PHYTOCHEMICAL AND BIOACTIVITY STUDIES OF

STROBILANTHES CRISPUS L.

by

KOAY YEN CHIN

Thesis submitted in the fulfillment of the requirements

for the degree of

Master of Science

UNIVERSITI SAINS MALAYSIA

January 2012

ACKNOWLEDGEMENT

First of all, I would like to sincerely thank my supervisor Assoc. Prof. Dr. Wong Keng

Chong, for providing me the opportunity to work in his research group. His valuable advice, guidance and support enormously contributed to the completion of this work. I am also grateful to my co-supervisor, Assoc. Prof. Dr. Hasnah Osman. Special thanks to the Dean, School of Chemical Sciences, USM, Prof. Wan Ahmad Kamil Mahmood for providing the facilities which ensured the completion of this work. I would like to acknowledge the financial supports provided by Universiti Sains Malaysia, which are

1001/PKIMIA/821070 and 1001/PKIMIA/821050. I would also like to thank the

Institute of Graduate Studies (IPS) for awarding me the Fellowship Scheme, which covered my tuition fees and my allowance.

I would like to thank Dr. Ibrahim M. Eldeen from the School of Pharmaceutical

Sciences, USM for his help in carrying out the bioactivity studies. I would also like to thank Mr. V. Shunmugam from herbarium of the School of Biological Sciences for his help in identifying the plant in this study.

I am thankful to the technical and laboratory staffs of the School of Chemical Sciences, in particular, Mr. Chow Cheng Por, Mr. Clement D'Silva and Mr. Megat Hasnul, for their constructive help in carrying out the research throughout the period. I gratefully thank Mr. Zahari Othman for his help in the NMR data acquisition. I thank my friends and colleagues for supporting me throughout my graduate program. I would like to

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convey my deepest gratitude to my father and my mother, along with my sister for their endless support and encouragement.

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TABLE OF CONTENTS

ACKNOWLEDGEMENT i

TABLE OF CONTENTS iii

LIST OF TABLES vii

LIST OF FIGURES viii

LIST OF SCHEMES xii

LIST OF ABBREVIATIONS xii

ABSTRAK xv

ABSTRACT xvii

CHAPTER ONE INTRODUCTION 1

1.1 Medicinal plants 1

1.2 Acanthaceae Family 1

1.2.1 The Genus Strobilanthes 2

1.2.1.1 Strobilanthes crispus L. Bremek 2

1.3 Biological properties of Strobilanthes crispus 3

1.4 Previous studies on S. crispus 5

1.4.1 Phytochemical studies on S. crispus 5

1.4.2 Studies on the volatile constituents of S. crispus 9

1.5 Studies on other Strobilanthes species 10

1.5.1 Strobilanthes auriculatus 10

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1.5.2 Strobilanthes callosus 10

1.5.3 Strobilanthes ciliatus 12

1.5.4 Strobilanthes cusia 12

1.5.5 Strobilanthes dyeriana 16

1.5.6 Strobilanthes formosanus 16

1.5.7 Strobilanthes ixiocephala 17

1.5.8 Strobilanthes japonicas 18

1.6 Biological properties for other species of the genus Strobilanthes 19

1.7 Objectives 21

CHAPTER TWO MATERIALS AND METHODS 22

2.1 Collection of plant material 22

2.2 Extraction procedure 22

2.3 Chromatography 23

2.3.1 Thin layer chromatography 23

2.3.2 Column chromatography 23

2.4 Instrumental 23

2.4.1 Specific optical rotation measurement 23

2.4.2 Melting point determination 24

2.4.3 Infrared spectroscopy 24

2.4.4 Direct-probe mass spectrometry 24

2.4.5 Gas chromatography 24

iv

2.4.6 GC-MS 25

2.4.7 Nuclear magnetic resonance spectroscopy 25

2.5 Isolation and purification of compounds in the hexane extract 25

2.5.1 1-Heptacosanol (Compound c-1) 26

2.5.2 Lignoceric acid (Compound c-2) 26

2.5.3 Stigmasterol (Compound c-3) 27

2.6 Isolation and purification of dichloromethane extract 28

2.6.1 A mixture of β-amyrin 3-docosanoate, β-amyrin 3-tetracosanoate, 29

β-amyrin 3-eicosanoate and β-amyrin 3-tricosanoate (c-4)

2.6.2 Taraxerone (Compound c-5) 31

2.6.3 Taraxerol (Compound c-6) 32

2.6.4 A mixture of taraxerol 3-docosanoate and taraxerol 3- 33

tetracosanoate (c-7)

2.7 Isolation and purification of methanol extract 35

2.7.1 4-Acetyl-2, 7-dihydroxy-1, 4, 8-triphenyloctane-3, 5-dione 35

(Compound c-8)

2.7.2 Stigmasterol 3-O-β-D-glucopyranoside (Compound c-9) 36

2.8 Antibacterial activity 38

2.8.1 Micro-dilution antibacterial assay 38

2.9 Anti-cholinesterase activity 39

2.9.1 Acetylcholinesterase enzyme inhibitory activity 39

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CHAPTER THREE RESULTS AND DISCUSSION 41

