chemical constituents and bioactivity of...

CHEMICAL CONSTITUENTS AND BIOACTIVITY OF MALAYSIAN AND

INDONESIAN KAEMPFERIA ROTUNDA

YAU SUI FENG

UNIVERSITI TEKNOLOGI MALAYSIA

i

CHEMICAL CONSTITUENTS AND BIOACTIVITY OF MALAYSIAN AND INDONESIAN KAEMPFERIA ROTUNDA

YAU SUI FENG

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

DECEMBER 2009

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Dedicated to Buddha, Dharmma, Sangha,

my beloved father, mother, sisters, brother and friends.

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ACKNOWLEDGEMENTS

First of all, I would like to express my deepest appreciation to my supervisor,

Prof. Dr. Hasnah Mohd. Sirat, for her guidance, motivation, and more crucially,

precious advise to all my research. I am truly grateful for her kindness, all the time

and effort she devoted to me.

I would like to extend special thanks to Assoc. Prof. Dr. Farediah Ahmad, Dr.

Shajarahtunnur Jamil and Mrs. Norazah Basar for their precious help and guidance.

I would also like to express my gratitude to Universiti Teknologi Malaysia

for granting me scholarship and to Department of Chemistry for allowing me to get

access to the GC, GC-MS, IR, UV and NMR facility.

I would like to express greatly appreciation to Ms. Mala, Mr. Emrizal and Mr.

Oh Boon Thai for their patience, support and discussions. Sincerely thanks to Mr.

Farriz, Mr. Syamil, Ms. Azlin, Mrs. Ummu and Mr. Salihin for the friendship and

help during the work.

Sincerely thanks to lab assistants especially Mrs. Mekzum, Mr. Azmi and Mr.

Hanan who helped me throughout these years.

Special thanks to my parents, my sisters, my brother and Mr. Ting Chee Ming

for their love, support and understanding.

v

PREFACE

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

Universiti Teknologi Malaysia between July 2007 and June 2009 under the

supervision of Prof. Dr. Hasnah Mohd. Sirat. Parts of my work described in this

thesis have been reported in the following publications:

1. Yau Sui Feng and Hasnah Mohd. Sirat (2009). Chemical Constituents of

Kaempferia rotunda. Mal. J. Sci. 2009. 28 (special edition): 81-88.


Kaempferia rotunda. Paper presented at International Conference on Molecular

Chemistry 2008 at University of Malaya, Kuala Lumpur. 25-26 November 2008.


Kaempferia rotunda. Poster presented at the 2nd Penang International Conference

for Young Chemists 2008 at Universiti Sains Malaysia, Penang. 18-20 June

2008.

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ABSTRACT

The essential oils and the phytochemicals of the rhizomes of Kaempferia rotunda cultivated in Malaysia and Indonesia have been studied. Hydrodistillation of the fresh rhizomes of K. rotunda gave 0.09% and 0.23% oils respectively. These oils were analyzed by GC (Kovats Indices) and GC-MS. The main constituents found in the rhizome oil of Malaysia were bornyl acetate (9.6%), benzyl benzoate (8.4%) and camphor (5.6%), while the rhizome oil from Indonesia was rich in benzyl benzoate (87.7%) and n-pentadecane (4.2%). Extractions by soxhlet apparatus were carried out on the dried samples to get the crude extracts. Fractionation and purification on the crude extracts resulted in the isolation of three new cyclohexane oxides and ten known compounds, comprising of cyclohexane oxides, esters, carboxylic acid, labdane diterpene, and flavonoids. Two new compounds identified as 2-(benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside and 3-debenzoyl-rotepoxide A, together with seven known compounds, crotepoxide, 4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate, 1,6-desoxypipoxide, curcumrinol C, 2'-hydroxy-4,4',6'-trimethoxychalcone and naringenin 4',7-dimethyl ether were isolated from the Malaysian species, while a new compound identified as 3-acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)-cyclohexa-4,6-diene with the seven known compounds namely crotepoxide, 4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate, 1,6-desoxypipoxide, 6-acetylzeylenol, trans-docosyl ferulate, benzyl benzoate and benzoic acid were isolated from the Indonesian species. The structures of all compounds were established based on spectral studies using MS, IR, UV and NMR spectroscopies. Naringenin 4',7-dimethyl ether, curcumrinol C, trans-docosyl ferulate, and benzoic acid were found for the first time from K. rotunda and also the genus of Kaempferia. Antibacterial and antioxidant screening assays using disc-diffusion method and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) method, respectively were carried out on the crude extracts and essential oils. The crude extracts and essential oils of K. rotunda from Malaysia and Indonesia did not show activities on antibacterial and antioxidant assay.

