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
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To my beloved Father Late Alhaji Muhammad Sarki and my beloved mother Hajiya Hauwa Muhammad Sarki
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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). vii
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 viii
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 ix
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 73 (150) 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 x
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 xi
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 xii
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 118 Extracts and Isolated Compounds xiv
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 xv
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 xvii
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. 126 platyphylla 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 xx
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 xxi
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 1 F2 H 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].
OH HO N
O N COOMe N H H H HO N (2)
HO COOMe COOMe H MeO N H O CHO O (1) H H H H H N O O N (3) O COOH (4)
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
8 REFERENCES
1. Çakılcıoğlu, U., Sengün, M. T., and Türkoğlu, I. An ethnobotanical survey of medicinal plants of Yazıkonak and Yurtba districts of Elazığ province,Turkey. Journal of Medicinal Plants Research. 2010. 4(7):567-572.
2. Devendra, B. N., Srinivas, N., and Solmon, K. S. A comparative pharmacological and phytochemical analysis of in vivo and in vitro propagated Crotalaria species. Asian Pacific Journal of Tropical Medicine. 2012. 5(1):37-41.
3. Balunas, M. J. and Kinghorn, A. D. Drug discovery from medicinal plants. Life Sciences. 2005. 78(5):431-441.
4. Samuelsson, G. Drugs of natural origin: a textbook of pharmacognosy. 4th ed ed. Stockholm: Swedish Pharmaceutical Press. 1999.p. 15-36.
5. Cragg, G. M., Newman, D. J., and Snader, K. M. Natural Products in Drug Discovery and Development. Journal of Natural Products. 1997. 60(1):52- 60.
6. Shoeb, M. Anticancer agents from medicinal plants Bangladesh Journal of Pharmacology. 2006. 1:35-41
7. Satyajit, S. D. and Lutfun, N. Chemistry for Pharmacy students: General, Organic and Natural Product Chemistry. Chichester, West Sussex, England: John Wiley and Sons. 2007.p. 283-359.
8. World Health, O., Traditional Medicine Strategy. 2005: Geneva. p. 1-10.
158
9. Beghyn, T., Deprez-Poulain, R., Willand, N., Folleas, B., and Deprez, B. Natural Compounds: Leads or Ideas? Bioinspired Molecules for Drug Discovery. Chemical Biology and Drug Design. 2008. 72(1):3-15.
10. Jurna, I. Sertürner und Morphin – eine historische Vignette. Schmerzforum. 2003. 17(4):280-283.
11. Lewis, G. B., Schrire, B. M., and Lock , M., Legumes of the world 2005, Royal Botanical Gardens, Kew, UK. p. 7-10.
12. McKey, D., ed. Legumes and nitrogen: the evolutionary ecology of a nitrogen-demanding lifestyle. 1994 Royal Botanic Gardens, Kew, UK. Pg 211–228
13. Graham, P. H. and Vance, C. P. Legumes: importance and constraints to greater use. Plant Physiology 2003. 131:872 – 877.
14. Gupta, D., Bleakley, B., and Gupta, R. K. Dragon's blood: Botany, chemistry and therapeutic uses. Journal of Ethnopharmacology. 2008. 115(3):361-380.
15. Gao, T., Sun, Z., Yao, H., Song, J., Zhu, Y., Ma, X., and Chen, S. Identification of Fabaceae plants using the DNA barcode matK. Planta Medica. 2011. 77(1):92-94.
16. Sanjib, S., Jamil A., S., Himangsu, M., Faroque, H., Md., A., Md., M. H., and Geoffrey A., C. Ethnomedicinal, phytochemical, and pharmacological profile of the genus Dalbergia L. (Fabaceae) Phytopharmacology. 2013. 4(2):291- 346.
17. Li, W., Asada, Y., and Yoshikawa, T. Flavonoid constituents from Glycyrrhiza glabra hairy root cultures. Phytochemistry. 2000. 55(5):447-456.
18. Ma, X., Zheng, C., Hu, C., Rahman, K., and Qin, L. The genus Desmodium (Fabaceae) traditional uses in Chinese medicine, phytochemistry and pharmacology. Journal of Ethnopharmacology. 2011. 138(2):314-332. 159
19. Ayo, R. G. and College, S. Phytochemical constituents and bioactivities of the extracts of Cassia nigricans Vahl : A review. Journal of Medicinal Plants. 2010. 4(14):1339-1348.
20. Silva, K. L. and Cechinel, F. V. Plantas do gênero Bauhinia: composição química e potencial farmacológico. Química Nova. 2002. 25:449-454.
21. Robertson, K. R. and Sytsma, K. J., Rosales in Encyclopædia Britannica Online. 2013. p. pg 213.
22. Jagtap, U. B. and Bapat, V. A. Artocarpus: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology. 2010. 129(2):142-166.
23. Jackson, R. G. S. A., Grasielly, R. S., Edigênia, C. C. A., Fabrício, S. S., Julianeli, T. L., Luciano, A. A. R., Xirley, P. N., José, M. B. F., Lucindo, J. Q. J., and Márcio, R. V. S. Medicinal Plants and Natural Compounds from the Genus Morus (Moraceae) with Hypoglycemic Activity: A Review. in Glucose Tolerance.2012. p. 189-206.