3.1 Chemical constituents isolated from the hexane extract of S. crispus 41

3.1.1 1-Heptacosanol (Compound c-1) 41

3.1.2 Lignoceric acid (Compound c-2) 48

3.1.3 Stigmasterol (Compound c-3) 55

3.2 Chemical constituents isolated from the dichloromethane extract of S. crispus 71

3.2.1 A mixture of β-amyrin 3-docosanoate, β-amyrin 3-tetracosanoate, 71

β-amyrin 3-eicosanoate and β-amyrin 3-tricosanoate (c-4)

3.2.2 Taraxerone (Compound c-5) 91

3.2.3 Taraxerol (Compound c-6) 106

3.2.4 A mixture of taraxerol 3-docosanoate and taraxerol 3- 120

tetracosanoate (c-7)

3.3 Chemical constituents isolated from the methanol extract of S. crispus 138

3.3.1 4-Acetyl-2, 7-dihydroxy-1, 4, 8-triphenyloctane-3, 5-dione 138

(Compound c-8)

3.3.2 Stigmasterol 3-O-β-D-glucopyranoside (Compound c-9) 153

3.4 Antibacterial activity 167

3.5 Acetylcholinesterase enzyme inhibitory activity 170

CHAPTER FOUR CONCLUSION 172

REFERENCES 174

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LIST OF TABLES

Table 3.1 GC-MS of trans-esterified products of c-4 72

Table 3.2 GC-MS of trans-esterified products of c-7 121

Table 3.3 Antibacterial activity of the extracts and compounds from S. crispus 167

as detected using the minimum inhibitory concentration assay.

Results obtained are presented as MIC values (µg mL-1) ± SD.

Table 3.4 Inhibition (percentage and IC50 values) of acetylcholinesterase 170

enzyme activity by crude extracts and the isolated compounds (100

μg mL-1) obtained from S. crispus as determined by the micro-plate

assay. Results are expressed as means ± SD.

