CHEMICAL CONSTITUENTS AND BIOACTIVITY STUDIES OF Bauhinia rufescens Lam. (FABACEAE) AND Ficus platyphylla Del. (MORACEAE)
AMINU MUHAMMAD
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Doctor of Philosophy (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
JUNE 2014
iii
To my beloved Father Late Alhaji Muhammad Sarki and my beloved mother Hajiya
Hauwa Muhammad Sarki
iv
ACKNOWLEDGEMENT
Praise belongs to almighty Allah, the lord of all being. May his peace be upon his
messenger Muhammad (PBUH). My appreciation goes to my Supervisor Prof. Dr.
Hasnah Mohd Sirat for her guidance, patience, motivation, support, and advices
during my research.
A special thanks to Assoc. Prof. Dr. Farediah Ahmad for her kindness and her
precious help. I would like to thank Mr. Rasydi and Mr. Azmi for their assistance in
obtaining the NMR spectra at Faculty of Science, UTM.
My appreciation to all academic and technical staffs of Chemistry Department, UTM,
as well as my fellow postgraduate colleagues; Mr. Salam, Mr. Nuzul, Mr. Shamsul,
Mr. Nazifi, Mrs. Athira, Mrs. Shariha, Syafiqah, Ernie, Iman, Edeline, for their
advice and encouragement. Special thanks to my parents, family, siblings and my
friends for their constant support and encouragement.
Last but not the least, I would also acknowledge BUK-MacArthur Grant, Bayero
University Kano, Nigeria for my doctoral fellowship. International Doctoral
fellowship, UTM for partial financial support, and to the Faculty of Science
Universiti Teknologi Malaysia for facilities. Thank you.
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ABSTRACT
The chemical constituents and bioactivities of the stem barks and leaves of
Bauhinia rufescens and the stem barks of Ficus platyphylla have been studied. Extraction of the plant materials using Soxhlet method with petroleum ether, ethyl acetate, and methanol afforded nine crude extracts. Fractionation and purification of the ethyl acetate and methanol extracts of stem bark of B. rufescens yielded two new compounds identified as 4-(2′-hydroxyphenethyl)-5-methoxy-2-methylphenol and bauhiniside. Compounds identified as 6-methoxy-7-methyl-8-hydroxydibenz [b,f]oxepin, α-amyrin acetate, β-sitosterol 3-O-β-D-xylopyranoside, menisdaurin, sequoyitol, 6-hydroxy-7,7a-dihydrobenzofuran-2(6H)-one, glyceryl trilinoleate, linoleic acid, stigmasterol and β-sitosterol were also isolated from the stem barks. The leaves extract yielded glyceryl trilinoleate, sequoyitol and bauhiniside. F. platyphylla yielded two new phenolic compounds from its methanolic extract which was identified as ficuside A and ficuside B. In addition, hordenine was isolated via alkaloid extraction, and also 3,4-dihydroxybenzoic acid anhydride, epicatechin, lupeol, lupeol acetate and α-amyrin acetate were identified from F. platyphylla. Bioactivities including antioxidant, antimicrobial, inhibitory studies on the brine shrimp larvae, tyrosinase, acetylcholinesterase and cyclooxygenase-2 were investigated. Among the nine crude extracts from the two plants, the methanol extract from the stem barks of B. rufescens showed a strong DPPH radical scavenging activity (14.31 µg/mL), and similar extract from F. platyphylla showed higher total phenolic content (719.58 µg GAE/mg), while the methanol extract from the leaves of B. rufescens showed the highest antioxidant value based on β-carotene bleaching assay (78.75%). The antimicrobial activity revealed the methanol extract from F. platyphylla was active against Gram-positive bacteria, Staphylococcus aureus (113 µg/mL) and a fungus, Aspergillus niger (56 µg/mL). Meanwhile, toxicity study indicated that, ethyl acetate from the leaves of B. rufescens was toxic against brine shrimp larvae (LC50 0.06 mg/mL). The methanol extract from F. platyphylla was found active against tyrosinase enzyme (49.2%). Evaluation of acetylcholinesterase inhibition using the microplate and bioautographic assays showed the methanol extract from F. platyphylla inhibited the enzyme activity with percent inhibition of 44.7% (LD 30.0 µg). In addition, among the isolated compounds, oxepin from the stem barks of B. rufescens was found to be active against Staphylococcus aureus (113 µg/mL) and lupeol acetate from F. platyphylla was found active against Candida glabrata (56 µg/mL). It was also found that, hordenine inhibited acetylcholinesterase activity at 58.6% (LD 33.0 µg). Lupeol and 3,4-dihydroxybenzoic acid anhydride from F. platyphylla showed the highest inhibitory activity against tyrosinase (67.7%) and COX-2 (IC50 0.15 µM) enzymes, respectively.
