CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF Garcinia...

CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF

Garcinia griffithii T. ANDERSON

NIK SHAZWANI AFIFAH BINTI NIK SAZALI

UNIVERSITI TEKNOLOGI MALAYSIA

CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF

Garcinia griffithii T. ANDERSON

NIK SHAZWANI AFIFAH BINTI NIK SAZALI

A thesis submitted in fulfillment of the

requirements for the award of the degree of

Master of Science (Chemistry)

Faculty of Science

Universiti Teknologi Malaysia

APRIL 2014

iii

Dedicated to

My most beloved mother and father

My dearest sisters and brothers

My friends

Who always supportand inspire me all the time.

iv

ACKNOWLEDGEMENTS

First and foremost, I thank Allah S.W.T. for His love and care which kept me

going forward and made me strong to complete this research. I would like to convey

my gratefulness and appreciationto my supervisor, Assoc. Prof. Dr. Farediah Ahmad

for her constant encouragement, generosity, guidance and patient throughout the

completion of this research.

I want to express my gratitude to all Organic Chemistry lectures, thanks for

the opinion and knowledge in helping out in this project. I would like to

acknowledge all the organic master students for all their guidance and assistance in

completion some part of this research. I also insist to thanks all the laboratory

assistances for their helps and cooperation.

I also like to extend my sincere appreciation and heartfelt gratitude to all my

laboratory mates. I am greatly thankful for their willingness to share useful

knowledge, help, ideas, support and guidance given. The moments we spent together

in overcoming the challenges throughout our project have truly making us to know

more each other.

My deepest appreciation goes to my beloved family members especially to

my parents, Nik Sazali Nik Ibrahim and Norizah Busu, who have been my constant

inspirational source to further my studies. Abundle of thousand thanks for their

continuous advices, support and motivationfor me for finish this research well. Not

forgetting to my siblings Nadhirah, Naim, Izzat, Izyan and Fadhli for motivated me.

Last but not least, thank you to those being with me during my master’s study

and also to the entire individual who directly and indirectly helped me in

accomplishing this research.

v

PREFACE

This thesis is the result of my work carried out in the Department of chemistry,

Universiti Teknologi Malaysia under the supervision of Assoc. Prof. Dr. Farediah

Ahmad. Parts of my work described in this thesis have been reported in the

following publications:

1. Nik Shazwani Afifah Nik Sazali and Farediah Ahmad (2011). Chemical

Constituents and Biological Activities of The Leaves of Garcinia Griffithii

(Guttiferae). Poster presented at International Conference on Natural

Products (ICNP) 2011, Palm Garden Hotel, IOI Resort, Putrajaya. 13 – 16

November 2011.

vi

ABSTRACT

Chemical and bioactivity investigations were carried out on the leaves and stem barks of Garcinia griffithii T. Anderson from Guttiferae family. Sample for each part of the plant was extracted consecutively with increasing polarity of solvents by Soxhlet method. Vacuum liquid chromatography and column chromatography were used to purify the crude extracts. The pure compounds were elucidated by using combined spectroscopic techniques which include UV, IR, NMR (1D and 2D) and MS. Chromatographic purification of the leaves extracts have afforded nine pure compounds identified as squalene, 28-hydroxyfriedelan-3-one, friedooleanan-3-one, olean-12-en-3-ol, amento-4'-methyl ether, 3,8''-binaringenin, 3,8''-binaringenin-7''-O-glucoside, morelloflavone and morelloflavone-7''-O-glucoside. Chromatographic purification of the ethyl acetate and methanol extracts of the stem barks yielded two compounds identified as amento-4'-methyl ether and morelloflavone. The crude extracts and pure compounds isolated from methanol crude extract of the leaves were screened for various types of antioxidant assay and tyrosinase inhibition activities. The antioxidant assay on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical showed that the crude n-hexane extract of the stem barks had the highest radical scavenging activity with IC50 = 96.43 ± 2.69 µg/mL, while morelloflavone was found to be the strongest antioxidant compound with IC50 = 57.57 ± 0.53 µg/mL compared to other compounds. The crude methanol extract of the stem barks showed the highest total antioxidant with 260.81 ± 2.21 mg/g of ascorbic acid equivalent (AAE/L) and 871.43 ± 6.62 mg/g of butylated hydroxyl toluene equivalent (BHTE/L) while the crude methanol extract of the leaves showed the highest total phenolic content with 444.10 ± 6.67 mg/g of gallic acid equivalent (GAE/L) and 423.10 ± 6.67 mg/g of (± )-cathechin equivalent (CE/L). Morelloflavone showed the highest value for both assays with values 58.50 ± 3.15 mg/g of AAE/L and 264.50 ± 9.45 mg/g of BHTE/L; and 841.33 ± 38.28 mg/g of GAE/L and 822.97 ± 33.93 mg/g of CE/L, respectively. The crude extracts and all compounds were found to have weak anti-tyrosinase activity. The antimicrobial assay of all the crude extracts were carried out by using disc diffusion method, followed by minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC). The methanol crude extract of the leaves showed the most significant antimicrobial activity towards E. faecalis and K. pneumoniae with MIC and MBC value ranged between 225 – 450 µg/mL compared to the other crude extracts.

vii

ABSTRAK

Kajian kimia dan bioaktiviti telah dijalankan ke atas daun dan batang pokok Garcinia griffithii T. Anderson daripada keluarga Guttiferae. Sampel untuk setiap bahagian pokok telah diekstrak secara berturutan dengan menggunakan pelarut mengikut kekutuban menaik melalui kaedah Soxhlet. Kromatografi turus vakum dan