vii

LIST OF FIGURES

Figure 1.1 Strobilanthes crispus L. Bremek 3

Figure 3.1 IR spectrum of c-1 43

Figure 3.2 EIMS of c-1 44

1 Figure 3.3 H NMR spectrum of c-1 (500 MHz, CDCl3) 45

13 Figure 3.4 C NMR spectrum of c-1 (125 MHz, CDCl3) 46

Figure 3.5 DEPT 135 spectrum of c-1 (125 MHz, CDCl3) 47

Figure 3.6 IR spectrum of c-2 50

Figure 3.7 EIMS of c-2 51

1 Figure 3.8 H NMR spectrum of c-2 (400 MHz, CDCl3) 52

13 Figure 3.9 C NMR spectrum of c-2 (100 MHz, CDCl3) 53

Figure 3.10 DEPT 135 spectrum of c-2 (100 MHz, CDCl3) 54

Figure 3.11 IR spectrum of c-3 56

Figure 3.12 EIMS of c-3 57

1 Figure 3.13 H NMR spectrum of c-3 (400 MHz, CDCl3) 62

13 Figure 3.14 C NMR spectrum of c-3 (100 MHz, CDCl3) 63

Figure 3.15 DEPT 135 spectrum of c-3 (100 MHz, CDCl3) 64

Figure 3.16 DEPT 90 spectrum of c-3 (100 MHz, CDCl3) 65

Figure 3.17a HMQC spectrum of c-3 (400 MHz, CDCl3) 66

Figure 3.17b HMQC spectrum of c-3 (expanded) (400 MHz, CDCl3) 67

Figure 3.18a HMBC spectrum of c-3 (400 MHz, CDCl3) 68

Figure 3.18b HMBC spectrum of c-3 (expanded) (400 MHz, CDCl3) 69

viii

1 1 Figure 3.19 H- H COSY spectrum of c-3 (400 MHz, CDCl3) 70

Figure 3.20 IR spectrum of c-4 73

Figure 3.21 EIMS of c-4 74

Figure 3.22 GC-MS of trans-esterified products of c-4 (a) TIC (b) Methyl 75

eicosanoate (c) Methyl docosanoate (d) Methyl tricosanoate

(e) Methyl tetracosanoate

1 Figure 3.23 H NMR spectrum of c-4 (500 MHz, CDCl3) 82

13 Figure 3.24 C NMR spectrum of c-4 (125 MHz, CDCl3) 83

Figure 3.25 DEPT 135 spectrum of c-4 (125 MHz, CDCl3) 84

Figure 3.26 DEPT 90 spectrum of c-4 (125 MHz, CDCl3) 85

Figure 3.27a HMQC spectrum of c-4 (500 MHz, CDCl3) 86

Figure 3.27b HMQC spectrum of c-4 (expanded) (500 MHz, CDCl3) 87

Figure 3.28a HMBC spectrum of c-4 (500 MHz, CDCl3) 88

Figure 3.28b HMBC spectrum of c-4 (expanded) (500 MHz, CDCl3) 89

1 1 Figure 3.29 H- H COSY spectrum of c-4 (500 MHz, CDCl3) 90

Figure 3.30 IR spectrum of c-5 92

Figure 3.31 EIMS of c-5 93

1 Figure 3.32 H NMR spectrum of c-5 (400 MHz, CDCl3) 97

13 Figure 3.33 C NMR spectrum of c-5 (100 MHz, CDCl3) 98

Figure 3.34 DEPT 135 spectrum of c-5 (100 MHz, CDCl3) 99

Figure 3.35 DEPT 90 spectrum of c-5 (100 MHz, CDCl3) 100

Figure 3.36a HMQC spectrum of c-5 (400 MHz, CDCl3) 101

Figure 3.36b HMQC spectrum of c-5 (expanded) (400 MHz, CDCl3) 102

ix

Figure 3.37a HMBC spectrum of c-5 (400 MHz, CDCl3) 103

Figure 3.37b HMBC spectrum of c-5 (expanded) (400 MHz, CDCl3) 104

1 1 Figure 3.38 H- H COSY spectrum of c-5 (400 MHz, CDCl3) 105

Figure 3.39 IR spectrum of c-6 107

Figure 3.40 EIMS of c-6 108

1 Figure 3.41 H NMR spectrum of c-6 (400 MHz, CDCl3) 111

13 Figure 3.42 C NMR spectrum of c-6 (100 MHz, CDCl3) 112

Figure 3.43 DEPT 135 spectrum of c-6 (100 MHz, CDCl3) 113

Figure 3.44 DEPT 90 spectrum of c-6 (100 MHz, CDCl3) 114

Figure 3.45a HMQC spectrum of c-6 (400 MHz, CDCl3) 115

Figure 3.45b HMQC spectrum of c-6 (expanded) (400 MHz, CDCl3) 116

Figure 3.46a HMBC spectrum of c-6 (400 MHz, CDCl3) 117

Figure 3.46b HMBC spectrum of c-6 (expanded) (400 MHz, CDCl3) 118

1 1 Figure 3.47 H- H COSY spectrum of c-6 (400 MHz, CDCl3) 119

Figure 3.48 IR spectrum of c-7 122

Figure 3.49a EIMS of c-7 123

Figure 3.49b FABMS of c-7 123

Figure 3.50 GC-MS of trans-esterified products of c-7 (a) TIC (b) Methyl 124

docosanoate (c) Methyl tetracosanoate

1 Figure 3.51 H NMR spectrum of c-7 (400 MHz, CDCl3) 128

Figure 3.52 1H NMR spectrum of a) triterpene moiety of c-7 after 129

trans-esterification b) taraxerol (500 MHz, CDCl3)

13 Figure 3.53 C NMR spectrum of c-7 (100 MHz, CDCl3) 130

x

Figure 3.54 DEPT 135 spectrum of c-7 (100 MHz, CDCl3) 131

Figure 3.55 DEPT 90 spectrum of c-7 (100 MHz, CDCl3) 132

Figure 3.56a HMQC spectrum of c-7 (400 MHz, CDCl3) 133

Figure 3.56b HMQC spectrum of c-7 (expanded) (400 MHz, CDCl3) 134

Figure 3.57a HMBC spectrum of c-7 (400 MHz, CDCl3) 135

Figure 3.57b HMBC spectrum of c-7 (expanded) (400 MHz, CDCl3) 136

1 1 Figure 3.58 H- H COSY spectrum of c-7 (400 MHz, CDCl3) 137

Figure 3.59 IR spectrum of c-8 142

Figure 3.60 EIMS of c-8 143

1 Figure 3.61 H NMR spectrum of c-8 (500 MHz, CDCl3) 144

13 Figure 3.62 C NMR spectrum of c-8 (125 MHz, CDCl3) 145

Figure 3.63 DEPT 135 spectrum of c-8 (125 MHz, CDCl3) 146

Figure 3.64 DEPT 90 spectrum of c-8 (125 MHz, CDCl3) 147

Figure 3.65a HMQC spectrum of c-8 (500 MHz, CDCl3) 148

Figure 3.65b HMQC spectrum of c-8 (expanded) (500 MHz, CDCl3) 149

Figure 3.66a HMBC spectrum of c-8 (500 MHz, CDCl3) 150

Figure 3.66b HMBC spectrum of c-8 (expanded) (500 MHz, CDCl3) 151

1 1 Figure 3.67 H- H COSY spectrum of c-8 (500 MHz, CDCl3) 152

Figure 3.68 IR spectrum of c-9 154

Figure 3.69 FABMS of c-9 155

1 Figure 3.70 H NMR spectrum of c-9 (400 MHz, C5D5N) 158

13 Figure 3.71 C NMR spectrum of c-9 (100 MHz, C5D5N) 159

Figure 3.72 DEPT 135 spectrum of c-9 (100 MHz, C5D5N) 160

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Figure 3.73 DEPT 90 spectrum of c-9 (100 MHz, C5D5N) 161

