ANALYTICAL, BIOACTIVITY AND STABILITY …eprints.usm.my/9532/1/ANALYTICAL,_BIOACTIVITY_AND_STABILITY_S… · ON STROBILANTHES CRISPUS L. BREMEK ... Professor Dr Zhari Ismail and co

ANALYTICAL, BIOACTIVITY AND STABILITY STUDIES ON STROBILANTHES CRISPUS L. BREMEK AND

SONCHUS ARVENSIS L. EXTRACTS

AFRIZAL

UNIVERSITI SAINS MALAYSIA

2008

ANALYTICAL, BIOACTIVITY AND STABILITY STUDIES ON STROBILANTHES CRISPUS L. BREMEK AND

SONCHUS ARVENSIS L. EXTRACTS

by

AFRIZAL

Thesis submitted in fulfillment of the requirements

for the Degree of Doctor of Philosophy

June 2008

DEDICATED TO

Beloved

My parents, Itam St. Pamenan and Tinun

My wife, Hartati

My children, Indah Permata, Ridho Sahary Adha and Nadya Chantika

ii

ACKNOWLEDGMENTS

Alhamdulillah, praise to Almighty Allah SWT, who is creator and sustainer of the

universe. His blessings be upon Prophet Muhammad SAW.

I respectfully express my sincere and deepest gratitude to my supervisor

Professor Dr Zhari Ismail and co supervisor Dr Amin Malik Shah Abdul Majid for

their helpful advice, generous help and guidance throughout the course of this

my study.

I am grateful to university authorities and Dean of School of Pharmaceutical

Sciences, University Sains Malaysia, for the facilities provided during the course

of this work.

I am thankful to all of my fellow colleagues who assisted me to complete my

work, especially, Nor Amin, Mohammad Abdul Razak, Suhaimi, Azizan,

Rosidah, Saravanan and Khalid Hussain.

In this occasion, I would like to express my special thanks to Mr. Hider who has

assisted me doing on antiangiogenic work.

Finally, I am grateful to my university for providing me study leave to complete

my education.

iii

TABLE OF CONTENTS Page

DEDICATION i

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iii

LIST OF TABLES ix

LIST OF FIGURES xi

LIST OF PLATES xxi

LIST OF APPENDICES xxii

LIST OF ABBREVIATIONS xxv

ABSTRAK xxvi

ABSTRACT xxviii

CHAPTER 1 INTRODUCTION 1

1.1 The Usage of Medicinal Plants 1

1.2 Strobilanthes crispus Plant 3

1.2.1 Botanical Description 3

1.2.2 Biological Activity 5

1.2.3 Phytochemistry 5

1.3 Sonchus arvensis Plant 7

1.3.1 Botanical Description 7

1.3.2 Biological Activity 8

1.3.3 Phytochemistry 9

1.4 Analysis and Isolation of the Chemical Constituents 10

1.5 Kidney Stone Disease 11

1.5.1 Aspects of Kidney Stone Disease 11

1.5.2 Natural Product Inhibitor of Urinary Calculi 12

1.6 Antioxidant 13

1.6.1 Aspect and Process of Antioxidation 13

iv

1.6.2 Systems of Antioxidation 14

1.6.3 Nutritional Antioxidants 17

1.7 Angiogenesis 18

1.7.1 Definition and Process of Angiogenesis 18

1.7.2 The Relationship between Antioxidants and Antiangiogenic

Agents 19

1.8 Drug Stability 22

1.8.1 General Concept in Drug Stability 22

1.8.2 Guideline for Stability Testing of Drug Substance and Drug

Product

24

1.8.3 Assessing Factors Influencing Drug Stabilities 25

1.8.4 Stability-Indicating Assay Method 27

1.8.5 Interpretation of the Chemical Decomposition of Drugs Using

Reactions Kinetic

28

1.8.6 Determination of the Reaction Order and Calculation of the

Rate Constants

28

1.8.7 Isothermal Processes in Determination of Expire Date 29

1.9 Objectives of the Study 30

CHAPTER 2 ANALYTICAL AND PHYTOCHEMICAL STUDIES ON STROBILANTHES CRISPUS AND SONCHUS

ARVENSIS EXTRACTS

31

2.1 Introduction 31

2.2 Materials and Methods 35

2.2.1 Chemicals 35

2.2.2 Instruments 35

2.2.3 Samples 36

2.2.4 Extraction of S. crispus and S. arvensis Leaves Using

Maceration Method

36

v

2.2.5 Analysis by Gas Chromatography/Time of Flight Mass

Spectrometry (GC/TOF-MS)