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ABSTRAK
Komponen kimia dan bioaktiviti terhadap kulit batang dan daun Bauhinia rufescens serta kulit batang Ficus platyphylla telah dikaji. Pengekstrakan tumbuhan menggunakan Soxhlet dengan petroleum eter, etil asetat dan metanol telah menghasilkan sembilan ekstrak mentah. Pengasingan dan penulenan ekstrak mentah etil asetat dan metanol daripada kulit batang B. rufescens berjaya menghasilkan dua sebatian baru yang dikenalpasti sebagai 4-(2′-hidroksifenetil)-5-metoksi-2-metilfenol dan bauhinisida. Sebatian dikenalpasti sebagai 6-metoksi-7-metil-8-hidroksidibenz[b,f]oksepin, α-amirin asetat, β-sitosterol 3-O-β-D-xilopiranosida, menisdaurin, sekuoyitol, 6-hidroksi-7,7a-dihidrobenzofuran-2(6H)-on, gliseril trilinoleat, asid linoleik, stigmasterol dan β-sitosterol turut diasingkan daripada kulit batang. Ekstrak daun menghasilkan gliseril trilinoleat, sekuoyitol dan bauhinisida. F. platyphylla menghasilkan dua sebatian fenolik baru daripada ekstrak metanol dan dikenalpasti sebagai fikusida A dan fikusida B. Di samping itu, hordenin diasingkan melalui pengekstrakan alkaloid dan 3,4-dihidroksibenzoik asid anhidrida, epikatekin, lupeol, lupeol asetat dan α-amyrin asetat juga dikenalpasti daripada F. platyphylla. Bioaktiviti termasuk antioksidan, antimikrob, kajian perencatan ke atas larva udang air masin, tirosinase, asetilkolinesterase dan siklooksigenase-2 turut dikaji. Antara sembilan ekstrak mentah daripada dua tumbuhan, ekstrak metanol daripada kulit batang B. rufescens menunjukkan aktiviti perencatan radikal yang tinggi (14.31 µg/mL) dan ekstrak yang sama daripada F. platyphylla menunjukkan kandungan fenolik keseluruhan yang lebih tinggi (719.58 µg GAE/mg). Ekstrak metanol daripada daun B. rufescens menunjukkan nilai antioksidan tertinggi berdasarkan aktiviti pelunturan β-karotena (78.75%). Aktiviti antimikrob menunjukkan ekstrak metanol daripada F. platyphylla adalah aktif terhadap bakteria Gram-positif, Staphylococcus aureus (113 µg/mL) dan kulat Aspergillus niger (56 µg/mL). Sementara itu, kajian ketoksikan menunjukkan etil asetat daripada daun B. rufescens adalah toksik terhadap larva udang air masin (LC50 0.06 mg/mL). Ekstrak metanol daripada F. platyphylla didapati aktif terhadap enzim tirosinase (49.2%). Penilaian perencatan asetilkolinesterase menggunakan plat mikro dan bioautografi menunjukkan ekstrak metanol daripada F. platyphylla merencat aktiviti enzim dengan peratus perencatan 44.7% (LD 30.0 µg). Selain itu, antara sebatian yang diasingkan, oksepin daripada kulit batang B. rufescens didapati aktif terhadap Staphylococcus aureus (113 µg/mL) dan lupeol asetat daripada F. platyphylla didapati aktif terhadap Candida glabrata (56 µg/mL). Hordenin turut didapati menrencat aktiviti asetilkolinesterase sebanyak 58.6% (LD 33.0 µg). Lupeol dan 3,4-dihidroksibenzoik asid anhidrida daripada F. platyphylla menunjukkan penrencatan aktiviti tertinggi masing-masing terhadap enzim tirosinase (67.7%) and COX-2 (IC50 0.15 µM).