Figure 3.74a HMQC spectrum of c-9 (400 MHz, C5D5N) 162

Figure 3.74b HMQC spectrum of c-9 (expanded) (400 MHz, C5D5N) 163

Figure 3.75a HMBC spectrum of c-9 (400 MHz, C5D5N) 164

Figure 3.75b HMBC spectrum of c-9 (expanded) (400 MHz, C5D5N) 165

1 1 Figure 3.76 H- H COSY spectrum of c-9 (400 MHz, C5D5N) 166

LIST OF SCHEMES

Scheme 3.1 Mass fragmentation patterns of c-3 59

Scheme 3.2 Mass fragmentation patterns of c-4 72

Scheme 3.3 Mass fragmentation patterns of c-5 95

Scheme 3.4 Mass fragmentation patterns of c-6 109

LIST OF ABBREVIATIONS

Chromatography CC Column chromatography GC Gas chromatography GC-MS Gas chromatography-Mass spectrometry

Rf Retention factor TLC Thin layer chromatography tR (min) Retention time (minutes) TIC Total ion chromatogram FID Flame ionization detector

xii

Instrumental and experimental IR Infrared FID Flame ionization detector EI-MS Electron ionization mass spectrometry FAB-MS Fast atom bombardment mass spectrometry NMR Nuclear magnetic resonance COSY Correlation spectroscopy DEPT Distortionless enhancement by polarization transfer HMBC Heteronuclear multiple bond correlation HMQC Heteronuclear multiple quantum correlation 1D, 2D one- or two- dimensional

Symbols m/z mass/charge eV electron volt ppm part per million J coupling constant Hz hertz br broad s singlet d doublet t triplet m multiplet dd doublet of doublets ddd doublet of doublet of doublets

[α]D specific optical rotation at the sodium D line

δC carbon-13 chemical shift in parts per million

xiii

δH proton chemical shift in parts per million

IC50 concentration of inhibitor required to inhibit a target by 50%

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KAJIAN FITOKIMIA DAN BIOAKTIVITI TERHADAP STROBILANTHES CRISPUS L.

ABSTRAK

Kajian fitokimia terhadap ekstrak heksana, diklorometana dan metanol daripada daun

Strobilanthes crispus telah berjaya mengasingkan sembilan sebatian, iaitu 1- heptakosanol (c-1), asid lignoserik (c-2) dan stigmasterol (c-3) daripada ekstrak heksana, campuran empat ester β-amirin (c-4), tarakseron (c-5), tarakserol (c-6) dan campuran dua ester tarakserol (c-7) daripada ekstrak diklorometana, dan 4-asetil-2,7-dihidroksi-

1,4,8-trifeniloktana-3,5-dion (c-8) serta stigmasterol 3-O-β-D-glukopiranosida (c-9) daripada ekstrak metanol. Struktur sebatian c-1 sehingga c-9 dikenalpasti melalui kaedah spektroskopi dan kromatografi. Pengasingan c-3 daripada daun S. crispus pernah dilaporkan dalam kajian terdahulu, tetapi lapan sebatian lagi merupakan pengasingan pertama kali daripada daun S. crispus. Ekstrak diklorometana dan metanol daripada daun serta sebatian yang diasingkan daripada ekstrak tersebut telah diuji untuk aktiviti antibakteria terhadap bakteria Gram-positif: Bacillus subtilis, Staphylococcus aureus, dan bakteria Gram-negatif: Klebsiella pneumoniae, Escherichia coli dan Salmonella typhimurium dengan menggunakan esei pencairan mikro. Hasil kajian mencadangkan bahawa kebanyakan sampel yang diuji mempunyai aktiviti terhadap Staphylococcus aureus dan Bacillus subtilis (7.8-62.5 μg mL-1), manakala aktiviti yang sederhana telah diperhatikan terhadap Salmonella typhimurium and Escherichia coli (31.0-125.0 μg mL-

1). Sebatian c-8 merupakan sebatian yang jarang ditemui dan didapati paling berkesan dalam perencatan pertumbuhan kedua-dua bakteria Gram-positif dan Gram-negatif dengan nilai MIC terendah (antara 7.8 dan 62.5 μg mL-1). Dalam kajian aktiviti

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perencatan enzim asetilkolinesterase, sebanyak 85.0% perencatan enzim telah

-1 ditunjukkan oleh estrak diklorometana dengan nilai IC50 46.0 µg mL pada kepekatan

100.0 µg mL-1. Sebatian yang diasingkan daripada ekstrak tersebut masing-masing menunjukkan aktiviti perencatan asetilkolinesterase pada tahap yang berbeza, dengan c-

8 menunjukkan perencatan enzim yang ketara dengan nilai IC50 terendah, iaitu 31.0 µg mL-1, diikuti oleh tarakseron dan campuran dua ester tarakserol, masing-masing ialah

42.0 dan 44.0 µg mL-1. Kajian ini adalah yang pertama melaporkan potensi S. crispus terhadap aktiviti perencatan enzim asetilkolinesterase. Keputusan positif yang diperolehi daripada kajian ini telah mencadangkan bahawa tumbuhan ini mempunyai aktiviti antibakteria dan anti-kolinesterase.

xvi

PHYTOCHEMICAL AND BIOACTIVITY STUDIES OF STROBILANTHES CRISPUS L.

