IN VITRO ANTICANCER ACTIVITY GUIDED INVESTIGATION

OF THE PHYTOCHEMICALS OF TYPHONIUM

FLAGELLIFORME (LODD.) BLUME (ARACEAE)

by

LAI CHOON SHEEN

Thesis submitted in fulfilment of the requirements

for the degree of

Doctor of Philosophy

June 2010

ii

ACKNOWLEDGEMENTS

Praise be unto God the creator of heaven and earth, for His grace and mercy,

that along with all other things, have faithfully and graciously provided me the

capability to complete all the work required in fulfilment of this degree.

I would like to express my gratitude to my main supervisor, Professor

Emeritus Dr. V. Navaratnam, for giving me the opportunity to work in the area of my

interest and also his constant support, help and encouragement throughout the course

of my study. I thank my co-supervisor, Associate Professor Dr. Mas Rosemal Hakim

Mas Haris, for all the inspiring stories, motivation, guidance and support particularly

in the area of structure elucidation. I would also like to thank Professor Dr. Sharif

Mahsufi Mansor for co-supervising this project and helping me to go through the

many hiccups I faced throughout the course of my study. I am truly grateful to

Professor Nair for his invaluable advice and guidance on this work.

I thank all the support staff of Centre for Drug Research, for things would not

have turned out smoothly if it were not for their help. Special thanks to Mr. Asokan

Muniandy and Mr. Arunacalam for maintaining a cheerful environment in the lab

and rendering helping hands to make things right. Many thanks to Mr. Hilman for his

assistance in acquiring the GC-MS data, Mr. Zamri, Mr. Nahari, Mr. Khoo, Mr.

Rahim and all the other lab staff and administrative staff for helping me in one way

or another.

My sincere gratitude also goes to HISCO (M) Sdn Bhd and Varian, Germany

for NMR spectral acquisition. Thanks are due to Research Instruments Sdn Bhd and

Waters Asia Ltd, Singapore too for obtaining the LC-MS data. I also thank Professor

iii

Dr. Alex Matter from Novartis Institute for Tropical Disease (NITD) for his advice

in this project and his staff, Dr. Sejal Patel and Ms. Jeasie Tan for their technical

assistance in compound separation.

I thank my dear friends, Shubhadra, Peng Lin, Venkatesh and Vanessa for

accompanying me through this part of the journey of my life, for sharing my ups and

down and providing me encouragement in tough times. I would also like to thank all

my church friends from Sungai Nibong Gospel Hall for their constant prayer and

support.

Last but not least, I thank my dearest mom and dad, my brother Choon

Shyang, my sister Jun Shi and most importantly my beloved husband En Haur for all

their encouragement, understanding and sacrifices made throughout these years so

that I could complete my studies smoothly.

iv

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iv

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF SYMBOLS AND ABBREVIATIONS xvii