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ABSTRAK

Kajian terhadap komposisi minyak pati dan fitokimia ke atas Kaempferia rotunda yang ditanam di Malaysia dan Indonesia telah dilakukan. Sebanyak 0.09% dan 0.23% hasil minyak pati masing-masing diperolehi daripada penyulingan hidro terhadap rizom segar K. rotunda. Minyak pati ini seterusnya dianalisis dengan menggunakan KG kapilari (Indeks Kovat) dan KG-SJ. Sebatian utama dalam minyak pati rizom dari Malaysia terdiri daripada bornil asetat (9.6%), benzil benzoat (8.4%), dan kamfor (5.6%), manakala minyak pati rizom dari Indonesia didapati kaya dengan benzil benzoat (87.7%) dan n-pentadekana (4.2%). Pengekstrakan soxhlet telah dijalankan terhadap rizom kering untuk mendapatkan ekstrak mentah. Pengasingan daripada ekstrak telah menemukan tiga sebatian baru dan sepuluh sebatian yang telah diketahui, merangkumi sebatian sikloheksana oksida, ester, asid karboksilik, diterpena labdana dan flavonoid. Dua sebatian baru yang diasingkan daripada spesies Malaysia dikenali sebagai 2-(benzoiloksimetil)fenil (3-O-asetil)-β-glukopiranosida dan 3-debenzoilrotepoksida A, bersama dengan tujuh sebatian yang diketahui, iaitu krotepoksida, 4-benzoiloksimetil-3,8-dioksatrisiklo[5.1.0.02,4]oktana-5,6-diol 5-asetat, 1,6-desoksipipoksida, kurkumrinol C, 2'-hidrosi-4,4',6'-trimetosikalkon and naringenin 4',7-dimetil eter. Satu sebatian baru dikenali 3-asetoksi-2-benzoiloksi-1-(benzoiloksimetil)sikloheksa-4,6-diena, manakala sebatian-sebatian yang telah diketahui, iaitu krotepoksida, 4-benzoiloksimetil-3,8-dioksatrisiklo[5.1.0.02,4]oktana-5,6-diol 5-asetat, 1,6-desoksipipoksida, 6-asetil-zeilenol, trans-dokosil ferulat, benzil benzoat dan asid benzoik telah diasingkan daripada spesies Indonesia. Struktur bagi semua sebatian telah dikenalpasti secara spektroskopi melalui MS, IR, UV dan NMR. Naringenin 4',7-dimetil eter, kurkumrinol C, trans-dokosil ferulat, dan benzoik asid telah ditemui pertama kali daripada K. rotunda khususnya daripada genus Kaempferia. Ujian antibakteria dan antioksidan melalui teknik pembauran cakera dan radikal bebas DPPH telah dijalankan ke atas ekstrak mentah dan minyak pati. Ekstrak mentah dan minyak pati K. rotunda dari Malaysia dan Indonesia didapati tidak menunjukkan perencatan aktiviti terhadap bakteria dan radikal bebas DPPH.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION OF THE STATUS OF THESIS

