ix
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENT
iv
PREFACE
v
ABSTRACT
vii
ABSTRAK
viii
TABLE OF CONTENTS
ix
LIST OF TABLES
xiii
LIST OF SCHEMES
xv
LIST OF FIGURES
xvi
LIST OF ABBREVIATIONS/SYMBOLS
xvii
LIST OF APPENDICES
xix
INTRODUCTION
1.1 General Introduction
1
1.2 Family of Thymelaeaceae
2
1.3 Genus Phaleria
3
1.4 Statement of Problem
3
1.5 Research Objectives
4
1.6 Scope of Study
4
1.7 Significance of Study
5
LITERATURE REVIEWS
2.1 Occurrence of Phaleria macrocarpa (Scheff.) Boerl.
6
x
2.2 Botanical Description of Phaleria macrocarpa
6
(Scheff.) Boerl.
2.3 Traditional Usage of Phaleria macrocarpa (Scheff.)
8
Boerl.
2.4 Phytochemicals Studies of Phaleria macrocarpa
8
(Scheff.) Boerl.
2.5 Bioactivities Studies of Phaleria macrocarpa
14
(Scheff.) Boerl.
3
2.5.1 Cytotoxicity and Anticancer Activity
14
2.5.2 Antidiabetic Activities
16
2.5.3 Anti-Inflammatory Activities
17
2.5.4 Antioxidant Activities
17
2.5.5 Antifungal and Antibacterial Activities
18
2.5.6 Others Bioactivities Studies
18
PHYTOCHEMICAL
STUDIES
OF
Phaleria
macrocarpa (Scheff.) Boerl. FRUITS AND LEAVES
3.1 Phytochemical Studies of the Fruits of Phaleria
20
macrocarpa
3.1.1 2,6,4'-Trihydroxy-4-methoxybenzophenone
(8)
20
3.1.2 6,4'-Dihydroxy-4-methoxybenzophenone-2-
24
O-β-D-glucopyranoside (7)
3.1.3 24-Methylenecycloartan-3-one (37)
28
3.1.4 24-Methyl-9,19-cyclolanost-25-en-3-ol (38)
30
3.2 Phytochemical Studies of the Leaves of Phaleria
33
macrocarpa
4
3.2.1 Stigmasta-5,23,dien-3β-ol (12)
33
3.2.2 β-Sitosterol (39)
36
BIOACTIVITIES OF Phaleria macrocarpa (Scheff.)
Boerl. FRUITS AND LEAVES
4.1 Antibacterial Activity
39
xi
4.1.1 Disc Diffusion Method
39
4.1.2 Minimum Inhibitory Concentration (MIC) and
43
Minimum Bactericidal Concentration (MBC)
4.2 Antioxidant Activity
4.2.1 DPPH Free Radical Scavenging Activity
4.3 Cytotoxic Activity
4.3.1 MTT Assay
5
45
45
48
49
EXPERIMENTAL
5.1 General Experimental Procedures of Phytochemicals
52
Study
5.1.1 Instrumentations for Phytochemicals Study
52
5.1.2 Chemicals and Reagents for Phytochemicals
53
Study
5.1.3 Plants Materials
5.2 Extraction and Isolation of Chemical Constituents
53
54
from the Fruits of Phaleria macrocarpa
5.2.1 2,6,4'-Trihydroxy-4-methoxybenzophenone
(8)
5.2.2 6,4'-Dihydroxy-4-methoxybenzophenone-2-
55
55
O-β-D-glucopyranoside (7)
5.2.3 24-Methylenecycloartan-3-one (37)
56
5.2.4 24-Methyl-9,19-cyclolanost-25-en-3-ol (38)
56
5.3 Extraction and Isolation of Chemical Constituents
57
from the Leaves of Phaleria macrocarpa
5.3.1 Stigmasta-5,23,dien-3β-ol (12)
58
5.3.2 β-Sitosterol (39)
58
5.4 Antibacterial Assay
59
5.4.1 Bacterial Strains
59
5.4.2 Chemicals and Reagents for Antibacterial
60
Assay
5.4.3 Bacteria Stock Solution
60
5.4.4 Disc Diffusion Method
60
xii
5.4.5 Minimum Inhibition Concentration (MIC)
61
5.4.6 Minimum Bactericidal Concentration (MBC)
62
5.5 Antioxidant Activity
5.5.1 Chemicals and Reagents for Antioxidant
62
63
Assay
5.5.2 DPPH Free Radical Scavenging Assay
5.6 Cytotoxic activity
5.6.1 Chemicals and Reagents for Cell Cultures and
63
64
64
MTT Assay
6
5.6.2 Cell Lines
64
5.6.3 Cell Cultures
65
5.6.4 Subculture of Monolayer Cells
67
5.6.5 Optimizing Seeding Density
68
5.6.7 MTT Assay
69
5.7 Statistical Analysis
70
CONCLUSION AND RECOMMENDATIONS
6.1 Conclusion
71
6.2 Recommendations
72
REFERENCES
73-85
Appendices 1 - 54
86-139
xiii
LIST OF TABLES
TABLE NO.