ABSTRAK xxi

ABSTRACT xxiii

CHAPTER 1 INTRODUCTION 1

1.1 Medicines from Plants 1

1.2 Plants as a Source of Anti-cancer Agents 4

1.3 Typhonium flagelliforme (Lodd.) Blume 8

1.3.1 Taxonomy and Distribution 8

1.3.2 Ethnomedical Background 10

1.3.3 Pharmacological Studies 12

1.3.4 Phytochemicals 13

1.4 Methods of Plant Extraction 15

1.4.1 Maceration 16

1.4.2 Soxhlet Extraction 16

1.4.3 Ultrasonic-assisted Solvent Extraction 17

1.5 Techniques of Separation 17

1.5.1 Thin-layer Chromatography 17

1.5.2 Column Chromatography 18

v

1.5.3 High Performance Liquid Chromatography 20

1.6 Compound Identification and Structure Elucidation 22

1.6.1 Ultraviolet-visible Spectroscopy 22

1.6.2 Infrared Spectroscopy 22

1.6.3 Nuclear Magnetic Resonance Spectroscopy 23

1.6.4 Mass Spectrometry 26

1.7 In Vitro Anticancer Assay 28

1.7.1 MTT Antiproliferative Assay 28

1.7.2 Apoptotic Cell Death 29

1.7.3 Photodynamic Therapy 30

1.8 Aims of the Study 31

CHAPTER 2 EXPERIMENTAL 33

2.1 Research Materials 33

2.1.1 List of Chemicals 33

2.1.2 List of Equipments and Instruments 35

2.2 Plant Material 35

2.2.1 Collection of Raw Material 35

2.2.2 Identification of Plant and Preparation of Voucher Specimen 36

2.3 Experimental Methods 36

2.3.1 Plant Extraction 36

2.3.1.1 Maceration 37

2.3.1.2 Ultrasonic-assisted Solvent Extraction 39

2.3.1.3 Soxhlet Extraction 39

2.3.1.4 Traditional Medicine Preparative Method 39

2.3.2 Comparison of Plant Extracts 40

vi

2.3.2.1 Extraction Yields 40

2.3.2.2 Antiproliferative Assay 40

2.3.2.3 Chemical Composition 40

2.3.3 Scaled-up Production 41

2.3.4 Flash Column Chromatography 41

2.3.4.1 Column Packing 41

2.3.4.2 Fractionation of Crude Hexane Extract 42

2.3.4.3 Fractionation of Crude DCM Extract 43

2.3.5 Isolation and Characterisation of the Chemical Constituents of

Fraction D/F19 44

2.3.6 Identification of the Chemical Constituents of Fraction D/F21 46

2.3.7 Purification and Identification of the Chemical Constituents of

Fraction D/F15 48

2.3.8 Compound Characterisation and Identification 49

2.3.8.1 Physical Characterisation 49

2.3.8.2 Ultraviolet-visibile Spectroscopy 50

2.3.8.3 Infrared Spectroscopy 50

2.3.8.4 Nuclear Magnetic Resonance Spectroscopy 50

2.3.8.5 Gas Chromatography-Mass Spectrometry 51

2.3.8.6 Liquid Chromatography-Tandem Mass Spectrometry 51

2.3.9 Animal Cell Culture 52

2.3.9.1 Preparation of Culture Media 53

2.3.9.2 Preparation of Phospate Buffer Saline 53

2.3.9.3 Resuscitation of Cryopreserved Cells 53

2.3.9.4 Subculture 54

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2.3.9.5 Cryopreservation 54

2.3.9.6 Cell Counting 55

2.3.10 MTT Antiproliferation Assay 56

2.3.10.1 Determination of Optimum Seeding Concentration for

MTT Assay 56

2.3.10.2 Cell Plating 62

2.3.10.3 Test Sample Preparation 63

2.3.10.4 End Point Measurement 65

2.3.10.5 Screening Strategy and Criteria for Activity 66

2.3.11 Apoptosis 68

2.3.11.1 Cell Morphological Examination 68

2.3.11.2 DNA Nick-End Labelling 68

2.3.12 Photosensitizing Activity 69

CHAPTER 3 RESULTS 71

3.1 Evaluation of the Extraction Methodology 71

3.1.1 Extraction Yields 71

3.1.2 Antiproliferative Activity 73

3.1.3 Chemical Constituents 76

3.2 Fractionation of the Active Extracts 79

3.2.1 Hexane Extract 79

3.2.2 DCM Extract 84

3.2.3 Selectivity Studies on Several Active Fractions 88

3.3 Isolation and Identification of Active Constituents 89

3.3.1 Isolation of the Constituents of Fraction D/F19 89

3.3.2 Characterisation and Structure Elucidation 92

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3.3.2.1 D/F19/03 92

3.3.2.1.1 Physical Characteristic 92

3.3.2.1.2 Ultraviolet-visible Spectroscopy 96

3.3.2.1.3 Infrared Spectroscopy 97

3.3.2.1.4 Nuclear Magnetic Resonance Spectroscopy 100

3.3.2.1.5 Liquid Chromatography-Tandem Mass

Spectrometry 117

3.3.2.2 D/F19/05 122

3.3.2.2.1 Physical Characteristic 122

3.3.2.2.2 Ultraviolet-visible Spectroscopy 124

3.3.2.2.3 Infrared Spectroscopy 125

3.3.2.2.4 Nuclear Magnetic Resonance Spectroscopy 128

3.3.2.2.5 Liquid Chromatography-Tandem Mass

Spectrometry 143

3.3.3 Identification of the Chemical Constituents of D/F21 147

3.3.3.1 Gas Chromatogrgaphy-Mass Spectrometry Analysis 147

3.3.3.2 NMR Analysis 151

3.3.3.3 Further Purification and Analysis of Subfractions of

D/F21 153

3.3.4 Evaluation of Fraction D/F18 154

3.3.5 Identification of Pheophytine-a and Pheophytine-b 155

3.3.6 Purification and Identification of the Chemical Constituents in

Fraction D/F15/03 156

3.3.7 Purification and Identification of the Chemical Constituents

in D/F15/02 161

ix

3.3.7.1 Infrared Spectroscopy 161

3.3.7.2 Gas Chromatography-Mass Spectrometry 164

3.4 In Vitro Anticancer Activity of the Chemical Constituents of

Typhonium flagelliforme 168

3.4.1 Antiproliferative and Photosensitizing Activity of the Subfractions

of D/F19 168

3.4.2 Antiproliferative Activity of the Subfractions of D/F21 171

3.4.3 Antiproliferative Activity of the White Solid Obtained from

D/F15/03 172

3.4.4 Antiproliferative Activity of the Light Yellowish Oil Purified

from D/F15/02 174

3.4.5 Apoptotic Effect of D/F19/03, D/F19/05, D/F19 and D/F21 174

3.4.5.1 Microscopic Observation 174

3.4.5.2 DNA Nick-End Labelling 177

CHAPTER 4 DISCUSSION 180

CHAPTER 5 CONCLUSION 198

REFERENCES 201

APPENDICES 214

LIST OF PUBLICATIONS 234

x

LIST OF TABLES

Page Table 3.1 IC50 of the extracts obtained by various methods of

extraction

76

Table 3.2 Mass yield of fractions obtained from the hexane extract and their chemical composition based on TLC.

81

Table 3.3 Mass yield of subfractions of H/F1 and H/F13 and their chemical composition based on TLC.

82

Table 3.4 Mass yield of fractions obtained from the DCM extract and their chemical composition based on TLC.

85

Table 3.5 Single bond correlation between 13C and 1H in pheophorbide-a (1) determined using the HSQC data.

106

Table 3.6 1H, 13C and 2D NMR data of pheophorbide-a (1) in CDCl3 acquired at 600 MHz.

116

Table 3.7 Single bond correlation between 13C and 1H in pyropheophorbide-a (2) determined using the HSQC data.

132

Table 3.8 1H, 13C and 2D NMR data of pyropheophorbide-a (2) in CDCl3 acquired at 600 MHz.

142

Table 3.9 Four major chemical constituents of D/F21 determined using GC-MS.

149

Table 3.10 Chemical constituents of D/F21 subfractions as suggested by GC-MS.

154

Table 3.11 GC-MS data of the chemical constituents of the white solid from D/F15/03.

159

Table 3.12 Four major chemical constituents of the oil sample obtained from fraction D/F15/02 determined using GC-MS.

166

Table 3.13 Inhibitory effect of D/F19/03, D/F19/05 and their respective standards on NCI-H23 and HS578T cells in the case where it was irradiated and not irradiated.

171

Table 3.14 Antiproliferative activity of D/F21 and its subfractions

171

xi

LIST OF FIGURES

Page Figure 1.1 Drugs approved by FDA from Jan 1981 to June 2006 by

source (total number of drugs, N = 1184).

3

Figure 1.2 Anticancer agents derived from plants.

7

Figure 1.3 Picture of Typhonium flagelliforme.

9

Figure 1.4 Geographical distribution of Typhonium flagelliforme.

10

Figure 1.5 Chemical structures of the compounds isolated from Typhonium flagelliforme.

14

Figure 1.6 Cleavage of MTT structure by the dehydrogenase enzyme to form MTT formazan.

28

Figure 2.1 Flow chart of sample preparation procedure for extraction to be carried out using various extraction methodologies.

38

Figure 2.2 Prep-HPLC chromatogram of D/F19 detected at 365 nm.

46

Figure 2.3 Optical density corresponding to the various cell seeding concentrations of NCI-H23 cells at 0, 48 and 72 h.

58

Figure 2.4 NCI-H23 cell growth profile at various seeding concentrations.

59

Figure 2.5 Optical density corresponding to the various cell seeding concentrations of BALB/c 3T3 cells at 0, 48 and 72 h.

60

Figure 2.6 BALB/c 3T3 cell growth profile at various seeding concentrations.

60

Figure 2.7 Optical density corresponding to the various cell seeding concentrations of HS578T cells at 0, 48 and 72 h.

61

Figure 2.8 HS578T cell growth profile at various seeding concentrations.

61

Figure 2.9 Schematic diagram of the layout of a 96-well plate arranged for a six-concentration assay.

63

Figure 2.10 Percentage inhibition versus concentration profile of vincristine sulphate (N = 3 × 3)

65

xii

Figure 3.1 Extraction yields of fresh plant and dried plant obtained using various extraction methods (Mean ± S.D., N = 3).

71

Figure 3.2 Total extraction yield of all the extracts obtained using various extraction methods (N = 3).

73

Figure 3.3 Percentage inhibition versus concentration profile of (A) hexane extracts, (B) DCM extracts, (C) MeOH extracts, obtained using various extraction methods and (D) juice extract (N = 3 × 3).

74

Figure 3.4 Thin-layer chromatogram of hexane extracts obtained by various extraction methods.

77

Figure 3.5 Thin-layer chromatogram of DCM extracts obtained by various extraction methods.

78

Figure 3.6 Thin-layer chromatogram of MeOH extracts obtained by various extraction methods.

79

Figure 3.7 Growth inhibitory effect shown by the fractions and subfractions of hexane extract at 100 µg/ml on NCI-H23. Dark coloured bars indicate >85 % inhibition (Mean ± S.E.M., N = 3 × 3).

83

Figure 3.8 IC50 of the active fractions and subfractions of hexane extract. Dark coloured bars indicate samples that inhibited NCI-H23 cells at IC50 <15 µg/ml (Mean ± S.E.M., N = 3 × 3).

84

Figure 3.9 Growth inhibitory effect shown by the fractions of DCM extract at 100 µg/ml on NCI-H23. Dark coloured bars indicate >85 % inhibition (Mean ± S.E.M., N = 3 × 3).

87

Figure 3.10 IC50 of the active fractions of DCM extract. Dark coloured bars indicate samples that inhibited NCI-H23 cells at IC50 <15 µg/ml (Mean ± S.E.M., N = 3 × 3).

88

Figure 3.11 Inhibitory effect of the active fractions on BALB/c 3T3 and NCI-H23 (Mean ± S.E.M., N = 3 × 3).

89

Figure 3.12 HPLC chromatogram of (A) D/F19/00, (B) D/F19/01, (C) D/F19/02, (D) D/F19/03, (E) D/F19/04 and (F) D/F19/05 obtained on an Atlantis dC18, 4.6 × 150 mm, 5 µm column.

91

xiii

Figure 3.13 UPLC chromatograms of D/F19/03 obtained by UV detection (top) and MS (bottom).

92

Figure 3.14 Expanded mass spectrum and elemental composition of the first constituent of D/F19/03.

94

Figure 3.15 Expanded mass spectrum and elemental composition of the second constituent of D/F19/03.

95

Figure 3.16 Chemical structure of pheophorbide-a (1).

96

Figure 3.17 UV-Vis spectrum of D/F19/03.

97

Figure 3.18 Infrared spectrum of D/F19/03 prepared in KBr pellet.

99

Figure 3.19 13C NMR spectrum of D/F19/03 in CDCl3 obtained at 150 MHz.

101

Figure 3.20 1H NMR spectrum of D/F19/03 in CDCl3 obtained at 600 MHz.

104

Figure 3.21 Part I (top) and part II (bottom) of the expanded COSY spectra of D/F19/03.

105

Figure 3.22 Part I (top), part II (bottom) and part III (next page) of the expanded HMBC spectrum of D/F19/03.

108-109

Figure 3.23 NOESY spectrum of D/F19/03.

110

Figure 3.24 Structures of (132R)-pheophorbide-a (top) and (132S)-pheophorbide-a (bottom) with arrows indicating NOESY correlation.

115

Figure 3.25 UPLC chromatogram of D/F19/03 (top) and pheophorbide-a standard (bottom) at approximately 3 µg/ml.