SUPERVISOR’S DECLARATION

CERTIFICATION OF EXAMINATION

TITLE PAGE i

DECLARATION OF ORIGINALITY AND ii

EXCLUSIVENESS

DEDICATION iii

ACKNOWLEDGEMENTS iv

PREFACE v

ABSTRACT vi

ABSTRAK vii

TABLE OF CONTENTS viii

LIST OF TABLES xii

LIST OF SCHEMES xiii

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xvi

LIST OF APPENDICES xviii

1 INTRODUCTION

1.1 General Introduction 1

1.2 Objectives 2

1.3 Scope of Study 3

2 LITERATURE REVIEWS

2.1 The Zingiberaceae Family 4

ix

2.2 Botany and Distribution of Kaempferia 5

2.3 The Usages of Kaempferia Species 6

2.4 Phytochemicals of Kaempferia Species 7

2.4.1 Chalcones 7

2.4.2 Flavones 10

2.4.3 Flavanones 11

2.4.4 Pimarane Diterpenes 12

2.4.5 Cyclohexane Oxides 14

2.4.6 Cinnamates 16

2.5.7 Phenolics 17

2.4.8 Esters 18

2.4.9 Monoterpene 19

2.5 Bioactivity Studies on Kaempferia Species 19

2.6 Essential Oil Studies on Kaempferia Species 21

2.7 Biogenetic Pathway on Cyclohexane Oxides 22

3 CHEMICAL COMPOSITIONS OF KAEMPFERIA ROTUNDA OILS

3.1 The Essential Oils of Kaempferia rotunda 27

3.1.1 Rhizome Oil of Malaysian Kaempferia rotunda 27

3.1.2 Rhizome Oil of Indonesian Kaempferia rotunda 31

3.1.3 Comparison of the Compositions of Malaysian 33

and Indonesian Kaempferia rotunda

4 PHYTOCHEMICAL AND BIOACTIVITY STUDIES OF

KAEMPFERIA ROTUNDA

4.1 Phytochemical Study of Malaysian Kaempferia rotunda 36

4.1.1 Crotepoxide (54) 37

4.1.2 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 38

4.1.3 4-Benzoyloxymethyl-3,8-dioxatricyclo- 39

[5.1.0.02,4]octane-5,6-diol 5-acetate (57)

4.1.4 1,6-Desoxypipoxide (69) 41

4.1.5 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 42

β-glucopyranoside (131)

x

4.1.6 5-Hydroxy-7,4'-dimethoxyflavanone 51

(Naringenin 4',7-dimethyl ether) (133)

4.1.7 12-Acetoxy-8α,13-dihydroxylab-14-en-7-one 52

(Curcumrinol C) (134)

4.1.8 4-Benzoyloxymethyl-3-oxabicyclo[4.1.0]heptane- 56

1,5,6,7-tetrol (3-Debenzoylrotepoxide A)(132)

4.2 Phytochemical Studies of Indonesian Kaempferia rotunda 64


4.2.2 Benzyl Benzoate (82) 65

4.2.3 trans-Docosyl ferulate (137) 66

4.2.4 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)- 69

cyclohexa-4,6-diene (136)

4. 2.5 6-Acetylzeylenol (68) 78

4. 2.6 Benzoic Acid (138) 80

4. 3 The Distribution of Compounds in Malaysian and 81

Indonesian Kaempferia rotunda

4.4 Bioactivity Studies on Kaempferia rotunda 82

4.4.1 Antibacterial Activity 83

4.4.2 Antioxidant Activity 85

5 EXPERIMENTAL

5.1 General Experimental Procedures 87

5.2 Chemicals 88

5.3 Plant Materials 88

5.4 Essential Oil Extraction and Analysis 89

5.5 Extraction and Isolation of Malaysian Kaempferia 90

rotunda


5.5.2 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 91

5.5.3 4-Benzoyloxymethyl-3,8-dioxatricyclo- 92


5.5.4 1,6-Desoxypipoxide (69) 92

5.5.5 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 93


xi

5.5.6 Naringenin 4',7-dimethyl ether (133) 94

5.5.7 Curcumrinol C (134) 94

5.5.8 3-Debenzoylrotepoxide A (132) 95

5.6 Extraction and Isolation of Indonesian Kaempferia 96

rotunda


5.6.2 Benzyl benzoate (82) 96

5.6.3 trans-Docosyl ferulate (137) 97

5.6.4 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)- 97

cyclohexa-4,6-diene (136)