ABSTRACT

Phytochemical investigation of the hexane, dichloromethane and methanol extracts of the leaves of Strobilanthes crispus has led to the isolation of nine compounds, comprising 1-heptacosanol (c-1), lignoceric acid (c-2) and stigmasterol (c-3) from the hexane extract, a mixture of four esters of β-amyrin (c-4), taraxerone (c-5), taraxerol (c-

6) and a mixture of two esters of taraxerol (c-7) from the dichloromethane extract, as well as 4-acetyl-2,7-dihydroxy-1,4,8-triphenyloctane-3,5-dione (c-8) and stigmasterol 3-

O-β-D-glucopyranoside (c-9) from the methanol extract. Structures of compounds c-1 to c-9 were elucidated with the aid of chromatographic and spectroscopic techniques.

Compound c-3 has previously been reported from the leaves of S. crispus, however, the rest of the compounds were isolated for the first time from this plant. The dichloromethane and methanol leaf extracts, together with the isolated compounds were tested against Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus, and

Gram-negative bacteria: Klebsiella pneumoniae, Escherichia coli and Salmonella typhimurium, using micro-dilution assay. The majority of the samples tested indicated promising inhibitory activities against Staphylococcus aureus and Bacillus subtilis (7.8-

62.5 μg mL-1), while appreciable activity was observed against Salmonella typhimurium and Escherichia coli (31.0-125.0 μg mL-1). Compound c-8, a rare natural occurring compound, exhibited the strongest inhibitory effect against both Gram-positive and

Gram-negative bacteria, with low MIC values (between 7.8 and 62.5 μg mL-1). In the acetylcholinesterase enzyme inhibitory test, the dichloromethane extract showed activity

xvii

-1 (85.0%) against acetylcholinesterase with an IC50 value of 46.0 µg mL at the concentration of 100.0 µg mL-1. The isolates exhibited different levels of acetylcholinesterase inhibitory activities with c-8 being significantly active in this

-1 bioassay with an IC50 of 31.0 µg mL , followed by taraxerone and the mixture of the

-1 two fatty esters of taraxerol, with IC50 values of 42.0 and 44.0 µg mL , respectively, with reference to galanthamine (control). This study is the first report describing the potential of S. crispus in the acetylcholinesterase enzyme inhibitory activity. The results of this study suggested that this plant possesses both antibacterial and anti-cholinesterase activities.

xviii CHAPTER ONE

INTRODUCTION

1.1 Medicinal plants

A recent report shows that approximately 420,000 plant species exist in nature (Vuorela et al., 2004). Medicinal plants are known as plants that are able to produce active constituents to prevent diseases, maintain health or cure ailments. Traditionally, medicinal plants are being explored therapeutically to alleviate ailments in humans for several millennia (Chen et al., 2003; Chattopadhyay et al., 2004; Radad et al., 2006).

Numerous studies have been conducted to isolate and identify novel compounds for further investigation of their biological activities (Zheng & Wang, 2001; Cai et al.,

2004; Surveswaran et al., 2007). Medicinal plant drug discovery continues to supply crucial leads against assorted targets including cancer, HIV/AIDS, Alzheimer’s, malaria and pain (Balunas & Kinghorn, 2005). Among over 15,000 species of higher plants found in Malaysia, 1200 are reported to possess pharmaceutical value, and of which can be classified as medicinal plants (Soepadmo, 1991; Bakar et al., 2006).

1.2 Acanthaceae Family

Acanthaceae family, or Acanthus family, is a taxon of dicotyledonous flowering plants containing almost 346 genera and about 4300 species. Most of them are tropical herbs, shrubs, or twining vines; some are epiphytes. Only a few species are distributed in temperate regions. The four main centers of distributions are Indonesia and Malaysia,

Africa, Brazil, and Central America. The representatives of the family can be found

1 nearly in every habitat, including dense or open forests, scrublands, wet fields, valleys, sea coast, marine areas, and swamps, as an element of mangrove woods (Sasidharan,

2004).

1.2.1 The Genus Strobilanthes

Strobilanthes is the second largest genus in the Acanthaceae family after Justicia L., comprising approximately 350 species of perennial herbs and subshrubs. It can be found mostly in Asia’s tropical region with a few species extending north into Asia’s temperate region (Scotland & Vollesen, 2000). It is known as one of the most interesting genera owing to its diversified habits, gregarious nature and scant but elegant flowering.

Strobilanthes plants typically have opposite leaves which are unequal in size (Liamas,

2003).

1.2.1.1 Strobilanthes crispus L. Bremek

Strobilanthes crispus L. Bremek (Figure 1.1) is a bush-like plant that can be found in tropical countries ranging from Madagascar to Indonesia on riverbanks or abandoned fields (Baker & Bakhuizen, 1965). It is popularly known as “daun pecah beling” in

Jakarta or “enyoh kilo”, “kecibeling” or “kejibeling” in Java and “Hei Mian Jiang Jun” in Chinese. It is a woody spreading shrub that can grow up to 6 feet in height. Javanese would often use this plant as fence hedges due to their height. The leaves are dark green in colour, oblong-lanceolate, a little toothed and elliptical in shape. As the bottom of the leaves is covered with short hairs, the leaves are rough to touch from underside as compared to the top surface. The plant is panicled with cluster of yellow flowers budded

2 in leafy sheaths. This shrub can be propagated via stem cuttings (Sunarto, 1977;

Fadzelly et al., 2006).