117

Figure 3.26 MS/MS spectra of (A) second constituent of D/F19/03, (B) first constituent of D/F19/03, (C) second constituent of pheophorbide-a standard and (D) first constituent of pheophorbide-a standard.

119

Figure 3.27 Expanded MS/MS spectrum of the first constituent of D/F19/03.

120

Figure 3.28 Fragment ions of pheophorbide-a (1).

121

Figure 3.29 UPLC chromatograms of D/F19/05 obtained by UV detection (top) and MS (bottom).

122

xiv

Figure 3.30 Expanded mass spectrum and elemental composition of D/F19/05.

123

Figure 3.31 Chemical structure of pyropheophorbide-a (2).

124

Figure 3.32 UV-Vis spectrum of D/F19/05.

125

Figure 3.33 Infrared spectrum of D/F19/05 prepared in KBr pellet.

127

Figure 3.34 13C NMR spectrum of D/F19/05 in CDCl3 obtained at 150 MHz.

129

Figure 3.35 1H NMR spectrum of D/F19/05 in CDCl3 obtained at 600 MHz.

133

Figure 3.36 COSY spectrum of D/F19/05.

134

Figure 3.37 Part I (top), part II (bottom) and part III (next page) of the expanded HMBC spectrum of D/F19/05.

136-137

Figure 3.38 NOESY spectrum of D/F19/05.

138

Figure 3.39 Structure of pyropheophorbide-a (2) with arrows indicating NOESY correlation.

141

Figure 3.40 UPLC chromatogram of D/F19/05 (top) and pyropheophorbide-a standard (bottom) at approximately 8 µg/ml.

143

Figure 3.41 Expanded MS/MS spectrum of D/F19/05.

145

Figure 3.42 Fragments ions of D/F19/05.

146

Figure 3.43 Total ion chromatogram (TIC) of fraction D/F21 obtained on a HP-5MS column (30 m × 0.25 mm).

148

Figure 3.44 Chemical structures of methyl hexadecanoate (3), 1-hexadecene (4) and phytol (5).

150

Figure 3.45

1H NMR spectrum of fraction D/F21 in CDCl3 acquired at 400 MHz.

152

Figure 3.46 TLC of the subfractions of D/F21 observed at (A) 254 nm and (B) 365 nm using a mobile phase of CHCl3-EtOAc, 19:1 (v/v).

153

Figure 3.47

HPLC chromatogram of (A) fraction D/F19 and (B) fraction D/F18 obtained on Nucleosil C18, 4.6 × 250 mm, 5 µm, using ACN-0.1% acetic acid, 95:5 (v/v).

155

xv

Figure 3.48 Chemical structures of pheophytine-a and pheophytine-b

156

Figure 3.49 Overlay of the chromatograms of the white solid purified from D/F15/03 and the stigmasterol standard.

158

Figure 3.50 Chemical structures of campesterol (6), stigmasterol (7) and β-sitosterol (8).

160

Figure 3.51 Infrared spectrum of the oil sample obtained from D/F15.

162

Figure 3.52 Infrared spectral data match for the oil sample from D/F15/02 (top) and linoleic acid (bottom) obtained from the Thermo Nicolet Sampler library.

163

Figure 3.53 Total ion chromatogram (TIC) of the oil sample obtained from D/F15/02.

165

Figure 3.54 Chemical structures of palmitic acid (9), oleic acid (10), linoleic acid (11) and linolenic acid (12).

167

Figure 3.55 Inhibitory effect of D/F19 and its subfractions on NCI-H23 in the case where it was irradiated and not irradiated (Mean ± S.E.M., N = 3 × 2).

169

Figure 3.56 Inhibitory effect of D/F19, D/F19/03 and D/F19/05 on HS578T in the case where it was irradiated and not irradiated (Mean ± S.E.M., N = 3 × 2).

170

Figure 3.57 Growth inhibition profile of the sterol mixture and the stigmasterol standard on NCI-H23 (Mean ± S.E.M., N = 3 × 2).

173

Figure 3.58 Growth inhibition profile of the sterol mixture and stigmasterol standard on HS578T (Mean ± S.E.M., N = 3 × 2).

173

Figure 3.59 Morphology of NCI-H23 after 24 h of incubation with (A) control cells, 12.50 µg/ml of (B) D/F19/03 (pheophorbide-a), C) D/F19/05 (pyropheophorbide-a) and D) D/F19 (arrows show cell shrinkage and membrane blebbing).

175

Figure 3.60 Morphology of NCI-H23 after 24 h of incubation with (A) control cells and (B) 50 µg/ml of fraction D/F21 (original magnification: 32 ×).

176

xvi

Figure 3.61 Morphology of HS578T after incubation for 24 h with (A) control cells, (B) 50.00 µg/ml of D/F19/03, (C) 25.00 µg/ml of D/F19/05 and (D) 25.00 µg/ml of D/F19 (arrows indicate accumulation of sample in cell).

177

Figure 3.62 Images of NCI-H23 treated with (A) control cells, (B) 3.13 µg/ml of D/F19, (C) 12.50 µg/ml of D/F19/03 and (D) 6.25 µg/ml of D/F19/05 (arrows show stained cell nuclei which mark DNA fragmentation).

178

Figure 3.63 Images of NCI-H23 (A) control cells and (B) cells treated with 12.50 µg/ml of D/F21 (arrows show stained cell nuclei which mark DNA fragmentation).

179

Figure 3.64 Images of HS578T (A) control cells and (B) cells treated with 12.50 µg/ml of D/F19 (arrows show stained cell nuclei which mark DNA fragmentation).

179

xvii

LIST OF SYMBOLS AND ABBREVIATIONS

δ Chemical shift

J Coupling constant

I Spin quantum number

1D One dimensional

2D Two dimensional

ACN Acetonitrile

AR Analytical grade reagent

ATCC American Type Culture Collection

BPI Base peak intensity

BuOH Butanol

CDCl3 Deuterated chloroform

CHCl3 Chloroform

COSY Homonuclear Shift Correlation Spectroscopy

DAB Diaminobenzidine

DCM Dichloromethane

dd Doublet of doublet

DEPT Distortionless Enhancement by Population Transfer

DMEM Dulbeco’s Modified Eagle’s Medium

DMSO Dimethyl sulphoxide

DNA Deoxyribonucleic acid

dq Doublet of quadruplet

DQF Double quantum filtered

dUTP Biotin-16-deoxyuridine triphosphate

xviii

EDTA Ethylenediaminetetracetic acid

EI Electron impact ionisation

EtOAc Ethyl acetate

EtOH Ethanol

FBS Fetal bovine serum

FDA Food and Drug Administration, United States

FT Fourier Transform

GC Gas chromatography

GR General purpose reagent, for analysis

HEPES N-2 hydroxyethylpiperazine-N'-2-ethanesulphonic acid

HMBC Heteronuclear Multiple Bond Coherence

HPLC High performance liquid chromatography

HSQC Heteronuclear Single Quantum Correlation

IC50 Concentration of a test substance that is required for 50 %

inhibition in vitro

ICCVAM Interagency Coordinating Committee on the Validation of

Alternative Methods

i.d. Internal diameter

IR Infrared

IUPAC International Union of Pure and Applied Chemistry

m Multiplet

MEM Eagle’s Minimal Essential Medium

MeOH Methanol

MS Mass spectrometry

MS/MS Tandem mass spectrometry

xix

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NCI National Cancer Institute, United States

NIST National Institute of Standards and Technology

NMR Nuclear magnetic resonance

NOE Nuclear Overhauser effect

NOESY NOE difference spectroscopy

OD Optical density

PBS Phosphate buffer saline

PDA Photodiode array

PDT Photodynamic therapy

ppm Parts per million

prep-TLC Preparative thin-layer chromatography

prep-HPLC Preparative high performance liquid chromatography

q Quadruplet

Q-TOF Quadrupole-Time of flight

Rf Retention factor

rpm Revolution per minute

RPMI Rosewell Park Memorial Institute

rTdT Recombinant terminal deoxynucleotidyl transferase

s Singlet

S.E.M. Standard error mean

Streptavidin-HRP Horseradish-peroxidase-labeled streptavidin

t Triplet

TIC Total ion chromatogram

TLC Thin layer chromatography

xx

TMS Tetramethylsilane

TUNEL rTdT mediated dUTP Nick-End Labelling

UV Ultraviolet

v/v Volume by volume

Vis Visible

WHO World Health Organisation

xxi

KAJIAN FITOKIMIA TYPHONIUM FLAGELLIFORME (LODD.) BLUME

(ARACEAE) BERPANDUKAN AKTIVITI ANTIKANSER IN VITRO

ABSTRAK

Typhonium flagelliforme (Lodd.) Blume merupakan sejenis tumbuhan

tempatan yang digunakan sebagai ubat herba tradisional terhadap berbagai jenis

penyakit kanser. Untuk mengetahui tindakan farmakologi, keselamatan dan

keberkesanan tumbuhan herba ini dengan lebih terperinci, kandungan kimia dan

kesan biologinya perlu dikenalpasti dan dicirikan terlebih dahulu.