3.1
PAGE
TITLE
Comparison on 1H and
13
C NMR Data of 2,6,4'-
24
Trihydroxy-4-methoxybenzophenone and Compound
(8)
3.2
Comparison on
1
H and
13
C NMR data of 6,4'-
28
Dihydroxy-4-methoxybenzophenone-2-O-β-D-glucopyranoside [38] and Compound (7)
3.3
The 1H and 13C NMR Data of Compound (37)
30
3.4
Summary of 1H and
C NMR Data of Compound
32
Comparison of NMR Data of Stigmasterol [67] and
35
13
(38)
3.5
Compound (12)
3.6
Comparison of NMR Data of β-Sitosterol [69] and
38
Compound (39)
4.1
Antibacterial Activity of the Extracts and Isolated
40
Compounds from P. macrocarpa Fruits and Leaves
by Disc Diffusion Method
4.2
Minimum Inhibitory Concentration (MIC) and
44
Minimum Bactericidal Concentration (MBC) of the
Extracts
and
Isolated
Compounds
from
P.
macrocarpa Fruits and Leaves
4.3
Summary of SC50 Values of Extracts and Isolated
46
Compounds from P. macrocarpa against DPPH
4.4
Summary of IC50 Values of Extracts and Isolated
50
Compounds against Normal and Cancer Cell Lines
5.1
Extracts from the Fruits of P. macrocarpa
54
xiv
5.2
Extracts from the Leaves of P. macrocarpa
57
5.3
The Specific Seeding Density of Each Cell Lines for
69
MTT Assay
xv
LIST OF SCHEMES
SCHEME NO.
TITLE
PAGE
3.1
Mass Fragmentation Pattern of Compound (7)
25
4.1
Mechanism on the Reduction of DPPH
46
4.2
Reduction of MTT to Formazan Crystal [89]
49
xvi
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
2.1
P. macrocarpa tree with unripened green fruits
7
2.2
(a) green tapering leave; (b) red fruit; (c) seed of P.
7
macrocarpa
3.1
COSY Correlations of Compound (8) with Ortho
22
Coupling
3.2
HMBC Correlations of Compound (8)
23
3.3
HMBC Correlations of Compound (7)
27
5.1
Arrangement of Sample Discs on the Petri Dish
61
5.2
Morphology of CHO cells observed by inverted
65
microscopy with 10× magnification
5.3
Morphology of MCF-7 cells observed by inverted
65
microscopy with 10× magnification
5.4
Morphology of MDA-MB-231 cells observed by
66
inverted microscopy with 10× magnification
5.5
Morphology of MDA-MB-468 cells observed by
66
inverted microscopy with 10× magnification
5.6
Morphology of HeLa cells observed by inverted
66
microscopy with 10× magnification
5.7
Morphology of HT-29 cells observed by inverted
67
microscopy with 10× magnification
5.8
Morphology of 3T3 cells observed by inverted
67
microscopy with 10× magnification
5.9
Summary of Subculture of Monolayer Cells [101]
68
xvii
LIST OF ABBREVIATIONS/SYMBOLS
acetone- d6
- Deuterated acetone
br
- Broad
°C
- Degree celcius
13
- Carbon Nuclear Magnetic Resonance
CC
- Column Chromatography
CDCl3
- Deuterated chloroform
cm-1
- Per centimeter
CO2
- Carbon dioxide
COSY
- Correlation Spectroscopy
δ
- Chemical shift
d
- Doublet
dd
- Doublet of doublet
DEPT
- Distortionless Enhancement by Polarization Transfer
DMEM
- Dulbeccos Modified Eagles Medium
DMSO
- Dimethyl sulfoxide
DPPH
- 2,2-Diphenyl-1-picrylhydrazyl
EIMS
- Electron Impact Mass Spectrometry
FBS
- Fetal bovine serum
FTIR
- Fourier Transform Infrared
GC
- Gas Chromatography
GC-MS
- Gas Chromatography-Mass Spectrometry
1
- Proton Nuclear Magnetic Resonance
C NMR
H NMR
HMBC
- Heteronuclear Multiple Bond Correlation
HMQC
- Heteronuclear Multiple Quantum Coherence
Hz
- Hertz
IC50
- Concentration of substrate that causes 50% growth
inhibition of cell
IR
- Infrared
J
- Coupling constant
xviii
KBr
- Potassium bromide
L
- Liter
lit.