37

2.2.6 Soxhlet Extraction of S. crispus and S. arvensis 37

2.2.7 Isolation of Tritriacontane from Hexane Extract of S. crispus 38

2.2.7.1 Ultraviolet Spectroscopic Analysis 39

2.2.7.2 Infrared Spectroscopic Analysis 39

2.2.7.3 1H-NMR and 13C-NMR Spectroscopic Analysis 39

2.2.7.4 Mass Spectroscopic Analysis 39

2.2.8 Isolation of Stigmasterol from Hexane Extract of S. crispus 40

2.2.9 Isolation of Lupeol from Hexane Extract of S. arvensis 40

2.2.10 Isolation of Quercetin from Methanol Extract of S. arvensis 41

2.3 Results and Discussion 41

2.3.1 Extraction of S. crispus and S. arvensis Leaves Using

Maceration Method

41

2.3.2 Analysis by Gas Chromatography/Time of Flight Mass

Spectrometry (GC/TOF-MS)

42

2.3.3 Extraction of S. crispus and S. arvensis Using Soxhlet 57

2.3.4 Isolation of Tritriacontane from Hexane Extract of S. crispus 57

2.3.5 Isolation of Stigmasterol from Hexane Extract of S. crispus 59

2.3.6 Isolation of Lupeol from hexane extract of S. arvensis 61

2.3.7 Isolation of Quercetin from the Methanol Extract of S.

arvensis 62

2.4 Conclusion 63

CHAPTER 3 CRYSTAL GROWTH INHIBITION AND ANTIOXIDANT ACTIVITY STUDIES ON STROBILANTHES CRISPUS AND SONCHUS ARVENSIS EXTRACTS