Suatu kaedah pengekstrakan yang sesuai telah dipilih dengan teliti pada

peringkat awal kajian ini. Tiga jenis kaedah pengekstrakan yang mempunyai cara

tindakan yang berlainan, iaitu soxhlet, pengekstrakan yang dibantui ultrasonik dan

rendaman telahpun dikaji. Kaedah rendaman merupakan kaedah pengekstrakan yang

paling sesuai kerana ekstrak yang diperolehi adalah lebih aktif dan mempunyai

kepelbagaian kandungan kimia yang tinggi, walaupun ia mempunyai peratusan hasil

yang lebih rendah berbanding dengan kaedah-kaedah lain. Kajian juga menunjukkan

bahawa pelarut-pelarut tidak polar seperti heksana dan diklorometana paling sesuai

digunakan untuk mengekstrak sebatian-sebatian aktif dalam tumbuhan ini.

Tiga fraksi daripada ekstrak diklorometana, iaitu D/F19, D/F21 dan D/F15

telah dikaji. D/F19 merupakan fraksi yang paling aktif terhadap turunan sel NCI-H23

(kanser paru-paru manusia) tetapi ia juga didapati aktif terhadap turunan sel bukan

kanser BALB/c 3T3. D/F21 merupakan fraksi yang kedua paling aktif dan

mempunyai spesifikasi yang bagus terhadap NCI-H23 berbanding dengan BALB/c

3T3. D/F15 merupakan fraksi utama Typhonium flagelliforme yang mempunyai

xxii

kesan antiproliferat yang sederhana. Feoforbid-a dan pirofeoforbid-a merupakan dua

sebatian utama yang dipencilkan daripada fraksi D/F19. Metil heksadekanoat, 1-

heksadekena, fitol dan suatu terbitan fitol merupakan kandungan kimia yang

mungkin terdapat dalam D/F21. Kampesterol, stigmasterol, β-sitosterol, asid

palmitik, asid oleik, asid linoleik dan asid linolenik merupakan kandungan kimia

D/F15.

Kesan antiproliferat D/F19 didapati bukan dihasilkan oleh komponen tunggal

tetapi adalah hasil tindakan sinergistik daripada campuran komponen-komponen.

D/F19 megaruhkan kematian sel mengikuti laluan apoptosis. Di samping itu, D/F19

juga didapati mempunyai kesan pengfotopekaan terhadap NCI-H23 dan suatu

turunan sel payu dara manusia, HS578T, yang dihasilkan daripada aktiviti feoforbid-

a, pirofeoforbid-a dan suatu lagi subfraksinya, iaitu D/F19/04. Feoforbid-a dan

pirofeoforbid-a merupakan agen pengfotopekaan cahaya yang telah dibuktikan

berkesan secara in vitro dan in vivo untuk digunakan dalam terapi fotodinamik.

Fraksi D/F21 didapati mempunyai aktiviti antiproliferat dan dapat mengaruhkan

apoptosis. Kesan ini mungkin adalah disebabkan oleh aktiviti fitol. Walaupun

kandungan kimia D/F15 merupakan sebatian-sebatian yang biasa ditemui dalam

tumbuh-tumbuhan, sebatian-sebatian ini dipercayai dapat menyumbang kepada kesan

rawatan Typhonium flagelliforme terhadap kanser berdasarkan kepada hasil-hasil

penyelidikan lain yang telah dilaporkan.

Secara keseluruhan, kandungan kimia Typhonium flagelliforme yang

mempunyai aktiviti antikanser secara in vitro telah berjaya dikenalpasti melalui

pendekatan bioaktiviti berpandu. Kajian ini menyokong penggunaan Typhonium

flagelliforme secara etnoperubatan terhadap kanser.

xxiii

IN VITRO ANTICANCER ACTIVITY GUIDED INVESTIGATION OF THE

PHYTOCHEMICALS OF TYPHONIUM FLAGELLIFORME (LODD.)

BLUME (ARACEAE)

ABSTRACT

Typhonium flagelliforme (Lodd.) Blume is an indigenous plant of Malaysia

used as a traditional herbal medicine to treat various types of cancer. In order to

effectively understand its pharmacological action, safety and efficacy, it is essential

to firstly identify and characterise its chemical constituents and their biological

relevance to the healing effect of the plant.

A suitable extraction methodology was carefully selected at the preliminary

stage of this study. Three types of extraction methods with different modes of action,

namely soxhlet, ultrasonic-assisted extraction and maceration methods were

evaluated. The maceration method proved to be the most suitable extraction

methodology for the purpose of this study with regard to the better activity and

greater diversity of the chemical constituents obtained in the extract although the

extraction yields were lower. The study also found that extraction carried out using

non-polar solvents like hexane and dichloromethane is most suitable in obtaining the

active chemical constituents.

Three fractions of the dichloromethane extracts, namely D/F19, D/F21 and

D/F15 were evaluated. D/F19 was the most active fraction on NCI-H23 (human lung

cancer) cell line but the fraction was also active on BALB/c 3T3 non-tumourous cell

line. D/F21 was the second most active fraction with good specificity for NCI-H23

as compared to BALB/c 3T3. D/F15 was the major fraction of Typhonium

xxiv

flagelliforme with moderate antiproliferative effect on NCI-H23. Pheophorbide-a and

pyropheophorbide-a were the two major constituents isolated from D/F19. Methyl

hexadecanoate, 1-hexadecene, phytol and a derivative of phytol were tentatively

suggested to be the constituents in D/F21. Campesterol, stigmasterol, β-sitosterol,

palmitic acid, oleic acid, linoleic acid and linolenic acid were the chemical

constituents of D/F15.

The antiproliferative effect of D/F19 did not originate from a single

component but was due to synergistic action from a mixture of components. Cell

death was induced by D/F19 via the apoptotic pathway. In addition, D/F19 was also

found to possess photosensitizing effect on NCI-H23 and a breast cancer cell line,

HS578T, mainly due to the activity of pheophorbide-a, pyropheophorbide-a and

another of its subfraction, D/F19/04. Pheophorbide-a and pyropheophorbide-a are

photosensitizers proven in vitro and in vivo to be effective for use in photodynamic

therapy. Fraction D/F21 was found to be antiproliferative and apoptosis inducing.

This effect may be due to the result of the activity of phytol. Although the chemical

constituents of D/F15 were commonly found in plants, these compounds are believed

to also contribute to the healing effect of Typhonium flagelliforme against cancer as

supported by many reported studies.

In short, the chemical constituents of Typhonium flagelliforme which possess

in vitro anticancer activity have been identified based on a bioactivity guided

approach. The study supports the ethnomedical use of Typhonium flagelliforme as a

herbal remedy towards cancer.

1

CHAPTER 1

INTRODUCTION

1.1 Medicines from Plants

Plants have formed the basis for treatment of diseases in traditional medicine

for thousands of years. It is the oldest and most natural form of medicine even at the

present time. The World Health Organisation (WHO) estimated that about 80% of

the world’s inhabitants today still rely on plants as their primary source of medicine

and health care agents (Farnsworth et al., 1985). Early drugs or so called ‘folk

medicine’ took the form of crude drugs prepared as tinctures, teas, poultices,

powders, infusions and inhalants (Geddes & Grosset, 2000). The drug discovery

trend then evolved into the isolation of their active constituents such as codeine,

aspirin, quinine and morphine of which some are still in use today (Ng, 2004). Out of

119 chemical substances that were isolated as drugs, above 74 % were discovered as

a direct result of chemical studies which focused on the isolation of active substances

from plants used in traditional medicine (Farnsworth et al., 1985). However, despite

the many successes of plants as a source of medicine, the process of drug discovery

from natural products has been a historically slow process, and the traditional natural

product extract libraries were not compatible with high-throughput screening (Koehn

& Carter, 2005).