Figure 1.1 Strobilanthes crispus L. Bremek

1.3 Biological properties of S. crispus

Throughout history, natural products have afforded us a large number of compounds with antiviral, antibacterial, antimalarial, anti-inflammatory, antioxidant, and anticancer properties. More than 60% of the commercially available anticancer drugs were discovered from natural sources. Infusion of the dried leaves of S. crispus has been used in traditional Chinese medicine and folk medicine for their antidiabetic, diuretic, anticancer and blood pressure lowering properties (Perry & Metzger, 1980; Bakar et al.,

2006). A poultice of fresh leaves is reported for treatment of wounds and snake bites, and oral administration of the S. crispus juice for enhancement of the rate of wound

3 healing in normal and diabetic rats (Wijayakusuma, 2000; Norfarizan-Hanoon et al.,

2009).

The methanolic crude extract of S. crispus exhibited potent antibacterial activity against

Bacillus cereus (Muskhazli et al., 2009). Muslim et al. (2010) and Rahmat et al. (2006) reported the methanolic extract displayed a promising cytotoxic effect on certain human cancer cell lines such as colon carcinoma cell (HCT 116), non-small cell lung adenocarcinoma cell (NCI-H23) and human breast ductal carcinoma cell (T-47D).

Yaacob et al. (2010) also reported that a sub-fraction from the dichloromethane extract of S. crispus possessed potential as a cancer therapeutic agent which selectively killed breast and prostate cancer cell lines, but not the normal breast epithelial cell line. Not only did the leaves extracts kill the breast cancer cell lines, a significant anti hepatocarcinogenesis effect on rats was also confirmed. It was also found that the hot water extract of the fermented and unfermented leaves has the efficacious ability to reduce blood glucose level in hyperglycemic rats (Fadzelly et al., 2006).

Kusumoto et al. (1992) had confirmed that the water extract of S. crispus inhibited the proliferation of retrovirus; an agent in viral disease such as acquired immune deficiency syndrome (AIDS) and adult T-cell leukemia. Iqbal et al. (2010) reported that a dose- dependent of extracts S. crispus showed possible protection against lipid peroxidation and DNA damage induced by Fe-NTA and H2O2.

Rahmat et al. (2006) reported that there were high antioxidant activities showed by the extracts of S. crispus, using the ferric thiocyanate (FTC) and thiobarbituric acid (TBA)

4 methods. Cosmetics with S. crispus as an active ingredient further featured excellent skin brightening properties and also skin pigmentation relieving properties (Sawaki et al., 2002).

1.4 Previous studies on S. crispus

Despite being the second largest genus in the Acanthaceae family, only very few species within the genus Strobilanthes have been investigated for their phytochemical constituents. Although the literature survey revealed that the leaves of S. crispus are potent for treatment of several diseases, only very few reports on its phytochemical investigation are available.

1.4.1 Phytochemical studies on S. crispus

The earliest phytochemical investigation on S. crispus started in 1987, when Soedira et al. (1987) isolated verbacoside (1) and seven phenolic acids, namely, caffeic acid (2), ferulic acid (3), gentisic acid (4), p-coumaric acid (5), p-hydroxybenzoic acid (6), syryngic acid (7) and vanillic acid (8) from the leaves. Liza et al. (2010) reported the presence of eight bioactive flavonoids identified as apigenin (9), (+)-catechin (10), (−)- epicatechin (11), kaempferol (12), luteolin (13), myricetin (14), naringenin (15) and rutin (16). β-Sitosterol (17) and stigmasterol (18), were isolated from the leaf extracts of

S. crispus (Rahmat et al., 2006). In addition, the leaves were reported to be rich in minerals such as potassium and calcium and contained high levels of vitamins C, B1, B2 which contributed further to its total anti-oxidant activity (Ismail et al., 2000).

5

1

2 3 4

5 6 7

6

8 9

10 11

12 13

7

14 15

16

17

8

18

1.4.2 Studies on the volatile constituents of S. crispus

Rahmat et al. (2006) identified a total of 28 constituents among the volatile constituents of S. crispus leaves. They were identified by the GC-MS analysis and the major components were determined to be α-cadinol (19), 2,3-dihydrobenzofuran (20), eugenol

(21), megastigmatrienone (22) and trans-phytol (23).

19 20 21

22 23

9

1.5 Studies on other Strobilanthes species

1.5.1 Strobilanthes auriculatus

A total of twenty-three compounds were identified in the essential oil of S. auriculatus, of which the two major components were found to be borneol (24) and isoborneol (25)

(Zutshi, 1970; Weyerstahl et al., 1987; Weyerstahl et al., 1988).