24. Kuete, V., Metuno, R., Keilah, P. L., Tshikalange, E. T., and Ngadjui, B. T. Evaluation of the genus Treculia for antimycobacterial, anti-reverse transcriptase, radical scavenging and antitumor activities. South African Journal of Botany. 2010. 76(3):530-535.
25. Amponsah, I. K. Chemical constituents, antiinflammatory, antioxidant and antimicrobial activities of the stem bark and leaves of Ficus exasperata (vahl) PhD. Kwame Nkrumah University of Science and Technology 2012
26. Lansky, E. P., Paavilainen, H. M., Pawlus, A. D., and Newman, R. A. Ficus spp. (fig): Ethnobotany and potential as anticancer and anti-inflammatory agents. Journal of Ethnopharmacology. 2008. 119(2):195-213.
27. Burkill, H. M. The Useful Plants of West Tropical Africa. Vol. I Royal Botanic Gardens, Kew 1985.p. 1–6. 160
28. Ugwah-Oguejiofor, C. J., Bello, S. O., Okolo, R. U., Etuk, E. U., Ugwah, M. O., and Igbokwe, V. U. Ficus platyphylla promotes fertility in female Rattus norvegicus Wistar strain: a preliminary study. Reproductive Biology and Endocrinology. 2011. 9:145.
29. Chindo, B. A., Anuka, J. A., McNeil, L., Yaro, A. H., Adamu, S. S., Amos, S., Connelly, W. K., Lees, G., and Gamaniel, K. S. Anticonvulsant properties of saponins from Ficus platyphylla stem bark. Brain Research Bulletin. 2009. 78(6):276-282.
30. Sandermann, W. Chapter 11 - Terpenoids: Metabolism. in Comparative Biochemistry, F Marcel, Editor. Academic Press.1962. p. 591-630.
31. Iribarren, A. M. and Pomilio, A. B. Sitosterol 3-O-α-D-xyluronofuranoside from Bauhinia candicans. Phytochemistry. 1987. 26(3):857-858.
32. Iribarren, A. M. and Pomilio, A. B. Sitosterol 3-O-α-riburonofuranoside from Bauhinia candicans. Phytochemistry. 1985. 24(2):360-361.
33. Iribarren, A. M. and Pomilio, A. B. Sitosterol 3-O-β-D-xylopyranoside from Bauhinia candicans. Phytochemistry. 1984. 23(9):2087-2088.
34. Achenbach, H., Stöcker, M., and A. Constenla, M. Flavonoid and other constituents of Bauhinia manca. Phytochemistry. 1988. 27(6):1835-1841.
35. Viana, E. P., Santa-Rosa, R. S., Almeida, S. S. M. S., and Santos, L. S. Constituents of the stem bark of Bauhinia guianensis. Fitoterapia. 1999. 70(1):111-112.
36. Athikomkulchai, S., Ruangrungsi, N., Sekine, T., Sumino, M., Igarashi, K., and Ikegami, F. Chemical constituents of Bauhinia sirindhorniae. Natural Medicines. 2003. 57:150-153.
37. Rao, Y. K., Fang, S.-H., and Tzeng, Y.-M. Antiinflammatory activities of flavonoids and a triterpene caffeate isolated from Bauhinia variegata. Phytotherapy Research. 2008. 22(7):957-962. 161
38. Shang, X. Y., Li, S., Wang, S. J., Yang, Y. C., and Shi, J. G. Chemical constituents of Bauhinia aurea. Journal of Asian Natural Products Research. 2012. 14(10):966-972.
39. El-Sayyad, S. M., Sayed, H. M., and Mousa, S. A. Chemical constituents and preliminary anthelmintic activity of Ficus platyphylla (Del). Bulletin of Pharmaceutical Sciences. 1986. 9:164-77.
40. Li, Y. C. and Kuo, Y. H. A monoterpenoid and two simple phenols from heartwood of Ficus microcarpa. Phytochemistry. 1998. 49(8):2417-2419.
41. Tuyen, N. V., Kim, D. S. H. L., Fong, H. S., Soejarto, D. D., Khanh, T. C., Tri, M. V., and Xuan, L. T. Structure elucidation of two triterpenoids from Ficus fistulosa. Phytochemistry. 1999. 50(3):467-469.
42. Lee, E. H., Kim, H. J., Song, Y. S., Jin, C., Lee, K.-T., Cho, J., and Lee, Y. S. Constituents of the Stems and Fruits of Opuntia Ficus indica var. saboten Archives of Pharmaceutical Research. 2003. 26(12):1018-1023.
43. Singh, D., Singh, B., and Goel, R. K. Traditional uses, phytochemistry and pharmacology of Ficus religiosa: A review. Journal of Ethnopharmacology. 2011. 134(3):565-583.
44. Kiem, P. V., Cuong, N. X., Nhiem, N. X., Thu, V. K., Ban, N. K., Minh, C. V., Tai, B. H., Hai, T. N., Lee, S. H., Jang, H. D., and Kim, Y. H. Antioxidant activity of a new C-glycosylflavone from the leaves of Ficus microcarpa. Bioorganic and Medicinal Chemistry Letters. 2011. 21(2):633-637.
45. Somwong, P., Suttisri, R., and Buakeaw, A. New sesquiterpenes and phenolic compound from Ficus foveolata. Fitoterapia. 2013. 85:1-7.
46. Hanson, J. R., Martyn Berry, and Drayton, C. Natural Products:The Secondary Metabolites ed. Abel E W, Davies A G, Phillips, D, and Woollins, JD: Royal Society of Chemistry, Cambridge, UK. 2003.p. pg. 21-14.