5.6.5 6-Acetylzeylenol (68) 98

5.6.6 Benzoic Acid (138) 98

5.7 Bioactivity Studies 99

5.7.1 Chemicals 99

5.7.2 Microorganisms 99

5.7.3 Antimicrobial Assay 99

5.7.3.1 Microorganisms and Culture Media 99

5.7.3.2 Disc Diffusion Method 100

5.7.4 Antioxidant Screening (Free Radical 101

Scavenging Activity (DPPH))

6 CONCLUSION AND RECOMMENDATION 103

REFERENCES 105

Appendices 117

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LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Biological Properties of Several Kaempferia Species 19

2.2 Biological Properties of Compounds Isolated from 20

Kaempferia Species

3.1 Constituents of Malaysian of Kaempferia rotunda Rhizome Oil 29

3.2 Constituents of Indonesian Kaempferia rotunda Rhizome Oil 32

3.3 Comparison of the Rhizomes Oils of Malaysian and Indonesian 33

Kaempferia rotunda

4.1 1H, 13C NMR and COSY Data of Compound (69) 42

4.2 1H and 13C NMR Data of Compound (131) and 2-(Benzoyl- 44

oxymethyl)phenyl (3,6-di-O-acetyl)-β-glucopyranoside (135)

4.3 1H, 13C NMR, COSY and HMBC Data of Compound (134) 55





4.8 Compounds Isolated from Malaysian and Indonesian 82

Kaempferia rotunda

5.1 The Inhibition Zones of Tested Samples 101

5.2 Percentage Inhibitions of Tested Samples 102

xiii

LIST OF SCHEMES

SCHEME NO. TITLE PAGE

4.1 The Elimination of Water Molecule from Compound (134) and 53

the Formation of Acylium Ion

4.2 The Suggested Mass Fragmentation Pattern of Compound (137) 68

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LIST OF FIGURES

FIGURES NO. TITLE PAGE

2.1 Biogenetic Pathway for the Formation of Crotepoxide (54) 23

2.2 Biogenetic Pathway for the Formation of Senepoxide (95) and 24

Pipoxide (96)

2.3 Biogenetic Pathway for the Formation of (-)-Zeylenol (75), 25

Senepoxide (95), Pipoxide (63), Seneol (106) and Zeylena (107)

2.4 Biogenetic Pathway for the Formation of (+)-Zeylenol (74) and 26

(-)-Zeylenol (75)

4.1 IR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 45


4.2 1H NMR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 46


4.3 CIMS Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 47


4.4 13C NMR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 48


4.5 13C NMR and DEPT Spectra of 2-(Benzoyloxymethyl)phenyl 49

(3-O-acetyl)-β-glucopyranoside (131)

4.6 HMBC Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 50


4.7 IR Spectrum of 3-Debenzoylrotepoxide A (132) 57

4.8 1H NMR Spectrum of 3-Debenzoylrotepoxide A (132) 58

4.9 1H-1H COSY Spectrum of 3-Debenzoylrotepoxide A (132) 59

4.10 13C NMR Spectrum of 3-Debenzoylrotepoxide A (132) 60

4.11 13C NMR and DEPT Spectra of 3-Debenzoylrotepoxide A (132) 61

xv

4.12 CIMS Spectrum of 3-Debenzoylrotepoxide A (132) 63

4.13 1H NMR Spectrum of 3-Acetoxy-2-benzoyloxy-1- 72

(benzoyloxymethyl)cyclohexa-4,6-diene (136)