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TABLE OF CONTENTS CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xiii
LIST OF SCHEMES xv
LIST OF FIGURES xvi
LIST OF ABBREVIATIONS xviii
LIST OF APPENDICES xxi
1 INTRODUCTION 1
1.0 Introduction 1
1.1 Background of Study 1
1.2 Medicinal Plants 3
1.3 Statement of the Problem 4
1.4 Objectives of Research 4
1.5 Significance of the Research 4
1.6 Scope of Work 5
2 LITERATURE REVIEWS 6
2.1 Fabaceae Family 6
2.2 Bauhinia Species 8
2.3 Moraceae Family 9
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2.4 Ficus species 10
2.5 Chemical Components of Bauhinia and Ficus
species 11
2.5.1 Terpenoids 11
2.5.2 Alkaloids 16
2.5.3 Flavonoids 17
2.5.4 Phenolics 20
2.5.5 Stilbenoids 22
2.5.6 Coumarins 23
2.5.7 Cyclitols 24
2.5.8 Fatty Acids 25
2.6 Bioactivities of Bauhinia and Ficus species 26
2.6.1 Antimicrobial Activity 27
2.6.2 Antiinflammatory Activity 28
2.6.3 Antidiabetic Activity 29
2.6.4 Cytotoxic Activity 30
2.6.5 Bioactive Compounds from Bauhinia and
Ficus species 31
3 CHEMICAL CONSTITUENTS OF Bauhinia
rufescens Lam 34
3.1 Extraction of Leaves and Stem Bark of Bauhinia
rufescens Lam. 34
3.2 Chemical Constituents of Bauhinia rufescens
Lam. 35
3.2.1 4-(2′-Hydroxyphenethyl)-5-methoxy-2-
methylphenol (141) 35
3.2.2 6-Methoxy-7-methyl-8-hydroxydibenz[b,f]
oxepin (138) 44
3.2.3 Bauhiniside (142) 47
3.2.3.1 Biosynthetic Pathway: Bauhiniside
(142) 58
3.2.4 Menisdaurin (99) 59
3.2.5 Menisdaurilide (143) 62
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3.2.6 Sequoyitol (94) 63
3.2.7 α-Amyrin Acetate (139) 64
3.2.8 β-Sitosterol 3-O-β-D-xylopyranoside (7) 65
3.2.9 Mixture of β-Sitosterol (10) and
Stigmasterol (14) 67
3.2.10 Triglyceride: Glyceryl trilinoleate (140) 68
3.2.11 Linoleic Acid (104) 69
4 CHEMICAL CONSTITUENTS OF Ficus
platyphylla Del 71
4.1 Extraction of Stem Bark of Ficus platyphylla
Del. 71
4.2 Chemical Constituents of Ficus platyphylla Del. 71
4.2.1 3,4-Dihydroxybenzoic acid anhydride
(150) 72
4.2.1.1 Biosynthetic Pathway of 3,4-
Dihydroxybenzoic acid anhydride
(150)
73
4.2.2 Ficuside A (158) 74
4.2.3 Ficuside B (159) 85
4.2.3.1 Biosynthetic Pathway: Ficuside A
(158) and Ficuside B (159) 94
4.2.4 Epicatechin (62) 95
4.2.5 Hordenine (164) 97
4.2.6 Lupeol (16) 98
4.2.7 Lupeol Acetate (163) 100
4.2.8 α-Amyrin Acetate (139) 102
5 BIOACTIVITY STUDIES OF Bauhinia rufescens
Lam. AND Ficus platyphylla Del. 103
5.1 Bioactivity Studies 103
5.2 Antioxidant Activity 103
5.2.1 DPPH Radical Scavenging Activity 104
5.2.2 β-Carotene–Linoleic acid Bleaching 107
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Activity
5.2.3 Total Phenolic Content 108
5.3 Brine Shrimp Lethality Test 110
5.4 Antimicrobial Activity 111
5.4.1 Disc Diffusion Method 112
5.4.2 Microdilution Method 116
5.5 Tyrosinase Inhibitory Activity 121
5.6 Acetylcholinesterase (AChE) Inhibitory Activity 123
5.6.1 TLC Bioautographic Assay for
Acetylcholinesterase 123
5.6.2 Microplate Assay for acetylcholinesterase 125
5.7 Cyclooxygenase-2 (COX-2) Inhibitory Activity 128
6 EXPERIMENTAL 130
6.1 General Experimental Procedures 130
6.2 Plant Materials 131
6.3 Isolation of Chemical Constituents of Bauhinia
rufescens Lam. 131
6.3.1 Isolation of 4-(2′-Hydroxyphenethyl)-5-
methoxy-2-methylphenol (141) 133
6.3.2 Isolation of 6-Methoxy-7-methyl-8-
hydroxydibenz[b,f] oxepin (138) 134
6.3.3 Isolation of Bauhiniside (142) 134
6.3.4 Isolation of Menisdaurin (99) 135
6.3.5 Isolation of Menisdaurilide (143) 135
6.3.6 Isolation of Sequoyitol (94) 136
6.3.7 Isolation of α-Amyrin Acetate (139) 136
6.3.8 Isolation of β-Sitosterol 3-O-β-D-
xylopyranoside (7) 137
6.3.9 Isolation of Mixture of β-Sitosterol (10)
and Stigmasterol (14) 137
6.3.10 Isolation of Triglyceride: Glyceryl
trilinoleate (140) 138
6.3.11 Isolation of Linoleic Acid (104) 138
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6.4 Isolation of Chemical Constituents of Ficus
platyphylla Del 139
6.4.1 Isolation of 3,4-Dihydroxybenzoic acid
anhydride (150) 140
6.4.2 Isolation of Ficuside A (158) 141
6.4.3 Isolation of Ficuside B (159) 141