47. Iribarren, A. M. and Pomilio, A. B. Components of Bauhinia candicans. Journal of Natural Products. 1983. 46(5):752-753. 162
48. Zhao, Y. Y., Cui, C. B., Cai, B., Han, B., and Sun, Q. S. A new phenanthraquinone from the stems of Bauhinia variegata L. Journal of Asian Natural Products Research. 2005. 7(6):835-838.
49. Neto, M. M., Neto, M. A., Filho, R. B., Lima, M. A. S., and Silveira, E. R. Flavonoids and alkaloids from leaves of Bauhinia ungulata L. Biochemical Systematics and Ecology. 2008. 36(3):227-229.
50. Lim, H., Kim, M. K., Lim, Y., Cho, Y. H., and Lee, C. H. Inhibition of cell- cycle progression in HeLa cells by HY52, a novel cyclin-dependent kinase inhibitor isolated from Bauhinia forficata. Cancer Letters. 2006. 233(1):89- 97.
51. Braca, A., Nunziatina, D. T., Lorenzo, D. B., Pizza, C., Politi, M., and Morelli, I. Antioxidant Principles from Bauhinia tarapotensis. Journal of Natural Products. 2001. 64(7):892-895.
52. Damu, A. G., Kuo, P. C., Shi, L. S., Li, C. Y., Kuoh, C.-S., Wu, P. L., and Wu, T. S. Phenanthroindolizidine Alkaloids from the Stems of Ficus septica. Journal of Natural Products. 2005. 68(7):1071-1075.
53. Simo, C. C. F., Kouam, S. F., Poumale, H. M. P., Simo, I. K., Ngadjui, B. T., Green, I. R., and Krohn, K. Benjaminamide: A new ceramide and other compounds from the twigs of Ficus benjamina (Moraceae). Biochemical Systematics and Ecology. 2008. 36(3):238-243.
54. Subramaniam, G., Ang, K. K. H., Siewbee, N. G., Buss, A. D., and Butler, M. S. A benzopyrroloisoquinoline alkaloid from Ficus fistulosa. Phytochemistry Letters. 2009. 2(2):88-90.
55. Ueda, J. Y., Takagi, M., and Shinya, K. Aminocaprophenone- and Pyrrolidine-Type Alkaloids from the Leaves of Ficus septica. Journal of Natural Products. 2009. 72(12):2181-2183.
56. Havsteen, B. H. The biochemistry and medical significance of the flavonoids. Pharmacology and Therapeutics. 2002. 96(2–3):67-202. 163
57. Kuo, Y. H., Yeh, M. H., and Huang, S. L. A novel 6-butyl-3- hydroxyflavanone from heartwood of Bauhinia purpurea. Phytochemistry. 1998. 49(8):2529-2530.
58. Yadava, R. N. and Tripathi, P. A novel flavone glycoside from the stem of Bauhinia purpurea. Fitoterapia. 2000. 71(1):88-90.
59. Ferreres, F., GiIzquierdo, A., Vinholes, J., Silva, S. T., Valentão, P., and Andrade, P. B. Bauhinia forficata Link authenticity using flavonoids profile: Relation with their biological properties. Food Chemistry. 2012. 134(2):894- 904.
60. Xu, W., Chu, K., Li, H., Zhang, Y., Zheng, H., Chen, R., and Chen, L. Ionic liquid-based microwave-assisted extraction of flavonoids from Bauhinia championii (Benth.) Benth. Molecules. 2012. 17:14323-14335.
61. Gaspar, D. M., Arruda, A. C., Arruda, M. S. P., and Müller, A. H. Methoxyflavones from Ficus maxima. Phytochemistry. 1997. 45(8):1697- 1699.
62. Pistelli, L., Chiellini, E. E., and Morelli, I. Flavonoids from Ficus pumila. Biochemical Systematics and Ecology. 2000. 28(3):287-289.
63. Yarmolinsky, L., Huleihel, M., Zaccai, M., and Benshabat, S. Potent antiviral flavone glycosides from Ficus benjamina leaves. Fitoterapia. 2012. 83(2):362-367.
64. Choo, C. Y., Sulong, N. Y., Man, F., and Wong, T. W. Vitexin and isovitexin from the Leaves of Ficus deltoidea with in-vivo α-glucosidase inhibition. Journal of Ethnopharmacology. 2012. 142(3):776-781.
65. Donia, A. E. R. M., Soliman, G. A., Zaghloul, A. M., Alqasoumi, S. I., Awaad, A. S., Radwan, A. M., and Basodan, O. A. Chemical constituents and protective effect of Ficus ingens (Miq.) on carbon tetrachloride-induced acute liver damage in male Wistar albino rats. Journal of Saudi Chemical Society. 2013. 17(1):125-133. 164
66. Boudet, A. M. Evolution and current status of research in phenolic compounds. Phytochemistry. 2007. 68(22–24):2722-2735.
67. Stafford, H. A. Chapter Two The evolution of phenolics in plants. in Recent Advances in Phytochemistry, JT Romeo, Varin, RIL, and Luca, VD, Editors. Elsevier.2000. p. 25-54.
68. Nageshwar, G., Anuradha, S., Radhakrishnaiah, M., and Narayana, L. Distribution pattern of phenolic constituents in species of Bauhinia; Linn. and its taxonomic significance. Proceedings: Plant Sciences. 1986. 96(1):1-7.
