 produced by separating the total lipid fraction on a Bond Elut column, were initially examined by GC on a Carlo Erba 5300 capillary column gas chromatography as non-saponifred and saponified samples. Free fatty acids from Bond Elut fraction 2 and fatty acids relased from the conjugates in Bond Elut fraction 3 are shown in Tables 4.4 and 4.5, respectively.
The total lipid sample both non-saponifred and saponified, together with the saponifred acidic phospholipid fraction (Bond Elut fraction 4) were further examined by GC-MS on a Hewlett Packard 6890 instrument to identify the unknown compounds present in fraction 4 and their possible detection in the total lipid sample. Results are shown in
Tables 4.6-4.8, respectively.
Table 4.3
GC analysis of the saponified Bond Elut fraction 1 (non polar lipids) obtained from whole Olive; The conditions for the GC analysis are described in method 4.2.6.2.
Fatty acid Rel. %
Palmitic (16:0) 17 Palmitoleic (16:1) n-9 2 Stearic (18:0) 2 Oleic (18:1) n-9 cis 61 Vaccenic (18:1) n-11 trans 4 Linoleic (18:2) n-9,12 14 Linolenic (18:3)n-9,12,15 <0.5 Arachidic (20:0) <1 Behenic (22:0) <0.5
2 0 5 C h a p t e r 4 P hospholipid a n a l y sis
Table 4.4
GC analysis of the non-saponified Bond Elut fraction 2 (non-esterifled fatty acids)obtained from whole Olive; The conditions for the GC analysis are described in method 4.2.6.2.
Fatty acid______Rel. %
Palmitic (16:0) 28 Palmitoleic (16:1) n-9 <1 Stearic (18:0) 4 Oleic (18:1) n-9 cis 54 Vaccenic (18:1) n-11 trans 6 Linoleic (18:2) n-9,12 7
Table 4.5
GC analysis of the saponified Bond Elut fraction 3 (non-acidic phospholipids)obtained from whole Olive; The conditions for the GC analysis are described in method 4.2.6.2.
Fatty acid______Rel. %
Palmitic (16:0) 30 Palmitoleic (16:1) n-9 2 Stearic (18:0) 5 Oleic (18:1) n-9 cis 50 Vaccenic (18:1) n-11 trans 6 Linoleic (18:2) n-9,12 6 Arachidic (20:0) 1
2 0 6 C h a p t e r 4 P hospholipid a n a l y s is
Table 4.6
GC-MS analysis of the non-saponifled total lipids obtained from whole Olive; The conditions for the GC-MS analysis are described in method 4.2.6.3
Instrument: Hewlett Packard HP 6890/5973 GC-MS Column: 10m X 0.10mm ID x 0.2um HP-WAX Carrier gas/flow rate: Helium. 0.5ml/min Injection mode/volume: Manualisplitless, O.Spl Oven temperature: 60(04)-16-240 Ionization mode: Electron Impact (El) Scan analysis: 40-400 amu
Carbon chain Detected as Rel. %
Palmitic acid (16:0) FAME 8.95 Palmitoleic acid (16:1) n-9 FAME 0.7 Linoleic acid (18:2) n-9,12 FAME 0.63 Stearic acid (18:0) FAME 1.17 Oleic acid (18:1) n-9 FAME 27.76 Octadecadienoic acid (18:2) n-10,13 FAME 4.31 Arachidic acid (20:0) FAME 0.98 Behenic acid (22:0) FAME 11.83 Palmitic acid (16:0) FFA 5.44 Stearic acid (18:0) FFA 1.54 Oleic acid (18:1) n-9 FFA 37.01
a) FAME Fatty acid methyl ester b) FADME Fatty acid dimethyl ester c) FAEE Fatty acid ethyl ester d) FF A Free fatty acid
207 C h a p t e r 4 P hospholipid a n a l y sis
Table 4.7
GC-MS analysis of the saponified total lipids obtained from whole Olive; The conditions for the GC-MS analysis are described in method 4.2.6.3
Instrument: Hewlett Packard HP 6890/5973 GC-MS Column: 10m X 0.10mm ID x 0.2um HP-WAX Carrier gas/fiow rate: Helium.0.5ml/min Injection mode/volume: Manual:splitless, 0.5pi Oven temperature: 60(04)-16-240 Ionization mode: Electron Impact (El) Scan analysis: 40-400 amu
Carbon chain Detected as Rel. %
Laurie acid (12:0) FAME 0.01 Myristic acid (14:0) FAME 0.03 Octanedioic acid (8:0) FADME 0.01 Pentadecanoic acid (15:0) FAME 0.01 Nonanedioic acid (9:0) FADME 0.05 Palmitic acid (16:0) FAME 20.88 Palmitoleic acid (16:1) n-9 FAME 2.8 Linoleic acid (18:2) n-9,12 FFA 0.01 Heptadecanoic acid (17:0) FAME 0.06 Oleic acid (18:1) n-9 FAME 60.0 Linoleic acid (18:2) n-9,12 FAME 13.4 Nonadecenoic acid (19:1) n-10 FAME 0.04 Linolenic acid (18:3) n-9,12,15 FAEE 0.59 Octadecadienoic acid (18:2) n-10,13 FAME 0.07 Arachidic acid (20:0) FAME 0.69 Eicosenoic acid (20:1) n-11 FAME 0.63 Eicosadienoic acid (20:2) n-11,14 FAME 0.03 Heneicosanoic acid (21:0) FAME 0.11 Behenic acid (22:0) FAME 0.23 Tricosanoic acid (23:0) FAME 0.12 Penyacosanoic acid (25:0) FAME 0.27 Oleic acid (18:1) n-9 FFA 0.12
a) FAME Fatty acid methyl ester b) FADME Fatty acid dimethyl ester c) FAEE Fatty acid ethyl ester d) FFA Free fatty acid
2 0 8 C h a p t e r 4 P hospholipid a n a l y sis
Table 4.8
GC-MS analysis of the saponified Bond Elut fraction 4 (acidic phospholipids) obtained from whole Olive; The conditions for the GC-MS analysis are described in method 4.2.6.3
Instrument: Hewlett Packard HP 6890/5973 GC-MS Column: 10m X 0.10mm ID x 0.2um HP-WAX Carrier gas/flow rate: Helium.0.5ml/min Injection mode/volume: Manualisplitless, O.Spl Oven temperature: 60(04)-16-240 Ionization mode: Electron Impact (El) Scan analysis: 40-400 amu
Carbon chain Detected as Rel. %
Laurie acid (12:0) FAME 0.08 Myristic acid (14:0) FAME 0.23 Palmitic acid (16:0) FAME 26.42 Palmitoleic acid (16:1) n-9 FAME 1.28 Stearic acid (18:0) FAME 6.75 Oleic acid (18:1) n-9 FAME 57.76 Linoleic acid (18:2) n-9,12 FAEE 6.05 Linoleic acid (18:2) n-9,12 FAME 0.02 Linolenic acid (18:3) n-9,12,15 FAME 0.22 Arachidic acid (20:0) FAME 0.89 Eicosenoic acid (20:1) n-11 FAME 0.41 Behenic acid (22:0) FAME 0.35
a) FAME Fatty acid methyl ester b) FADME Fatty acid dimethyl ester c) FAEE Fatty acid ethyl ester d) FFA Free fatty acid
2 0 9 C h a p t e r 4 P hospholipid a n a l y sis
4.3.3 Determination of phospholipids by ESI-MS electrospray
Electrospray ionisation mass spectrometry can be performed directly on an extract, with no prior chromatographic separation. This technique was used after Bond Elut column separation because of the low phospholipid-sample concentration and high fatty acid chain concentration.
The mass spectrometer operating parameters in the electrospray ionisation mode were optimised to detect either PC or PE. Specific chain length combinations were considered on the basis of the predominant fatty acids identified by GC and GC-MS.
The mass spectrometer was set to monitor the masses of PC and PE substituted with combinations of these fatty acid. The masses monitored are shown in Table 4.9:
Table 4.9 Phospholipid masses monitored by MS
Phospholipid Fatty acid chains MW
PC (16:0)2 733.6
PC (18:1)2 786.6
PC (16:0) 759.6 (18:1) PE (16:0)2 691.5
PE (18:1)2 743.5
PE (16:0) 717.5 (18:1)
210 C h a p t e r 4 P hospholipid a n a l y sis
Bond Elut fraction 3 was examined by positive electrospray ionisation and a mass ion was detected at m/z 760.6, indicative of the presence of a PC substituted with palmitic and oleic acid.
When Bond Elut fraction 4 was examined by negative electrospray ionisation, strong mass ions at m/z 690.5, 742.5 and 716.5 were detected, indicative of the presence of a
PE substituted with two palmitic acids, a PE substituted with two oleic acids and a PE substituted with a palmitic and oleic acid respectively.
211 C h a p t e r 4 P hospholipid a n a l y sis
4.4 Discussion
Separation by solid phase chromatography has been employed as a rapid and effective
method for isolating compounds of interest from complex mixtures, including blood,
urine, environmental samples and pharmaceutical formulations [Jones, 1986]. Bond Elut
ion-exchange chromatography has also been efficiently used to separate lipids into 4
fractions: neutral lipids (fraction 1), non-esterified fatty acids (fraction 2), non-acidic
phospholipids (fraction 3) and acidic phospholipds (fraction 4).
Although NMR analysis has proved to be a quick and comprehensive technique it does
have drawbacks; fatty acid analysis can be incomplete and some low abundance lipids
even in 2D NMR can be difficult to identify and quantify.