kromatografi turus graviti telah digunakan untuk penulenan ke atas ekstrak mentah. Struktur sebatian tulen dikenalpasti dengan menggunakan kaedah spektroskopi UV, IR, NMR (1D dan 2D) dan MS. Penulenan dan pencirian ke atas ekstrak mentah n-heksana daripada daun telah Berjaya menemukan Sembilan sebatian tulen yang dikenalpasti sebagai skualena, 28-hidroksifriedelan-3-on, friedooleanan-3-on, olean-12-en-3-ol, amento-4'-metil eter, 3,8''-binaringenin, 3,8''-binaringenin-7''-O-glukosida, morelloflavon dan morelloflavon-7''-O-glukosida. Penulenan secara kromatografi ke atas ekstrak etil asetat dan methanol batang pokok menghasilkan dua sebatian yang dikenalpasti sebagai amento-4'-metil eter dan morelloflavon. Ekstrak mentah dan sebatian tulen yang telah diasingkan daripada ekstrak mentah methanol daun disaring untuk beberapa ujian antioksidan dan aktiviti rencatan tirosinase. Ujian antioksidan ke atas radikal bebas 2,2-difenil-1-pikrilhidrazil (DPPH) menunjukkan bahawa ekstrak mentah n-heksana daripada batang pokok mempunyai aktiviti radikal tertinggi dengan nilai IC50 = 96.43 ± 2.69 µg/mL, manakala sebatian morelloflavon ditemui sebagai sebatian antioksidan terkuat dengan nilai IC50 = 57.57 ± 0.53 µg/mL berbanding dengan sebatian lain. Ekstrak mentah methanol daripada batang pokok menunjukkan nilai jumlah antioksidan yang paling tinggi dengan nilai 260.81 ± 2.21 mg/g setara dengan asid askorbik (AAE/L) dan 871.43 ± 6.62 mg/g setara dengan butil hidroksitoluena (BHTE/L) manakala ekstrak mentah methanol daripada daun mempunyai jumlah kandungan fenol yang paling tinggi, iaitu 444.10 ± 6.67 mg/g setara dengan asid galik (GAE/L) and 423.10 ± 6.67 mg/g setara dengan (± )-katekin (CE/L). Sebatian morelloflavon menunjukkan nilai tertinggi bagi kedua-dua saringan masing-masing dengan nilai 58.50 ± 3.15 mg/g AAE/L dan 264.50 ± 9.45 mg/g BHTE/L; dan 841.33 ± 38.28 mg/g GAE/L dan 822.97 ± 33.93 mg/g CE/L. Ekstrak mentah dan kesemua sebatian tulen didapati mempunyai aktiviti anti-tirosinase yang lemah. Saringan antimikrob telah dijalankan ke atas ekstrak mentah dengan menggunakan kaedah pembauran cakera, diikuti dengan penentuan nilai rencatan minimum (MIC) dan kepekatan bakterisida minimum (MBC). Ekstrak mentah methanol daripada daun menunjukkan aktiviti antimikrob yang paling signifikan terhadap E. faecalis and K. pneumonia dengan nilai MIC dan MBC antara 225 – 450 µg/mL berbanding dengan ekstrak mentah yang lain.

viii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION

ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

PREFACE v

ABSTRACT vi

ABSTRAK vii

TABLE OF CONTENTS viii

LIST OF TABLES xiii

LIST OF FIGURES xv

LIST OF SCHEMES xvi

LIST OF ABBREVIATIONS xvii

LIST OF APPENDICES xix

1 INTRODUCTION

1.1 General Introduction 1

1.2 Guttiferae Family 2

1.3 Garcinia griffithii 4

1.4 Problem Statement 5

1.5 Objectives of Research 5

1.6 Scope of Research 5

2 LITERATURE REVIEW

2.1 Phytochemical Studies on Genus Garcinia 7

2.1.1 Xanthones 7

ix

2.1.2 Biflavanoids 15

2.1.3 Benzophenones 17

2.1.4 Triterpenoids 19

2.1.5 Other Phytochemicals 24

2.2 Bioactivity Studies on Genus Garcinia 25

3 RESULTS AND DISCUSSION

3.1 The Leaves of Garcinia griffithii 27

3.1.1 Squalene (116) 28

3.1.2 28-Hydroxyfriedelan-3-one (117) 30

3.1.3 Friedooleanan-3-one (73) 32

3.1.4 Olean-12-en-3-ol (118) 35

3.1.5 Amento-4'-methyl ether (119) 38

3.1.6 3,8''-Binaringenin (53) 43

3.1.7 3,8''-Binaringenin-7''-O-glucoside (120) 47

3.1.8 Morelloflavone (51) 49

3.1.9 Morelloflavone-7''-O-glucoside (55) 54

3.2 The Stem Barks of Garcinia griffithii 58

4 BIOACTIVITY STUDIES

4.1 Antioxidant Assay (DPPH Free Radical

Scavenging Activity)

58

4.2 Total Antioxidant Assay 61

4.3 Total Phenolic Content Assay 62

4.4 Tyrosinase Inhibitor Activity 64

4.5 Antimicrobial Assay 66

4.5.1 Disc Diffusion Susceptibility 66

4.5.2 Minimum Concentration Inhibitory

(MIC) and Minimum Bactericidal

Concentration (MBC)

67

5 EXPERIMENTAL

5.1 Apparatus and Chemicals 70

x

5.2 Plant Material 71

5.3 Extraction and Isolation of Chemical

Constituents from the Leaves of G. griffithii

72

5.3.1 Fractionation and Purification of the

n-Hexane Crude Extract

72

5.1.1.1 Squalene (116) 72

5.3.1.2 28-Hydroxyfriedelan-3-one (117) 73

5.3.1.3 Friedooleanan-3-one (73) 73

5.3.1.4 Olean-12-en-3-ol (118) 74


CH2Cl2 Crude Extract

75


MeOH Crude Extract

75

5.3.3.1 Amentoflavone-4'-methyl ether

(119)

76

5.3.3.2 3,8''-Binaringenin(53) 76

5.3.3.3 Morelloflavone(51) 77

5.3.3.4 3,8''-Binaringenin-7''-O-glucoside

(120)

78

5.3.3.5 Morelloflavone-7''-O-glucoside

(55)

79

5.4 Extraction and Isolation of Chemical

Constituents from the Stem Barks of

G. griffithii

80


EtOAc Crude Extract

80


MeOH Crude Extract

81

5.5 Antioxidant Assay (DPPH Free Radical

Scavenging Activity)

81

5.5.1 Preparation of 50 µM DPPH Solution 82

5.5.2 Preparation of Control and Samples 82

5.5.3 DPPH Radical Scavenging 83

xi

5.6 Total Antioxidant Assay 86

5.6.1 Preparation of Reagent Solution 86

5.6.2 Preparation of Standard Solutions 88

5.6.3 Preparation of Samples 89

5.6.4 Total Antioxidant Assay 89

5.7 Total Phenolic Content Assay 91

5.7.1 Preparation of 20% Na2CO3 Solution

and Standard Solutions

92

5.7.2 Preparation of Samples 92

5.7.3 Total Phenolic Content Assay

Screening

93

5.8 Tyrosinase Inhibitor Activity 95

5.8.1 Preparation of 0.1 M Phosphate

Buffer Solution (pH 6.8 in 1L)

95

5.8.2 Preparation of Tyrosinase Enzyme 96

5.8.3 Preparation of 2.5 mM L-Dopa 96

5.8.4 Preparation of Standard Solution and

Samples

97

5.8.5 Tyrosinase Assay Screening Method 97

5.9 Antimicrobial Assay 100

5.9.1 Apparatus and Microorganism 100

5.9.2 Preparation of Agar, Broth and Samples 100

5.9.3 Culturing Bacteria 101

5.9.4 Preparation of Agar Plate 101

5.9.5 Preparation of McFarland Solution 101

5.9.6 Disc Diffusion Assay 102

5.9.7 Minimum Inhibition Concentration

(MIC)

102

5.9.8 Minimum Bactericidal Concentration

(MBC)

103

6 CONCLUSION

6.1 Conclusion 104

xii

6.2 Future Works 106

REFERENCES 107

Appendices 1-79 118

xiii

LIST OF TABLES

TABLE NO. TITLE PAGE

3.1

The Comparison of 13C NMR Data of Olean-12-en-3-ol

(118)

36

3.2 The NMR Data of Amentoflavone-4'-methyl ether (119) 41

3.3 The NMR Data of 3,8''-Binaringenin (53) 45

3.4 The NMR Data of 3,8''-Binaringenin-7''-O-glucoside

(120)