64

3.1 Introduction 64


3.2.1 Chemicals 67

vi


3.2.3 Determination of Inhibition on Calcium Oxalate Crystal

Growth

68

3.2.4 Determination of Antioxidant Activity 69

3.2.4.1. Free Radical Scavenging Activity 69

3.2.4.2. Inhibition on Xanthine Oxidase Activity 70

3.2.4.3. Antioxidant Assay Using β-Carotene-Linoleate

Model System

71

3.2.5. Determination of Total Phenolics Content 72

3.2.6. Determination of Total Polysaccharides Content 72

3.2.7. Determination of Protein and Nitrogen Compounds Content 73

3.2.8. Statistical Analysis 74

3.3. Results and Discussion 75

3.3.1 Determination of Inhibition on Calcium Oxalate Crystal

Growth

75

3.3.1.1 Inhibition Index 75

3.3.1.2 Crystal Size Distribution 78

3.3.1.3 Profile of Calcium Oxalate Crystal Growth 81

3.3.1.4 Self-Organizing Map 83

3.3.2 Determination of Antioxidant Activity 86

3.3.2.1 Free Radical Scavenging Activity 86

3.3.2.2 Inhibition on Xanthine Oxidase Activity 92

3.3.2.3 Antioxidant Assay Using β-carotene-linoleate Model

System

95

3.3.3 Determination of Total Phenolics Content 99

3.3.4 Determination of Total Polysaccharides Content 100

3.3.5 Determination of Protein and Nitrogen Compounds Content 101

3.4 Conclusion 102

vii

CHAPTER 4 STABILITY STUDIES ON STROBILANTHES CRISPUS AND SONCHUS ARVENSIS EXTRACTS

105

4.1 Introduction 105


4.2.1 Chemicals 106


4.2.3 Extraction and Preparation of Samples 107

4.2.4 Ultraviolet Spectroscopic Analysis 108

4.2.5 Infrared Spectroscopic Analysis 108

4.2.6 High Performance Thin Layer Chromatographic Analysis 108

4.2.7 High Performance Liquid Chromatographic Analysis 108

4.2.7.1 Chromatographic Conditions 109

4.2.7.2 Validation of the HPLC Methods 109

4.2.7.3 Analysis of Extracts 110

4.2.8 Chemometric Data Analysis 110


4.3.1 Ultraviolet Spectroscopic Analysis 110

4.3.2 Infrared Spectroscopic Analysis 116

4.3.3 High Performance Thin Layer Chromatographic Analysis 119

4.3.4 High Performance Liquid Chromatographic Analysis 123

4.3.4.1 Validation of the HPLC Methods 123

4.3.4.2 Analysis of Extracts 125

4.3.5 Determination of the kinetic parameters of degradation

reaction for marker compounds

131

4.3.5.1 Kinetic of Stigmasterol and Lupeol Degradation 132

4.3.5.2 Determination of the Order of the Degradation

Reaction

135

viii

4.3.5.3 Determination of the Rate Constant for Marker

Compound Degradation

138

4.3.5.4 Determination of the Activation Energy 140

4.3.5.5 Determination of the Shelf life (t90) 142

4.3.6 Chemometric Data Analysis of FT-IR Spectra 145

4.4 Conclusion 168

CHAPTER 5 ANTIANGIOGENIC STUDIES ON STROBILANTHES

CRISPUS AND SONCHUS ARVENSIS EXTRACTS 170

5.1 Introduction 170


5.2.1 Chemicals 172


5.2.3 Rat Aorta Assay 172


5.4 Conclusion 179

CHAPTER 6 GENERAL CONCLUSION AND SUGGESTION 180

6.1 General Conclusion 180

6.2 Suggestion 183

REFERENCES 184

APPENDICES 200

LIST OF CONFERENCES 241

ix

LIST OF TABLES Page

Table 1.1 Plants used for treatment of kidney stone and related

diseases

13

Table 1.2 Storage conditions of the stability studies of drug

substance and drug product

25

Table 2.1 Comparison of compounds having 1% or greater

percentage in one of the leaf S. crispus extracts detected

by GC/TOF-MS

43

Table 2.2 Comparison of compounds having 1% or greater

percentage in one of the leaf S. arvensis extracts detected

by GC/TOF-MS

46

Table 2.3 Selected compounds having the highest percentage in S.

crispus extracts

53

Table 2.4 Selected compounds having the highest percentage in S.

arvensis extracts

54

Table 2.5 Comparison of the chemical shift of the isolated compound

and tridecane, CH3(CH2)11CH3 (Breitmaier, 1979)

58

Table 3.1 The ability to inhibit the calcium oxalate crystal growth of

the water extracts compared to that of 25 mM sodium

citrate

78

Table 3.2 Crystal numbers of blank, positive control, and sample at

various incubation times

83

Table 3.3 Effective concentration 50% (EC50) of S. crispus extracts 91

Table 3.4 Effective concentration 50% (EC50) of S. arvensis extracts 91

Table 3.5 Effective concentration 50% (EC50) of reference

compounds

91

Table 3.6 Polysaccharide contents in S. crispus and S. arvensis

water extracts

100

x

Table 3.7 Protein and nitrogen compounds content in S. crispus and

S. arvensis water extracts

102

Table 4.1 Storage conditions for stability studies on S. crispus and S.

arvensis extracts with controlled relative humidity (RH)

using saturated salt solution

107

Table 4.2 Calibration data of marker compounds using Agilent HPLC

system

124

Table 4.3 Analytical precision of stigmasterol and lupeol as marker

and in samples

124

Table 4.4 Recovery test for marker in S crispus and S. arvensis

extract

125

Table 4.5 Activation Energy (Ea) of stigmasterol and lupeol in

extracts, respectively

142

xi

LIST OF FIGURES Page

Fig. 1.1 Chemical structures of S. crispus constituents 6

Fig. 1.2 Chemical structures of S. arvensis constituents 9

Fig. 1.3 Chemical structures of a variety of phytochemicals

exhibiting antiangiogenic activity

21

Fig. 2.1 Diagram of soxhlet extraction method for S. crispus and S.