6.4.4 Isolation of Epicatechin (62) 142
6.4.5 Isolation of Hordenine (164) 143
6.4.6 Isolation of Lupeol (16) 143
6.4.7 Isolation of Lupeol Acetate (163) 144
6.4.8 Isolation of α-Amyrin Acetate (139) 144
6.5 Bioactivity Studies 145
6.5.1 Chemicals and Instrumentation for
Bioactivity Assay 145
6.5.2 Microorganisms for Bioactivity Assay 145
6.5.3 Antioxidant Assay 146
6.5.3.1 2,2-Diphenyl-2-picrylhydrazyl
(DPPH) Radical-Scavenging Assay 146
6.5.3.2 β-Carotene–Linoleic Acid Assay 146
6.5.3.3 Determination of Total Phenolic
Content 147
6.5.4 Cytotoxicity Assay against Brine Shrimp 148
6.5.4.1 Hatching Shrimp 148
6.5.4.2 Brine shrimp assay 148
6.5.5 Antimicrobial Assay 148
6.5.5.1 Antimicrobial Disc Diffusion
Assay 148
6.5.5.2 Microdilution Assay 149
6.5.6 Tyrosinase Inhibitory Assay 149
6.5.7 Acetylcholinesterase Inhibitory Assay 150
6.5.7.1 TLC Bioautographic Method for
Acetylcholinesterase Inhibition 150
6.5.7.2 Microplate Method for 151
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Acetylcholinesterase Inhibition
6.5.8 Cyclooxygenase-2 Inhibitory Assay 151
7 CONCLUSIONS AND RECOMMENDATIONS 153
7.1 Phytochemical Studies 153
7.2 Bioactivity Studies 154
7.3 Recommendations 156
REFERENCES 157
Appendices A - S 177 – 267
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LIST OF TABLES TABLE NO. TITLE PAGE
2.1 Bioactivities and Phytochemicals from Bauhinia and
Ficus species 31
3.1 Percentage Yield and Appearance of Crude Extracts of
B. rufescens 34
3.2 NMR Spectroscopic Data of Compound (138) 46
3.3 NMR Spectroscopic Data of Compound (142) 48
3.4 NMR Spectroscopic Data of Compound (7) 66
4.1 Percentage Yield and Appearance of Crude Extracts of
F. platyphylla 71
4.2 NMR Spectroscopic Data of Compound (158) 85
4.3 NMR Spectroscopic Data of Compound (62) 96
4.4 NMR Spectroscopic Data of Compound (163) 101
5.1 Antioxidant Activity of the Extracts of B. rufescens and
F. platyphylla 107
5.2 β-Carotene Bleaching Assay of the Extracts of B.
rufescens and F. platyphylla 108
5.3 Total Phenolic Content assay of the Extracts of B.
rufescens and F. platyphylla 109
5.4 Antioxidant Correlation 110
5.5 Brine Shrimp Assay of B. rufescens and F. platyphylla 111
5.6 Diameter of Zone of Inhibition for the Crude Extracts
and Isolated Compounds 114
5.7 Minimum Inhibition Concentration (MIC) and
Minimum Microbicidal Concentration (MMC) of Crude
Extracts and Isolated Compounds
118
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5.8 TLC Bioautographic Assay of The Crude Extracts and
Isolated Compounds 124
5.9 Acetylcholinesterase Inhibitory Activity 127
5.10 COX-2 Inhibitory Activity 129
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LIST OF SCHEMES SCHEME NO. TITLE PAGE
3.1 The EIMS Fragmentation Pattern of Compound (138) 45
3.2 Fragment ions from Compound (142) 56
3.3 Proposed Biosynthetic Pathway for Compounds (142) 59
3.4 The ESIMS Fragmentation Pattern of Compound (99) 61
3.5 Condensation of Cyclohexenecyanomethylene (148) to
Menisdaurin (99) 61
4.1 The EIMS Fragmentation Pattern of Compound (150) 73
4.2 Biosynthetic Pathway for Compounds (150) 74
4.3 The ESIMS Fragmentation Pattern of Compound (158) 75
4.4 Biosynthetic Pathway for Compounds (158) and (159) 95
4.5 The EIMS Fragmentation Pattern of Compound (62) 96
4.6 The EIMS Fragmentation Pattern of Compound (164) 98
4.7 The EIMS Fragmentation Pattern of Compound (16) 99
6.1 Flow Chart for the Isolation Process of B. rufescens 132
6.2 Flow Chart for the Isolation Process of F. platyphylla 140
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LIST OF FIGURES FIGURE NO. TITLE PAGE
3.1 IR Spectrum of Compound (141) 37
3.2 1H NMR Spectrum of Compound (141) 38
3.3 1H-1H COSY Spectrum of Compound (141) 39
3.4 13C NMR and DEPT Spectra of Compound (141) 40
3.5 HREIMS Spectrum of Compound (141) 41
3.6 HMQC Spectrum of Compound (141) 42
3.7 HMBC Spectrum of Compound (141) 43
3.8 HMBC Correlations of Compound (141) 44
3.9 IR Spectrum of Compound (142) 49
3.10 1H NMR Spectrum of Compound (142) 50
3.11 1H-1H COSY Spectrum of Compound (142) 51