4.14 1H-1H COSY Spectrum of 3-Acetoxy-2-benzoyloxy-1- 73


4.15 IR Spectrum of 3-Acetoxy-2-benzoyloxy-1- 74


4.16 13C NMR Spectrum of 3-Acetoxy-2-benzoyloxy-1- 75


4.17 13C NMR and DEPT Spectra of 3-Acetoxy-2-benzoyloxy-1- 76


4.18 EIMS Spectrum of 3-Acetoxy-2-benzoyloxy-1- 77


4.19 Graft of Percentage Scavenging Capacity of DPPH by Vitamin C, 86

5-Deoxyquercetin, Crude Extracts and Essential Oils from

Kaempferia rotunda Measured by UV Spectrometric Assay

5.1 Kaempferia rotunda cultivated in Kempas, Johor 90

5.2 Kaempferia rotunda imported from Indonesia 90

5.3 The Arrangement of the Sample Discs and Control Discs in 101

Petri Dish

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LIST OF ABBREVIATIONS

br broad

CC Column Chromatography

COSY Correlation Spectroscopy 13C Carbon-13

CDCl3 Deuterated chloroform

CD3COCD3 Deuterated acetone

CHCl3 Chloroform

CIMS Chemical Ionization Mass Spectrometry

DPPH 2,2-Diphenyl-1-picrylhydrazyl

d doublet

dd doublet of doublet

ddd doublet of doublet of doublet

DCM Dichloromethane

DEPT Distortionless Enhancement by Polarization Transfer

D2O Deuterium oxide

EtOAc Ethyl acetate

EIMS Electron Impact Mass Spectrometry

Et2O Diethyl ether

GC Gas Chromatography

GC-MS Gas Chromatography-Mass Spectrometry 1H Proton

HMBC Heteronuclear Multiple Bond Correlation

HMQC Heteronuclear Multiple Quantum Coherence

Hz Hertz

IR Infrared

IC50 Inhibition Concentration at 50%

J coupling constant

xvii

KBr Potassium bromide

KI Kovats Index

lit. Literature

LWT Lebensm.-Wiss. u.-Technol / Food Science and Technology

MIC Minimum Inhibition Concentration

MS Mass Spectrometry

mM millimolar

m/z mass to charge ion

MeOH Methanol

m.p. melting point

MgSO4 Magnesium sulphate

MHz Megahertz

m multiplet

NMR Nuclear Magnetic Resonance

nm nanometer

NaOH Sodium hydroxide

NaCl Sodium chloride

Ph Phenyl

PE Petroleum ether

ppm parts per million

q quartet

Rf retention factor

RP-VLC Reversed Phase Vacuum Liquid Chromatography

SD Standard Deviation

s singlet

t triplet

tr trace

TLC Thin Layer Chromatography

UV Ultraviolet

VLC Vacuum Liquid Chromatography

μM micromolar

δ chemical shift

c concentration

xviii

LIST OF APPENDICES

APPENDIX TITLE PAGE

1 GC Chromatogram of Malaysian Kaempheria rotunda 117

(Rhizome) Oil

2 GC Chromatogram of Indonesian Kaempheria rotunda 118

(Rhizome) Oil

3 CIMS Spectrum of Crotepoxide (54) 119

4 1H NMR Spectrum of Crotepoxide (54) 120

5 1H-1H COSY Spectrum of Crotepoxide (54) 121

6 HMBC Spectrum of Crotepoxide (54) 122

7 IR Spectrum of Crotepoxide (54) 123

8 13C NMR Spectrum of Crotepoxide (54) 124

9 13C NMR and DEPT Spectra of Crotepoxide (54) 125

10 HMQC Spectrum of Crotepoxide (54) 126

11 UV Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 127

12 IR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 128

13 1H NMR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 129

14 13C NMR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 130

15 13C NMR and DEPT Spectra of 2'-Hydroxy-4,4',6'- 131

trimethoxychalcone (1)