arvensis leaves

38

Fig. 2.2 Comparing percentages of total extracts from S. crispus

and S. arvensis leaves using different macerating solvents

42

Fig. 2.3 Structure of phytol 52

Fig. 2.4 Structures of stigmasterol (a) and α-sitosterol (b) 54

Fig. 2.5 Structure of lupeol 56

Fig. 2.6 Comparing percentages of total extracts from S. crispus

and S. arvensis leaves with n-hexane, chloroform and

methanol solvents, using continuous soxhlet extractor.

57

Fig. 2.7 Structure of tritriacontane 59

Fig. 2.8 Structure of quercetin 62

Fig. 3.1 Microscope slide gel of Schneider's method 69

Fig. 3.2 Comparing inhibition indices of S. crispus extracts and

sodium citrate on calcium oxalate crystal growth at various

incubation times

77

Fig. 3.3 Comparing inhibition indices of S. arvensis extracts and

sodium citrate on calcium oxalate crystal growth at various

incubation times

77

Fig. 3.4 The histogram of the crystal size distribution of calcium

oxalate in blank, control of sodium citrate and sample of

water extract from S. crispus (Ew-Sc)

79

xii

Fig. 3.5 The histogram of the crystal size distribution of calcium

oxalate in blank, control of sodium citrate and sample of

water extract from S. arvensis (Ew-Sa)

80

Fig. 3.6 The growth profile of calcium oxalate crystal for blank,

positive control and S. crispus extract (Ew-Sc) at 2, 4, 8,

and 24 hours of incubation time

82

Fig. 3.7 The growth profile of calcium oxalate crystal for blank,

positive control and S. arvensis extract (Ew-Sa) at 2, 4, 8,

and 24 hours of incubation time

82

Fig. 3.8 The U matrix and the variable information for the particles 85

Fig. 3.9 The crystal data on the map for blank (white), sodium citrate

as control (yellow) and 10,000 ppm water extract of S.

crispus (violet)

86

Fig. 3.10 Comparing FRSA of S. crispus extracts using DPPH

method

89

Fig. 3.11 Comparing FRSA of S. arvensis extracts using DPPH

method

90

Fig. 3.12 Comparing FRSA of reference compounds using DPPH

method

90

Fig. 3.13 Comparing xanthine oxidase inhibitory activities of 100 ppm

S. crispus extracts to xanthine substrate

94

Fig. 3.14 Comparing xanthine oxidase inhibitory activities of 100 ppm

S. arvensis extracts to xanthine substrate

94

Fig. 3.15 Comparing antioxidant activities of S. crispus extracts and

quercetin, BHA, and BHT as reference compounds using β-

carotene-linoleic acid method

97

Fig. 3.16 Comparing antioxidant activities of S. arvensis extracts and

quercetin, BHA, and BHT as reference compounds using β-

carotene-linoleic acid method

97

xiii

Fig. 3.17 Comparing total phenolic contents of S. crispus extracts 100

Fig. 3.18 Comparing total phenolic contents of S. arvensis extracts 100

Fig. 4.1 Ultraviolet spectra of acetone, Eac (1), methanol, Em (2)

and water, Ew (3) extracts from S. crispus at 25 0C/65%

RH (a) and 600C/85%RH (b) with period 0 to 6 months in

storage (Red, black, green, pink, maroon, dark green, and

blue colours are 0, 1, 2, 3, 4, 5 and 6 months storage

period, respectively).