69. Shang, X. Y., Li, S., Wang, S. J., Yang, Y. C., and Shi, J. G. Chemical constituents of Bauhinia aurea. Zhongguo Zhong Yao Za Zhi. 2006. 31(23):1953-1955.
70. Qiao, l. Z., Wu, Z., Zheng, Z., Lu, X., Liang, H., Cheng, W., Zhang, Q., and Zhao, Y. Phenolic acid derivatives from Bauhinia glauca subsp. pernervosa Acta Pharmaceutica Sinica. 2011. 46(8).
71. Lee, T. H., Kuo, Y. C., Wang, G. J., Kuo, Y. H., Chang, C. I., Lu, C. K., and Lee, C. K. Five New Phenolics from the Roots of Ficus beecheyana. Journal of Natural Products. 2002. 65(10):1497-1500.
72. Riviere, C., Pawlus, A. D., and Merillon, J. M. Natural stilbenoids: distribution in the plant kingdom and chemotaxonomic interest in Vitaceae. Natural Product Reports. 2012. 29(11):1317-1333.
73. Anjaneyulu, A., Reddy, A., Reddy, D., Ward, R., Adhikesavalu, D., and Cameron, T. Pacharin: a new dibenzo(2,3-6,7) oxepin derivative from Bauhinia racemosa Lamk. Tetrahedron 1984. 40:4245–4252.
74. Kittakoop, P., Kirtikara, K., Tanticharoen, M., and Thebtaranonth, Y. Antimalarial preracemosols A and B, possible biogenetic precursors of racemosol from Bauhinia malabarica Roxb. Phytochemistry. 2000. 55(4):349-352. 165
75. Boonphong, S., Puangsombat, P., Baramee, A., Mahidol, C., Ruchirawat, S., and Kittakoop, P. Bioactive Compounds from Bauhinia purpurea Possessing Antimalarial, Antimycobacterial, Antifungal, Anti-inflammatory, and Cytotoxic Activities. Journal of Natural Products. 2007. 70(5):795-801.
76. Sardari, S., Nishibe, S., and Daneshtalab, M. Coumarins, the bioactive structures with antifungal property. in Studies in Natural Products Chemistry, R Atta ur, Editor. Elsevier.2000. p. 335-393.
77. Elgamalps, M. H. A., Elewa, N. H., Elkhrisy, E. A. M., and Duddeck, H. 13C NMR chemical shifts and carbon-proton coupling constants of some furocoumarins and furochromones. Phytochemistry. 1979. 18:139-143.
78. Khattab, A. A. Coumarin constituents of some Egyptian Ficus species. Bulletin of Faculty of Pharmacy. 1993. 31:19-20.
79. Gribble, G. W. Chapter 5b - Natural products containing a cyclohexane, cyclohexene, or cyclohexadiene subunit. in Second Supplements to the 2nd Edition of Rodd's Chemistry of Carbon Compounds, S Malcolm, Editor. Elsevier: Amsterdam.1991. p. 375-445.
80. Chen, C. C., Chen, Y. P., Hsu, H. Y., Lee, K. H., Tani, S., and McPhail, A. T. Bauhinin, a new nitrile glucoside from Bauhinia championii. Journal of Natural Products. 1985. 48:933-7.
81. Fort, D. M., Jolad, S. D., and Nelson, S. T. Lithospermoside from Bauhinia fassoglensis (Fabaceae). Biochemical Systematics and Ecology. 2001. 29(4):439-441.
82. Kuo, Y. H. and Yeh, M. H. Chemical constituents of heartwood of Bauhinia purpurea. Journal of the Chinese Chemical Society. 1997. 44:379-383.
83. Sahu, G. and Gupta, P. A review on Bauhinia variegata. International Research Journal of Pharmacy. 2012. 3(1):48-51.
84. Sashidhara, K. V., Singh, S. P., Misra, S., Gupta, J., and Misra-Bhattacharya, S. Galactolipids from Bauhinia racemosa as a new class of antifilarial agents 166
against human lymphatic filarial parasite, Brugia malayi. European Journal of Medicinal Chemistry. 2012. 50:230-235.
85. Jeong, W. S. and Lachance, P. A. Phytosterols and Fatty Acids in Fig (Ficus carica, var. Mission) Fruit and Tree Components. Journal of Food Science. 2001. 66(2):278-281.
86. Hosamani, K. M. and Pattanashettar, R. S. Occurrence of unusual fatty acids in Ficus benghalensis seed oil. Industrial Crops and Products. 2003. 18(2):139-143.
87. Eddy, N. O., Ameh, P., Gimba, C. E., and Ebenso, E. E. Chemical information from GCMS of Ficus platyphylla gum and its corrosion inhibition potential for mild steel in 0.1 M HCl. International Journal of Electrochemical Science. 2012. 7:5677-5691.
88. Pettit, G. R., Numata, A., Iwamoto, C., Usami, Y., Yamada, T., Ohishi, H., and Cragg, G. M. Antineoplastic Agents. 551. Isolation and Structures of .Journal of Natural Products .Bauhiniastatins 1−4 from Bauhinia purpurea٣ 2005. 69(3):323-327.
89. Hassan, H. S., Sule, M. I., Usman, M. A., Usman, M., and Ibrahim, A. Preliminary phytochemical and antimicrobial screening of the stem bark extracts of Bauhinia rufescens Lam. Bayero Journal of Pure and Applied Sciences. 2009. 2(2):53-55.