16 EIMS Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 132

17 HMBC Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 133

18 CIMS Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo- 134


19 IR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo- 135


xix

20 1H NMR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo- 136


21 13C NMR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo- 137


22 HMBC Spectrum of Benzoyloxymethyl-3,8-dioxatricyclo- 138


23 HMQC Spectrum of Benzoyloxymethyl-3,8-dioxatricyclo- 139


24 EIMS Spectrum of 1,6-Desoxypipoxide (69) 140

25 13C NMR Spectrum of 1,6-Desoxypipoxide (69) 141

26 13C NMR and DEPT Spectra of 1,6-Desoxypipoxide (69) 142

27 IR Spectrum of 1,6-Desoxypipoxide (69) 143

28 1H NMR Spectrum of 1,6-Desoxypipoxide (69) 144

29 1H-1H COSY Spectrum of 1,6-Desoxypipoxide (69) 145

30 IR Spectrum of Naringenin 4',7-dimethyl ether (133) 146

31 1H NMR Spectrum of Naringenin 4',7-dimethyl ether (133) 147

32 1H-1H COSY Spectrum of Naringenin 4',7-dimethyl ether (133) 148

33 13C NMR Spectrum of Naringenin 4',7-dimethyl ether (133) 149

34 13C NMR and DEPT Spectra of Naringenin 4',7- 150

dimethyl ether (133)

35 EIMS Spectrum of Naringenin 4',7-dimethyl ether (133) 151

36 IR Spectrum of Curcumrinol C (134) 152

37 13C NMR Spectrum of Curcumrinol C (134) 153

38 13C NMR and DEPT Spectra of Curcumrinol C (134) 154

39 EIMS Spectrum of Curcumrinol C (134) 155

40 1H NMR Spectrum of Curcumrinol C (134) 156

41 1H NMR Spectrum of Curcumrinol C (134) (Expansion) 157

42 1H-1H COSY Spectrum of Curcumrinol C (134) 158

43 1H-1H COSY Spectrum of Curcumrinol C (134) (Expansion) 159

44 HMBC Spectrum of Curcumrinol C (134) 160

45 1H NMR Spectrum of Benzyl Benzoate (82) 161

46 IR Spectrum of Benzyl Benzoate (82) 162

47 13C NMR Spectrum of Benzyl Benzoate (82) 163

48 13C NMR and DEPT Spectra of Benzyl Benzoate (82) 164

xx

49 EIMS Spectrum of Benzyl Benzoate (82) 165

50 13C NMR Spectrum of trans-Docosyl ferulate (137) 166

51 13C NMR and DEPT Spectra of trans-Docosyl ferulate (137) 167

52 IR Spectrum of trans-Docosyl ferulate (137) 168

53 1H NMR Spectrum of trans-Docosyl ferulate (137) 169

54 1H-1H COSY Spectrum of trans-Docosyl ferulate (137) 170

55 HMBC Spectrum of trans-Docosyl ferulate (137) 171

56 EIMS Spectrum of trans-Docosyl ferulate (137) 172

57 IR Spectrum of 6-Acetylzeylenol (68) 173

58 1H NMR Spectrum of 6-Acetylzeylenol (68) 174

59 CIMS Spectrum of 6-Acetylzeylenol (68) 175

60 13C NMR Spectrum of 6-Acetylzeylenol (68) 176

61 13C NMR and DEPT Spectra of 6-Acetylzeylenol (68) 177

62 1H-1H COSY Spectrum of 6-Acetylzeylenol (68) 178

63 HMBC Spectrum of 6-Acetylzeylenol (68) 179

64 IR Spectrum of Benzoic Acid (138) 180

65 1H NMR Spectrum of Benzoic Acid (138) 181

66 13C NMR Spectrum of Benzoic Acid (138) 182

CHAPTER 1

INTRODUCTION

1.1 General Introduction

Malaysia with its tropical forest is blessed with high biological diversity,

which enclosed over 10% of the world’s total number of species, with some of them