48

3.5 The 1H NMR Data of Morelloflavone (51) and Its

Isomer

51

3.6 The NMR Data of Morelloflavone (51) 52

3.7 The NMR Data of Morelloflavone-7''-O-glucoside (55) 56

4.1 The IC50 of Ascorbic Acid and Crude Extracts of

The Leaves and Stem Barks of G. griffithii and Pure

Compounds

60

4.2 Average (AAE/L) and (BHTE/L) of Crude Extracts

and Pure Compounds of The Leaves and Stem Barks

of G. griffithii

62

4.3 Average (GAE/L) and (CE/L) of Crude Extracts and

Pure Compounds of The Leaves and Stem Barks of

G. griffithii

63

4.4 Inhibition Percentage (I%) of the Crude Extracts and

Pure Compounds of The Leaves and Stem Barks of

G. griffithii

65

xiv

4.5 Inhibition Zone of The Crude Extracts of The

Leaves and Stem Barks of G. griffithii for Gram-

Positive and Gram-Negative Bacteria

66

4.6 MIC of The Crude Extract of The Leaves and Stem

Barks of G. griffithii

67

4.7 MBC of The Crude Extract of The Leaves and Stem

Barks of G. griffithii

67

5.1 Inhibiton Percentage (I%) of Acid Ascorbic and

Samples of G. griffithii for Each Concentration

84

5.2 Average Absorbance (A) of Ascorbic acid and BHT

for Each Concentration

90

5.3 (AAE/L) and (BHTE/L) of Crude Extracts and Pure

Compounds from The Leaves and Stem Barks of

G. griffithii

91

5.4 Average Absorbance (A) of Gallic acid and

(±)-Cathechin for Each Concentration

93

5.5 (GAE/L) and (CE/L) of Crude Extracts and Pure


G. griffithii

94

5.6 Absorbances (A) of Crude Extracts and Pure


G. griffithii

99

xv

LIST OF FIGURES

FIGURE NO. TITLE PAGE

3.1

The HMBC Correlation of Compound (119)

(a) between ring B and ring D; (b) between

ring A and ring C; and (c) between ring E and

ring F.

41

3.2 The HMBC Correlation of Compound (53) (a)

between protons at ring A; (b) between ring B

and ring C; and (c) between ring E and ring F.

46

3.3 The HMBC Correlation of Compound (51) (a)

between ring C, D and F; (b) between ring A,

B and ring C; and (c) between ring E and ring

F.

54

3.4 The HMBC Correlation of Compound (55) 56

5.1 Linear Equation Obtained from the Graph of

Average Absorbance Against Concentration of

Ascorbic Acid and BHT

90

5.2 Linear Equation Obtained from the Graph of

Average Absorbance Against Concentration

for Gallic Acid and (±)-Catechin

93

5.3 Arrangement Inside A 96-Wells Microtiter

Plate for One Sample

98

5.4 Disc Arrangement Inside A Petri Disc 102

6.1 Summary of the Chemical Constituent Isolated

from The Leaves and Stem Barks of G. griffithii

105

xvi

LIST OF SCHEMES

SCHEME NO. TITLE PAGE

3.1

Mass Fragmentation Patterns of Compound (116)

28

3.2 Mass Fragmentation Patterns of Compound (117) 31



4.1 The Reduction Reaction of DPPH 60

xvii

LIST OF ABBREVIATIONS

AAE/L - Ascorbic acid equivalent

BHTE/L - Butylated hydroxytoluene equivalent

br - Broad

CE/L - (±)-Catechin equivalent

°C - Degree celcius 13C - Carbon 13

CC - Column Chromatography

CDCl3 - Deuterated Chloroform

CHCl3 - Chloroform

CH2Cl2 - Dichloromethane

CIMS - Chemical Ionization Mass Spectrometry

COSY - Correlation spectroscopy

δ - Chemical shift

cm-1 - Per centimeter

d - Doublet

dd - Doublet of doublet

DEPT - Distortionless Enhancement by Polarization Transfer

DMSO - Dimethyl sulfoxide

DPPH - 2,2-Diphenyl-1-picryhydrazyl

EIMS - Electron Impact Mass Spectrometry

Et2O - Diethyl ether

EtOAc - Ethyl acetate

EtOH - Ethanol

FTIR - Fourier Transform Infrared

GAE/L - Gallic acid equivalent 1H - Proton

xviii

H2O - Water

H3BO3 - Boric acid

HMBC - Heteronuclear Multiple Bond Correlation

HMQC - Heteronuclear Multiple Quantum Coherence

Hz - Hertz

IC50 - Inhibition concentration for 50%

IR - Infrared

J - Coupling constant

KBr - Potassium bromide

L - Liter

λ - Lambda

m/z - Mass to charge ratio

m - Multiplet

m.p. - Melting point

µg - Microgram

mg - Miligram

mL - Mililiter

MBC - Minimum bactericidal concentration

MeOH - Methanol

MIC - Minimum inhibitory concentration

nm - Nanometer

NA - Nutrient agar

NB - Nutrient broth

NMR - Nuclear Magnetic Resonance

ppm - Parts per million

quint - Quintet

Rf - Retention factor

s - Singlet

SiO2 - Silica gel

t - Triplet

TLC - Thin Layer Chromatography

UV - Ultraviolet

VLC - Vacuum Liquid Chromatography

xix

LIST OF APPENDICES

APPENDIX NO. TITLE PAGE

1

GCMS Spectrum of Squalene (116)

119

2 IR Spectrum of Squalene (116) 120

3 1H NMR Spectrum of Squalene (116) 121

4 COSY Spectrum of Squalene (116) 122

5 13C NMR Spectrum of Squalene (116) 123

6 DEPT Spectra of Squalene (116) 124

7 IR Spectrum of 28-Hydroxyfriedelan-3-one (117) 125

8 1H NMR Spectrum of 28-Hydroxyfriedelan-3-one (117) 126

9 COSY Spectrum of 28-Hydroxyfriedelan-3-one (117) 127

10 HMQC Spectrum of 28-Hydroxyfriedelan-3-one (117) 128

11 13C NMR Spectrum of 28-Hydroxyfriedelan-3-one (117) 129

12 DEPT Spectra of 28-Hydroxyfriedelan-3-one (117) 130

13 EIMS Spectrum of 28-Hydroxyfriedelan-3-one (117) 131

14 IR Spectrum of Friedooleanan-3-one (73) 132

15 1H NMR Spectrum of Friedooleanan-3-one (73) 133

16 COSY Spectrum of Friedooleanan-3-one (73) 134

17 13C NMR Spectrum of Friedooleanan-3-one (73) 135

18 DEPT Spectra of Friedooleanan-3-one (73) 136

19 HMQC Spectrum of Friedooleanan-3-one (73) 137

20 GCMS Spectrum of Friedooleanan-3-one (73) 138

21 13C NMR Spectrum of Olean-12-en-3-ol (118) 139

22 DEPT Spectra of Olean-12-en-3-ol (118) 140

23 1H NMR Spectrum of Olean-12-en-3-ol (118) 141

24 COSY Spectrum of Olean-12-en-3-ol (118) 142

xx

25 HMQC Spectrum of Olean-12-en-3-ol (118) 143

26 GCMS Spectrum of Olean-12-en-3-ol (118) 144

27 IR Spectrum of Olean-12-en-3-ol (118) 145

28 CIMS Spectrum of Amentoflavone-4'-methyl ether (119) 146

29 UV Spectrum of Amentoflavone-4'-methyl ether (119) 147

30 IR Spectrum of Amentoflavone-4'-methyl ether (119) 148

31 1H NMR Spectrum of Amentoflavone-4'-methyl ether

(119)