3.12 13C NMR and DEPT Spectra of Compound (142) 52
3.13 HMBC Correlations of Compound (142) 53
3.14 HMQC Spectrum of Compound (142) 54
3.15 HMBC Spectrum of Compound (142) 55
3.16 ESIMS Spectrum of Compound (142) 57
4.1 HMBC Correlations of Compound (150) 73
4.2 IR Spectrum of Compound (158) 77
4.3 HRESIMS Spectrum of Compound (158) 78
4.4 ESI Spectrum of Compound (158) 79
4.5 1H NMR Spectrum of Compound (158) 80
4.6 1H-1H COSY Spectrum of Compound (158) 81
4.7 13C NMR and DEPT Spectra of Compound (158) 82
4.8 HMQC Spectrum of Compound (158) 83
4.9 HMBC Spectrum of Compound (158) 84
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4.10 HMBC Correlations of Compound (158) 85
4.11 IR Spectrum of Compound (159) 87
4.12 HRESIMS Spectrum of Compound (159) 88
4.13 1H NMR Spectrum of Compound (159) 89
4.14 1H-1H COSY Spectrum of Compound (159) 90
4.15 13C NMR and DEPT Spectra of Compound (159) 91
4.16 HMQC Spectrum of Compound (159) 92
4.17 HMBC Spectrum of Compound (159) 93
4.18 HMBC Correlations of Compound (159) 94
5.1 DPPH Reaction 105
5.2 Percentage Inhibition of DPPH Radical Assay of Crude
Extracts from B. rufescens 105
5.3 Percentage Inhibition of DPPH Radical Assay of Crude
Extracts from F. platyphylla 106
5.4 Oxidation of Linoleic acid 108
5.5 Oxidation Process of Tyrosinase 121
5.6 Tyrosinase Inhibitory Activity of B. rufescens 122
5.7 Tyrosinase Inhibitory Activity of F. platyphylla 122
5.8 Hydrolysis of Acetycholinesterase 123
5.9 Acetylcholinesterase Inhibitory Activity of B. rufescens 126
5.10 Acetylcholinesterase Inhibitory Activity of F. platyphylla 126
5.11 Cyclization of Arachidonic acid 128
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LIST OF ABBREVIATIONS
α - Alpha
AA - Ascorbic Acid
Abs - Absorbance
Ac - Acetone
β - Beta
br - broad
BHT - Butylated hydroxytoluene
BCB - β-carotene bleaching
BaCl2 - Barium chloride
BSLT - Brine Shrimps Lethality Test
c - concentration 13C - Carbon-13
CC - Column Chromatography
COSY - Correlation Spectroscopy
CDCl3 - Deuterated chloroform
CD3COCD3 - Deuterated acetone
CHCl3 - Chloroform
cm - Centimeter
cm-1 - Per centimeter
1D - 1 Dimension
2D - 2 Dimension
δ - Chemical shift
d - doublet
dd - doublet of doublets
ddd - doublet of doublets of doublets
dq - doublet of quartet
dt - doublet of triplet
xix
DCM - Dichloromethane
DEPT - Distortionless Enhancement by Polarization Transfer
DMSO - Dimethyl sulfoxide
DPPH - 2,2-Diphenyl-1-picrylhydrazyl
EtOAc - Ethyl acetate
EIMS - Electron Impact Mass Spectrometry
ESIMS - Electrospray Ionization Mass Spectrometry
Et2O - Diethyl ether
EtOH - Ethanol
FT-IR - Fourier Transform Infrared
γ - Gamma
GC - Gas Chromatography
GC-MS - Gas Chromatography-Mass Spectrometry 1H - Proton
HRESIMS - High Resolution Electrospray Ionization Mass Spectrometry
HMBC - Heteronuclear Multiple Bond Correlation
HMQC - Heteronuclear Multiple Quantum Coherence
H2O - Water
H2SO4 - Sulfuric acid
Hz - Hertz
HCl - Hydrochloric acid
IR - Infrared
IC50 - Inhibition Concentration at 50%
J - coupling constant
KBr - Potassium bromide
K2HPO4 - Potassium phosphate dibasic anhydrous
λ - Lamda
L - Liter
LD - Detection Limit
lit. - Literature
MgSO4.7H2O - Magnesium sulfate heptahydrate
MIC - Minimum Inhibition Concentration
MMC - Minimum Microbicidal Concentration
MS - Mass Spectrometry
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M+ - Molecular ion
m/z - mass to charge ion
MeOH - Methanol
m.p - melting point
MgSO4 - Magnesium sulphate
MHz - Megahertz
mg - Miligram
m - multiplet
min. - Minute(s)
mm - milimeter
mL - mililiter
NA - Nutrient agar
NB - Nutrient broth
NMR - Nuclear Magnetic Resonance
nm - nanometer
NaOH - Sodium hydroxide
NaCl - Sodium chloride
O2 - Oxygen
PE - Petroleum ether
ppm - parts per million
q - quartet
Rf - retention factor
rpm - Revolutions per minute
SD - Standard Deviation
s - singlet
t - triplet
td - triplet of doublets
tr - Retention time
tr - trace
TLC - Thin Layer Chromatography
µM - Micro molar
UV - Ultraviolet
VLC - Vacuum Liquid Chromatography
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LIST OF APPENDICES