90. Sosa, S., Braca, A., Altinier, G., Della Loggia, R., Morelli, I., and Tubaro, A. Topical anti-inflammatory activity of Bauhinia tarapotensis leaves. Phytomedicine. 2002. 9(7):646-653.
91. Annan, K. and Houghton, P. J. Antibacterial, antioxidant and fibroblast growth stimulation of aqueous extracts of Ficus asperifolia Miq. and Gossypium arboreum L., wound-healing plants of Ghana. Journal of Ethnopharmacology. 2008. 119(1):141-144. 167
92. Ao, C., Li, A., Elzaawely, A. A., Xuan, T. D., and Tawata, S. Evaluation of antioxidant and antibacterial activities of Ficus microcarpa L. fil. extract. Food Control. 2008. 19(10):940-948.
93. Alimuddin, S., Hemlata, R., and Patel, N. M. Evaluation of Antimicrobial activity of stem bark of Ficus bengalensis Linn. Collected from different geographical regions. Pharmacognosy Journal. 2010. 2(7):178-180.
94. Taha, M. S., Fawkeya, A. A., Zeinab, I. E., and Ahmed, M. M. Two new polyphenolic compounds from Ficus retusa L."variegata" and the biological activity of the different plant extracts Journal of Pharmacognosy and Phytotherapy. 2011. 3(7):89-100.
95. Meyre, S. C., Yunes, R. A., Delle Monache, F., Santos, A. R., Schmeling, L. O., Gadotti, V. M., Liz, F., and Cechinel, F. V. Phytochemical and pharmacological analysis of Bauhinia microstachya (Raddi) Macbr. (Leguminosae). Zeitschrift für Naturforschung. 2001. 56(11-12):939-42.
96. Gupta, M., Mazumder, U. K., Kumar, R. S., Gomathi, P., Rajeshwar, Y., Kakoti, B. B., and Selven, V. T. Anti-inflammatory, analgesic and antipyretic effects of methanol extract from Bauhinia racemosa stem bark in animal models. Journal of Ethnopharmacology. 2005. 98(3):267-273.
97. Jung, H. W., Son, H. Y., Minh, C. V., Kim, Y. H., and Park, Y. K. Methanol extract of Ficus leaf inhibits the production of nitric oxide and proinflammatory cytokines in LPS-stimulated microglia via the MAPK pathway. Phytotherapy Research. 2008. 22(8):1064-1069.
98. Ajayi, A. M., Tanayena, J. K., Balogun, S. O., Ibrahim, A., Ezeonwumelu, J. O. C., Kiplagat, D., Oyewale, A. A., Oloro, J. O., Goji, A. D. T., and Adzu, B. Antiinflammatory and Analgesic Properties of Ethanolic Stem Bark Extract of Ficus trichopoda in Rats. Pharmacognosy Journal. 2011. 2(18):43- 47.
99. Menezes, C., Kunal, G., Reema, N., Satyanarayana, D., and Jagadish, K. Analgesic and anti-inflammatory activity of Ficus glomerata in experimental 168
animal models. International Journal of Pharmaceutical Sciences and Nanotechnology. 2011. 4:1501-1504, 4 pp.
100. Arunachalam, K. and Parimelazhagan, T. Anti-inflammatory, wound healing and in-vivo antioxidant properties of the leaves of Ficus amplissima Smith. Journal of Ethnopharmacology. 2013. 145(1):139-145.
101. Fuentes, O., Arancibia, A. P., and Alarcón, J. Hypoglycemic activity of Bauhinia candicans in diabetic induced rabbits. Fitoterapia. 2004. 75(6):527- 532.
102. Wu, Z. B., Zhao, Y. Y., Yang, X. W., and Liang, H. Flavonoids from Bauhinia glauca subsp. pernervosa. Chemical and Pharmaceutical Bulletin. 2009. 57(6):628-631.
103. Filho, V. C. Chemical composition and Biological Potential of plants from the Genus Bauhinia. Phytotheraphy Research. 2009. 23:1347-1354.
104. Aliyu, A. B., Ibrahim, M. A., Musa, A. M., Ibrahim, H., Abdulkadir, I. E., and Oyewale, A. O. Evaluation of antioxidant activity of leave extract of Bauhinia rufescens Lam. (Caesalpiniaceae). Journal of Medicinal Plants Research. 2009. 3(8):563-567.
105. Pepato, M. T., Keller, E. H., Baviera, A. M., Kettelhut, I. C., Vendramini, R. C., and Brunetti, I. L. Anti-diabetic activity of Bauhinia forficata decoction in streptozotocin-diabetic rats. Journal of Ethnopharmacology. 2002. 81(2):191- 197.
106. Gayathri, M. and Kannabiran, K. Antidiabetic and ameliorative potential of Ficus bengalensis bark extract in streptozotocin induced diabetic rats. Indian Journal of Clinical Biochemistry. 2008. 23:394-400.
107. Singh, R. K., Mehta, S., Jaiswal, D., Rai, P. K., and Watal, G. Antidiabetic effect of Ficus bengalensis aerial roots in experimental animals. Journal of Ethnopharmacology. 2009. 123(1):110-114. 169
108. Jahan, I. A., Nahar, N., Mosihuzzaman, M., Rokeya, B., Ali, L., Khan, A. K. A., Makhmur, T., and Choudhary, M. I. Hypoglycaemic and antioxidant activities of Ficus racemosa Linn. fruits. Natural Product Research. 2009. 23:399-408.