149

32 COSY Spectrum of Amentoflavone-4'-methyl ether (119) 150

33 13C NMR Spectrum of Amentoflavone-4'-methyl ether

(119)

151

34 DEPT Spectra of Amentoflavone-4'-methyl ether (119) 152

35 HMQC Spectrum of Amentoflavone-4'-methyl ether

(119)

153

36 Expansion of HMQC Spectrum of Amentoflavone-

4'-methyl ether (119)

154

37 HMBC Spectrum of Amentoflavone-4'-methyl ether

(119)

155

38 Expansion of HMBC Spectrum (I) of Amentoflavone-


156

39 HMBC Spectrum (II) of Amentoflavone-4'-methyl

ether (119)

157

40 Expansion of HMBC Spectrum (II) of Amentoflavone-


158

41 UV Spectrum of 3,8''-Binaringenin (53) 159

42 CIMS Spectrum of 3,8''-Binaringenin (53) 160

43 IR Spectrum of 3,8''-Binaringenin (53) 161

44 13C NMR Spectrum of 3,8''-Binaringenin (53) 162

45 DEPT Spectra of 3,8''-Binaringenin (53) 163

46 1H NMR Spectrum of 3,8''-Binaringenin (53) 164

47 COSY Spectrum of 3,8''-Binaringenin (53) 165

48 HMQC Spectrum of 3,8''-Binaringenin (53) 166

49 HMBC Spectrum of 3,8''-Binaringenin (53) 167

xxi

50 CIMS Spectrum of 3,8''-Binaringenin-7''-O-glucoside

(120)

168

51 UV Spectrum of 3,8''-Binaringenin-7''-O-glucoside (120) 169

52 IR Spectrum of 3,8''-Binaringenin-7''-O-glucoside (120) 170

53 13C NMR Spectrum of3,8''-Binaringenin-7''-O-glucoside

(120)

171

54 DEPT Spectra of 3,8''-Binaringenin-7''-O-glucoside

(120)

172

55 1H NMR Spectrum of 3,8''-Binaringenin -7''-O-glucoside

(120)

173

56 COSY Spectrum of 3,8''-Binaringenin-7''-O-glucoside

(120)

174

57 HMQC Spectrum of 3,8''-Binaringenin-7''-O-glucoside

(120)

175

58 UV Spectrum of Morelloflavone (51) 176

59 CIMS Spectrum of Morelloflavone (51) 177

60 IR Spectrum of Morelloflavone (51) 178

61 1H NMR Spectrum of Morelloflavone (51) 179

62 Expansion of 1H NMR Spectrum of Morelloflavone

(51)

180

63 COSY Spectrum of Morelloflavone (51) 181

64 13C NMR Spectrum of Morelloflavone (51) 182

65 DEPT Spectra of Morelloflavone (51) 183

66 HMQC Spectrum of Morelloflavone (51) 184

67 HMBC Spectrum of Morelloflavone (51) 185

68 HMBC Spectrum (I) of Morelloflavone (51) 186

69 Expansion of HMBCSpectrum (I) of Morelloflavone

(51)

187

70 HMBC Spectrum (II) of Morelloflavone (51) 188

71 DEPT Spectra of Morelloflavone-7''-O-glucoside (55) 189

72 CIMS Spectrum of Morelloflavone-7''-O-glucoside (55) 190

73 IR Spectrum of Morelloflavone-7''-O-glucoside (55) 191

74 1H NMR Spectrum ofMorelloflavone-7''-O-glucoside (55) 192

xxii

75 COSY Spectrum of Morelloflavone-7''-O-glucoside (55) 193

76 13C NMR Spectrum ofMorelloflavone-7''-O-glucoside

(55)

194

77 DEPT Spectra of Morelloflavone-7''-O-glucoside (55) 195

78 HMQC Spectrum ofMorelloflavone-7''-O-glucoside (55) 196

79 HMBC Spectrum ofMorelloflavone-7''-O-glucoside (55) 197

CHAPTER 1

INTRODUCTION

1.1 General Introduction

A natural product is a chemical compound or a substance produced by living

organisms, plants, animals, insects and microbes. These chemical constituents

usually have pharmacological or bioactivities that can be applied in pharmaceutical

and drug design [1]. They have, until recently, been the primary source for the

commercial medicines and drug research development [2]. Natural products

chemistry has always been concerned with the discovery of bioactive constituents

and it remains one of the main keys that play an important role in the continuous

research for new drugs in the industrial drug discovery process [3]. Besides, many

natural products have reached the market without chemical modification, thus the

potential to commercialize these small, drug-like molecules are very economical [4].

The abundance of natural renewable supply of plants and herbs indeed is a great

source of affordable drugs, which is complimentary to the modern medicine [5].

The practices of modern medicine have yielded numerous purified

compounds with medicinal properties as the result of the chemical investigations and

purification of extracts of plants. These compounds have been developed into

pharmaceutical agents [6]. To date about 25% of all available modern drugs such as

morphine and salicylates are derived directly or indirectly from higher plants. These

interesting compounds developed major classes of the analgesic drugs, namely,

opioids which are classified as central nervous system depressants and non-steroidal

anti-inflammatory drugs. Over the years, natural products and their derivatives are

2

not only used clinically, but also play an important role in discovery of new targets

such as receptors, enzymes, transporters or ion channels involved in relevant

physiological and pathological processes [7].

The analysis of plant components should thus begin with bioactivity-directed

screening and bioactivity-directed fractionation leading to the isolation and

characterization of pure biologically active compounds [8]. With the advancement of

spectroscopic methods, numerous types of active compounds can be isolated from

plants and are structurally characterized. In due course, many of these compounds

are synthesized in the laboratory. Sometimes, better-tolerated drugs are produced by

chemical modifications or by total synthesis of analogues of the active principles [9].

As a result, the findings from this area of research will assist in contributing to the

evolution in practice of traditional and modern medicineby utilizing them on a larger

commercial basis to eliminate health problems. Therefore, today, an increase of

global interest for industrial production concurrently meet the demand for

conserving biodiversity to enhance the development of renewable natural products

for medicine [10].

1.2 Guttiferae Family

Guttiferae family is one of the families in order Guttiferals. The family

contains about 48 genera and over 1000 species of perennial herbs, shrubs and trees,

widely distributed by the tropical and temperate regions of the world [11]. Most

members of the family are in view of their economical and medicinal purposes in

many parts of the world for treatment of different illnesses [12]. In Malaysia,

Guttiferae is an important component of the Malaysian Rain Forest as the second-

storey forest trees which include some well-known and important trees such as

ironwood tree, mangosteen and penaga laut. Whitemore [11] identified and

classified four genera and 121 species that can be found in Peninsular Malaysia

which are Calophyllum (45 species), Garcinia (49 species), Mammea (23 species)

and Mesua (4 species).