In the beginning of 1990s, with the advent of combinatorial chemistry, many

big pharmaceutical companies switched their drug discovery strategies to include a

large proportion of combinatorial chemistry (Balkenhohl et al., 1996). The ability of

combinatorial chemistry to generate huge amount of compound libraries which

would then undergo high-throughput screening to identify the lead compounds has

been thought to be an efficient approach and thus carries great promise (Lee &

2

Breitenbucher, 2003). In addition, most of the synthetic compound libraries have no

issues with intellectual property right and biodiversity conservation as those

experienced with natural products (Baker et al., 1995; Fischli et al., 2002). However,

by the mid to end of 1990s, results from the early combinatorial libraries had been a

great disappointment with almost no lead compounds ever generated despite a few

hits (Lahana, 1999). The inability of the synthetic libraries to generate lead

compounds of biological relevance is mainly due to the different chemical space

occupied by the synthetic compounds compared to natural products (Butler, 2004).

Synthetic compounds have lower molecular weights, higher number of nitrogen,

halogen and sulphur atoms but lower content of oxygen than natural products

(Henkel et al., 1999). In addition, these compounds have less chiral centres, less

complex structures and are greater in flexibility compared to natural products.

Structural flexibility results in entropic losses that weaken the binding with protein

molecules where the therapeutic effect is exerted (Feher & Schmidt, 2003).

In the present decade, there has been a renewed interest in natural product

research due to the failure of other alternative drug discovery methods to produce

lead compounds in key therapeutic areas particularly immunosuppresion,

antiinfectives, anticancer and metabolic diseases (Butler, 2004). Although natural

products isolated as the active compounds were sometimes not suitable for direct

development into an effective drug, it can provide a suitable lead for conversion into

clinically useful agents (Young, 1999; Hall et al., 2001). Some of the examples of

new drugs derived from natural sources are apomorphine, which is a morphine

derivative isolated from Papaver comniferum used to treat Parkinson’s disease;

arteether, an antimalarial drug developed from artemisinin isolated from Artemisia

annua; vinflunine, an improved anticancer agent derived from vinblastine isolated

3

from Catharanthus roseus; tiotropium, a derivative of atropine isolated from Atropa

belladonna for the treatment of bronchospasm associated with chronic obstructive

pulmonary disease and many others (Balunas & Kinghorn, 2005; Chin et al., 2006).

Statistics have shown that among the drugs approved by FDA from 1981 to 2006,

those of natural products origin contributed a substantial amount of 52 % (Cragg et

al., 1997; Newman et al., 2003; Newman & Cragg, 2007) as shown in Figure 1.1.

Thus this indicates that natural products still play an important and irreplaceable role

as a source of modern pharmaceuticals.

Figure 1.1: Drugs approved by FDA from Jan 1981 to June 2006 by source (total number of drugs, N = 1184). (Source: Newman & Cragg, 2007, p.472).

V4% B

14%N

5%

ND23%S

30%

S/NM10%

S*4%

S*/NM10%

Legends: V = vaccine B = biological such as peptide or protein N = natural product ND = derived from a natural product (semisynthetic) S = totally synthetic S* = synthetic but pharmacophore was from a natural product NM = natural product mimic

4

1.2 Plants as a Source of Anti-cancer Agents

According to the National Cancer Institute (NCI) of the United States, cancer

is defined as ‘a disease in which abnormal cells divide without control and are able to

invade other tissues’ (National Cancer Institute, 2009). This disease occurs mainly

due to the mutation of certain genes that resulted in the disruption of cell growth

regulation, repair of cell damage and growth inhibition (King & Robins, 2006).

Human cancers can generally be classified into several categories. Carcinomas are

malignant tumours that originated from epithelial tissues. Sarcoma refers to cancer

that is mesenchymal in origin, such as the bone, cartilage, fat, muscle, blood vessels,

or other connective or supportive tissue. Neoplasms of the hematopoietic system are

termed leukemia if it originates from the blood forming tissue, or lymphoma if it

begins from the cells in the immune system (Ruddon, 1995).

Cancer is one of the major diseases affecting mankind with approximately

10.9 million people around the world diagnosed with cancer every year of which

45% are from Asia (Ferlay et al., 2004). Mortality as a result of cancer is estimated to

be 6.7 million per year, which is 12% of total deaths that occur annually (Ferlay et al.,

2004; Ma & Yu, 2006). In Malaysia, cancer is the fourth leading cause of death

among the medically certified deaths with an annual incidence of 30,000 people (Lim,

2002). It is estimated that out of four Malaysians, one is expected to get cancer in

his/her lifetime. The most common cancers found among men are lung, nasopharynx,

colon, leukaemia, rectum and prostate while in women the common types of cancer

are breast, cervix, colon, ovary, leukaemia and lung (Lim et al., 2003).

The approaches to the treatment of cancer are surgery, radiotherapy,

immunotherapy and chemotherapy. Surgery is usually the first choice of treatment

with the goal to remove totally or at least reduce the mass of tumour. However, in

5

many cases, secondary tumours have established by the time the primary tumour is

detected, thus chemotherapy and radiotherapy usually follow after surgery (King &

Robins, 2006). Chemotherapy involves the use of cytotoxic or cytostatic drugs to kill

or prevent malignant cells from proliferating (Cragg et al., 1996). The efficacy of the

drug depends on the concentration of the drug that reaches the tumour, the duration

of exposure and the proportion of the population that is proliferating. In many cases

however, the action of chemotherapeutic drugs are not specific towards malignant

cells alone but affects proliferating cells throughout the body and can be toxic to vital

organs such as the heart and kidney, resulting in numerous untoward side effects. To

minimise these unwanted effect, many chemotherapists use combinations of drugs

which have additive effects against tumours but have less toxicities to normal cells

(Boyer & Tannock, 1998).

Alkylating agents are drugs that act through covalent bonding of the alkyl

groups to intracellular macromolecules. The formation of a positively charged

intermediate compound combines with the electron-rich nucleophilic groups such as

amino, phosphate, sulfhydryl or hydroxyl moiety that are present in the DNA,

resulting in lethal toxicity to the cell. Examples of anticancer drugs in this category

are such as the nitrogen mustards and nitrosoureas (Neidle & Thurston, 2005).

Antimetabolites are drugs that inhibit nucleic acid synthesis and prevent

normal cell division from occurring. Examples of drugs in this category are

methotrexate, 5-fluorouracil and cytosine arabinoside. These drugs are cell cycle-

specific and exert toxicity on other proliferating cells such as the bone marrow cells

and gastrointestinal mucosa (Highley et al., 2009).

The third category of anticancer drugs is the natural products. It covers a

substantial percentage of the total amount of anticancer drugs introduced in the time

6

frame of 1981–2006, whereby 77.8% were either natural products per se or derived

from natural products (Newman & Cragg, 2007). There are four major classes of

natural products based anticancer drugs which are derived from plants and these have

quite diverse mechanism of actions. The first anticancer drugs of this category are

the vinca alkaloids, vincristine and vinblastine, isolated from the Madagascar

periwinkle, Caranthus roseus. These compounds block mitosis with metaphase arrest

by binding specifically to tubulin resulting in its depolymerization (Okouneva et al.,

2003) and are useful for the treatment of leukaemia, lymphoma, breast and lung

cancers (Cragg & Newman, 2005). Another class of compounds which also affect

the microtubules by causing tubular bundling are the taxanes, paclitaxel and

docetaxel, extracted from pacific yew tree Taxus brevifolia (Hirose & Takiguchi,

1995). Paclitaxel is used primarily for the treatment of breast, ovarian and non-small

cell lung cancer (Wall & Wani, 1996). The epipodophyllotoxins, etoposide and

teniposide, derived from the mandrake plant Podophyllum peltatum are DNA

topoisomerase II inhibitors. These drugs are used against lymphoma, bronchial and

testicular cancers (Gordaliza et al., 2004). Camptothecin is a selective inhibitor of

topoisomerase I which was isolated from the stem bark of the Chinese tree

Camptotheca acuminate. It is used against breast, lung and colorectal cancers (Wall

& Wani, 1996). Many other drugs which are derived from these four major classes of

compounds are currently under clinical trials (Balunas & Kinghorn, 2005). The

chemical structures of some of the anticancer agents derived from plants are given in

Figure 1.2.