The efficiency of the Bond Elut separation was observed by the separation of non-acidic
phospholipids from acidic phospholipids; however their spectra were dominated by
polar steroid resonances. This was emphasised by the absence of choline and
ethanolamine head group resonances in the spectrum of fraction 4; as well as the
absence of acidic phospholipid head groups in fraction 3. This made it possible to
identify only PC and PE. The NMR spectrum of the acidic phospholipids (fraction 4)
was more difficult to analyse due to low concentrations and overlap of signal
resonances. PI was found to be the predominant acidic phospholipid with PG present at
a lower level. CAR and PA were difficult to identify due to overlaps in resonances.
The fatty acid content of the neutral lipid, non-acidic and acidic phospholipid fractions
were further investigated by GC-MS.
212 C h a p t e r 4 P hospholipid a n a l y s is
The total lipid fraction together with Bond Elut fractions 1,2,3 and 4 when initially examined by GC showed the presence of vaccenic acid [C (18:1) n-11 trans], a fatty acid that had not been previously found in olive oil.
The acids found in this study by GC are listed in table 4.10.
Table 4.10 Fattv acids found present in the total lipids from olive fruit bv GC
Fatty acid
Palmitic (16:0) Palmitoleic (16:1) Stearic (18:0) Oleic (18:1) n-9 cis Vaccenic (18:1) n-11 trans Linoleic (18:2) Arachidic (20:0) Behenic (22:0)
The total lipid sample was further examined, saponified and non-saponified by GC-MS.
The résultés are shown in Table 4.11.
213 C h a p t e r 4 P hospholipid a n a l y sis
Table 4.11 Fattv acids present in the total lipids from olive fruit bv GC-MS
Carbon chain Detected as Rel. %
Laurie acid (12:0) FAME 0.01 Myristic acid (14:0) FAME 0.03 Octanedioic acid (8:0) FADME 0.01 Pentadecanoic acid (15:0) FAME 0.01 Nonanedioic acid (9:0) FADME 0.05 Palmitic acid (16:0) FAME 20.88 Palmitoleic acid (16:1) n-9 FAME 2.8 Linoleic acid (18:2) n-9,12 FFA 0.01 Heptadecanoic acid (17:0) FAME 0.06 Oleic acid (18:1) n-9 FAME 60.0 Linoleic acid (18:2) n-9,12 FAME 13.4 Nonadecenoic acid (19:1) n-10 FAME 0.04 Linolenic acid (18:3) n-9,12,15 FAEE 0.59 Octadecadienoic acid (18:2) n-10,13 FAME 0.07 Arachidic acid (20:0) FAME 0.69 Eicosenoic acid (20:1) n-11 FAME 0.63 Eicosedienoic acid (20:2) n-11,14 FAME 0.03 Heneicosanoic acid (21:0) FAME 0.11 Behenic acid (22:0) FAME 0.23 Tricosanoic acid (23:0) FAME 0.12 Penyacosanoic acid (25:0) FAME 0.27 Oleic acid (18:1) n-9 FFA 0.12
Bond Elut separation produced lipid classes of the following categories: neutral lipids, non-esterified fatty acids, non-acidic phospholipids and acidic phospholpids.
The neutral lipids found in Bond Elut firaction 1 were diglicerides, triglecerides and sterodial lipids and reflected the general composition of the olive finit. Saponification of this fi*action gave nine fatty acid methyl esters; palmitic C (16:0); palmitoleic C (16:1)
214 C h a p t e r 4 P hospholipid a n a l y sis n-9; stearic C (18:0); oleic C (18:1) n-9; vaccenic C (18:1) n-11; linoleic C (18:2) n-
9,12; linolenic C (18:3) n-9,12,15; arachidic C (20:0) and behenic C (22:0), acids.
The free fatty acids found in Bond Elut fraction 2 are palmitic C (16:0); palmitoleic C
(16:1) n-9; stearic C (18:0); oleic C (18:1) n-9; vaccenic C (18:1) n-11 and linoleic C
(18:2) n-9,12. Vaccenic acid, as discussed, had not previously been found in olive fruit.
Non-acidic phospholipids found in Bond Elut fraction 3 were phosphatidylcholine (PC) and phosphatidylethanolamine (PE). This fraction on saponification gave the following fatty acids: palmitic C (16:0); palmitoleic C (16:1) n-9; stearic C (18:0); oleic C (18:1) n-9; vaccenic C (18:1) n-11; linoleic C (18:2) n-9,12 and arachidic (20:0); confirming the presence of conjugates containing these moieties.
The acidic phospholipids phosphatidylinositol and phosphatidylglycerol are thought to be present at trace levels in Bond Elut fraction 4 but cannot be quantified.
Saponification of Bond Elut fraction 4 produced the following additional fatty acids, released from conjugation with the acidic phospholipids: lauric C (12:0); myristic C
(14:0); palmitic C (16:0); palmitoleic C (16:1) n-9; stearic C (18:0); oleic (18:1) n-9; linoleic C (18:2) n-9,12: linoleic C (18:3) n-9,12,15; arachidic C (20:0); eicosanoic
(20:1) n-11 and behenic (22:0).
Although species of odd carbon numbers are rare in nature, six fatty acids of odd carbon number can be observed in the saponified total lipid sample probably having been released from conjugate moleciles. All of these fatty acids are saturated except nonadecenoic acid C (19:1) n-10.
In all of the samples vaccenic acid [C (18:1) n-11 trans] has been detected. Trans
isomers are extremely rare in nature and the presence of this compound has not been
215 C h a p t e r 4 P hospholipid a n a l y s is previously reported. It may therefore shed some understanding on the total metabolic process ongoing in the olive fruit.
Also of interest is the presence of two dicarboxylic acids: octanedioic C (8:0) and
nonanedioic C (9:0), in the saponified sample of total lipids. The presence of such
compounds has not been reported in olive fruit and clearly nonanedioic acid could be
the oxidised form of 9-oxo-nonanoic found in the saponified sample of olive skins.
Two phospholipids were found to be present at low concentration by NMR and were
subsequently detected by electro spray ionisation mass spectrometry. PC was identified
with oleic and palmitic acid side chains while three PE side chain combinations were
also identified. These are shown in Table 4.12:
Table 4.12 Phospholipids detected in olives bv ESI-MS
Phospholipid Fatty acid chain MW
PC (16:0) 759.6 (18:1) PE (16:0)2 691.5
PE (18:1)2 743.5
PE (16:0) 717.5 (18:1)
The presence of PC and PE in olives is well established [Williams et al., 1993].
However specific side chain combination have not previously been established. The
216 C h a p t e r 4 P hospholipid a n a l y sis
analysis of PC and PE demonstrates the use of electrospray mass spectrometry in the direct analysis of plant phospholipids.
The spectroscopic methods used in this study, together with developments in
chromatography could be of importance as tools in the study of plant lipid profiles and
therefore, plant diseases and toxicity and the monitoring of plant metabolic changes.
2 1 7 CHAPTER 5
ANTIINFLAMMATORY,ANTIMICROBIAL
and ANTIPROTOZOAL
ACTIVITIES OF OLIVE SKIN EXTRACTS
218 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O live sk in e x t r a c t
5 ANTIINFLAMMATORY, ANTIMICROBIAL and ANTIPROTOZOAL
ACTIVITIES OF OLIVE SKIN EXTRACTS
5.1 Introduction
Plants have been a source of important drugs in clinical use. There are 250,000 known species of higher plants but plant derivatives have only been produced from less than 90 species; therefore the number of potential chemical substances which can be derived from plants with biological activities is large and it is logical to presume that many more useful drugs will be found [Farnsworth, 1990].
Plant extracts have long been employed in folk remedies as anti-inflammatory agents and numerous plant species have been recommended for the therapy of inflammatory disorders. Recent studies show that bioflavonoids are the active components in a large number of aqueous (usually ethanol-water mixtures) plant extracts that are used to combat inflammation [Middleton & Kandaswami, 1992]. A study has been made of the aqueous and lipid extracts of camomile in their action against inflammation in the mouse [Loggia et al, 1986]. In these experiments, the aqueous extract (containing polyphenols) was twice as active compared to the lipid extract (containing the essential oils).
The distribution of bioflavonoids varies in different parts of the plant. Bioflavonoids in fruits and berries are for the main part, equally distributed throughout the skin and flesh, which explains the uniform colour seen in plums, cherries and raspberries. In contrast, other plants concentrate their flavonoids mainly in the skin, and to a lesser extent in the outer layer of the fruit flesh [Teng, 1989]. In apples, for example, the flavonoids are
2 1 9 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
almost exclusively found in the skin. All bioflavonoids are polyphenols, but not all polyphenols are bioflavonoids. Flavonoids are therefore a very large subgroup with
around 4000 identified to date. This group of molecules has generated much interest recently because of their efficacy and novelty with regard to disease prevention.
Table 5.1: Biochemical effects of bioflavonoids
1. Antioxidant (radical scavenger and metal chelator) 2. Inhibition of calcium-dependent ATPase 3. Inhibition of lipoxygenase 4. Inhibition of cyclic AMP phosphodiesterase 5. Inhibition of protein kinase C 6. Inhibition of aldose reductase
Recognition of the therapeutic applications of bioflavoids proceeds at a slow pace;
however, the pharmaceutical industry has synthesised, patented and frequently marketed
flavonoids for the treatment of human diseases. Allergy, asthma, and inflammation are
the best documented medical applications, although a growing body of data suggests
that flavonoids have some beneficial effect in a number of other conditions, including
cancer, impaired immune responses, viral and microbial infections, cardiovascular
diseases, diabetic cataracts, and liver disorders such as cirrhosis.