3

The trees or shrubs of this family have inner barks with yellow or white latex

in droplets. The bark is smooth, fissured or scaly patter and the bole rarely with stilt

roots or buttresses [13]. The leaves are mostly opposite without true stipule and the

secondary nerves often numerous, close and parallel. Flowers are bisexual or

sometimes unisexual, have scented and can be on the twigs behind the leaves. The

sepals and petals are four to five each and overlapping whereas, the stamens usually

connate in bundles. The fruits of this family can be a drupe for Calophyllum and

Mammea, a nut for Mesua or a berry with the seeds embedded in pulp, not splitting

with wall leathery of fleshy for Garcinia [11, 14].

Primarily, the genus Garcinia is the biggest genus with the common village

fruit-trees such as G. atroviridis (asam gelugor), G. cowa (village kandis) and

G. prainiana (kechupu). The trees are small to medium, rarely taller than 30 m;

therefore the trees are almost completely confined to the interior of the forest, in

shade. Many species of Garcinia have very similar leaves but differ in flower and

fruit characters [11]. The fruit hull of G. mangostana has found many uses in

traditional medicine such as for healing skin infections and wounds in Thai folk

medicine [15].

The second largest genus is Calophyllum called ‘bintangor’ by Malays. It

provides timber such as C. coriaceum (bintangor gunung daun besar), C. cuneatum

(bintangor gunung daun kecil) and C. inophylloide (bintangor batu). Trees of this

genus have the largest size among the other three genera with heights of up to

30 – 36m and are common of the lowland and mountain forest. C. inophyllum has

wide variety of cures found from its oil to its roots. Its gum, bark, leave, and all

other parts of it are found to be curative in medicine hence known as the 'All Heal'

plant [16]. The seed oil of C. inophyllumis reported to cure rheumatism and skin

affections in Indian traditional medicine [17].

The Malay name for genus Mesua is ‘penaga’ and also consists of valuable

timber with very hard and heavy wood such as M. grandis (penaga sabut),

M. lepidota (penaga tikus) and M. nuda (penaga lilin). The trees comprise small to

medium trees with height up to about 23 m [11]. M. ferreais a very well-known

4

species; the word ‘ferrea’ is from the Latin word, ‘ferrum’ which means ‘iron’

referring to its extremely hard wood [18]. The heartwood is very hard, dense, strong,

heavy and durable like ebony and used extensively for heavy construction, in

machinery work, vehicles and agricultural implements [19]. In Malaysia, the kernels

of M. ferrea are pounded with the seed oil and applied to wounds as poultice [18].

The last genus identified in Malaysia is Mammea consists of M. brevipes,

M. malayana, M. siamensis and M. odorata. The Malaysian wild Mammea species

are rare trees and restricted to the lowland. The genus has small trees with about

15 m height and has little important uses to human. M. siamensis has a big flower

which smell of violets and planted in temple. Its pollen is reputed to be used as a

cosmetic. The well-known species in this genus is M. americana, the sole American

species which produces large edible apricot-like fruits called as the Mammea apple

[11].

1.3 Garcinia griffithii

Species of Garcinia griffithii (apple-kandis or kandis gajah) is a small to

medium tree that may reach 23 m tall and conspicuous from the large leaves and

fruits. Its latex is yellowish white and the inner bark with opaque yellow exudate.

The sapwood and heartwood cannot be distinct and in dark red-brown colour. The

leaves have very large blade with size from 15×7 to 28×16 cm, broady elliptic,

strongly ribbed and pointed. The edges incurved and has base rounded. The young

leaves are pink and the drying leaves are blackish green with thin-texture. The

flowers mainly in short woody on the twigs behind the leaves and have four sepals

and petals with yellow flushed red colours at the base. The fruits are

characteristically globose, faintly ribbed, subsessile, fattened at the top, clustered on

the branched, edible and turning brownish yellow with watery acid flesh like green

apple. The stigma is usually sunken entirely, flat or slightly convex. The species is

common in Peninsular Malaysia at the low land forest [11, 16, 20].

5

1.4 Problem Statement

To date, there are two reports on the phytochemicals and antiplasmodial

activity of Garcinia griffithii collected from Indonesia [21] and Singapore [22].

However, the bioactivities of the plants were not studied thoroughly. Although the

climate and ecology of Singapore and Indonesia are similar with Malaysia, this

study is still being continued hoping to get variations of chemical constituents with

interesting bioactivities. The data obtained will be utilized for future research on the

chemical markers of Garcinia species from Malaysia.

1.5 Objectives of Research

The objectives of this research are:

• To isolate the chemical compounds from the leaves and stem barks of

G. griffithii.

• To elucidate the structures of the chemical compounds by combined

spectroscopic methods.

• To carry out the antioxidant, antimicrobial and tyrosinase inhibition

activities on the crude extracts and pure compounds of G. griffithii.

1.6 Scope of Research

This research will focus on the leaves and stem barks of G. griffithii. The

sample will be extracted by Soxhlet apparatus using n-hexane, dichloromethane,

ethyl acetate and methanol as the solvent to afford the crude extracts. Separation and

purification of the natural compounds from the crude extracts will be carried out

with chromatographic techniques such as vacuum liquid chromatography and

6

column chromatography. The structure of the pure compounds will be elucidated

with various spectroscopic techniques such as UV, IR, NMR (1D and 2D) and MS.

The crude extracts will be screened for bioactivity focusing on antioxidant,

tyrosinase inhibition and antimicrobial activities. The antioxidant assay by DPPH

method, the total antioxidant assay by formation of the green phosphomolybdenum

complex and the total phenolic content assay using Folin-Ciocalteaumicro method

will be used to screen the antioxidant activities of the samples. The antimicrobial

assay will be carried out by disc diffusion method against two Gram-positive

bacteria: Enterococcus faecalis and Bacillus subtilis and two Gram-negative

bacteria: Escherichia coli and Klebsiella pneumoniae. Further evaluation of the

antimicrobial activity will be carried out by determination of minimum inhibition

concentration (MIC) and minimum bactericidal concentration (MBC). The isolated

compounds will be screened for antioxidant as above and tyrosinase inhibition

activities only.

REFERENCES

1. Newman, D.J. and Cragg, G.M. (2007). Natural Products as Sources of New

Drugs over the Last 25 Years. Journal of Natural Products.70, 461-477.

2. Cseke, L.J., Kirakosyan, A., Kaufman, P.B., Warber, S.L., Duke, J.A.,

Brielman, H.L. (2006). Natural Products from Plants. Boca Raton, FL. CRC

Press Taylor & Francis Group. 2-3.

3. Steven, M.C. and Russell, J.M. (1993). Bioactive Natural Products: Detection,

Isolation and Structural Determination. Boca Raton, FL. CRC Press Taylor &

Francis Group. 2-8, 10-49.

4. Lixin, Z. and Arnold, L.D. (2005). Natural Products: Drug Discovery and

Therapeutic Medicine. Totowa, NJ. Humana Press. 4.