7

Figure 1.2: Anticancer agents derived from plants.

There are approximately 250,000 plant species distributed throughout the

world and more than half of these are found in the tropical rainforests (Wilson, 1988).

Vinblastine: R = CH3

Vincristine: R = CHO

O NH

O

O

OHO

O

O

H

OO

O

O

OH

O

OH

O

O NH

O

O

OHO

O

O

H

OO

O

O

OH

O

OH

NH

NOH

O

O

N

N

H

OH

OO

R

O

HO

O

Paclitaxel Docetaxel

N

N

O

O

OH O

Campthothecin

O

O

O

OH

O O

O

OO

OO

OHHO

H

O

O

O

OH

O O

O

OO

OO

OHHO

S

Etoposide Teniposide

8

So far, only 1% of tropical species have been explored for their pharmaceutical

potentials and there remain a substantial opportunity for finding more new

compounds of pharmaceutical interests (Zuhud, 1991; Fakim, 2006). The vast variety

of flora and fauna hosted by the tropical rainforests provide natural product chemists

with numerous compounds as starting point for the development of new anticancer

drugs.

Malaysia is home to about 40,000 species of higher plants of which about

36,000 are flowering plants, 3,600 are ferns and 87 are conifers (Soepadmo, 1998).

In addition to the magnificent array of plant species, Malaysia has a unique

confluence of traditional medicine system due to the diverse ethnic communities in

the country. The rich ethnobotanical and ethnopharmacological knowledge in the use

of plants has provided a good starting point for Malaysian scientists for locating

useful tropical plants for phytochemical investigation. To make use of these

advantages, the present study selected Typhonium flagelliforme as the subject of

study since it has been used locally as an alternative treatment for cancer but has not

been investigated in depth scientifically.

1.3 Typhonium flagelliforme (Lodd.) Blume

1.3.1 Taxonomy and Distribution

Typhonium flagelliforme (Lodd.) Blume is a herbal plant indigenous to

Malaysia which belongs to the family of Araceae under the subfamily of Aroid. The

tuber of the plant is nearly round and measures about 1 cm in diameter. The leaves

are green and smooth, typically narrowly hastate with spreading basal lobes but may

be elliptic sometimes. The size of the blade is about 6 × 4 cm. The petiole is

yellowish and thin, it measures about 6–20 cm long, partially enclosed in the spathe.

9

The colour of the spathe is green with brownish margin, the limb is linear acute,

dilated and round at the base. The flowers are unisexual (Hsuan et al., 1998;

Nicolson & Sivadasan, 1981). A picture of Typhonium flagelliforme is given in

Figure 1.3.

Figure 1.3: Picture of Typhonium flagelliforme.

Typhonium flagelliforme, which was previously mistakenly regarded as

Typhonium divaricatum, is distributed across IndoChina, Peninsular Malaysia,

Northern Queensland, South India, Sri Lanka and Luzon as shown in Figure 1.4

(Nicolson & Sivadasan, 1981). It grows wild in waste grounds and is native to

several parts of Peninsular Malaysia such as the ditches in Melaka town and the

Sepoy lines in Penang (Ridley, 1967). However, due to developments and

10

deforestations, these plants are no longer found in the mentioned areas but are mostly

cultivated in pots and garden yards for personal consumption.

Figure 1.4: Geographical distribution of Typhonium flagelliforme (Source: Nicolson

& Sivadasan, 1981, p.491).

1.3.2 Ethnomedical Background

Typhonium flagelliforme, known by its vernacular name of Keladi Tikus

(Malay) or Rodent Tuber (English), is described as toxic, warming and phlegm-

resolving in the Malaysian Traditional Chinese Medicine. The leaves or whole plant

is often applied over swellings and used against various kinds of malignancy. The

roots are used to eliminate expectoration (Lee & Wong, 2004). There have been

many accounts on the uses of Typhonium flagelliforme for treating cancer, including

colon, breast, prostrate, liver, lung and bone cancer. Many cancer patients who have

tried such alternative therapy have claimed successes while others who took this

11

during the course of chemotherapy have experienced significant improvement in

appetite, reduction in hair loss with no cases of nausea and vomiting (Teo & Ch’ng,

1999).

The use of three other species of Typhonium has also been documented in the

Traditional Chinese Medicine and Ayurvedic system for the treatment of cancer. In

China, the tuber of Typhonium giganteum, the major component of Herbal Medicine

Rhizoma Typhonii is applied externally in the form of paste to the lesion for the

treatment of reticulocytic sarcoma and lymphoma (Chang, 1992). In India, the

poultice of Typhonium trilobatum is used to treate Scirrhous tumours and cancerous

tumours, while Typhonium orixense is used by the Indians and Pakistanis to treat

Scirrhous tumours (Hartwell, 1982).

Each preparation of Typhonium flagelliforme requires the use of

approximately 50 g of the fresh whole plant. The plant is pounded briefly using a

mortar and pestle to break the cell walls and the juice is then forced out through a

thin cloth. The juice extract is then mixed with honey in order to prevent irritation to

the throat and is recommended to be consumed immediately. Initially, the juice is to

be drunk 3 times daily and can be reduced to a maintenance dose of twice a week if

improvement is observed. This can either be used as a single therapy or taken while

undergoing the course of chemotherapy or radiotherapy (Neoh, 1992; Teo & Ch’ng,

1999). There are also other less commonly used preparative methods of Typhonium

flagelliforme, such as wrapping 2–6 pieces of the plant leaves in Longan flesh for

direct consumption (Lee & Wong, 2004) and pulverising the dried plant material to

prepare as a drink which is not documented. Of late, encapsulated dried plant powder

of the plant is also available commercially (Global Bioscience, 2007).

12

1.3.3 Pharmacological Studies

In vitro studies carried out on the various extracts of Typhonium flagelliforme

(then wrongly referred as Typhonium divaricatum (Nicolson & Sivadasan, 1981)

revealed that the hexane, chloroform (CHCl3) and ethyl acetate (EtOAc) extracts

exhibited significant cytotoxic effect on a murine lymphoid cell line with the rate of

inhibition above 90%. However, the concentration of the extract tested and the cell

line designation was not specified (Neoh, 1992). Choo et al. (2001a) had also

observed cytotoxic activity for the hexane and CHCl3 extracts on P388 leukemia

cells and the IC50s were found to be 15 µg/ml and 6 µg/ml respectively for the root

extracts, 65 µg/ml and 8 µg/ml respectively for the stems/leaves extracts. Chan et al.

(2005) also found the hexane, CHCl3 and n-butanol (n-BuOH) extracts to be active

towards NCI-H23 human lung cancer cell line at IC50 1.8, 5.2 and 1.2 µg/ml,

respectively, and T-47D human breast cancer cell lines at IC50 12.2, 23.0 and 54.6

µg/ml, respectively.

A study on the effect of T. flagelliforme on the immune system of cat fish

found that the plant, given as intraperitoneal injections, was able to increase

phagocytic activity and other non-specific immune response. Resistance of the cat

fish against live Vibrio parahaemolyticus was also found to have increased (Lam,

1998). Another in vivo study conducted in rats showed that T. flagelliforme juice

extract had a similar effect as glycyrrhizin in preventing hepatocarcinogenesis

(Karuppiah et al., 1999). Other pharmacological studies also showed that the water,

alcohol and ester extracts of Typhonium flagelliforme is antiasthmatic,

antiinflamatory, analgesic, sedative, antiexpectoration and cough relieving (Zhong et

al., 2001).

13

1.3.4 Phytochemicals

Chen et al. (1997) reported the isolation of benzenetridecanoic acid (synonym:

13-phenyltridecanoic acid) and methyl benzenetridecanoate (synonym: methyl 13-

phenyltridecanoate) from the ethyl acetate extract of Typhonium flagelliforme. Four

other esters, namely methyl hexadecanoate, methyl octadecanoate, methyl 9-

octadecenoate and methyl 9, 12-octadecadienoate were also identified by Choo et al.