Olive fhiits contain a variety of simple and complex phenolic compounds which differ
in variety depending on the location. Several of these compounds have been isolated
220 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t from olive fruit and identified in recent years. Although the effects of chemical structure on the anti-oxidant activity of phenolic compounds has been discussed [Le Tutour &
Guedom, 1992; Dziedzic & Hudson, 1984] little has been done regarding individual chemical effects on pathogenic organisms and micro-organisms. Ruiz-barba et al., 1990 studied the effect of the total phenolics from green olive brine on the survival of
Lactobacillus plantarum. More recently Tuncel & Nergiz, 1993 investigated the effects of some phenolic compounds against Staphylococcus aureus. Bacillus cereus and
Escherichia coli. However their work did not include any species of Pseudomonas,
Salmonela, Klebsiella and the yeasts Saccharomyces and Candida. This present study is not exclusively with phenolic compounds but on the total extracts from olive skins, however in some of the extracts the major components are phenolics.
Compounds that have been extracted from olives and shown to inhibit or delay the rate of growth of a range of bacteria and microfiingi, include oleuropein and its hydrolysis products p-3,4-dihydroxyphenylethyl alcohol, elenolic acid and oleuropein aglycon.
The antimicrobial activity of many naturally occurring compounds raises considerable
interest in their use as additives in food to prevent or delay the growth of pathogens or to delay the onset of spoilage. In this use phenolic antioxidants are thought to have a preservative action.
Higher plants have yielded antiprotozoal agents with a wide variety of structures
including alkaloids, quinones, terpenes, flavonoids, aurones, xanthones and coumarins
and in addition these plant derived compounds have been used as templates for the
development of synthetic and semi-synthetic drugs [Phillipson & Wright, 1991].
Diseases caused by protozoan parasites cause considerable mortality throughout the
world. Malaria which is caused by Plasmodium spp. is the most prevalent of all these
221 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t diseases, afflicting 300-500 million people with a mortality of up to 2 million annually.
Trypanosoma brucei spp. is responsible for African trypanosomiasis in humans
(sleeping sickness) while Trypanosoma cruzi causes South American trypanosomiasis
(Chagas’ disease) Leishmania spp. is the etiological agent of leishmaniasis.
Worldwide estimations are that some 2 to 3 million people are infected by Leishmania parasites; about 18 million people in South America suffer from Chagas’ disease with
some 45,000 deaths per year, and around 250,000 cases of sleeping sickness occur each year.
There are other protozoan parasites like Entamoeba histolytica and Giardia intestinalis,
causing amoebiasis and giardiasis, responsible for 42 million and 200 million infections per year, respectively. Opportunistic protozoal parasites such as Cryptosporidium parvum. Toxoplasma gondii, Leishmania infantum and Microsporidia, are responsible
for a considerable number of deaths in immunocompromised patients (Croft, 1997).
The chemotherapy of protozoal diseases has been undermined in the last few years
because of the widespread development of resistance in the protozoal parasites to the
present therapies. Plasmodium falciparum {P. falciparum) has become resistant to
antimalarial drugs such as Quinine, Chloroquine, Mefloquine and
Sulfadoxinepyrimethamine. The drugs of choice for treating leishmaniasis, Pentostam
and Glucantime, have variable efficacy and side effects. Nitroheterocyclic drugs such as
nifurtimox and benzidazole used for the treatment of Chagas’ diseases have variable
efficacy against the early acute stage, but are not effective against the chronic phase of
the disease and are toxic. The only drug available to treat all forms of the late stage in
human sleeping sickness is the arsenic containing drug, Melarsoprol. This drug can
cause reactive encephalopathy and it is difficult to administer. In the 1980’s the
222 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t polyamine eflomithine was introduced for the treatment of West African sleeping
sickness caused by Trypanosoma brucei gambiense, but it is ineffective against
Trypanosoma brucei rhodesiense infections. The drugs Suramin and Pentamidine are
only useful in the early stage blood infections of sleeping sickness.
There is therefore a need for new drugs in the treatment of protozoal diseases.
In vitro screening procedures are used to provide an initial assessment of plant extracts
or pure compound activity. These tests are rapid, sensitive, inexpensive and require only
a small quantity of test material. Once in vitro activity has been found, the active plant
extract or a pure compound can be further tested in vivo as a secondary stage to the
screening procedure.
In this study Olive skin extracts have been screened in vitro, for antimicrobial,
antiprotozoal and cytotoxic activity and in vivo testing has been done for
antiinflamatory activity.
5.2 ANTIINFLAMATORY ACTIVITY
5.2.1 Materials and methods
Black Greek olives {Olea europaea L.) were hand picked from the Greek island of Kim.
TP A (tetradecanoyl phorbol acetate) was purchase from Sigma, UK. Test solutions of
each olive skin extract were prepared at concentrations of 100, 20 and 10 pg/ml in
acetone or water. TP A was used at a dose concentration of 0.1 pg per ear.
Female GDI mice weighting about 20 g, were used in groups of five to include three
223 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t test animals plus a negative and positive control.
5.2.2 Inhibition of inflammation
The olive skin extracts were applied using a Drummond microcap affixed to the outer surface of the right ear of each mouse. A dose of 5 pi of each extract concentration was used. The left ear of each mouse was used as the negative control to evaluate the degree of erythema. TP A alone was used as a positive control to show the maximum erythema.
TP A was applied fifteen minutes after each olive skin extract had been applied; the mice were then examined after 2, 3, 4, 6, 7 and 24 hours to estimate the inhibition of inflammation. TP A alone produces maximum redness at six hours after application.
Results were expressed as a percentage of inhibition in the number of animals affected.
5.2.3 Assessment of irritancy of Olive skin extracts alone
Applications of the extracts were made as above, without the subsequent application of
TP A as an inflammation inducer, and repeated daily to the same ear at the same dose.
Effects were monitored at the same time intervals but with the addition of monitoring daily for 2 weeks to see if the olive extracts alone exhibited proinflammatory effects.
5.2.4 Results
The four olive extracts were tested for antiinflammatory activity using the mouse erythema ear test. The percentage of reduction in the inflammation produced by each
224 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t extract at 100 lag/ear/mouse is shown in Table 5.2 and Figure 5.5, after the mice were treated with the Tridecanoyl phorbol acetate (TPA) as the irritant agent (0.1 pg per ear).
Table 5.2:
Sample Oh 2h 3h 4h 6h 7h 24 h
Hexane Mean 0 55 66 66 100 100 100 extract SEM 0 11 0 19 0 0 0
Chloroform Mean 0 0 0 0 66 22 33 extract SEM 0 0 0 0 19 11 0
Methanol Mean 0 44 55 0 100 100 100 extract SEM 0 11 11 0 0 0 0
Water Mean 0 11 0 66 100 100 100 extract SEM 0 11 0 19 0 0 0
TPA Mean 0 66 66 77 100 100 100 SEM 0 19 19 11 0 0 0
The results indicated that the chloroform extract of the Olive skin was the most active of the four samples tested. This extract completely reduced inflammation produced by the
TP A (77%) in the first 4 hours after the extract was applied. The same sample reduced
inflammation from 100% to 66% at 6 hours, and after 24 hours, reduction was to 33%.
The water extract demonstrated most activity in the first 3 hours and completely reduced the inflammation produced by the TP A (66%).
No redness was observed in any animals, for any olive extract, at any time during the
course of the experiment.
2 2 5 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
5.3 ANTIMICROBIAL ACTIVITY
5.3.1 Introduction
The four olive skin extracts were tested for antimicrobial activity against four Gram- negative bacteria {Escherichia coli KL 16, Pseudomonas aeruginosa NCTC 6750,
Salmonella typhimurium and Klebsiella aerogenes), two Gram-positive bacteria
{Staphylococcus aureus NCTC 6571 and Bacillus subtilis NCTC 8236 ) and two yeasts
{Saccharomyces cereviciae and Candida albicans).
5.3.2 Material and methods
Antibacterial activity was estimated by the agar diffusion technique. E. coli, P. aeruginosa, S. typhimurium, K. aerogenes, S. aureus and B. subtilis were grown overnight in nutrient broth (Oxoid No. 2, code CM67) at 37°C, and tested in nutrient agar (nutrient broth solidified with 1.5% Lab M agar) with incubation under aerobic conditions. Saccharomyces cereviciae and Candida albicans were grown in Sabouraud dextrose broth for 2 days at room temperature, and tested in Sabouraud dextrose agar
(Oxoid, code CM41). For all organisms, 100 ml molten sterile agar was cooled to 55°C and inoculated with 1 ml of a culture of the test organism; 20 ml quantities of inoculated media were then poured into 9 cm plastic Petri dishes and allowed to set. Wells of 6 mm diameter were bored in to the agar, using a sterile cork borer, and filled with a solution of the compound under test. Solutions of plant extracts were tested at 10, 30 and 100 pg/ml. The positive control. Chloramphenicol (Sigma, UK), was tested at 10, 1 and 0.1 p-g/ml. Solutions of both extracts and the control antibacterial were made in sterile
2 2 6 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t ethanol, methanol, DMSO (dimethylsulphoxide) and water. These solvents, when tested alone, had no significant inhibitory activity under the conditions used. After incubation, the diameters of the zones of inhibition were measured in three directions, and the average used to give a semi-quantitative indication of the antimicrobial activity.
5.3.3 Results
The four olive extracts were examined for antimicrobial and antifimgal activity. Six tests microorganisma were used to determine the antimicrobial activity; these were:
Escherichia coli. Pseudomonas aeruginosa. Salmonella typhimurium, Klebsiella aerogenes. Staphylococcus aerus and Bacillus subtilis. In addition two further microorganisms were used to evaluate any antifimgal activity; these were:
Saccharomyces cereviciae and Candida albicans. The results in Table 5.3 showed that only the hexane extract of olive skins had a weak activity at 100 pg/ml against Candida albicans, whereas the other extracts were devoid of any antimicrobial activity at the concentrations tested. It has been reported that olive oil has antimicrobial activity (Kubo et a l, 1995) and it is possible that olive skin may possess antimicrobial activity against other microorganisms.