112

Fig. 4.2 Ultraviolet spectra of acetone, Eac (1), methanol, Em (2)

and water, Ew (3) extracts from S. arvensis at 25 0C/65%

RH (a) and 600C/85%RH (b) with period 0 to 6 months in

storage (Red, black, green, pink, maroon, dark green, and

blue colours are 0, 1, 2, 3, 4, 5 and 6 months storage

period, respectively)

113

Fig. 4.3 Peak intensities at 415 nm of UV spectra of acetone

extracts (Eac) from S. crispus at various storage

temperatures and periods

114

Fig. 4.4 Peak intensities at 415 nm of UV spectra of methanol

extracts (Em) from S. crispus at various storage


114

Fig. 4.5 Peak intensities at 415 nm of UV spectra of acetone

extracts (Eac) from S. arvensis at various storage


114

Fig. 4.6 Peak intensities at 415 nm of UV spectra of methanol

extracts (Em) from S. arvensis at various storage

temperatures and time periods

115

Fig. 4.7 Peak intensities at 330 nm of UV spectra of water extracts

(Ew) from S. arvensis at various storage temperatures and

time periods

115

xiv

Fig. 4.8 FT-IR spectra of acetone, Eac (1), methanol, Em (2) and water, Ew (3) extracts from S. crispus stored 0-6 months (red, black, green, pink, maroon, dark green, and blue colours are 0, 1, 2, 3, 4, 5 and 6 months storage period, respectively) at 25 0C/65% RH (a) and 600C/85%RH (b)

117

Fig. 4.9 FT-IR spectra of acetone, Eac (1), methanol, Em (2) and

water, Ew (3) extracts from S. arvensis stored 0-6 months

(red, black, green, pink, maroon, dark green and blue

colours are 0, 1, 2, 3, 4, 5 and 6 months storage period,

respectively) at 25 0C/65% RH (a) and 600C/85%RH (b)

118

Fig. 4.10 3D-HPTLC chromatogram of stigmasterol (st) and various

extracts from S. crispus; methanol, Em (a), acetone, Eac

(b), and water, Ew (c) before storage

120

Fig. 4.11 3D-HPTLC chromatogram of lupeol (lu), and various

extracts from S. arvensis; methanol, Em (a); acetone, Eac

(b), and water, Ew (c) before storage

120

Fig. 4.12 3D-HPTLC chromatogram of stigmasterol (st), and various

extracts from S. crispus; acetone, Eac (maroon), methanol,

Em (brown), and water, Ew (black) after 6 months storage

period at 25 (a), 40 (b), 50 (c) and 60 oC (d)

121

Fig. 4.13 3D-HPTLC chromatogram of lupeol (lu), and various

extracts from S. arvensis; acetone, Eac (green), methanol,

Em (purple), and water, Ew (orange) after 6 months storage

period at 25 (a), 40 (b), 50 (c) and 60 oC (d)

122

Fig. 4.14 HPLC chromatograms of acetone extracts (Eac) from S.

crispus stored 0 (a), 1 month (b) and 2 months (c) at 25 0C/65% RH

126

Fig. 4.15 HPLC chromatograms of methanol extracts (Em) from S.


127

xv

Fig. 4.16 HPLC chromatograms of water extracts (Ew) from S.


128

Fig. 4.17 HPLC chromatograms of acetone extracts (Eac) from S.

arvensis stored 0 (a), 1 month (b) and 2 months (c) at 25 0C/65% RH

129

Fig. 4.18 HPLC chromatograms of methanol extracts (Em) from S.


130

Fig. 4.19 HPLC chromatograms of water extracts (Ew) from S.