(2001b) from the hexane extract of Typhonium flagelliforme in addition to methyl

13-phenyltridecanoate. However, despite the earlier report by Choo et al. (2001a)

which showed that the CHCl3 extracts were more active than the hexane extracts, no

study was carried out on the CHCl3 extracts. Huang et al. (2004a,b) had isolated

several glycosides, namely coniferin, methylconiferin, 12-hydroxy-N-(3-hydroxy-1-

O-β-glucopyranosyloctadeca-4E,8E-dienyl)octadecanamide and two sterols, β-

sitosterol and β-daucosterol from the n-BuOH fraction of Typhonium flagelliforme.

The chemical structures of the constituents are illustrated in Figure 1.5. Except for

methyl 13-phenyltridecanoate that was found to be inactive in vitro, none of the

chemical constituents had been tested for anticancer activity in the studies mentioned

and none was used to support its ethnopharmacological application against cancer.

14

Figure 1.5: Chemical structures of the compounds isolated from Typhonium flagelliforme.

2-Hydroxy-N-(3-hydroxy-1-O-β-glucopyranosyloctadeca-4E, 8E-dienyl)octadecanamide

O

OCH3

O

OH

13-Phenyltridecanoic acid

Methyl 13-phenyltridecanoate

O

H3CO

O

H3CO

Methyl hexadecanoate

Methyl octadecanoate

Methyl 9-octadecenoate

Methyl 9,12-octadecadienoate

Coniferin

Methyl coniferin OH

OO

H3CO

OH

HOHO OH

OCH3OO

H3CO

OH

HOHO OH

β-Sitosterol

OOOH

HOOH

OH

β-Daucosterol HO

H3CO

O

O

H3CO

O ONH

OOH

OH

OH

OHHO

HO

15

1.4 Methods of Plant Extraction

Extraction can be carried out on either fresh or dried plants. Ideally, fresh

plant tissues should be used for phytochemical investigation right after collection

(Harborne, 1998). However, if extraction is to be carried out a few days after

collection, it should be stored dry in a plastic bag (Harborne, 1998). Alternatively,

dried plants can also be used and has several advantages. Unlike in the case of fresh

plant, solubility of the chemical constituents in the dried plants is not affected by the

presence of water. It is also easier to handle when large scale extraction is carried out

due to the lower mass to plant ratio (Eloff, 1998). However, when dried plant is

intended for use, the material needs to be dried rapidly to prevent degradation of the

components by air or microorganisms. If a vacuum system is used for drying, care

should be taken in order to avoid excessive loss of the volatile constituents which

may possess interesting biological activity (Fakim, 2006).

Nowadays, many extraction methods are available. The more commonly used

methods are such as cold extraction, hot percolation, soxhlet, supercritical fluid

extraction, ultrasonic-assisted solvent extraction and pressurized liquid extraction

(Péres et al., 2006; Barnes et al., 2009). However, careful selection of the right

extraction methodology is needed so that it is adaptable to the intended study (Fakim,

2006). The improper extraction methods may result in degradation and loss of

bioactive natural products which seriously hampers the outcome of the study (Davey

et al., 2009). Three common and inexpensive extraction methodologies that were

evaluated in this study are briefly introduced in the following sections.

16

1.4.1 Maceration

The maceration method is the simplest but yet still widely used procedure.

Pulverised plant material is allowed to soak in solvents of differing polarity in a

closed container at room temperature (Fakim, 2006). The metabolites are allowed to

diffuse into the solvent and the period of extraction may differ from few hours to

several weeks. Occasional or constant stirring is used sometimes to speed up the

extraction. The disadvantage of this method is that it consumes large volumes of

solvents and some of the poorly soluble metabolites may not be extracted efficiently

since extraction is carried out at room temperature (Seidel, 2006).

1.4.2 Soxhlet Extraction

Soxhlet extraction is a classical and most widely used extraction method.

Pulverised plant material is placed in a cellulose thimble in an extraction chamber

which is located above a collecting flask but beneath a reflux condenser. A suitable

solvent is then added into the soxhlet apparatus and the solvent is heated to a boil.

The solvent vapour rises and condenses into the thimble. When the thimble is filled

with the solvent, it is siphoned back into the distillation flask and the whole cycle is

then repeated (Pavia et al., 1995). The main advantage of Soxhlet extraction is that

the solvent, when saturated with solubilized metabolites, is emptied into the

distillation flask and fresh solvent is recondensed into the extraction thimble

continuously, thus making the extraction procedure more efficient and solvent saving.

However, the major disadvantage of this method is that the extraction carried out at

high temperature may degrade thermolabile compounds and initiate the formation of

artefacts (Seidel, 2006).

17

1.4.3 Ultrasonic-assisted Solvent Extraction

The ultrasonic-assisted solvent extraction is a technique modified from the

maceration method. This solvent extraction method is facilitated by the use of

ultrasound (frequency pulses approximately 20 kHz) and is one of the well

established and commonly used methods today for extracting plant materials for

bioactive principles (Sališová et al., 1997; Trusheva et al., 2007). The pulverised

plant material is soaked in a suitable solvent in a container and ultrasound is used to

induce mechanical stress on the plant which results in the formation of cavitations in

the sample. The cellular breakdown allows easier access of the solvent into the cells

thereby increases the solubility of the metabolites in the solvent. In addition the

ultrasound also facilitates mass transfer and thus is a more efficient method of

extraction (Vinatoru et al., 1997). However, the huge amount of energy generated

increases extraction temperature and thus a cooling system is required in order to

control the temperature.

1.5 Techniques of Separation

1.5.1 Thin-layer Chromatography

Thin-layer chromatography (TLC) is a simple, cheap and quick method for

analysis and screening of natural products. Thin layers of adsorbents, usually silica

gel or alumina, is coated on glass, plastic and aluminium sheets. The TLC sheets can

be cut into strips of various sizes according to the desire of the user. A mixture of

natural products is then spotted near the bottom of the thin strip and developed in a

jar that contains saturated atmosphere of the eluting solvent. The eluting strength of

the solvent is varied by mixing two or more solvents with different polarity. As the

mobile phase moves up the plate through capillary action, the components of the

18

mixture are carried along at different rates and hence separate from one another. The

development of the plate is complete when the solvent front has advanced nearly to

the top of the plate. The separated compounds on the TLC plate can be detected by

ultraviolet (UV) illumination or spray with suitable detection reagent. TLC is

frequently used to determine the optimal combinations of solvent for preparative

column chromatography separations and to monitor the progress of separation by

column chromatography (Gilbert & Martin, 1998).

Preparative thin-layer chromatography (prep-TLC) is a scaled up procedure

of TLC and is used to separate compound mixtures at a larger scale. Prep-TLC is

normally used as a final purification step in compound isolation, most frequently

after column chromatography. The chromatographic conditions for prep-TLC can be

predetermined using a TLC plate by trial and error experimentation and then scale up

in proportion using a similar or slightly less polar solvent combination. The separated

compounds are either detected by the non-destructive UV method or by using a spray

reagent. When using a reagent, only a small section at the edge of the plate is

sprayed, and the unreacted section of a particular separation band is scraped from the

plate and desorbed from the silica sorbent using an appropriate solvent that is able to

dissolve the compound (Gibbons, 2006).

1.5.2 Column Chromatography

Column chromatography plays a very important role in the separation of

compounds from a complex mixture of natural products. The principle of separation

takes place through selective distribution of the components between the mobile

phase, which is the eluting solvent, and the stationary phase, which is usually the

silica gel. Different compounds have different adsorption affinities for the surface of

19

the stationary phase and the extent of adsorption is governed by the types of

interaction between the compound molecule and the stationary phase such as

hydrogen bonding, Van der Waals forces, dipole-dipole interactions, acid-base

properties and others (Gilbert & Martin, 1998). The column can be developed using

various eluting techniques such as gravity, application of nitrogen pressure at the

inlet, vacuum suction or controlled by a pump. A solvent gradient is applied for

separation of complex mixtures of compounds where the composition of the solvents

is varied in steps so as to gradually increase the eluting strength of the mobile phase.