- Literature
λ
- Lambda
M+
-
m
- Multiplet
m/z
- Mass to charge ion
mg
- Milligram
mL
- Milliliter
m.p.
- Melting point
μg
- Microgram
MBC
- Minimum Bactericidal Concentration
MIC
- Minimum Inhibitory Concentration
MTT
- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Molecular ion
bromide
NA
- Nutrient agar
NB
- Nutrient broth
NMR
- Nuclear Magnetic Resonance
nm
- Nanometer
ppm
- Part per million
Rf
- Retention factor
SiO2
- Silica gel
S
- Singlet
SC50
- Radical scavenging activity at concentration of 50%
RPMI 1640
- Roswell Park Memorial Institute 1640
t
- Triplet
tR
- Retention time
TLC
- Thin Layer Chromatography
UV
- Ultraviolet
VLC
- Vacuum Liquid Chromatography
xix
LIST OF APPENDICES
APPENDIX
NO.
TITLE
PAGE
1
HRESI-MS Spectrum of 2,6,4ꞌ-Trihydroxy-4-methoxy-
86
benzophenone (8)
2
UV(MeOH) Spectrum of 2,6,4ꞌ-Trihydroxy-4-methoxy-
87
benzophenone (8)
3
Infrared Spectrum of 2,6,4ꞌ-Trihydroxy-4-methoxy-
88
benzophenone (8)
4
1
H NMR Spectrum of 2,6,4ꞌ-Trihydroxy-4-methoxy-
89
benzophenone (8)
5
COSY Spectrum of 2,6,4ꞌ-Trihydroxy-4-methoxybenzo-
90
phenone (8)
6
13
C NMR Spectrum of 2,6,4ꞌ-Trihydroxy-4-methoxy-
91
benzophenone (8)
7
DEPT Spectra of 2,6,4ꞌ-Trihydroxy-4-methoxybenzo-
92
phenone (8)
8
HMQC
Spectrum
of 2,6,4ꞌ-Trihydroxy-4-methoxy-
93
of
2,6,4ꞌ-Trihydroxy-4-methoxy-
94
HRESI-MS Spectrum of 6,4'-Dihydroxy-4-methoxy-
95
benzophenone (8)
9
HMBC
Spectrum
benzophenone (8)
10
benzophenone-2-O-β-D-glucopyranoside (7)
11
UV(MeOH) Spectrum of 6,4'-Dihydroxy-4-methoxybenzo-
96
phenone-2-O-β-D-glucopyranoside (7)
12
Infrared Spectrum of 6,4'-Dihydroxy-4-methoxybenzophenone-2-O-β-D-glucopyranoside (7)
97
xx
13
1
H NMR Spectrum of 6,4'-Dihydroxy-4-methoxybenzo-
98
phenone-2-O-β-D-glucopyranoside (7)
14
COSY NMR Spectrum of 6,4'-Dihydroxy-4-methoxy-
99
benzophenone-2-O-β-D-glucopyranoside (7)
15
13
C NMR Spectrum of 6,4'-Dihydroxy-4-methoxy-
100
benzophenone-2-O-β-D-glucopyranoside (7)
16
DEPT Spectra of 6,4'-Dihydroxy-4-methoxybenzo-
101
phenone-2-O-β-D-glucopyranoside (7)
17
HMQC Spectrum of 6,4'-Dihydroxy-4-methoxybenzo-
102
phenone-2-O-β-D-glucopyranoside (7)
18
HMBC Spectrum of 6,4'-Dihydroxy-4-methoxybenzo-
103
phenone-2-O-β-D-glucopyranoside (7)
19
Infrared Spectrum of 24-Methylenecycloartan-3-one
104
(37)
20
1
H NMR Spectrum of 24-Methylenecycloartan-3-one
105
(37)
21
13
C NMR Spectrum of 24-Methylenecycloartan-3-one
106
(37)
22
DEPT Spectra of 24-Methylenecycloartan-3-one (37)
107
23
Gas
24-Methylene-