227 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O live sk in e x t r a c t
Table 5.3
Diameters of zones of inhibition tm m l produced bv test compounds
Sample Cone. E. coli P. aeruginosa K. aerogenes S. typhimurium B. subtilis S. aerus C albicaiis S. cereviciae jug/ml
Olive skin 10 Hexane 30 - Extract 100 - - 1 0
Olive skin 10
Chloroform 30 - - - - Extract 100
Olive skin 10 Methanol 30 - - Extract 100
Olive skin 10
Water 30 - - Extract 100
0.1
Chloramphenicol 1 11 - - - 10 10 17-14 15 -
5.4 ANTIPROTOZOAL ACTIVITIES
5.4.1 Materials and methods
5.4.1.1 Preparation of samples for biological testing
The chloroform and hexane extracts were first dissolved in DMSO and diluted with the appropriate culture medium prior to testing. The final concentration of DMSO never exceeded 0.1% in the test medium, a concentration which has no significant effect on
2 2 8 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t the growth of the cells. The methanol and aqueous extracts were diluted directly with culture medium.
5.4.1.2 Antiprotozoal activity
5.4.1.2.1 Parasites
The Chloroquine-resistant (Kl) and Chloroquine-sensitive (3D7) strains of P. falciparum were maintained in human M erythrocytes suspended in RPMI 1640 supplemented with
D-glucose, Naz CO 3, gentomycin and Albumax H, at 37 in a 5% CÜ 2-air mixture.
Trypanosoma brucei rhodesiense blood stream form (bsf) trypomastigotes were
maintained in HMI-18 medium supplemented with 0.1 % glucose, 1 % non essential
aminoacids, and 15 % of HIFCS at 37 in a 5% COz-air mixture. Trypanosoma brucei
brucei (S427) bsf trypomastigotes were maintained in HMI-18 medium with 15% of
HIFCS (heat inactivated fetal calf serum) at 37 in a 5% COz-air mixture. L. donovani
(MHOMJET/67/L82;LV9) promastigotes were maintained in RPMI 1640 with 10%
heat-inactivated foetal calf serum (HIFCS) at 26 ^C. L. donovani amastigotes were
maintained in male golden hamsters. From a freshly killed golden hamster, infected for
approximately 6-8 weeks with L. donovani the spleen was isolated and homogenised
with a sterile Potter crusher containing RPMI 1640 medium. The suspension was spun
and the pellets were washed three times with medium and resuspended in RPMI 1640 +
10% HIFCS.
2 2 9 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
5.4.1.2.2 Plasmodium falciparum test
In a 96-well microtitre plate three-fold serial dilutions of the samples were performed to obtain a series of 4 concentrations in triplicate starting from 300 pg/ml. To each well containing 50 pi of diluted sample was added 50 pi of P. /a/czpan/m-infected human erythrocytes (A^) suspended in RPMI 1640 supplemented with D-glucose, NazCOs gentomycin and Albumax H, and this was incubated at 37 in a 5% CO 2 air mixture.
After 24 h, 5 pi of ^H-hypoxanthine (40 pCi/ml) was added to each well and the incubation continued for a further 18-24 h. The plates were frozen, washed three times, with double distilled water using a semi automated cell harvester, and dried in an oven for 2-3 h. 50 pi of Ecoscint scintillation fluid was added into each well, and the radioactivity was measured by a scintillation counter (Beckman, LS 5000CE, USA).
Two series of controls were performed: (a) parasitised blood without sample, and (b) uninfected red blood cells without sample. Chloroquine diphosphate was used as the standard. The inhibitory concentration (IC 50 ) value which is the concentration of sample necessary to cause 50% inhibition of parasite or cell growth was calculated by linear regression analysis using an x/fit, MS Excel statistical program.
5.4.1.2.3 Trypanosoma brucei rhodesiense and Trypanosoma brucei brucei test
In a flat-bottomed 96 well microtitre plate each sample was diluted by three fold dilution in triplicate. Each well containing 100 pi of diluted sample was inoculated with
2 xlO"^ of r. b. rhodesiense or T. b. brucei bsf trypomastigotes suspended in 100 pi of
HMI-18 medium supplemented with 15 % of HIFCS and incubated at 37 in a 5%
2 3 0 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
CO2 air mixture for 72 h. Two series of controls were performed: (a) parasites in cultured medium without sample and (b) parasites in cultured medium with 0.1 % of
DMSO. Pentamidine was used as the antitrypanosomal standard. The minimum inhibitory concentration (MIC) value of each sample is the concentration of sample necessary to inhibit 100 % of parasite or cell growth. This was determined by observing each well under a microscope and comparing it with untreated controls.
5 .4 .1 .2 .4 Leishmania donovani promastigotes test
Each sample was diluted by three-fold serial dilutions in triplicate in a 96-well microtitre plate. To each well containing 100 pi of diluted sample was added 1x10^ promastigotes in 100 JAl of RPMI 1640 supplemented with 10 % HIFCS and this was then incubated at 26 °C for 72 h. Two series of controls were performed, (a) parasites in cultured medium without sample, (b) parasites in cultured medium with 0.1% of
DMSO. Pentamidine was used as the antileishmanial standard. The MIC value of each sample was determined by observing each well under a microscope and comparing it with untreated controls.
5 .4 .1 .2 .5 Leishmania donovani am astigote test
Macrophages obtained from the peritoneum of a freshly killed CD 1 male mouse were suspended in RPMI 1640 supplemented with 10% HIFCS and counted on a haemocytometer. 500 pi of a suspension of macrophages (5 x 10 were added into each glass-bottomed chamber of an eight chambered slide (Lab-Tek Products, Miles
231 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
Lab.) and incubated overnight at 37 in a 5% CO 2 -air mixture. The supernatant in each chamber was removed the following day, and the macrophages were infected with amastigotes in a ratio of 1:10. Incubation took place overnight at 37 in a 5% CÜ 2-air mixture. The following day the supernatant was removed and the infected macrophages were exposed to 4 concentrations in quadruplicate of each sample starting from 30 pg/ml. Incubation took place for 48 h at 37 in a 5 % C02-air mixture. The medium with the drug was replaced with freshly diluted sample and incubated for a further 72 h.
The supernatant was then removed and the slides fixed with methanol and stained with
10% Giemsa. The percentage of inhibition of amastigotes in each concentration was determined relative to the untreated control. Sodium stibogluconate was used as the standard. The IC 50 value for each sample was calculated by linear regression analysis
(using x/fit, MS Excel statistical program).
5.4.1.3. Cytotoxic activity
5.4.1.3.1 KB cytotoxicity
The KB cells (human epidermoid carcinoma from the nasopharynx) were cultured in
DMEM supplemented with 10 % HIFCS at 37 in a 5% C02-air mixture. The cells were removed from the flasks and made into a suspension. The number of cells per ml was determined on a haemocytometer under a microscope. For each sample ten fold serial dilutions in triplicate were performed in a 96-well microtitre plate. To each well containing 50 p,l of diluted sample was added 50 pi of cell (1 x 10 ^) suspension, and the plates were then incubated at 37 in a 5% CO 2 air mixture. After 72 h, 65 pl/ml of
232 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t trichloroacetic acid (25%) at 4 was added to each well and the plate refrigerated at 4
for 1 hour. The plates were rinsed (x5) with distilled water and left to dry at room temperature. Aqueous eosin B stain 1% (100 pi) was added to each well and left to
stand at room temperature for 1 h. The plates were washed (x5) with 1% acetic acid, and
allowed to dry at room temperature. Sodium hydroxide solution (200 pi, 5 mM) was
added to each well, and the optical density (O.D.) determined at 490 nm on a
microplate reader (Dynex, USA). Podophyllotoxin was used as standard. The ICso value
for each sample was determined by linear regression analysis (using x/fit, MS Excel
statistical program).
5.4.2 Results
The four olive extracts were examined for antiprotozoal activity using six antiparasite
tests together with a test for cytotoxic activity. The antiparasite tests were Plasmodium falciparum , Trypanosoma brucei rhodesiense!Trypanosoma brucei brucei , Leishmania
donovani promastigotes and Leishmania donovani amastigote . The results for these
tests are shown in Tables 5.4 - 5.6.
The cytotoxic test was performed using KB cells with the results of this test shown in
Table 5.7.
233 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
Table 5.4: Antiprotozoal activities of Olive skin extracts on P. falciparum 3D7 and Kl
Sample P. falciparum 3D7 P. falciparum K l Olive skins ICso (pg/ml - SEM) ICso (pg/ml - SEM) Hexane Extract >300 >300 Chloroform Extract 12.47-0.17 11.22-0.09 Methanol Extract 72.10-5.03 31.98-1.2 Water Extract 209.93 -41.91 >300 Chloroquine 0.0026 - 0.0001 0.27 - 0.004
Table 5.5: Antiprotozoal activity of Olive skin extracts on T.b. rhodesiense and T.b. brucei
Sample T. b. rhodesiense T.b. brucei Olive skins MIC MIC Hexane Extract 300 300 Chloroform Extract 33.33 33.33 Methanol Extract >300 >300 Water Extract >300 >300 Pentamidine 0.003 0.001
234 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O live sk in e x t r a c t
Table 5.6: Antiprotozoal activities of Olive skin extracts on L. donovani
Sample L.donovani L.donovani Olive skins MIC ICso (pg/ml - SEM) Hexane Extract >300 >300 Chloroform Extract >300 >300 Methanol Extract >300 >300 Water Extract 0.03 <1.0 Pentamidine 10 NA Pentostam NA 11,16 - 1.66
Table 5.7: Cytotoxic activities of Olive skin extracts on KB cells
Sample KB cells Olive skins ICso (|ag/ml - SEM) Hexane Extract >300 Chloroform Extract 186-3.1 Methanol Extract >300 Water Extract >300 Pentamidine 0.068 - 0.002
2 3 5 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
Tables 5.8-5.11 show the concentration dependence of the percentage inhibition for each active extract together with the plot of log concentration versus percentage inhibition.