APPENDIX TITLE PAGE
A Spectral Data of 6-Methoxy-7-methyl-8-hydroxydibenz[b,f] oxepin (138) 177
A1 IR spectrum of 6-Methoxy-7-methyl-8-hydroxydibenz[b,f] oxepin (138) 177
A2 EIMS spectrum of 6-Methoxy-7-methyl-8-hydroxydibenz[b,f] oxepin (138) 178
A3 13C NMR spectrum of 6-Methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin (138) 179
A4 DEPT spectra of 6-methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin (138) 180
A5 1H NMR spectrum of 6-methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin (138) 181
A6 1H-1H COSY spectrum of 6-Methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin (138) 182
A7 HMQC spectrum of 6-Methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin (138) 183
A8 HMBC spectrum of 6-Methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin (138) 184
B Spectral Data of Menisdaurin (99) 185
B1 IR spectrum of Menisdaurin (99) 185
B2 1H NMR spectrum of Menisdaurin (99) 186
B3 13C NMR spectrum of Menisdaurin (99) 187
B4 DEPT spectra of Menisdaurin (99) 188
B5 1H-1H COSY spectrum of Menisdaurin (99) 189
B6 HMQC spectrum of Menisdaurin (99) 190
B7 HMBC spectrum of Menisdaurin (99) 191
B8 ESIMS spectrum of Menisdaurin (99) 192
C Spectral Data of Menisdaurilide (143) 193
C1 IR spectrum of Menisdaurilide (143) 193
xxii
C2 EIMS spectrum of Menisdaurilide (143) 194
C3 13C NMR spectrum of Menisdaurilide (143) 195
C4 1H NMR spectrum of Menisdaurilide (143) 196
C5 1H-1H COSY spectrum of Menisdaurilide (143) 197
C6 DEPT spectra of Menisdaurilide (143) 198
C7 HMQC spectrum of Menisdaurilide (143) 199
D Spectral Data of Sequoyitol (94) 200
D1 IR spectrum of Sequoyitol (94) 200
D2 1H NMR spectrum of Sequoyitol (94) 201
D3 1H-1H COSY spectrum of Sequoyitol (94) 202
D4 13C NMR spectrum of Sequoyitol (94) 203
D5 DEPT spectra of Sequoyitol (94) 204
D6 HMQCspectrum of Sequoyitol (94) 205
D7 EIMS spectrum of Sequoyitol (94) 206
E Spectral Data of α-Amyrin acetate (139) 207
E1 EIMS spectrum of α-Amyrin acetate (139) 207
E2 IR spectrum of α-Amyrin acetate (139) 208
E3 1H NMR spectrum of α-Amyrin acetate (139) 209
E4 13C NMR spectrum of α-Amyrin acetate (139) 210
E5 DEPT spectra of α-Amyrin acetate (139) 211
E6 HMQC spectrum of α-Amyrin acetate (139) 212
F Spectral Data of β-Sitosterol 3-O-β-D-xylopyranoside (7) 213
F1 IR spectrum of β-Sitosterol 3-O-β-D-xylopyranoside (7) 213
F2 1H NMR spectrum of β-Sitosterol 3-O-β- D-xylopyranoside (7) 214
F3 1H-1H COSY spectrum of β-Sitosterol 3-O-β-D-xylopyranoside (7) 215
F4 13C NMR spectrum of β-sitosterol 3-O-β-D-xylopyranoside (7) 216
F5 DEPT spectra of β-Sitosterol 3-O-β-D-xylopyranoside (7) 217
G Spectral Data of β-Sitosterol (10) and Stigmasterol (14) 218
G1 GC spectrum of β-Sitosterol (10) and Stigmasterol (14) 218
G2 EIMS spectrum of β-Sitosterol (10) 219
xxiii
G3 EIMS spectrum of Stigmasterol (14) 220
G4 IR spectrum of β-Sitosterol (10) and Stigmasterol (14) 221
G5 1H NMR spectrum of β-Sitosterol (10) and Stigmasterol (14) 222
H Spectral Data of Glyceryl trilinoleate (140) 223
H1 IR spectrum of Glyceryl trilinoleate (140) 223
H2 13C NMR spectrum of Glyceryl trilinoleate (140) 224
H3 1H NMR spectrum of Glyceryl trilinoleate (140) 225
H4 HMQC spectrum of Glyceryl trilinoleate (140) 226
I Spectral Data of Linoleic acid (104) 227
I1 IR spectrum of Linoleic acid (104) 227
I2 1H NMR spectrum of Linoleic acid (104) 228
I3 13C NMR spectrum of Linoleic acid (104) 229
I4 EIMS spectrum of Linoleic acid (104) 230
J Spectral Data of 3,4-Dihydroxybenzoic acid anhydride (150) 231
J1 IR Spectrum of 3,4-Dihydroxybenzoic acid anhydride (150) 231
J2 1H NMR Spectrum of 3,4-Dihydroxybenzoic acid anhydride (150) 232
J3 Expansion of 1H-1H COSY Spectrum of 3,4-Dihydroxybenzoic acid anhydride (150) 233
J4 13C NMR Spectrum of 3,4-Dihydroxybenzoic acid anhydride (150) 234
J5 DEPT Spectra of 3,4-Dihydroxybenzoic acid anhydride (150) 235
J6 Expansion of HMQC Spectrum of 3,4-Dihydroxybenzoic acid anhydride (150) 236
J7 Expansion of HMBC Spectrum of 3,4-Dihydroxybenzoic acid anhydride (150) 237
J8 EIMS Spectrum of 3,4-Dihydroxybenzoic acid anhydride (150) 238