24 25

1.5.2 Strobilanthes callosus

Agarwal & Rangari et al. (2001) reported the presence of crassifolioside (26), lupeol

(27), stigmasterol 3-O-β-D-glucopyranoside (28), and few phenylpropanoid glycosides from the chloroform and ethyl acetate extracts of S. callosus. Singh et al. (2002) reported the presence of taraxerol (29) from the benzene extract S. callosus.

26

10

27

28

29

11

1.5.3 Strobilanthes ciliatus

Phytochemical investigations on the extracts of the root and stem of S. ciliatus have resulted in the isolation of six compounds, namely betulin (30), lupeol (27), stigmasterol (18), stigmasterol glycoside (28), taraxerol (29) and 4-acetyl-2,7- dihydroxy-1,4,8-triphenyloctane-3,5-dione (31) (Reneela & Sripathi, 2010).

30

31

1.5.4 Strobilanthes cusia

Chen et al. (1987) isolated β-sitosterol (17) and three triterpenoids, namely, betulin (30), lupeol (27) and lupenone (32) from the roots of S. cusia. Isolation by Li et al. (1993)

12 yielded two quinazolinone alkaloids, 4-quinazolinone (33), and 2,4-quinazolinedione

(34), and two indole alkaloids, idirubin (35) and indigo (36).

32 33 34

35 36

A total of eight compounds were isolated from S. cusia, a new lignan, namely, (+)- lyoniresinol 3α-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (37), (+)-9-O-β-D- glucopyranosyl lyoniresinol (38), two phenylethanoid glycosides, namely, [2-(3,4- dihydroxyphenylethyl)]-3-O-α-D-apiofuranosyl-(1→4)-4-O-caffeoyl)-β-D- glucopyranoside (cusianoside A) (39), and [2-(3,4-dihydroxyphenylethyl)]-3-O-β-D- xylopyranosyl-(1→3)-4-O-caffeoyl)-β-D-glucopyranoside (cusianoside B) (40), together

13 with lupeol (27), acetoside (41), (+)-5,5’-dimethoxy-9-O-β-D-glucopyranosyl lariciresinol (42) and (+)-5,5’-dimethoxy-9-O-β-D-glucopyranosyl secoisolariciresinol

(43) (Tomonori et al., 2004).

37 R = D-Apiofuranosyl

38 R = H

39 R = D-Apiofuranosyl

40 R = D-Xylopyranosyl

41 R = L-Rhamnopyranosyl

14

42

43

Kuntze et al. (1979) reported the presence of an alkaloid, namely tryptanthrin (44), from the leaves of S. cusia. A previous study conducted by Li et al. (2007) confirmed the presence of a seco-pregnane steroid glaucogenin C (45) and its monosugar-glycoside cynatratoside A (46) in the leaves of S. cusia.

44

15

45 R = H

46 R =

1.5.5 Strobilanthes dyeriana

Smith et al. (1981) isolated two anthocyanins, namely, cyanidin-3,5-diglucoside (47) and peonidin-3,5-diglucoside (48), from the leaves of S. dyeriana.

47 R = H

48 R = CH3

1.5.6 Strobilanthes formosanus

Kao et al. (2004) revealed the presence of two new flavone glycosides, 5,7- dimethoxyflavone-4'-O-[β-D-apiofuranosyl(1→5)-β-D-glucopyranoside] (49) and 3'-

16 hydroxy-5,7-dimethoxyflavone-4'-O-β-D-apiofuranoside (50) and together with four known compounds betulin (30), 2,6-dimethoxy-1,4-benzoquinone (51), 4'-hydroxy-5,7- dimethoxyflavone (52) and lupeol (27) from the stem and roots of S. formosanus.

49 R1 = H, R2 = -[β-D-apiofuranosyl(1→5)-β-D-glucopyranoside]

50 R1 = OH, R2 = -β-D-apiofuranoside

51 52

1.5.7 Strobilanthes ixiocephala

Agarwal & Rangari et al. (2001) isolated a rare triterpenic alcohol 19β-lupeol (53) from

S. ixiocephala. Investigation of the essential oil from the flowering tops of S. ixiocephala indicated the presence of cadinol (19), β-caryophyllene (54), α-fenchyl acetate (55) and a new sesquiterpene, ixiocephol (56) (Agarwal & Rangari, 2003a).

17

53 54 55

56

1.5.8 Strobilanthes japonicas

A new flavone glycoside, 5,7-dimethoxy-4'-hydroxyflavone-4'-O-apioside (57), namely, strobilanthin, together with two known compounds 5,7-dimethoxy-4'-hydroxyflavone

(58) and stigmasterol (18), were isolated from the extract of S. japonicas (Huang et al.,

1987).

57

18

58

1.6 Biological properties of other species of the genus Strobilanthes

An alkaloid, namely, tryptanthrin (46), isolated from the leaves of Strobilanthes cusia was found to possess a marked antifungal properties in treating dermatophytes (Kuntze et al., 1979). Tryptanthrin (46) is confirmed to display an inhibitory activity against the growth of Mycobacterium tuberculosis (Mitscher & Baker, 1998). The methanol extract of S. cusia leaves displayed potent anti-inflammatory and antipyretic effects (Ho et al.,

2003). The roots of S. cusia are popularly used as a traditional Chinese medicine for treating influenza, epidemic cerebrospinal meningitis, encephalitis B, viral pneumonia and mumps (Tomonori et al., 2004). The dichloromethane and ethyl acetate extracts of

S. cusia marked a significant cytotoxic activity on cell lines (Nguyen et al., 2006).

Steroids such as glaucogenin C (44) and cynatratoside A (45), isolated from S. cusia, were reported to exhibit antiviral activity (Li et al., 2007).