Table 5.8
In vitro activitv of Olive skin chloroform extract on P. falciparum K l
Cone, jig/ml Log conc. Chloroquine % of inhibition
0.1 -1 0 0 0.3 -0.522 41.2 0 1 0 78.47 0 3 0.477 86.45 0 10 1 91.86 49.28 30 1.477 100 95.78 100 2 100 98 09 300 2.477 100 98.95 IC 50 pg/ml 0.27 11.22
Figure 5.1:
In vitro activitv of Olive skin chloroform extract on P. falciparum K l
100 90 - 80 - 70 - 60 ■ -Chloroquine 50 - OL-CHCI3 40 - 30 ■
20 -
10 -
1 - 0.5 0 0.5 1 1.5 2 2.5
Log Concentration
236 Chapter 5 Anti inflammatory, antimicrobial, antiprotozoal activities of Olive skin extract
Table 5.9
In vitro activitv of Olive skin chloroform extract on P. falciparum 3D7
Cone. |o.g/ml Log conc. Chloroquine % of inhibition
0.003 -2.522 0 0 0.01 -2 95 0 0.03 -1.522 97 0 0.1 -1 99 0 0.3 -0.522 100 0 1 0 100 0 3 0.477 100 0 10 1 100 41 30 1.477 100 94 100 2 100 98 300 2.477 100 100 1C 50 ^ig/ml 0.0026 12.47
Figure 5.2:
In vitro activitv of Olive skin chloroform extract on P. falciparum 3D7
100 90 - 80 - TO GO ■ ■ -Chloroquine 50 - OL-CHCI3 40 - 30 -
20 ■
0 6- -2.5 2 -1.5 ■1 -0.5 0 1 1.50.5 2 2.47
Log Concentration
237 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal ,^a c t iv it ie s o f O l iv e s k in e x t r a c t
Table 5.10: In vitro activitv of Olive skin water extract on Leishmania donovani
Conc. |ag/ml Log conc. Pentostam % o f inhibition
1 0 2 58.25 3 0.477 26.63 78.5 10 1 44.49 87.5 30 1.477 75.12 99.5
IC 50 fig/ml 11.16-1.66 < 1.0
Figure 5.3
In vitro activitv of Olive skin water extract on Leishmania donovani
100
80
■ Pentostam 60 ■ 0L4H20 40
0 0.47 1 1.47
Log Concentration
2 3 8 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
Table 5.11 In vitro activitv of Olive skin chloroform extract on KB cells
Gone. |ig/ml Log conc. Podophyllotoxin % o f inhibition
0.01 -2 0 0 0.1 -1 75 0 1 0 79 0 3 0.477 80 0
30 1.477 82 4.4 300 2.477 93 68.9
IC 50 |Tg/ml 0.068 - 0.002 186.78
Figure 5.4:
In vilro activitv of Olive skin chloroform extract on KB cells
100
70 ■Podophyllotoxin 50 OL-CHCI3 40
20
2 1 0 0.5 1.5 2.5
Log Concentration
2 3 9 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O live sk in e x t r a c t
5.5 Discussion
The hexane, chloroform, methanol and water extracts from Olive skins were examined for antiinflammatory, antimicrobial and antiprotozoal activities.
The chloroform extract after 7 hours (78 % inhibition) was the most active extract in the antiinflammatory screen. Activity was detected in the water extract at 3 hours, when the total inhibition of the inflammation caused by TPA (66%) was recorded. Much smaller amounts of antiinflammatory activity were found in the other two extracts.
Figure 5.5 shows the results of the antiinflammatory test at 2, 3, 4, 6, 7 and 24 hours, for the chloroform and water extracts.
The two constituents isolated from the chloroform extract of the olive skin, P-oleanolic acid and p-maslinic acid have been shown to have antiinflammatory activity (Singh et al., 1992; Gupta, 1969; del Carmen Recio, 1995; Martinez-Vazquez et al., 1997;
Delporte et al., 1997; Shimizu et al, 1986). It is therefore possible that the activity observed in the chloroform extract is related to the presence of both of these triterpenes.
240 C h a p t e r 5 A ntiinfuvmmatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
Figure 5.5. Percentage of inhibition of inflammation (erythema) produced bv Olive skin extracts
a) Chloroform extract
1 0 0 -r
90
80
70
60
50 □ TPA □ OE-CHCI3 40
30
20
10
OH : i' 2(hr) 3(hr) 4(hr) 6(hr) 7(hr) 24(hr)
Time (hr) b) Water extract
100-/'
□ TPA H0E-H20
2(hr) 3(hr) 4(hr) 6(hr) 7(hr) 24(hr)
Time (hr)
241 C h a p t e r 5 A ntiinflammatory , antimicrobial , antiprotozoal a c t iv it ie s o f O l iv e s k in e x t r a c t
The antimicrobial screen involved six bacteria and tv^o fungi, only the hexane extract demonstrated activity and only against the fungi Saccharomyces serviciae. The results are shown in Table 4.3.
The antiprotozoal screens involved testing against the following protozoan parasites;
Plasmodium falciparum 3D7, Plasmodium falciparum K l, Trypanosoma brucei rhodesiense. Trypanosoma brucei brucei and Leishmania donovani. The chloroform extract was the most active against Plasmodium falciparum 3D7 and Plasm odium falciparum K l. The methanol extract also showed activity against both parasites but at a lower level. The results are shown in Table 5.4.
The water extract demonstrated considerable activity against Leishmania donovani showing a 10 fold increase in activity (ICso < 1.0 pg/ml) over the standard compound
(Pentostam, ICso 1.16 -1.66 p,g/ml). The other extracts demonstrated no activity in this test. The plot log concentration versus % inhibition for this fraction is reproduced in
Figure 5.3.
Figure 5.3:
100 90 - 80 - 70 - ■ Pentostam
50 - - ■0L-H20 40 - - 30- -
20 - -
10 - -
0 0.47 1 1.47
Log Concentration
242 CHAPTER 6
CONCLUSION
243 C h a p t e r 6 C o n c l u s io n
6 CONCLUSION
Previous studies have concentrated on whole olive and provided information on the chemical composition. From these studies it was shown that olives are a rich source of different chemical entities which could be important as food additives or in medical products. In this study we have endeavoured to search for specific chemical types, in particular phospholipids and to reveal the complexity of the glycerol conjugates by examining the lipids derived from these molecules.
This analysis of the olive fruit has confirmed the existence of compounds previously reported and has found other compounds of a similar type to be present. We have also examined (in this study) both olive pulp and olive skin in separate studies with a view to establishing which compound types are contained in each. A dedicated study of the phospholipid content was also made. In the course of this study compounds not previously detected have been identified in both pulp and skin (and compounds previously found have not been detected). In each study the method of extraction and the subsequent chromatography would have dictated which compounds were recovered.
In the analysis of the fhiit pulp, the following compounds were detected (Figure 6.1), which shows predominantly phenolic compounds of the iridoid type; while Table 2.16 shows both saturated and unsaturated fatty acids reflecting the complex lipid conjugations that can be present.
244 C h a p t e r 6 C o n c l u s io n
Ho,^rX ooc ÏÏ COOMe
OGlc
5’deoxy-oleuropein (MW: 524.19)
OH
3' r OOC COOMe
10
OGlc
Oleuropein (MW: 540.52)
HOOC COOMe
OGlc
Oleoside-l 1 -methyl ester (MW: 404.37)
245 C h a p t e r 6 C o n c l u s io n
CH2CH2OH
* ^ 0 H OGlc
2-(3’-hydroxy 4’-glucopyranosylphenyl)ethanol (MW: 316.31)
CH2CH2OH
CH 3
2-(4’-methylphenyl)ethanol (MW: 136.09)
CH, OGlc CH2CH3 CH
1,4 Dimethyl-3-ethyl-2-naptholglycoside (MW: 362,17)
Figure 6.1 Compounds identified in the pulp of Olive finit
2 4 6 C h a p t e r 6 C o n c l u s io n
Olive skins have not previously been analysed in detail. This study shows that compound types similar to these found in pulp are found in skin (Figure 6.2). However the compounds listed in Table 6.1 and Figure 6.2 are exclusive to Olive skin.
3-(glucopyranosyl)-cyanidol (MW: 449.39)
CH.OH
HO
12-01eanene-3,28-diol (MW: 442.72)
247 C h a p t e r 6 C o n c l u s io n
4,14-Dimethyl-9,19-cycloergost-23-en-3-ol (MW: 426.72)
.0. OMe
H™)— O-C-CHs
0 ■ ° 4' CHsO OMe
4’-methyl-8-acetyl 4-(glucopyranosyl)-pinoresinol (MW: 592.59)
Figure 6.2 Compounds identified in the skin of Olive fmit
248 C h a p t e r 6 C o n c l u s io n
Table 6.1 Fatty acids detected in Olive skins only
Carbon chain
Hexadecenoic acid (16:1) n-7 Lignoceric acid (24:0) Hexacosanoic acid (26:0) Octacosanoic acid (28:0) 9 - 0 X 0-nonanoic acid (9:0)
The presence of C (26:0) and C (28:0) in the saponified sample of the hexane extract from olive skins indicates the presence of these chains in the non-saponified conjugate molecules. This is the fist reported fiunding of such substituent chains in olives. 9-oxo- nonanoic acid is also a novel finding in olive skin and indicates again the possible presence of such a substituent chain in the non-saponified conjugate. A list, with relative percentages, of the total fatty acids found in olive skin and pulp is given in Table 6.2.