K Spectral Data of Epicatechin (62) 239
K1 IR spectrum of Epicatechin (62) 239
K2 EIMS spectrum of Epicatechin (62) 240
K3 1H NMR spectrum of Epicatechin (62) 241
K4 1H-1H COSY spectrum of Epicatechin (62) 242
K5 13C NMR spectrum of Epicatechin (62) 243
xxiv
K6 DEPT Spectra of Epicatechin (62) 244
K7 HMQC spectrum of Epicatechin (62) 245
L Spectral Data of Hordenine (164) 246
L1 IR spectrum of Hordenine (164) 246
L2 1H NMR spectrum of Hordenine (164) 247
L3 1H-1H COSY spectrum of Hordenine (164) 248
L4 13C NMR spectrum of Hordenine (164) 249
L5 DEPT spectra of Hordenine (164) 250
L6 HMQC spectrum of Hordenine (164) 251
L7 EIMS spectrum of Hordenine (164) 252
M Spectral Data of Lupeol (16) 253
M1 IR spectrum of Lupeol (16) 253
M2 EIMS spectrum of Lupeol (16) 254
M3 1H NMR spectrum of Lupeol (16) 255
M4 13C NMR spectrum of Lupeol (16) 256
M5 DEPT spectra of Lupeol (16) 257
N Spectral Data of Lupeol acetate (163) 258
N1 IR spectrum of Lupeol acetate (163) 258
N2 EIMS spectrum of Lupeol acetate (163) 259
N3 1H NMR spectrum of Lupeol acetate (163) 260
N4 13C NMR spectrum of Lupeol acetate (163) 261
N5 DEPT spectra of Lupeol acetate (163) 262
O Pearson’s Correlation for Antioxidant 263
P Brine Shrimps Lethality after 24 Hours 264
Q Test Samples Inhibitions against Acetylcholinesterase 265
R Test Samples Inhibitions against Cyclooxygenese 2 266
S Publications 267
1
1 CHAPTER 1
INTRODUCTION
1.1 Background of Study
Over the centuries human being has relied on plants for basic needs such as
food, clothing, and shelter, which are produced from matrices or storage parts of a
plant i.e. leaves, woods, fibers, fruits, tubers etc. Plants have also been utilized for
some other purposes, such as, poisons used for hunting, hallucinogens used for ritual
purposes, stimulants for endurance, and hunger suppression, as well as medicines.
The plant chemicals used for these latter purposes are largely the secondary
metabolites, which are derived biosynthetically from plant primary metabolites,
carbohydrates, amino acids, and lipids. The substances are also not directly involved
in the growth, development, or reproduction of plants [1, 2]. These secondary
metabolites can be classified into several groups according to their chemical classes,
such as flavonoids, alkaloids, terpenoids and coumarins.
The plant medicines initially took the form of crude drugs such as tinctures,
teas, poultices, powders, and other herbal formulations. The specific plants to be used
and the methods of application for particular ailments were passed down through oral
history. Eventually information regarding medicinal plants was recorded in herbals
[3]. The first written record on the medicinal uses of plants appeared in about 2600
BC from the Sumerians and Akkaidians [4]. The “Ebers Papyrus”, the best known
Egyptian pharmaceutical record, which documented over 700 drugs, represents the
history of Egyptian medicine dated from 1500 BC. The Chinese “Materia Medica”,
describes more than 600 medicinal plants, well documented dating from about 1100
BC [5]. Documentation of the Ayurvedic system recorded in Susruta and Charaka
2
date from about 1000 BC [6]. The Greeks also contributed substantially to the
rational development of the herbal drugs. Dioscorides, the Greek physician (100
A.D.), described more than 600 medicinal plants in his work “De Materia Medica”
[4].
Drug discovery from medicinal plants include numerous fields of inquiry and
various methods of analysis. The process typically begins with a botanist who
collects and identifies the plants of interest. Collection may involve species with
known biological activity for which active compounds have not been isolated or may
involve taxa collected randomly for a large screening program [3]. Consequently, a
number of drugs have been developed from natural products, for examples,
anticancer drug vincristine (1) from Vinca rosea, narcotic analgesic morphine (2)
from Papaver somniferum, antimalarial drug artemisinin (3) from Artemisia annua
and antibiotic penicillin G (4) from Penicillium sp [7].