Lupeol (27) and 19α-lupeol (52), isolated from the roots of Strobilanthus callosus and

Strobilanthus ixiocephala exhibit anti-inflammatory and antiarthritic activities (Agarwal

& Rangari, 2003b). Both benzene and ethanol extracts and taraxerol (29), isolated from

S. callosus, demonstrate anti-inflammatory and antimicrobial activities respectively

19

(Singh et al., 2002). Ngo et al. (1995) demonstrated that Strobilanthes flaccidifolius juice, when taken orally by normal guinea pigs and rabbits, possessed potent abortive properties.

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1.6 Objectives

1. To isolate and identify the chemical constituents of the leaves of S. crispus.

2. To characterize the isolated constituents with the aid of different spectroscopic methods, namely, IR, mass spectrometry and nuclear magnetic resonance spectroscopy, including 2D NMR techniques.

3. To evaluate the biological activities of the selected crude extracts and isolated compounds.

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CHAPTER TWO

MATERIALS AND METHODS

2.1 Collection of plant material

Strobilanthes cripus was obtained from a commercial supplier and was identified by Mr.

V. Shunmugam, a staff of the herbarium of the School of Biological Sciences, Universiti

Sains Malaysia, Penang, where a voucher specimen (Voucher No. 11246) has been deposited.

2.2 Extraction procedure

Fresh leaves (3.0 kg) collected from the plant were rinsed with distilled water and were air-dried at room temperature in an open space for 2 weeks. The air-dried leaves (1.0 kg) were powdered and macerated sequentially in hexane, dichloromethane and methanol.

Each of the different extractions was performed at room temperature three times (3 × 5

L), 24 h each time. All extracts after filtration were evaporated in vacuo using a rotary evaporator to give 10 g (0.33% w/w of fresh leaves), 15 g (0.50% w/w of fresh leaves) and 12 g (0.40% w/w of fresh leaves) of hexane, dichloromethane and methanol extracts, respectively.

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2.3 Chromatography

2.3.1 Thin Layer Chromatography

Thin layer chromatography (TLC) were performed on pre-coated TLC plates (20 × 20 cm, coated with 0.2 mm silica gel F254 on aluminium sheets, Merck). The spots on TLC plates were visualized with a UV lamp (Vilber Lournet, multiband UV-254/356 nm).

Triterpenes and plant sterols were detected with 95% methanolic sulphuric acid by heating the TLC plates with a heat gun after the plates were dipped in the reagent.

2.3.2 Column chromatography

Column chromatography (CC) was carried out using silica gel 60 (230-240 mesh

ASTM, Merck, 0.040-0.060 mm) and gradient elution was performed from less polar solvents to more polar solvents (hexane, chloroform, ethyl acetate and methanol).

Samples were dissolved in a minimum quantity of an appropriate solvent and taken up in a small quantity of silica gel. Upon drying on a rotary evaporator, a powdered material was produced which was loaded onto the column (Sharp et al., 1989).

2.4 Instrumental

2.4.1 Specific optical rotation measurement

Optical rotations were measured using an ATAGO AP-300 automatic polarimeter

(Japan). Sucrose solution (1.0%) was used as a standard and the sodium lamp was set at

589 nm. Samples were dissolved in chloroform and a cell with 100 mm length was used for the measurements.

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2.4.2 Melting point determination

Melting points were measured on a Stuart Scientific SMP-1 (United Kingdom) melting point apparatus.

2.4.3 Infrared spectroscopy

Infrared (IR) spectra were recorded on a Perkin-Elmer System 2000 FT-IR spectrometer

(England, United Kingdom). Spectra were obtained by the pressed disk technique using potassium bromide (KBr), and scanned in the range 4000-650 cm-1.

2.4.4 Direct-probe mass spectrometry

The electron impact mass spectra (EI-MS), 70 eV and fast atom bombardment mass spectra (FAB-MS) were determined on an Agilent 5975C MSD Mass Spectrometer and a Thermo Finnigan MAT95XL Mass Spectrometer, respectively.

2.4.5 Gas chromatography

GC analysis of the methyl esters from compounds c-4 and c-7 were carried out using a

Thermo Finnigan instrument, fitted with a Supelcowax 10 fused-silica capillary column

(30 m, 0.25 mm ID, 0.25 μm, Supelco Inc., USA) and equipped with a flame ionization detector (FID). The operating conditions were: initial oven temperature, 40°C (held for

10 min), then programmed at 5°C min-1 to 250°C, and held for 20 min at 250°C. The

-1 carrier gas was N2 at a flow rate of 2.0 mL min . Injector port and detector temperatures were set at 250°C and 275°C, respectively.

24