131

Fig. 4.20 Comparing percentage of remaining stigmasterol

concentration in acetone extracts (Eac) from S. crispus at

various storage temperatures and periods

133

Fig. 4.21 Comparing percentage of remaining stigmasterol

concentration in methanol extracts (Em) from S. crispus at

various storage temperatures and periods

133

Fig. 4.22 Comparing percentage of remaining lupeol concentration in

acetone extracts (Eac) from S. arvensis at various storage


134

Fig. 4.23 Comparing percentage of remaining lupeol concentration in

methanol extracts (Em) from S. arvensis at various storage


134

Fig. 4.24 Decrease of stigmasterol percentage in methanol and

acetone extracts from S. crispus stored for 6 months at

various temperatures

135

Fig. 4.25 Decrease of lupeol percentage in methanol and acetone

extracts from S. arvensis stored for 6 months at various

temperatures

135

xvi

Fig. 4.26 Plot of ln C of remaining stigmasterol in acetone extracts

(Eac) from S. crispus against time at various storage

conditions

136

Fig. 4.27 Plot of ln C of remaining stigmasterol in methanol extracts

(Em) from S. crispus against time at various storage

conditions

137

Fig. 4.28 Plot of ln C of remaining lupeol in acetone extracts (Eac)

from S. arvensis against time at various storage conditions

137

Fig. 4.29 Plot of ln C of remaining lupeol in methanol extracts (Em)

from S. arvensis against time at various storage conditions

138

Fig. 4.30 Comparing degradation rate constant of stigmasterol in S.

crispus extracts at various storage conditions

139

Fig. 4.31 Comparing degradation rate constant of lupeol in S.

arvensis extracts at various storage conditions

140

Fig. 4.32 Arrhenius plot for stigmasterol in acetone (a) and methanol

extracts (b) from S. crispus

141

Fig. 4.33 Arrhenius plot for lupeol in acetone (a) and methanol

extracts (b) from S. arvensis

141

Fig. 4.34 Comparing shelf life (t90) of stigmasterol in S. crispus

extracts at various storage conditions

143

Fig. 4.35 Comparing the shelf life (t90) of lupeol in S. arvensis extracts

at various storage conditions

144

Fig. 4.36 Comparing the shelf life (t90) of S. crispus extracts stored at

room temperature (250C/60%RH)

144

Fig. 4.37 Comparing shelf life (t90) of S. arvensis extracts stored at

room temperature (250C/60%RH)

144

Fig. 4.38 PCA of acetone extracts (Eac) from S. crispus stored at

room temperature (25 0C/65% RH) for 0-6 months storage

period in the spectral region 2000-900 cm-1 (PC2 vs PC1)

149

xvii

Fig. 4.39 PCA of acetone extracts (Eac) from S. crispus stored at 60 0C/85% RH for 0-6 months storage period in the spectral

region 2000-900 cm-1 (PC2 vs PC1)

149

Fig. 4.40 PCA of methanol extracts (Em) from S. crispus stored at



150

Fig. 4.41 PCA of methanol extracts (Em) from S. crispus stored at 60 0C/85% RH for 0-6 months storage period in the spectral


150

Fig. 4.42 PCA of water extracts (Ew) from S. crispus stored at room

temperature (25 0C/65% RH) for 0-6 months storage period

in the spectral region 2000-850 cm-1 (PC3 vs PC1)

151

Fig. 4.43 PCA of water extracts (Ew) from S. crispus stored at 60 0C/85% RH for 0-6 months storage period in the spectral


151

Fig. 4.44 PCA of acetone extracts (Eac) from S. arvensis stored at



152

Fig. 4.45 PCA of acetone extracts (Eac) from S. arvensis stored at 60 0C/85% RH for 0-6 months storage period in the spectral


152

Fig. 4.46 PCA of methanol extracts (Em) from S. arvensis stored at



153


60 0C/85% RH for 0-6 months storage period in the

spectral region 1900-900 cm-1 (PC3 vs PC1)

153

Fig. 4.48 PCA of water extracts (Ew) from S. arvensis stored at room

temperature (25 0C/65% RH) for 0-6 months storage period

in the spectral region 1800-1200 cm-1 (PC2 vs PC1)

154

xviii

Fig. 4.49 PCA of water extracts (Ew) from S. arvensis stored at 60 0C/85% RH for 0-6 month storage period in the spectral


154

Fig. 4.50 PCA of acetone extracts (Eac) from S. crispus stored at

various temperatures for 1 month storage period (S1-oC) in

the spectral region 2000-1200 cm-1 (PC2 vs PC1)