249 C h a p t e r 6 C o n c l u s io n
Table 6.2
Fatty acids found in Olive fruit
Carbon chain Detected as Rel. %
Laurie acid (12:0) FAME 0.01 Myristic acid (14:0) FAME 0.03 Octanedioic acid (8:0) FADME 0.01 Pentadecanoic acid (15:0) FAME 0.01 9-oxo-nonanoic acid (9:0) FAME 0.23 Nonanedioic acid (9d% FADME 0.05 Palmitic acid (16:0) FAME 20.21 Palmitoleic acid (16:1) n-9 FAME 2.71 Hexadecanoic acid (16:1) n-7 FAME 0.23 Linoleic acid (18:2) n-9,12 FFA 0.01 Heptadecanoic acid (17:0) FAME 0.06 Oleic acid (18:1) n-9 FAME 58.0 Octadecenoic acid (18:1) n-8 FAME 0.29 Linoleic acid (18:2) n-9,12 FAME 12.9 Nonadecenoic acid (19:1) n-10 FAME 0.04 Octadecatrienoic acid (18:3) n-9,12,15 FAME 0.80 Octadecatrienoic acid (18:3)n-6,9,12 FAME 0.15 Linolenic acid (18:3) n-9,12,15 FAEE 0.59 Octadecadienoic acid (18:2) n-10,13 FAME 0.07 Arachidic acid (20:0) FAME 0.69 Eicosenoic acid (20:1) n-11 FAME 0.63 Eicosedienoiccid (20:2) n-11,14 FAME 0.03 Heneicosanoic acid (21:0) FAME 0.11 Behenic acid (22:0) FAME 0.23 Tricosanoic acid (23:0) FAME 0.12 Penyacosanoic acid (25:0) FAME 0.27 Oleic acid (18:1) n-9 FFA 0.12 Lignoceric acid (24:0) FAME 0.92 Hexacosanoic acid (26:0) FAME 1.45 Octacosanoic acid (28:0) FAME 0.28
2 5 0 C h a p t e r 6 C o n c l u s io n
The presence of PC and PE phospholipids in olive finit has been reported. In this study we have reported specific side chain combinations in these phospholipids. These are
shown respectively for PC and PE in Table 6.3.
Table 6.3 Phospholipids detected in olives bv ESI-MS
Phospholipid Fatty acid chain MW
PC (16:0) 759.6 (18:1) PE (16:0)2 691.5
PE (18:1)2 743.5
PE (16:0) 717.5 (18T)
Acidic phospholipids are also thought to be present but at a much smaller level. Further
development of pre-concentration techniques would enable such compounds to be
detected and quantified. Cardiolipin, PS, PA PI and PG would benefit from such a
development and further studies by ESI-MS could identify possible side chain
combinations in these phospholipids. These side chain options are shown in Table 6.4 which lists the fatty acids found in the GC-MS analysis of the saponified acidic and non-acidic phospholipids obtained from olive finit.
251 C h a p t e r 6 C o n c l u s io n
Table 6.4 Fatty acids from phoshpolipids obtained from Olive fruit
Carbon chain Detected as Rel. %
Laurie acid (12:0) FAME 0.08 Myristic acid (14:0) FAME 0.22 Palmitic acid (16:0) FAME 24.8 Palmitoleic acid (16:1) n-9 FAME 1.20 Stearic acid (18T0 FAME 6.34 Oleic acid (18:1) n-9 FAME 54.2 Vaccenic acid (18:1) n-11 trans FAME 5.65 Linoleic acid (18:2) n-9,12 FAEE 5.68 Linoleic acid (18:2)n-9,12 FAME 0.02 Linolenic acid (18:3) n-9,12,15 FAME 0.21 Arachidic acid (20:0) FAME 0.83 Eicosenoic acid (20:1) n-11 FAME 038 Behenic acid (22:0) FAME 033
ID and 2D NMR conclusions were consistent with the extraction of principally triglicerides and polar sterols with very low concentrations of phospholipids. This indicates that olives would probably not be a very useful “biotech” source of phospholipids, but could be used for sterol or related compounds.
In this study on olive skins parallel activity testing was conducted on the solvent extracts for antiinflammatory, antimicrobial and antiprotozoal activities.
The chloroform and water extracts showed activities in the antiinfalmmatory test with the chloroform extract showing most activity (see Figure 5.5).
252 C h a p t e r 6 C o n c l u s io n
The antimicrobial screen indicated that the hexane extract was active only against the
fungi Saccharomyces serviciae (Table 5.3). No other fraction was active against the six bacteria and two fungi used in this study.
The antiprotozoal screen showed that the chloroform extract was the most active against
Plasmodium falciparum K1 and Plasmodium falciparum 3D7. The methanol extract
showed a lower activity in this screen (see Table 5.4). The water extract demonstrated a
ten fold increase in activity over the standard compound when tested against
Leichmania donovani ( see Table 5.10 and Figure 5.3).
No testing has yet been performed on the HPLC fractions or on the compounds
identified. The isolation of the compounds from the methanol and water extractions may
not have been complete due to the complexity of the mixture extracted and its stability.
The water extract may require a more detailed study particularly on the isolation and
separation of this complex mixtures as indicated by the NMR and MS analysis carried
out in this study. This would form a natural progression from the work described in this
thesis.
2 5 3 APPENDIX 1
254 APPENDIX 1
APPENDIX 1
A.1 MEMBRANE LIPIDS
A.1.1 Plant membrane: history and functional aspects of plant membranes
Biological membranes play a crucial role in almost all cellular organisms.
The major role of membrane lipids was understood in 1925 and was further defined in
1935. This work was done on red blood cells and lipids were seen as a bilayer matrix surrounding the blood cells. In 1972 Singer and Nicholson [Singer & Nicolson, 1972] refined a version of the fluid mosaic model, which envisaged a basic structure consisting of a lipid bilayer in which proteins may be embedded as well as attached to the outer surface. Membrane lipids have amphipatic characteristics, so they can naturally form bilayers in aqueos media with their acyl chains shielded from the aqueous enviroment. The presence of polar or hydrophilic head groups enables the appropriate interactions with extrinsic proteins, ions and other molecules. The chemical nature of these groups can be varied.
Membrane lipids meet the demands of a membrane structure which are fluidity and permeability, and can provide a matrix with which membrane proteins are associated.
Today we recognize that a membrane has three major classes of components which are proteins, lipids and oligosaccharides.
For a complete understanding of biological membrane systems it is necessary to know the nature and influence of lipid-protein interactions. There are two classes of interactions. The first, concerns proteins with hydrophobic segments which have penetrated into the lipid bilayer and span the membrane. They are called intrinsic or
255 APPENDIX 1 integral proteins, and they constitute the major fraction of membrane proteins. It is assumed that the integral proteins are critical to the structural integrity of membranes.
Protein can also be embedded in only one half of the bilayer. This is the second class of membrane proteins, and concerns water soluble proteins which interact electrostatically with negatively charged groups at the lipid interface. These are known as extrinsic or peripheral proteins. They are held to the membrane only by week noncovalent interactions and are not strongly associated with membrane lipids. These proteins have effects on membrane lipid fluidity [Wiley et a l, 1982].
A “fluid” membrane is needed for the functioning of membrane proteins and not only permits movement of substrates and products of enzymes but permits the lateral and transverse movement of proteins (used in transport, electron transport and light- harvesting). Several kinds of experiments indicate [Singer& Nicolson, 1972], that protein-lipid interactions play a direct role in a variety of membrane functions. Many membrane-bound enzymes and antigens require specific phospholipids, for expression of their activities. The nature of the fatty acids incorporated into phospholpids affects the function of certain membrane-bond proteins in bacterial membranes [Schairer &
Overath, 1969].
Lipids as the other half of a biological membrane also showed sidedness, where the distribution of lipids in the outside and inside of a plant membrane are not the same.
There is a considerable movement in the hydrocarbon chain as well as considerable rotation of individual carbons in the acyl chains together with the transverse movement of a whole lipid. This rapid movement allows lipids, like plastoquinone to function
efficiently (as an electron carriers) in photosynthesis. In contrast to this sideways movements, lipids are constrained from crossing from one side of the bilayer to the
256 APPENDIX 1 other. This process known as a flip-flop, is not possible because of the hydrophilic nature of the head groups which cannot cross the hydrophobic membrane interior. This membrane lipid sidedness has important functional significance.
The ability of membranes to provide a bilayer permeability between external and internal environments is one of its most important functions. The nature and selectivity of this barrier to various molecules and ions of biological interest allows cells and their organelles to maintain unique environments in which metabolic and related processes can take place. Specific transport mechanisms are needed to allow substances such as water, uncharged water-soluble nonelectrolytes and ionised solutes, to move across membranes as required.
Membranes provide a site for enzymatic and related activities of proteins dealing with hydrophobic molecules present in the membrane.
Membranes may be used for packaging materials and in secretory processes and can contain receptors which serve to mediate the influence of external signals.
257 r o y O O
( /
'Sure 4 ./ H^">b % ft APPENDIX 1
A. 1.2 Membrane lipids : occurrences and distribution of lipids
A fluid mosaic model of a membrane is presented in Figure A.l showing the organization and structure of the proteins and lipids in biological membranes. Where proteins are embedded in a matrix of phospholipids, the phospholipids are organized as a discontinuous fluid bilayer.