5. Sujata, V.B., Nagasasampagi, B.A., Meeenakshi, S. (2009). Natural Products:

Chemistry and Applications. Oxford, UK. Alpha Science International Ltd. 3.

6. Balandarin, M.F., Kinghorn, A.D., Fransworth, N.R. (1993). Chapter 1: Plant-

Derived Natural Products in Drugs Discovery and Development, An

Overview. Human Medical Agents from Plants. Washington, DC. American

Chemical Society. 2-12.

7. Calixto, J.B., Kassuya, C.A., Andre, E., Ferreira, J. (2005). Contribution of

Natural Products to the Discovery of the Transient Receptor Potential (TRP)

Channels Family and Their Functions. Pharmacology & Therapeutics.

106(2), 179-208.

108

8. Anderson, J.E., Goetz, C.M., McLaughlin, J.L., Suffness, M. (1991). A Blind

Comparison of Simple Bench-Top Bioassays and Human Tumor Cell

Cytotoxicities as Tumor Antitumor Prescreens. Phytochemical Analysis. 2,

107-111.

9. Dewick, P.M. (2009). Medical Natural Products: A Biosynthetic Approach.

3rd Edition. University of Nottingham, UK. John Wiley & Sons, Ltd. 7-8.

10. Tringali, C. (2012). Bioactive Compounds from Natural Sources: Natural

Products as Lead Compounds in Drug Discovery. 2nd Edition. Boca Raton,

FL. CRC Press Taylor & Francis Group. 2.

11. Whitemore, T.C. (1972). Malayan Forest Records No. 26: Tree of Malaya: A

Manual for Foresters. Kuala Lumpur. Longman.2, 162-236.

12. Sultanbawa, M.U.S. (1980). Xanthonoids of Tropical Plants. Tetrahedron. 36,

1465-1506.

13. Kochummen, K.M. (1997). Malay Forest Records No. 44: Tree Flora of

Pasoh Forest. Kuala Lumpur. Forest Research Institute Malaysia. 255.

14. Hsuan, K. (1978). Orders and Family of Malayan Seed Plants. Singapore.

Singapore University Press. 88.

15. Parveen, M., Khan, N.U., Acchari, B., Dutta, P.K. (1991). A Triterpene from

Garcinia mangostana. Phytochemistry. 30, 361-362.

16. Corner, E.J.H. (1940). Wayside Trees of Malaya in Two Volumes. Singapore.

Government Printing Office. 310-321.

17. Govindachari, T.R., Viswanathan, N., Pai, B.R., Rao, U.R., Srinivasan, M.

(1967). Triterpenes of Callophyllum inophyllum Lin. Tetrahedron. 23, 1901-

1910.

109

18. Wee, Y.C. (2003). Tropical Trees and Shrubs: A Selection for Urban

Planting. Singapore. Suntree Marketing Pte Ltd. 93.

19. Foxworthy, F.W. (1927). Malayan Forest Records No. 3: Commercial Timber

Trees of The Malay Peninsula. Singapore. Fraser & Neave, Ltd. 131-137.

20. Desch, H.E. (1941). Malayan Forest Records No. 15: Manual of Malayan

Timbers. Kuala Lumpur. Caxton Press Ltd. 218-220.

21. Nilar, L.-H., Nguyen, D., Ganpathi, V., Sim, K.-Y., Harrison, L.J. (2005).

Xanthones and Benzophenones from Garcinia griffithii and Garcinia

mangostana. Phytochemistry. 66, 1718-1723.

22. Elfita, E., Muharni, M., Madyawati, L., Darwati, D., Ari, W., Supriyatna, S.,

Husein, H.B., Dachriyanus, D., Paul, C., Louis, M., Kenne, F., Sandra, A.,

Luc, P. (2009). Antiplasmodial and Other Constituents from Four Indonesian

Garcinia spp. Phytochemistry. 70, 907-912.

23. Gautam, B. (2009). Natural Product: Chemistry, Biochemistry and

Pharmocology. Oxford, UK. Alpha Science International Ltd. 326-327.

24. Bennett, G.J. and Lee, H.J. (1989). Review Article Number 43: Xanthones

from Guttiferae. Phytochemistry. 28(4), 967-998.

25. Waterman, P.G. and Hussain, R.A. (1983). Systematic Significance of

Xanthones, Benzophenones and Biflavonoids in Garcinia. Biochemical

Systematics and Ecology. 11, 21-28.

26. Hill, J.R. (1915). Mangostin: A Crystalline Substance Allied to The Resins.

Journal of the Chemical Society, Transactions. 107, 595-601.

27. Arunrattiyakorn, P., Suksamrarn, S., Suwannasai, N., Kanzaki, H. (2011).

Microbial Metabolism of α-Mangostin Isolated from Garcinia mangostana L.

Phytochemistry. doi: 10.1016/j.phytochem.2011.02.007.

110

28. Nilar, L.-H. and Harrison, L.J. (2002). Xanthones from The Heartwood of

Garcinia mangostana. Phytochemistry. 60, 541-548.

29. Kinghorn, A.D., Chai, H.-b., Sung, C.K., Keller, W.J. (2011). The Classical

Drug Discovery Approach to Defining Bioactive Constituents of Botanicals.

Fitoterapia. 82, 71-79.

30. Pedraza-Chaverri, J.,Cardenas-Rodriguez, N., Orozco-Ibarra, M.,Perez-Rojas,

J.M. (2008). Review: Medicinal Properties of Mangosteen (Garcinia

mangostana). Food and Chemical Toxicology. 46, 3227-3239.

31. Xu, Y.-J., Cao, S.-G., Wu, X.-H., Lai, Y.-H., Tan, B.H.K., Pereira, J.T., Goh,

S.H., Venkatraman, G., Harrison, L.J., Sim, K.-Y. (1998). Griffipavixanthone,

A Novel Cytotoxic Bixanthone from Garcinia griffithii and G. parvifolia.

Tetrahedron Letters. 39, 9103-9106.

32. Rukachaisirikul, V., Trisuwan, K., Sukpondma, Y., Phongpaichit, S. (2008).

A New Benzoquinone Derivative from The Leaves of Garcinia parvifolia.

Archives of Pharmacal Research. 31(1), 17-20.

33. Rukachaisirikul, V., Naklue, W., Phongpaichit, S., Towatana, N.H.,

Maneenoon, K. (2006). Phloroglucinols, Depsidones and Xanthones from The

Twigs of Garcinia parvifolia. Tetrahedron. 62, 8578- 8585.

34. Klaiklay, S., Sukpondma, Y., Rukachaisirikul, V., Phongpaichit, S. (2013).

Friedolanostanes and Xanthones from The Twigs of Garcinia hombroniana.

Phytochemistry. 85, 161-166.

35. Hui, Y., Mario, F., Satoshi, T., Scott, B., Bei, J., Margaret, J.B., Bernard,

I.W., Edward, J.K. (2010). Benzhophenone and Biflavonoids from Garcinia

livingstonei Fruits. Journal of Agriccultural and Food Chemistry. 58, 4749-

4755.