The different rate of movement of the chemical constituents along the column then

results in the separation of the compound mixture (Reid & Sarker, 2006). However,

for a complex mixture of natural products such as crude extracts, compounds with

similar polarity often co-elute in the same fraction and thus, chromatography may be

carried out several times using different chromatographic conditions in order to

obtain reasonably pure compounds.

Nowadays, the use of flash column chromatography has largely replaced

classic column chromatography techniques. Flash column chromatography is an air

pressure driven column chromatography which has been optimized for rapid

separations. The smaller particle size of the silica gel and rapid flow rate was able to

overcome poor separation recovery which is a problem faced in classic column

chromatography due to band tailing (Landgrebe, 1993). In addition, the technique is

inexpensive, easy to operate and allows separation of samples weighing between

0.01–10.0 g in a short period of time (Still et al., 1978). This method is currently a

preferred technique for routine large scale purification of natural products.

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1.5.3 High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a modern day analytical

technique derived from the same working principle as for column chromatography.

Compared to classical column chromatography, HPLC offers major advantages in

convenience, speed, accuracy, selectivity and the ability to carry out difficult

separations owing to the improvements in equipment sophistication, material,

technique and the application of theory (Rouessac & Rouessac, 2007). The general

setup of a HPLC system consists of a high-pressure pump, an injection port, a

column which is the stationary phase, and a detector. The sample is introduced into

the injection port and is carried by the stream of mobile phase delivered by the pump

which then passes through the column where the separation takes place. Separation is

then monitored with a detector.

Two modes of adsorption chromatography are available in HPLC, i.e.

normal-phase and reversed-phase chromatography. Reversed-phase HPLC is

generally preferred by many researchers as it is applicable to a wide range of

chemical compounds. This mode of separation utilises the combination of a non-

polar stationary phase and a polar mobile phase which is usually a combination of

acetonitrile and/or methanol with an aqueous solution. Acids, bases or buffers are

frequently used as the aqueous solution with the purpose of suppressing compound

ionisation and to control the degree of ionisation of free unreacted silanol groups in

the column that can result in peak tailing which in turn affect the separation (Snyder

& Kirkland, 1979).

For a long time the size of the HPLC column have been standardised around

the internal diameter of 3–4.6 mm, which requires a mobile phase flow rate of 0.5–2

ml/min. However, a wide choice of column now exists such as narrow-bore (2–4 mm

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i.d.), microbore (1–2 mm i.d.) and even packed capillaries (<1 mm), for which the

flow rate descends to a few µl/min and thus requires special pumps and detectors.

Such developments were necessary as there has been increased demand for the

determination of analyte at trace level concentrations over the recent years (Rouessac

& Rouessac, 2007). Ultra high-performance liquid chromatography with the trade

name of UPLC (Waters) or UHPLC (Agilent) is an improved version of HPLC. With

the UPLC or UHPLC pump capable of operating at extremely high pressure limit,

the system is designed to be used with sub-2 µm particle columns leading to

exceptionally better resolution, sensitivity and separation speed due to the much

improved column efficiency. Furthermore, when the system is integrated with a mass

spectrometer, often termed as ‘hyphenation’, it allows provisional identification of an

unknown natural product sample, thus further expand the potential and versatility of

the analytical instrument (Niessen, 1999).

While an analytical HPLC is used for qualitative and quantitative

determination of a compound, a preparative HPLC (prep-HPLC) is meant for

isolation and purification of a compound. Purification of natural products using prep-

HPLC has been widely adopted by many synthetic and natural product chemists to

date as the demand for higher throughput could be better met in an automated prep-

HPLC system. Moreover, the reproducibility, efficiency and consistency in

compound separation are better in prep-HPLC compared to conventional column

chromatography. Depending on the amount of sample intended to be purified, a wide

range of prep-HPLC columns are available to cater for sample from µg scale up to kg

level (Huber & Majors, 2004).

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1.6 Compound Identification and Structure Elucidation

1.6.1 Ultraviolet-visible Spectroscopy

Natural products absorb light in the ultraviolet or visible (UV-Vis) region of

the electromagnetic spectrum. Absorption of the UV-Vis light occurs when the

energy of the incident radiation is the same as the electronic excitation of the

molecule (Lambert et al., 1976). An unknown natural product can be identified

according to the complexity of its spectrum and the general position of the

wavelength maxima. Different classes of compounds have their characteristic light

absorption spectra and the identification of the class of compounds can be enhanced

by performing measurements on the sample solution at different pH or in the

presence of a particular inorganic salt and then observing the shift of the absorption

band (Harborne, 1998).

1.6.2 Infrared Spectroscopy

Infrared (IR) radiation is the part of electromagnetic spectrum in between the

visible and microwave regions. The portion which is of greatest practical use for a

natural product chemist is the mid-IR region, which ranges from 4000 – 400 cm-1. A

molecule that possesses an oscillating dipole absorbs energy from the oscillating

electric field of a beam of infrared radiation when the IR frequency of the applied

electric field is similar to the frequency of the oscillating dipole. Except for

homonuclear diatomics, practically all molecules have some vibrations that produce

a change of dipole moment (Lambert et al., 1976). The wavelength of absorption

depends on the relative masses of the atoms, the force constants of the bonds

between the atoms and the geometry of the atoms (Silverstein, et al., 2005).

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A mid-IR spectrum can generally be evaluated in two parts. The region

between 4000 – 1300 cm-1 is called the functional group region where characteristic

stretching frequencies for important functional groups, such as O-H, N-H, C-H, C=O

and C=C, are observed. The region between 1300–900 cm-1 is referred to as the

“fingerprint” region where the complex absorption bands originated from interacting

vibrational modes. Absorption at this region is unique for every type of molecule and

can be used to cross examine the types of vibration in reference to the other regions.

Due to its simplicity and reliability in identifying the functional groups, IR

spectroscopy is an extremely useful method of assigning a compound to its class

(Silverstein et al., 2005; Harborne, 1998).

1.6.3 Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance (NMR) Spectroscopy is practically an

indispensable tool used for the identification and structure elucidation of natural

products. Akin to IR and UV spectroscopy, it is another form of absorption

spectroscopy that takes place in the radio frequency region. All nuclei are charged

particles and some in addition have spin angular momentum. The nuclei of interest

are those that have appropriate spin properties (spin quantum number, I ≠ 0) and

generate a magnetic field. 1H and 13C nuclei (both having I = ½) have the widest

application for identification of organic molecules.

In the presence of an external magnetic field, a spinning nucleus absorbs

radiofrequency radiation, which has a magnitude that is identical to the Larmor

frequency, resulting in nuclear transition to a higher energy spin state (Silverstein et

al., 2005). However, a nucleus within a molecule experiences a lesser magnetic field

than the applied external field. This phenomenon is due to the presence of electron

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cloud around the nucleus which generates an induced magnetic field opposed to the

external field termed as the “shielding effect”. The density of the shielding varies

with the chemical environment of the nucleus and the variation gives rise to

differences in chemical shift positions and the ability to distinguish among individual

absorptions, hence the usefulness of NMR spectroscopy (Macomber, 1998).

Chemical shift is measured relative to a reference compound, which is usually

tetramethylsilane (TMS).

Based on a 1H NMR spectrum, the relative positions of the various types of

protons in a molecule can be determined by evaluation of its chemical shifts, spin-

spin couplings and peak integral. Similarly, a 13C NMR spectrum provides

information on the relative positions of various types of carbons in a molecule. Due

to the low abundance of 13C nucleus and high abundance of 1H nucleus in nature as

well as the large C-H coupling constants, a proton-coupled carbon spectrum usually

shows complex overlapping multiplets which are difficult to interpret. Thus, for

routine analysis of sample, a broadband proton-decoupled 13C spectrum is frequently

used, where each type of carbon nucleus is represented by a single peak, making the

interpretation much simpler (Silverstein et al., 2005). Distortionless enhancement by

population transfer (DEPT) 13C NMR displays separate subspectra for CH, CH2 and

CH3 carbon signals. Three types of DEPT experiments using three different pulse

angles, θ, at 45°, 90° and 135° are available. A combination of DEPT experiments

helps distinguish the types of protonated carbons. Any remaining carbon signal in the

normal 13C spectrum which is not present in the DEPT subspectra would belong to

the quaternary carbons (Macomber, 1998).

Two-dimensional (2D) NMR spectroscopy is also referred to as correlation

spectroscopy where the correlation between two or more types of the same or