Biological membranes contain a wide variety of lipids. To clarify the functional roles of individual lipids is a difficult problem.
Membrane lipids exclude lipids which are poorly soluble in bilayer membrane systems, like fats (triglycerides) and cholesterol esters. Plants contain the same types of phosphoglycerides as animals but in different proportions. The lipid composition of membranes varies widely in different cells or organelles or in the same membrane systems of different species. Different sides or monolayers of the same membrane can contain different lipid species [Harwood, 1989]. Argument favouring lipid asymmetry comes from the fact that the chemical environment of both layers is, in most cases, completely different and may consequently induce or maintain a specific localization of the various phospholpids. Most membranes contain a large variety of lipids differing not only in their apolar parts, but also in size, charge and chemical reactivity of their head groups.
In eucaryotic membranes the glycerol-based phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and cardiolipin predominate. Phosphatidylglycerol, is a minor component in animals, but is a major lipid in photosynthetic membranes. Phosphotidylcholine and phosphotidylethanolamine are the main lipids in the extracloroplastid membranes. In the plasma membranes.
259 APPENDIX 1 phosphatidylinositol and its phosphorylated derivatives, function in signal transmission.
Phosphatidylserine is a minor lipid in all membranes, while cardiolipin is located in the inner mitochondrial membrane. Sphingosine-based lipids, including sphingolipids and glycosphingolipids are another major group. The glycolipids with carbohydrate containing glycerol based lipids have major roles as cell surface associated antigens.
There are a large number of minor lipids to be included. Cholesterol is a major lipid in mammalian membrane systems but there is a significant difference in the cholesterol contents of plants, where the endoplasmatic reticulum or the inner mitochondria membrane contains little or no cholesterol. This distribution correlates with sphyngomyelin distribution.
260 A P P E N D IX 2
261 APPENDIX 2
APPENDIX 2
ANALYTICAL TECHNIQUES
A.2.1 Thin-layer chromatography
Thin-layer chromatography (TLC) has long been used for the separation of sample mixtures into their constituent components classes. Analyses are performed rapidly and samples can be analysed simultaneously alongside standards. The separated components can be visualised and the chromatograms kept as a permanent record, or, alternatively, samples can be recovered for further analysis. The mixture is resolved into its components by differential migration, as a stream of solvent passes through the layer of adsorbent by capillary action. In a given solvent system each component has a characteristic mobility which can be described as its Rf value. The Rf value is defined as the distance travelled by the component divided by the distance travelled by the solvent front, both distances being measured from the origin. Since lipids and phenolics are generally colourless, the separated components have to be rendered visible by chemical reagents. For quantitative analysis, the proportion of the individual components is then determined by reference to controlled amounts of standards.
Silica gel is the adsorbent most frequently used for the analysis of lipids and phenolics by TLC. The particle size of the silica gel used for standard TLC is in the range of 10-15 pm with the particles manufactured to contain pores of a definite diameter, 40, 60, 80,
100 or 150 A. Silica gel 60 is the most commonly used adsorbent in the TLC of lipids and phenolics. The use of pre-coated TLC plates rather than self-prepared plates can be better for several reasons. The analyses performed with pre-coated plates can be more
262 APPENDIX 2 reproducible than with self-prepared plates, because batch variations are much less due to carefully controlled manufacturing processes. The pre-coated layers are also of superior optical quality when quantitative densitrometry is to be applied; and finally, the layers of pre-coated plates are more resistant to abrasion.
The maximum amount of compound that can be applied as a spot or a full-width streak to a TLC plate depends on the nature of the components as well as the thickness of the adsorbent layer.
A.2.2 High-performance TLC
The silica gel adsorbent used for high-performance TLC (HPTLC) is only available on pre-coated plates and consists of silica gel 60 with a particle size of only 4.5-5.0 pm and an adsorbent layer thickness of 0.15-0.2 mm. By using this technique, less sample is needed, the time of analysis is shorter, less solvent is used, and the resolution of the separated components is superior, which makes this method very useful with a separation efficiency close to that of HPLC.
A.2.3 High-performance liquid chromatography
High-performance liquid chromatography (HPLC) is, now the primary technique used in the analysis of different compound types. However, in the area of lipid analysis its use is still relatively uncommon, and other techniques are more often used. There are several different types of HPLC separation; adsorption, partition (both normal-phase and reversed-phase), ion-exchange, ion-pairing, and exclusion chromatography The differences in functional group composition within molecules must be exploited to choose the right chromatographic system. Modem liquid chromatography or HPLC is
263 APPENDIX 2 based on the development of the chromatographic separation in several areas. HPLC now provides a highly efficient separation process with instrumention where the conditions can be controlled and are reproducible.
The actual choice of the mobile phase will be determined by the nature of the sample and the type of chromatography being employed. An important property of the. mobile phase is its UV cut-off. Most lipids absorb weakly in the UV range 200 to 210nm, but so do many of the solvents used in lipid analysis, especially chloroform. Even solvents, which are transparent in this, range, like methanol, water and acetonitrile, often show considerable absorption, due to traces of impurities. Isocratic elution can be used for simpler separations, but for the analysis of more complex mixtures, gradient elution has to be used.
The designs of a successful HPLC separation depends on matching the right mobile phase to a given column and sample. Many different properties of the solvent must be considered, including solvent strength and selectivity. Two forms of partition chromatography can be recognised according to the relative polarities of the mobile and stationary phases: normal-phase partition chromatography where the stationary phase is more polar than the mobile phase, and reversed-phase chromatography where the stationary phase is less polar than the mobile phase. Most of the earlier work on HPLC used silica columns, but now more often bonded-type phases are used. Silica C l8 is the most commonly used stationary phase of this type. The name, reversed-phase chromatography, was introduced with the appearance of chemically bonded non-polar stationary phases using polar, aqueous mobile phases. The popularity of the technique lies in its versatility. The separation can be designed more easily and takes place according to the lipophilicity of the sample components. A major requirement for HPLC
264 APPENDIX 2 is a sensitive detector for monitoring the column effluent. A good detector should satisfy the following major requirements: a) To be equally sensitive to all molecular types; b) Not to be affected by changes in mobile phase composition or temperature; c) To be able to monitor small amounts of compounds (in trace analysis);
The most widely used HPLC detectors are refractometers, UV spectrometers, fluorescence spectrometers, electrochemical detectors, radioactivity detectors, and mass spectrometers.
The most commonly used detection method in HPLC is the variable wavelength UV spectrometer. Modem detectors have small volume flow cells with relatively long light paths to give a high sensitivity and can be used at variable wavelengths to be able to monitor the UV maximum of the compound of interest. Chemical derivatization to form compounds, which do absorb in the UV region, is a way of extending the use of this detector to lipid molecules.
A.2.4 Gas chromatography
Gas chromatography (GC) is used extensively in the analysis of all major lipid classes.
It is used to separate mixtures of compounds which exhibit appreciable vapour pressures
(volatilities). The GC analysis of lipids involves: a) Sample Injection
Introducing a small volume (1 pi) of a solution of microgram to nanogram per microlitre concentration dissolved in a suitable organic solvent. Injections can be in split
265 APPENDIX 2 or splitless mode. Split injection is used where concentrated sample solutions are involved, to avoid overloading the capillary GC column. Splitiless injection is best suited to the analysis of dilute solutions. In common to both techniques, samples are introduced using syringes equipped with robust stainless steel needles, via a rubber septum inlet, into a heated zone. In split injections the sample solution is rapidly volatilised by the high temperature of the injector and the vapour delivered into the GC column by the high flow rate of the carrier gas. By contrast in splitless injections the sample solution is volatilised and the vapour delivered, by a lower carrier gas flow, on to the GC column, which is held at a relatively low temperature. This ensures that the solvent and sample components condense as a narrow plug at the head of the column before volatilising of the solvent and dissolved compounds into the inert carrier gas, flowing continuously through the GC. The split injection technique is adequate for most lipid analyses but splitless injections when used are less reliable for quantitative analysis. b) Separation
Temperature programming is commonly used in the GC analysis of lipids mixtures. In temperature programming the analysis starts at a low GC oven temperature, which allows elution of the more volatile substances. The oven temperature is then increased gradually, usually at a constant rate. GC run times are typically 20 to 60 min., depending upon the complexity of the mixture under investigation and the nature of the separation required. c) Detection
The flame ionisation detector (FID) is the most commonly used technique for the detection of lipids. Compounds eluting from GC columns are combusted in a hydrogen
266 APPENDIX 2 air flame to yield ions, which produce an increase in the potential between the FID jet tip and the collector electrode. The resulting signal is amplified and recorded as the chromatogram. The FID detector has a fast response, which is linear over a wide range, and is very reliable in long-term operation. Mass spectrometry (MS) is very widely used as a detector in the GC analysis of lipids. The advantage of combined GC-MS is that mass spectra can be recorded continuously during the GC analysis, with the result that, in many instances, complete structural identification can be made on eluting components, without recourse to other physico-chemical techniques.
GC columns can be either packed or capillary with flexible-fused silica capillary columns now available commercially. Selection of an appropriate stationary phase is essential if the desired separation is to be achieved but the high efficiencies of capillary columns means that they will generally be successful in resolving compounds that are otherwise difficult to separate.
Gas chromatography (GC) is used extensively in the analysis of the major lipid classes found in biological materials. Fatty acids are the class of lipid most commonly analysed by GC with carbon numbers ranging from C 4 to Cso, but the most commonly occurring are those of chain length from Cm to C 22. Gas chromatography (GC) analysis of fatty acids is most commonly performed after they have been converted to their, methyl esters derivatives [Metcalfe & Schmitz, 1961].