N
N
H
N
N
OH
COOMe
H
HO COOMe
CHOMeO
COOMe
(1)
O
HO
HO
NH H
(2)
N
N
OO
H H
COOH
H
(4)
O
H
H
H
OO
(3)
Natural products are used directly in the ‘natural’ pharmaceutical industry,
consequently traditional medicine programmes are incorporated into the primary
health care systems in some parts of the world, such as Mexico, China, Nigeria and
Malaysia [8]. Conventional methods of drug discovery from natural products could
be viewed based on two approaches. Bioassay-guided isolation, where a drug targets
3
are exposed to crude extracts, any evidence of activity in an extract will then go
through fractionation and isolation of the active compound. Secondly, is the isolation
of a number of natural compounds (compounds library) suitable for undergoing any
bioactivity screening [7].
Modern drug discovery approach involving High-Throughput Screening
(HTS), where, applying full automation and robotics in which hundreds of molecules
can be screened using several assays within a short time, and with very little amounts
of compounds. In order to incorporate natural products in the modern HTS
programmes, a natural product library (a collection of dereplicated natural products)
is required. This will allow dereplication; a process by which one can eliminate re-
isolation of similar compounds from various extracts. In an attempt to improve the
dereplication, a number of techniques are to be developed, including liquid
chromatography–photo-diode-array detector (LC-PDA), liquid chromatography–
mass spectrometry (LC-MS) and liquid chromatography–nuclear magnetic resonance
spectroscopy (LC-NMR) [9].
1.2 Medicinal Plants
Medicinal plants have had a sort of continuous and critically controlled
clinical trial, represent a primary source for the discovery of new drugs. It is,
therefore, amazing that many medicinal plants from the Mediterranean (Greek–
Latin–Arabic) tradition were over looked for so long by Modern medicine.
The birth of drug discovery is closely connected to the study of plant natural
products and was shaped by two seminal events, the isolation of morphine (2) from
opium by the pharmacist Serturner in 1817 and the introduction in the clinics of
Antipyrin (phenazone) 70 years later, in 1887. The isolation of a pure compound
responsible for the medicinal properties of a crude drug marked the beginning of
medicinal chemistry, triggering the transition from botanical extracts to pure
molecules and eventually leading to the isolation of the active substance of most
drugs. The importance of medicinal plants can be ascertained from the fact that
4
according to the World Health Organization (WHO) estimates, 80% of the World’s
population fulfill their healthcare needs from phytomedicinal sources [10].
1.3 Statement of the Problem
The plants from Fabaceae and Moraceae families have been claimed to have
medicinal applications which include antidiabetic, antitumor, antihypertensive,
antimicrobial infections. The bioactivities exhibited are associated to the presence of
various classes of secondary metabolites in the plants, such as flavonoids, alkaloids,
terpenoids, phenyl propanoids and glycosides. Thus, there are need to investigate the
bioactive compounds present in the Bauhinia rufescens (Fabaceae) and Ficus
platyphylla (Moraceae).
1.4 Objectives of Research
The objectives of this research are to isolate the chemical constituents of
Bauhinia rufescens (Fabaceae) and Ficus platyphylla (Moraceae) and to evaluate
bioactivity of the crude extracts and the isolated compounds for antioxidant,
antimicrobial, antityrosinase, brine shrimp lethality test, antiacetylcholinesterase and
antiinflammatory inhibitors. The research is divided into two parts. The first part is to
isolate and identify the phytochemicals from the dried samples followed by the study
of bioactivity of crude extracts and pure compounds.
1.5 Significance of the Research
This study is to ascertain the medicinal values of Bauhinia rufescens
(Fabaceae) and Ficus platyphylla (Moraceae) through phytochemicals and bioactivity
studies and make necessary recommendations that could lead to a scientific evidence
of their medicinal applications.
5
1.6 Scope of Work
This research is focused on the Bauhinia rufescens (Fabaceae; leaves and
stem bark) and Ficus platyphylla (Moraceae; stem bark) for the studies on their
chemical constituents and bioactivities. The dried samples will be extracted using
soxhlet extraction with different organic solvents in an increasing polarity gradient.
The crude extracts will be fractionated using vacuum liquid chromatography (VLC).
Purification of the fractions will be carried out by gravity column
chromatography (CC) to obtain the pure compounds. An elucidation of chemical
structure for the isolated compounds in pure forms will be conducted using
spectroscopic techniques, which includes; UV, IR, 1H NMR, 13C NMR, 2D NMR
(COSY, HMQC, HMBC) and mass spectrometry.
Finally, the bioactivity studies, including DPPH radical scavenging assay,
total phenolic content assay, β-carotene/linoleic acid bleaching assay, brine shrimp
lethality test, antibacterial, antifungal, antityrosinase, acetylcholinesterase, anti-
inflammatory assays will be carried out on the crude extracts and pure compounds.
157
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