109. Hassan, F., El-Razek, A., and Hassan, A. Nutritional Value and Hypoglycemic Effect of Prickly Cactus Pear (Opuntia Ficus indica) Fruit Juice in Alloxan-Induced Diabetic Rats. Australian Journal of Basic and Applied Sciences. 2011. 5(10):356-377.
110. Barata, L. Empirismo e ciência: fonte de novos medicamentos. Ciên Cult 2005. 57:1–4.
111. Kumar, R. S., Sivakumar, T., and Sunderam, R. S. Antioxidant and antimicrobial activities of Bauhinia racemosa L. stem bark. Brazilian Journal of Medical and Biological Research. 2005. 38:1015–1024.
112. Damu, A. G., Ping-Chung, K., Li-Shian, S., Chia-Ying, L., Chung-Ren, S., and Tian-Shung, W. Cytotoxic Phenanthroindolizidine Alkaloids from the Roots of Ficus septica. Planta Medica. 2009. 75:1152–1156.
113. Xu, H., Wang, X. M., Wei, X., Li, J. Y., and Liu, K. A new chalcone from the aerial roots of Ficus microcarpa. Chinese Chemical Letters. 2009. 20(5):576- 578.
114. Bernhoft, A. Bioactive compounds in plants – benefits and risks for man and animals in The Norwegian Academy of Science and Letters. 2008. Norway: Novus forlag, Oslo.
115. de Sousa, E., Zanatta, L., Seifriz, I., Creczynski-Pasa, T. B., Pizzolatti, M. G., Szpoganicz, B., and Silva, F. R. M. B. Hypoglycemic Effect and Antioxidant Potential of Kaempferol-3,7-O-(α)-dirhamnoside from Bauhinia forficata Leaves. Journal of Natural Products. 2004. 67(5):829-832.
116. Perez G, R. M., Zavala S, M. A., Perez G, S., and Perez G, C. Antidiabetic effect of compounds isolated from plants. Phytomedicine. 1998. 5(1):55-75. 170
117. Yadava, R. N. and Reddy, V. M. S. Anti-Inflammatory Activity of a Novel Flavonol Glycoside from the Bauhinia Variegata Linn. Natural Product Research. 2003. 17(3):165-169.
118. Zhao, Y., Cui, C., Cai, B., Han, B., and Sun, Q. Study on the constituents with anticancer activities of alkaloids from Bauhinia variegata L. Zhongguo Yaowu Huaxue Zazhi. 2004. 14:169-171.
119. Chiang, Y. M., Chang, J. Y., Kuo, C. C., Chang, C. Y., and Kuo, Y. H. Cytotoxic triterpenes from the aerial roots of Ficus microcarpa. Phytochemistry. 2005. 66(4):495-501.
120. Maillard, M. P., Recio-Iglesias, M. C., Saadou, M., Stoeckli-Evans, H., and Hostettmann, K. Novel antifungal tetracyclic compounds from Bauhinia rufescens Lam. Helvetica Chimica Acta. 1991. 74:791-9.
121. Wang, X., Wei, X., Tian, Y., Shen, L., and Xu, H. Antifungal Flavonoids from Ficus sarmentosa var. henryi (King) Corner. Agricultural Sciences in China. 2010. 9(5):690-694.
122. Gadotti, V. M., Santos, A. R. S., Meyre-Silva, C., Schmeling, L. O., Machado, C., Liz, F. H., and Filho, V. C. Antinociceptive action of the extract and the flavonoid quercitrin isolated from Bauhinia microstachya leaves. Journal of Pharmacy and Pharmacology. 2005. 57(10):1345-1351.
123. Aderogba, M. A., McGaw, L. J., Ogundaini, A. O., and Eloff, J. N. Antioxidant activity and cytotoxicity study of the flavonol glycosides from Bauhinia galpinii. Natural Product Research. 2007. 21(7):591-599.
124. Wang, X., Wei, X., Huang, X., Shen, L., Tian, Y., and Xu, H. Insecticidal Constructure and Bioactivities of Compounds from Ficus sarmentosa var. henryi. Agricultural Sciences in China. 2011. 10(9):1402-1409.
125. Kuete, V., Nana, F., Ngameni, B., Mbaveng, A. T., Keumedjio, F., and Ngadjui, B. T. Antimicrobial activity of the crude extract, fractions and compounds from stem bark of Ficus ovata (Moraceae). Journal of Ethnopharmacology. 2009. 124(3):556-561. 171
126. Baumgartner, B., Erdelmeier, C. A. J., Wright, A. D., Rali, T., and Sticher, O. An antimicrobial alkaloid from Ficus septica. Phytochemistry. 1990. 29(10):3327-3330.
127. Dok-Go, H., Lee, K. H., Kim, H. J., Lee, E. H., Lee, J., Song, Y. S., Lee, Y. H., Jin, C., Lee, Y. S., and Cho, J. Neuroprotective effects of antioxidative flavonoids, quercetin, (+)-dihydroquercetin and quercetin 3-methyl ether, isolated from Opuntia Ficus indica var. saboten. Brain Research. 2003. 965(1–2):130-136.