are unique only to Malaysia. Among more than 7,000 species of angiosperms and

600 species of ferns in Malaysia, about 12 to 18% of trees, shrubs and herbs are

reported to have medicinal properties [1]. The usage of medicinal plant products has

attracted interest since the past decade. The beneficial medicinal effects of plant

materials typically result from the secondary products in the plant [2]. Many

biologically active plant-derived compounds were discovered as a result of chemical

studies through isolation of active compounds from traditional medicine [3]. Our

Malaysian flora represents a huge, barely untapped reserve of natural resources

which is believed to contain substances with therapeutic potentials that yet to be

explored.

Research projects related to the natural products carried out in Malaysia

included: the chemistry and technology of palm oil, natural pesticides, natural

flavours and pharmalogical testing of medicinal plants [4]. Development of organic

chemistry has been closely associated with the chemistry of natural products. Many

techniques of extraction, separation, structure determination and synthesis have been

developed to understand the structural variation among the natural products.

Research in this area has certainly led to better understanding of the structural

2

requirements for a variety of physiological activities, leading to the synthesis and

modification of several lead compounds and analogues.

The study of bioactive natural products constituents is the first step in drug

discovery programs, while the eventual outcomes of blockbuster drugs may not be

that easily realised in view to the high cost and research effort [5]. Despite all these,

natural products drug discovery programs are developed all over the world, mainly

because of the high chemical diversity from natural products as compared to

synthetics. The potential of these natural products is largely unknown and

endangered plants have added the urgency for more vigorous screening programs.

With the currently available tools of extraction, chromatographic separation and

structure identification, a fresh look at the well-studied plants may also be rewarding.

In Malaysia, local universities and research institutes are embarking on their own

programs which involve a concerted multidisciplinary approach in the discovery of

bioactive agents from plant-derived natural products. Certain novel strategies have

been carried out based on the expertise and funds available. The screening programs

implemented and administered are aimed to discover new compounds from the

Malaysian flora for use in the pharmaceutical and related industries. This in turn,

will help in the development and transfer of technology, which can be achieved

through collaborative programs.

1.2 Objectives

Phytochemical studies reported in the literature review are mostly carried out

on the Kaempferia species of Thailand. Literature search revealed only a few reports

on the studies of K. rotunda compared to other species. Therefore, this research will

focus on the chemical compositions of essential oil and chemical constituents the

rhizomes of K. rotunda cultivated in Kempas, Johor and Indonesia. In addition,

antioxidant and antimicrobe activities of the rhizomes of K. rotunda will also be

investigated.

3

The first objective is to extract the essential oils from the fresh rhizomes of

Kaempferia rotunda and to analyze the chemical compositions of the essential oils.

The second objective is to extract phytochemicals from the dried rhizomes, to purify

and identify their structures. The third goal is to evaluate the antioxidant and

antimicrobe activities of the essential oils, crude extracts and the pure isolated

compounds.

1.3 Scope of Study

This research focuses on the study of the essential oils compositions of the

fresh rhizomes and phytochemicals from the dried rhizomes of Kaempferia rotunda

from Malaysia and Indonesia. The fresh rhizomes will be extracted by

hydrodistillation technique to obtain the essential oil. The dried rhizomes will be

extracted using soxhlet with different polarity of solvents. The crude extracts will be

fractionated by using vacuum liquid chromatography (VLC), followed by

purification of the fractions using gravity column chromatography (CC) or

chromatotron or versa-flash column chromatography and or recystallization to obtain

the pure compounds. The compositions of the essential oils will be analyzed using

GC and GC-MS, while the chemical constituents from dried samples will be

characterized using spectroscopic methods including MS, IR, 1H NMR, 13C NMR,

2D NMR (COSY, HMQC, HMBC) and UV. The biological activities such as

antioxidant and antibacterial will be carried out on the crude extracts, essential oils

and the pure isolated compounds.

chemical constituents and bioactivity of...

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