2 6 7 APPENDIX 2
A.2.5 Nuclear magnetic resonances spectroscopy
By continuosly spinning nuclei with angular momentum, certain isotopes which exhibit a paramagnetic spin property (non-even spin number) can give rise to an associated magnetic field. If one of such nuclei is subjected to a very powerful external magnetic field, being perturbed with a pulse of radio-ffequency energy, the nuclei will resonate between different quantised energy levels at specific frequencies, absorbing some of the applied energy. The change of energy of the system as it relaxes back towards equilibrium after the radio-frequency pulse, although very small, can be detected, amplified and displayed in a spectrum format. The trace obtained, of the variations in the intesity of the resonance signal with increasing applied magnetic field, is the nuclear magnetic resonances (NMR) [Sander and Hunter, 1990].
Depending on the locations, resonance signals from the same nuclear species can be varied within one molecule, due to the slight differentiation of chemical-magnetic environments (shielding) that each chemical group is subjected to. In other words, NMR resonance signals are dependent on the structure of the molecule. Such a phenomena is defined as the chemical shift. In Fourier transform (FT) NMR experiments, all nucleii are excited simultaneously by the application of an intensive energy pulse. In FT NMR following the perturbation (radio-frequency) pulse, the subsequent time domain relaxation response, namely the free induction decay (FID), is converted into digital form and stored as data poins in a computer for analysis. In order to display the spectrum in a frequency manner, the time domain FID is converted to the frequency domain spectrum via a Fourier transformation. Like all other spectroscopic techniques the resolution of an NMR spectrum is by the signal-to-noise ratio. If the signal-to-noise ratio is low, it can be improved by the accumulation of FIDs. The final signal-to-noise
268 APPENDIX 2 ratio is proportional to the square root of the number of FIDs which are co-added. There are limitations to the dynamic range of signals which can be successfully avareged.
One of the most exciting advances in recent NMR technology is the introduction of two- dimensional (and multi-dimensional) experiments [Freeman & Morris, 1979]. A 2D spectrum is a map containing two frequency axes. One axis F2, contains conventional chemical shifts. The other axis FI, may contain shift or coupling information, or both.
The position of a signal on the map reveals both the chemical shift of that signal, and another piece of information which depends on the nature of FI. The magnetization that is experimentally detected by collecting FIDs appears along F2, while FI reports on the behavior of the spins during the evolution period, labelled as t. A picture of the evolution period is indirectly built by acquiring a series of spectra with increasing evolution times. All 2D experiments have a ID equivalent using a single value for the evolution time. The effect of many different functional groups has been determined by the examination of a large number of molecular types.
The most widely used NMR techniques are ’H and in ID experiments followed by
2D COSY and 2D J-resolve applications. ’H spectra contain less information than spectra, but do provide better quantitative data.
A.2.6 Mass spectrometry
Mass spectrometry (MS) is used in the elucidation of compound structure and detection and quantification at trace levels. The mass spectrometer can produce molecular ions and fragments from these ions. First, molecules are subjected to bombardment by a stream of high-energy electrons, converting some of the molecules into ions. These ions are accelerated in an electric field and then separated according to their mass-to-charge
269 APPENDIX 2 ratio in a magnetic or electric field. Finally, the ions with a particular mass-to-charge ratio are detected by a device which is able to count the number of ions which strike it.
The detector’s output is amplified and fed to a recorder. The trace from the recorder is a mass soectrum, a graph of the number of ions as a fimction of the mass-to-charge ratio.
Fast atom bombartment (FAB) is the most commonly used mass specrometric technique for producing gas phase ions from thermally-unstable biological substances, such as polar lipids. FAB mass spectrometry uses a beam of high-energy atoms, commonly xenon, produced by accelerating xenon ions through an electric field, to bombard a solution of the analyte dissolved in a relatively involatile liquid matrix (e.g. glycerol).
Electron ionization (El) and chemical ionization (Cl) are the two most widely used ionization techniques in the mass spectrometric analysis of lipids. El is produced by accelerating electrons from a hot filament through a potential differences, usually of 70 eV. Organic molecules will be ionized and fragmented, in the presence of this beam.
The initial product of El in the ion source is a radical cation , resulting from the removal of an electron from the analyte molecule. The is frequently weak or absent from spectra, so chemical ionization (Cl) is used to produce a molecular ion adduct, so providing molecular weight information. The lack of fragment ions in Cl spectra results from the low energy transfer involved in the Cl process compared to EL [M+H] ions are energetically more stable than the radical ions produced by EL
The coupling of chromatographic techniques, such as GC and HPLC, to mass spectrometers allows separation of the individual molecular spicies with on-line analysis of the eluting components. GC and MS are especially complementary because both are gas phase techniques, displaying optimum performance with sample sizes in the nanogram range. Flexible fused silica capillary columns are now preferred for GC-MS
270 APPENDIX 2 work since they can be fed directly from the GC oven into the ion source of the mass
spectrometer. The temperature of the transfer line between the GC and MS must be maintaned at, or slightly above, the maximum temperature used in the GC analysis, in order to avoid sample components condensing in this region.
Liquid chromatography (LC) with MS is useful for the analysis of mixtures of compounds that are intractable for analysis by GC-MS without prior chemical or enzymatic degradation, e.g. phospholipids. Combining HPLC with MS is much more difficult than with GC. The fundamental problem is the enormous volume of solvent vapour that is generated from the evaporation of the liquid eluent in the MS. Different types of MS interface are now available commercially.
Atmosferic pressure ionisation (API) has proved to be invaluable in meeting sensitivity requirements in quantitative methods. It also can provide further structural information together with the molecular weight. Electrospray Mass Spectrometry (ES-MS) is regarded as a soft ionisation technique providing a rapid and accurate means of
analysisng a wide range of polar molecules. Electrospary ionization applied to smaller molecules up to 1000 Daltons in molecular mass, results in either a protonated, [M+H] ^ or deprotonated, [M-H] ', molecular ion depending on whether positive or negative ionisation had been selected. The optimisation of this selection would depend on the type of molecule being analysed. Some fragmentation can be induced to provide structural information.
271 APPENDIX 2
Sample cone HV lens ATMOSPHERIC PRESSURE Skimmer lens
Capillary Skimmer
SECOND VACUUM REGION
Heated nebuliser Corona pin FIRST VACUUM REGION
Figure A.2.1 Schematic of the APCI source showing
the principle components and pressure regions
Atmosferic Pressure Chemical ionization, APCI is a very soft ionisation technique and has many similarities to electrospary ionisation. Ionisation takes place at atmospheric pressure and the ions are extracted into the mass detector in the same way as in electrospray. Similarly, [M+H]^ and [M-H] ' ions are usually formed which give molecular weight information. Fragmentation can be induced in the source by increasing the cone voltage to give structural information.
2 7 2 APPENDIX 3
273 APPENDIX 3
APPENDIX 3
Table A.3.1:
BIOLOGICAL ACTIVITIES REPORTED FOR MASLINIC ACID
ACTIVITY REFERENCE
Inhibitory activity against HIV-protease X\xetal, 1996
Effect on glucose transport activity on Ehrlich Murakami et al, 1993 tumour cells Cytotoxic activity Numataeia/., 1989
Inhibition of skin-tumour promotion on Epstein-Barr Konoshima et al, 1987 virus activation
Table A.3.2
BIOLOGICAL ACTIVITITES REPORTED FOR OLEANOLIC ACID
ACTIVITY REFERENCE
Anti-complementary activity Kapil & Sharma, 1994; Knaus & Wagner, 1996
Anti-viral Serra et al, 1994
Differentiation-inducing activity on mouse myeloid Umehara et al, 1992 leukaemia cell line (Ml).
Hypoglycemic activity Liu et al., 1994; Yoshikawa et al, 1996
Adryamycin-induced histamine release Balanehru & Nagaraian, 1994
Anti-inflammatory activity Singh et al, 1992; Gupta, 1969; del Carmen Recio, 1995; Martinez-Vazquez et al, 1997; Delporte et al., 1997
Inhibition of skin cancer Tokuda et al, 1986; Ishida et al, 1998
Testosterone-5 c(-reductase )inhibitor Ohyo Seikagaku Kenkyusho, 1998pat.
274 APPENDIX 3
Protective effect against chemical-induced necrotic lÀMetal., 1993; 1995a,b liver injury in mice
Induction of differentiation in the culture Fg Lee et al, 1994 teratocarcinoma stem cells
Antitumor activity Ryu et al., 1994; Hsu et al., 1996; Ohigashi et al., 1986; Delporate et al., 1997
Inhibition of Streptococcus pyogenes Oosawa & Yasuda, 1997; 1998
Effect on CC14-induced liver injury in mice Ye, et al., 1996
Antifungal activity Anisov et al., 1979
Antidiabetic activity Yamahara e? a/., 1981
Deodorant and antiperspirant effects Jean & Cariel, 1998
Ulcer prevention Snyckers & Fourie, 1998
Hypolipidemic properties Voskanyan e? a/., 1983
Protection against lipid peroxidation Balanehru & Nagarajan, 1994
Cytotoxic activities Montagnac et al., 1997
Hair growth stimulants Tsuji et al., 1998
Inhibitor of glycerol phosphate dehydrogenase Wada et al., 1998
Phytotoxicity Hoagland et al., 1996
Feeding-deterrence effect Shukla et al., 1997
Antihemolitic activity Osawa et al., 1997
Antimicrobial activity Verma et al., 1997
275 REFERENCES
2 7 6 Re f e r e n c e s
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