128. Sermboonpaisarn, T. and Sawasdee, P. Potent and selective butyrylcholinesterase inhibitors from Ficus foveolata. Fitoterapia. 2012. 83(4):780-784.
129. Mu, L. H., Li, J. B., Yang, J. Z., and Zhang, D. M. New dibenz[ b, f] oxepins from Cercis chinensis Bunge. Journal of Asian Natural Products Research. 2007. 9(7):649-653.
130. Van, B. M., Daenens, P., Cokelaere, M. M., and Janssen, G. Isolation and Structure Elucidation of the Major Simmondsin Analogs in Jojoba Meal by Two-Dimensional NMR Spectroscopy. Journal of Agricultural Food Chemistry. 1994. 42:2684-7.
131. Baumgartner, S., Genner-Ritzmann, R., Haas, J., Amado, R., and Neukom, H. Isolation and identification of cyclitols in carob pods (Ceratonia siliqua L.). Journal of Agricultural Food Chemistry. 1986. 34:827-9.
132. Binder, R. G. and Haddon, W. F. Cyclitols of soybean leaves. Journal of Agricultural Food Chemistry. 1984. 32:685-7.
133. Seigler, D. S., Pauli, G. F., Fröhlich, R., Wegelius, E., Nahrstedt, A., Glander, K. E., and Ebinger, J. E. Cyanogenic glycosides and menisdaurin from Guazuma ulmifolia, Ostrya virginiana, Tiquilia plicata, and Tiquilia canescens. Phytochemistry. 2005. 66(13):1567-1580.
134. Bachmann, T. L., Ghia, F., and Torssell, K. B. G. Lignans and lactones from Phyllanthus anisolobus. Phytochemistry. 1993. 33(1):189-191. 172
135. Mukherjee, R. and Axt, E. M. Cyclitols from Croton celtidifolius. Phytochemistry. 1984. 23(11):2682-2684.
136. Krishnaswamy, N. R., Prasanna, S., Seshandri, T. R., and Vedantham, T. N. C. α- and β-Amyrin esters and sitosterol glucoside from Spilanthes acmella. Phytochemistry. 1975. 14(7):1666-1667.
137. Miranda, R. R. S., Silva, G. D. F., Duarte, L. P., Fortes, I. C. P., and Filho, S. A. V. Structural determination of 3β-stearyloxy-urs-12-ene from Maytenus salicifolia by 1D and 2D NMR and quantitative 13C NMR spectroscopy. Magnetic Resonance Chemistry. 2006. 44(2):127-131.
138. Saxena, V. K. and Albert, S. β-Sitosterol-3-O-β-D-xylopyranoside from the flowers of Tridax procumbens Linn. Journal of Chemical Sciences. 2005. 117(3):263-266.
139. Yokosuka, A., Kawakami, S., Haraguchi, M., and Mimaki, Y. Seven new triterpene glycosides from the pericarps of Stryphnodendron fissuratum. Phytochemistry Letters. 2011. 4:259-266.
140. Anjoo Kamboj and Ajay Kumar Saluja. Isolation of stigmasterol and β- sitosterol from petroleum ether extract of aerial parts of Ageratum conyzoides (Asteraceae). International Journal of Pharmacy and Pharmaceutical Sciences. 2011. 3(1):94-96.
141. Kumar, R., Bansal, V., Tiwari, A. K., Sharma, M., Puri, S. K., Patel, M. B., and Sarpal, A. S. Estimation of Glycerides and Free Fatty Acid in Oils Extracted From Various Seeds from the Indian Region by NMR Spectroscopy. Journal of the American Oil Chemists' Society. 2011. 88(11):1675-1685.
142. Zaka, S., Saleem, M., Shakir, N., and Khan, S. A. Fatty Acid Composition of Bauhinia variegata and Bauhinia malabarica Seed Oils - Comparison of their Physico-Chemical Properties. Fette, Seifen, Anstrichmittel. 1983. 85(4):169- 170. 173
143. Termentzi, A., Zervou, M., and Kokkalou, E. Isolation and structure elucidation of novel phenolic constituents from Sorbus domestica fruits. Food Chemistry. 2009. 116(1):371-381.
144. Wilfred, V. and Ralph, N. Phenolic Compound Biochemistry Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. 2006.p. pg 15-18.
145. Yang, F. and Cao, Y. Biosynthesis of phloroglucinol compounds in microorganisms - A review. Applied Microbiology and Biotechnology. 2012. 93(2):487-495.
146. Achkar, J., Xian, M., Zhao, H., and Frost, J. W. Biosynthesis of phloroglucinol. Journal of the American Chemical Society. 2005. 127(15):5332-5333.
147. Buhlmann, P. Structure determination of organic compounds: tables of spectral data: Springer. 2009.p.
148. Zeng, X., Qiu, Q., Jiang, C., Jing, Y., Qiu, G., and He, X. Antioxidant flavanes from Livistona chinensis. Fitoterapia. 2011. 82(4):609-614.
149. Martin, T., Kikuzaki, H., Hisamoto, M., and Nakatani, N. Constituents of Amomum tsao-ko and their radical scavenging and antioxidant activities. Journal of the American Oil Chemists' Society. 2000. 77(6):667-673.
150. Neal, J. M., Sato, P. T., and McLaughlin, J. L. Cactus alkaloids. XI. Isolation of tyramine, N-methyltyramine, and hordenine from Obregonia denegrii. Economic Botany. 1971. 25(4):382-384.