108
GC-MS Spectrum of 24-Methylenecycloartan-3-one
109
Chromatogram
Spectrum
of
cycloartan-3-one (37)
24
(37)
25
Infrared Spectrum of 24-Methyl-9,19-cyclolanost-25-
110
en-3-ol (38)
26
1
H NMR Spectrum of 24-Methyl-9,19-cyclolanost-25-
111
en-3-ol (38)
27
13
C NMR Spectrum of 24-Methyl-9,19-cyclolanost-25-
112
en-3-ol (38)
28
DEPT Spectra of 24-Methyl-9,19-cyclolanost-25-en-3-
113
ol (38)
29
Gas Chromatogram Spectrum of 24-Methyl-9,19cyclolanost-25-en-3-ol (38)
114
xxi
30
GC-MS Spectrum of 24-Methyl-9,19-cyclolanost-25-en-
115
3-ol (38)
31
Infrared Spectrum of Stigmasta-5,22,dien-3β-ol (12)
116
32
1
117
33
13
118
34
DEPT Spectra of Stigmasta-5,23,dien-3β-ol (12)
119
35
GC-MS Spectrum of Stigmasta-5,22-dien-3β-ol (12)
120
36
Infrared Spectrum of β-Sitosterol (39)
121
37
1
122
38
13
123
39
DEPT Spectra of β-Sitosterol (39)
124
40
Gas Chromatogram Spectrum of β-Sitosterol (39)
125
41
GC-MS Spectrum of β-Sitosterol (39)
126
42
Free Radical Scavenging Activity of Extracts of
127
H NMR Spectrum of Stigmasta-5,22-dien-3β-ol (12)
C NMR Spectrum of Stigmasta-5,22-dien-3β-ol (12)
H NMR Spectrum of β-Sitosterol (39)
C NMR Spectrum of β-Sitosterol (39)
P. macrocarpa at Different Concentrations
43
Free
Radical
Scavenging
Activity
of
Isolated
128
Compounds from P. macrocarpa and Positive Control at
Different Concentrations
44
Cytotoxic Study of Extracts against CHO Cell Line at
129
Different Concentrations
45
Cytotoxic Study of Isolated Compounds against CHO
130
Cell Line at Different Concentrations
46
Cytotoxic Study of Extracts against MCF-7 Cell Line at
131
Different Concentrations
47
Cytotoxic Study of Isolated Compounds against MCF-7
132
Cell Line at Different Concentrations
48
Cytotoxic Study of Extracts against MDA-MB-231 Cell
133
Line at Different Concentrations
49
Cytotoxic Study of Isolated Compounds against MDAMB-231 Cell Line at Different Concentrations
134
xxii
50
Cytotoxic Study of Extracts against MDA-MB-468 Cell
135
Line at Different Concentrations
51
Cytotoxic Study of Isolated Compounds against MDA-
136
MB-468 Cell Line at Different Concentrations
52
Cytotoxic Study of Fruits Extracts and Isolated
137
Benzophenones against 3T3 Cell Line at Different
Concentrations
53
Cytotoxic Study of Fruits Extracts and Isolated
138
Benzophenones against HT-29 Cell Line at Different
Concentrations
54
Cytotoxic Study of Fruits Extracts and Isolated
Benzophenones against HeLa Cell Line at Different
Concentrations
139