111

36. Sordat-Diserens, I., Rogers, C., Sordat, B., Hostettmann, K. (1992).

Prenylated Xanthones from Garcinia livingstonei. Phytochemistry. 30(1),

313-316.

37. Heim, F., Maheu, J., Matrod, L. (1919). The Value of The Pericarps of

Garcinia mangostana L. in The Tanning Industry. Monthly Bulletin of

Agriculture Intellgence and Plant Disease. Monthly Bulletin of Agriculture

Intelligence and Plant Diesease. 10, 1256-1257.

38. Murakami, M. (1932). Constitution of Fukugetin and Garcinin. Proceedings

of The Imperial Academy (Tokyo). 8, 500-502. ISSN: 0369-9846.

39. Gunatilaka, A.A.L., Silva, A.M.Y.J.D., Sotheeswaran, S., Balasubramaniam,

S., Wazeer, M.I.M. (1984). Terpenoid and Biflavonoid Constituents of

Calophyllum calaba and Garcinia spicata From Sri Lanka. Phytochemistry.

23(2), 323-328.

40. Konoshima, M. and Ikeshiro, Y. (1970). Fukugiside, The First Biflavonoid

Glycoside from Garcinia spicata. Tetrahedron Letters. 20, 1717-1720.

41. Konoshima, M., Ikeshiro, Y., Nishinaga, A., Matsuura, T., Kubota, T.,

Sakamoto, H. (1969).The Constitution of Flavonoids from Garcinia spicata

Hook. Tetrahedron Letters. 2, 121-124.

42. Nguyen, H.D., Trinh, T.D., Tran, Q.N., Nguyen, H.D., Pham, H.D., Hansen,

P.E., Duus, F., Connolly, J.D., Nguyen, L.-H.D. (2011). Friedolanostane,

Friedocycloartane and Benzophenone Constituents of The Bark and Leaves of

Garcinia benthami. Phytochemistry. 72, 290-295.

43. Elya, B., He, H.P., Kosela, S., Hanafi, M., Hao, X.J. (2006). A New

Benzophenone from The Stem Bark of Garcinia benthami. Natural Product

Research. 20(12), 1059-1062.

112

44. Rukachaisirikul, V., Adair, A., Dampawan, P., Taylor, W.C., Turner, P.C.

(2000). Lanostane and Friedolanostane from The Pericarp of Garcinia

hombroniana. Phytochemistry. 55, 183-188.

45. Rukachaisirikul, V., Saelim, S., Karnsomchoke, P., Phongpaichit, S. (2005).

Friedolanostanes and Lanostanes from The Leaves of Garcinia hombroniana.

Journal of Natural Products. 68, 1222-1225.

46. Vieira, L.M.M., Kijjoa, A., Silva, A.M.S., Mondranondra, I.-O., Kengthong,

S., Gales, L., Damas, A.M., Herz, W. (2004). Lanostanes and Friedolastones

from The Barks of Garcinia speciosa. Phytochemistry. 65, 393-398.

47. Vieira, L.M.M., Kijjoa, A., Wilairat, R., Nascimento, M.S.J., Gales, L.,

Damas, A.M., Silva, A.M.S., Mondranondra, I.-O., Herz, W. (2004).

Bioactive Friedolanostanes and 11(10→8)-Abeolanostanes from The Bark of

Garcinia speciosa. Journal of Natural Products. 67, 2043-2047.

48. Lee,L.-T., Tsai, Y.-F., Hu, N.-Y., Wang, C.-W., Huang, K.-K., Hsiao, J.-K.,

Shih, Y.-C., Munekazu, L. (2013). Anti-Arthritis Effect of Mangostins from

Garcinia mangostana. Biomedicine & Preventive Nutrition.3(3), 227-232.

49. Keiser, J., Vargas, M., Winter, R. (2012). Anthelminthic Properties of

Mangostin and Mangostin Diacetate. Parasitology International. 61, 369-

371.

50. Lee, L.-T., Tsai, H.-P., Wang, C.-C., Chang, C.-N., Liu, W.-C., Hsu, H.-C.,

Hsieh, C.-T., Chen, Y.-C., Tseng, H.-W., Gau, R.-J., Liu, S.-H., Chen, I-S.,

Iiluma, M. (2013). Guttiferone F from The Fruit of Garciniamultiflora and Its

Anti-Hepatocellular Carcinoma Activity. Biomedicine & Preventive Nutrition.

3, 247-222.

51. Wu, Y.-P., Zhao, W., Xia, Z.-Y., Kong, G.-H., Lu, X.-P., Hu, Q.-F., Gao, X.-

M. (2013). Three New Xanthones from The Stems of Garcinia oligantha and

Their Anti-TMV Activity. Phytochemistry Letters. 6, 629-632.

113

52. Pavia, D.L., Lampman, G.M., Kriz, G.S., Vyvyan, J.R. (2009). International

Student Edition: Introduction to Spectroscopy. 4th Edition. United State.

Brooks/Cole, Cengage Learning. 33, 55.

53. Suga, T. and Shishibori, T. (1975). The Stereospecificity of Bisynthesis of

Squalene and β-amyrin in Pisumsativum. Phytochemistry.14, 2411-2417.

54. Ragasa, C.Y., Levida, R.M., Don, M.-J., Shen, C.-C. (2012). Cytotoxic

Isothiocyanates from Moringa oleifera Lam Seeds. Philippine Science

Letters. 5(1), 46-52.

55. Nozaki, H., Suzuki, H., Hirayama, T., Kasai, R., Wu, R.-Y., Lee, K.-H.

(1986). Antitumor Triterpenes of Maytenus diversifolia. Phytochemistry.

25(2), 479-485.

56. Shaiq Ali, M., Mahmud, S., Perveen, S., Uddin Ahmad, V., Hafeez Rizwani,

G. (1999). Epimers from The Leaves of Calophyllum inophyllum.


57. Hui, W.-H., Ko, P.D.S., Lee, Y.-K., Li, M.-M., Arthur, H.R. (1975).

Triterpenoids from The Ten Lithocarpus Species of Hong Kong.


58. Betancor, C., Freire, R., Gonzalez, A.G., Salazar, J.A., Pascard, C., Prange, T.

(1980). Three Triterpenes and Other Terpenoids from Catha cassinoides.


59. Quieroga, C.L., Silva, G.F., Dias, P.C., Possenti, A., Carvalho, J.E. (2000).

Short Communication: Evaluation of the Antiulcerogenic Activity of

Friedelan-3β-ol and Friedelin Isolated from Maytenus ilicifolia (Celastraceae).

Journal of Ethnopharmacology. 72, 465-468.

114

60. Tahany, M.A.A.-R., Hegazy, A.K., Sayed, A.M., Kabiel, H.F., El-Alfy, T.,

El-Komy, S.M. (2010). Full Length Research Paper: Study on Combined

Antimicrobial Activity of Some Biologically Active Constituents from Wild

Moringa peregrine Forssk. Journal of Yeast and Fungal Research. 1(1), 15-

24.

61. Vazquez, L.H., Palazon, J. and Navarro-Ocana, A. (2012). The Pentacyclic

Triterpenesα, β-amyrins: A Review of Resources and Biological Activities.

Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

by Dr. Venketeshwer Rao (Ed). ISBN: 978-953-51-0296-0. InTech.

62. Ryu, Y.B., Jeong, H.J., Kim, J.H., Kim, Y.M., Park, J.-H., Kim, D., Naguyen,

T.T.H., Park, S.-J., Chang, J.S., Park, K.H., Rho, M.-C., Lee, W.S. (2010).

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CLpro Inhibition.

Bioorganic & Medicinal Chemistry.18, 7940-7947.

63. Markham, K.R. (1982). Techniques of Flavonoid Identification. UK, London.

Academic Press. 39, 42-48.

64. Markham, K.R., Sheppard, C., Geiger, H. (1987). 13C NMR Studies of Some

Naturally Occuring Amentoflavone and Hinokiflavone Biflavonoids.

Phytochemistry. 72 (12), 3335-3337.

65. Feng, W.-S., Zhu, B., Zheng, X.-K., Zhang, Y.-L., Yang, L.-G., Li, Y.-J.

(2011). Chemical Constituents of Selaginella stautoniana. Chinese Journal of

Natural Medicines. 9 (2), 108-111.

66. Chen, F.-C., Lin, Y.-M., Hung, J.-C. (1975). Phenolic Compounds from The

Heartwood of Garcinia multiflora. Phytochemistry.14, 300-303.

67. Crichton, E.G. and Waterman, P.G. (1979). Manniflavanone, A New 3,8-

Linked Flavanone Dimer from The Stem Bark of Garcinia manni.


115

68. Duddeck, H., Snatzke, G., Yemul, S.S. (1978). 13C NMR and CD of Some

3,8''-Biflavanoids From Garcinia Species And of Related Flavanones.


69. Chen, F.-C., Lin, Y.-M., Hung, J.-C. (1975). A New Biflavanone Glucoside

from Garcinia multiflora. Phytochemistry. 14, 818-820.

70. Chiruvella, K.K., Mohammed, A., Dampuri, G., Ghanta, R.G., Raghavan,

S.C. (2007). Phytochemical and Antimicrobial Studies of Methyl Angolensate

and Luteolin-7-O-glucoside Isolatef from Calluc Cultures of Soymida

febrifuga. International Journal of Biomedicinal Science. 3(4), 269-278.

71. Li, X.-C., Joshi, A.S., Tan, B., ElSohly, H.N., Walker, L.A., Zjawiony, J.K.,

Ferreira, D. (2002). Absolute Configuration, Conformation and Chiral

Properties of Flavanone-(3→8'')-Flavone Biflavonoids from Rheedia

acumianata. Tetrahedron. 58, 8709-8717.

72. Konoshima, M. and Tkeshiro, Y. (1970). Fukugiside, The First Biflavonoid

Glycoside from Garcinia spicata Hook. Tetrahedron Letters. 20, 1717-1720.

73. Padmaa, M.P. and Ravichandra, V.D. (2012). Antioxidant Activity of Some

Plants Native to Karnataka: A Review. Medicinal Plants as Antioxidant

Agents: Understanding Their Mechanism of Action and Therapeutic Efficacy.

Research Signpost. 59-81. ISBN: 978-81-308-0509-2.

74. Prakash, A. (2001). Antioxidant Activity. Analytical Progress. 19, 1-6.

75. Rice-Evans, C.A., Miller, N.J., Paganga, G. (1997). Antioxidant Properties of

Phenolic Compounds. Trends in Plant Science Reviews. 2(4), 152-159.

PII S1360-1385(97)01018-2.

76. Gandhiappan, J. and Rengasamy, R. (2012). Comparative Study On

Antioxidant Activity of Different Species of Solanaceae Family. Pelagia

116

Research Library. Advances in Applied Science Research. 3(3), 1538-1544.

ISSN: 0976-8610.

77. Kyoung, O.C. and Kim, D.-O. (2004). Consideration On Equivalent

Chemicals In Total Phenolic Assay of Chlorogenic Acid-Rich Plums. Food

Research International. 37, 337-342.

78. Tsong-Min, C. (2012). Tyrosinase and Tyrosinase Inhibitors. Journal of

Biocatalysis & Biotransformation. 1:2. doi: 10.4172/2324-9099.1000e106

79. Ohad, N., Ramadan, M., Soliman, K., Snait, T., Jacob, V. (2004). Chalcones

as Potent Tyrosinase Inhibitors: The Effect of Hydroxyl Positions and

Numbers. Pytochemistry. 65, 1389-1395.

80. Te-Sheng, C. (2009). Review: An Updated Review of Tyrosinase Inhibitors.

International Journal of Molecular Sciences. 10, 2440-2475.

81. Salie, F., Eagles, P.F.K., Leng, H.M.J. (1996). Preliminary Antimicrobial

Screening of Four South African Asteraceae Species. Journal of

Ethnopharmacology. 52, 27-33.

82. Magina, M.D.A., Dalmarco, E.M., Wisniewski Jr., A., Siminatto, E.L.,

Dalmacro, J.B., Pizzolatti, M., Brighente, I.M.C. (2009). Chemical

Composition and Antibacterial Activity of Essential Oils of Eugenia Species.

Journal of Natural Medicines. 63, 345-350.

83. Tagashira, M. and Ohtake, Y. (1998). A New Antioxidative 1,2-Benzodioxole

from Melissa officinalis. Planta Medica. 64, 555-558.

84. Prieto, P., Pineda, M., Aguilar, M. (1999). Spectrophotometric Quantitation

of Antioxidant Capacity Through The Formation of A Phosphomolybdenum

Complex: Specific Application to The Determination of Vitamin E1.

Analytical Biochemistry. 269, 337–341.

117

85. Slinkard, K. and Singleton, V.L. (1977).Total Phenol Analysis: Automation

and Comparison with Manual Methods. American Journal of Enology and

Viticulture. 28,49-55.

86. Likhitwitayawuid, K. and Sritularak, B. (2001). A New Dimeric Stilbene with

Tyrosinase Inhibitory Activity from Artocarpus gomezianus. American

Chemical Society and American Society of Pharmacognosy. doi:

10.1021/np0101806.

87. Hsing-Tan, L., Syun-Wun, R., Jin-Cherng, H., Hsin-Liang, C., Chung-Yi, C.

(2012). Antioxidant and Tyrosinase Inhibitor from Leucaena leucocephala.

African Journal of Biotechnology. 11(77), 14182-14185. ISSN: 1684-5315.

88. Zavala, M.A., Perez, G.S., Perez, G.R.M. (1997). Antimicrobial Screening of

Some Medicinal Plants. Phytotherapy Research. 11, 368-371.

89. Petrus, E.M., Tinakumari, S., Chai, L.C., Ubong, A., Tunung, R., Elexson, N.,

Chai, L.F., Son, R. (2011). A Study on The Minimum Inhibitory

Concentration and Minimum Bactericidal Concentration of Nano Colloidal

Silver on Food-Borne Pathogens. International Food Research Journal. 18,

55-66.

CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF Garcinia...

Documents

Transcript of CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF Garcinia...