151. Supaluk, P., Puttirat, S., Rungrot, C., Somsak, R., and Virapong, P. New bioactive triterpenoids and antimalarial activity of Diospyros rubra Lec. EXCLI Journal. 2009. 9:1-10.
152. Dai, J. and Mumper, R. J. Plant Phenolics: Extraction, Analysis and Their Antioxidant and Anticancer Properties Molecules. 2010. 15:7313-7352. 174
153. Aktumsek, A., Zengin, G., Guler, G. O., Cakmak, Y. S., and Duran, A. Antioxidant potentials and anticholinesterase activities of methanolic and aqueous extracts of three endemic Centaurea L. species. Food and Chemical Toxicology. 2013. 55:290-296.
154. Argolo, A. C. C., Sant'Ana, A. E. G., Pletsch, M., and Coelho, L. C. B. B. Antioxidant activity of leaf extracts from Bauhinia monandra. Bioresource Technology. 2004. 95(2):229-233.
155. Torey, A., Sasidharan, S., Latha, L. Y., Sudhakaran, S., and Ramanathan, S. Antioxidant activity and total phenolic content of methanol extracts of Ixora coccinea. Pharmaceutical Biology. 2010. 48(10):1119-1123.
156. Karagözler, A. A., Erdağ, B., Emek, Y. Ç., and Uygun, D. A. Antioxidant activity and proline content of leaf extracts from Dorystoechas hastata. Food Chemistry. 2008. 111(2):400-407.
157. Compaoré, M., Lamien, C. E., Lamien-Meda, A., Vlase, L., Kiendrebeogo, M., Ionescu, C., and Nacoulma, O. G. Antioxidant, xanthine oxidase and lipoxygenase inhibitory activities and phenolics of Bauhinia rufescens Lam. (Caesalpiniaceae). Natural Product Research. 2011. 26(11):1069-1074.
158. Alimi, H., Hfaiedh, N., Bouoni, Z., Hfaiedh, M., Sakly, M., Zourgui, L., and Rhouma, K. B. Antioxidant and antiulcerogenic activities of Opuntia Ficus indica f. inermis root extract in rats. Phytomedicine. 2010. 17(14):1120-1126.
159. Othman, A., Ismail, A., Ghani, N. A., and Adenan, I. Antioxidant capacity and phenolic content of cocoa beans. Food Chemistry. 2007. 100:1523–1530.
160. Asuero, A., Sayago, A., and Gonzalez, A. The correlation coefficient: An overview. Critical reviews in analytical chemistry. 2006. 36(1):41-59.
161. Taylor, R. Interpretation of the correlation coefficient: a basic review. Journal of diagnostic medical sonography. 1990. 6(1):35-39. 175
162. Manilal, A., Sujith, S., Seghal, G. K., Selvin, J., and Shakir, C. Cytotoxic Potentials of Red Alga, Laurencia brandenii Collected from the Indian Coast. Global Journal of Pharmacology. 2009. 3(2):90-94.
163. Mukherjee, P. K., Kumar, V., Mal, M., and Houghton, P. J. Acetylcholinesterase inhibitors from plants. Phytomedicine. 2007. 14(4):289- 300.
164. Heinrich, M. and Lee Teoh, H. Galanthamine from snowdrop—the development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. Journal of Ethnopharmacology. 2004. 92(2–3):147- 162.
165. Bertanha, C. S., Braguine, C. G., Moraes, A. C. G., Gimenez, V. M. M., Groppo, M., Silva, M. L. A., Cunha, W. R., Januário, A. H., and Pauletti, P. M. Cyclooxygenase inhibitory properties of nor-neolignans from Styrax pohlii. Natural Product Research. 2012. 26(24):2323-2329.
166. Blobaum, A. L. and Marnett, L. J. Structural and Functional Basis of Cyclooxygenase Inhibition. Journal of Medicinal Chemistry. 2007. 50(7):1425-1441.
167. Oja, V., Chen, X., Hajaligol, M. R., and Chan, W. G. Sublimation Thermodynamic Parameters for Cholesterol, Ergosterol, β-Sitosterol, and Stigmasterol. Journal of Chemical and Engineering Data. 2008. 54(3):730- 734.
168. Jo, S., Lachumy, T., Sasidharan, S., Sumathy, V., and Zuraini, Z. Pharmacological activity, phytochemical analysis and toxicity of methanol extract of Etlingera elatior (torch ginger) flowers. Asian Pacific Journal of Tropical Medicine. 2010. 3(10):769-774.
169. Karaman, İ., Şahin, F., Güllüce, M., Ögütçü, H., Şengül, M., and Adıgüzel, A. Antimicrobial activity of aqueous and methanol extracts of Juniperus oxycedrus L. Journal of Ethnopharmacology. 2003. 85(2–3):231-235. 176
170. Kubo, I., Kinst-Hori, I., Chaudhuri, S. K., Kubo, Y., Sánchez, Y., and Ogura, T. Flavonols from Heterotheca inuloides: Tyrosinase Inhibitory Activity and Structural Criteria. Bioorganic and Medicinal Chemistry. 2000. 8(7):1749- 1755.
171. Yang, Z., Zhang, D., Ren, J., Yang, M., and Li, S. Acetylcholinesterase inhibitory activity of the total alkaloid from traditional Chinese herbal medicine for treating Alzheimer’s disease. Medicinal Chemistry Research. 2012. 21(6):734-738.