155

Fig. 4.51 PCA of methanol extracts (Em) from S. crispus stored at



155

Fig. 4.52 PCA of water extracts (Ew) from S. crispus stored at



156

Fig. 4.53 PCA of acetone extracts (Eac) from S. arvensis stored at



156




157

Fig. 4.55 PCA of water extracts (Ew) from S. arvensis stored at



157

Fig. 4.56 3D plot of FT-IR spectra (4000–400 cm-1) of acetone

extracts (Eac) from S. crispus stored at room temperature

(25°C/65% RH) for 0-6 month storage period

159


extracts (Eac) from S. crispus stored at 60°C/85% RH for

0-6 month storage period

159

Fig. 4.58 3D plot of FT-IR spectra (4000–400 cm-1) of methanol

extracts (Em) from S. crispus stored at room temperature

(25°C/65% RH) for 0-6 months storage period

160

xix


extracts (Em) from S. crispus stored at 60°C/85% RH for 0-

6 months storage period

160

Fig. 4.60 3D plot of FT-IR spectra (4000–400 cm-1) of water extracts

(Ew) from S. crispus stored at room temperature


161


(Ew) from S. crispus stored at 60°C/85% RH for 0-6

months storage period

161


extracts (Eac) from S. arvensis stored at room temperature


162


extracts (Eac) from S. arvensis stored at 60°C/85% RH for

0-6 months storage period

162


extracts (Em) from S. arvensis stored at room temperature


163


extracts (Em) from S. arvensis stored at 60°C/85% RH for

0-6 months storage period

163


(Ew) from S. arvensis stored at room temperature


164


(Ew) from S. arvensis stored at 60°C/85% RH for 0-6

months storage period

164


extracts (Eac) from S. crispus stored at various

165

xx

temperatures for 1 month storage period


extracts (Em) from S. crispus stored at various


165


(Ew) from S. crispus stored at various temperatures for 1

month storage period

166


extracts (Eac) from S. arvensis stored at various


166


extracts (Em) from S. arvensis stored at various


167


(Ew) from S. arvensis stored at various temperatures for 1

month storage period

167

Fig. 5.1 Inhibition percentages of S. crispus and S. arvensis extracts

on angiogenic using rat aorta ring assay.

175

Fig. 5.2 Images of rat aorta with water (a) and methanol extracts

from S. crispus as angiogenesis inhibitor (b)

176

Fig. 5.3 Images of rat aorta with water (a) and methanol extracts

from S. arvensis as angiogenesis inhibitor (b)

177

Fig. 5.4 Images of rat aorta and control (a) and betulinic acid (b) 178

xxi

LIST OF PLATES

Page

Plate 1.1 Picture of S. crispus plant

4

Plate 1.2 Picture of S. arvensis plant

8

xxii

LIST OF APPENDICES Page

Appendix 2.1 GC / TOF-MS chromatograms of the acetone, Eac (a),

70% acetone, E7ac (b), methanol, Em (c), water, Ew (d),

and n-hexane, Eh (e) extracts of S. crispus L leaves

200

Appendix 2.2 GC / TOF-MS chromatograms of the acetone, Eac (a),

70% acetone, E7ac (b), methanol, Em (c), water, Ew (d),

and n-hexane, Eh (e) extracts of S. arvensis L leaves

201

Appendix 2.3 Spectroscopic data of isolated tritriacontane 202

Appendix 2.4 HPLC chromatograms of standard stigmasterol (a) and

isolated stigmasterol (b)

204

Appendix 2.5 TLC profiles of the standard stigmasterol (1) and isolated

stigmasterol (2) using CHCl3 [a, Rf = 0.16], CHCl3:

MeOH (95: 5) [b, Rf = 0.73] and n-hexane: CHCl3:

MeOH (5: 4: 1) [c, Rf = 0.67] as mobile phase,

respectively under 365 nm

204

Appendix 2.6 3D-HPTLC of the standard stigmasterol (green) and

isolated stigmasterol (blue) using CHCl3 [a], CHCl3:

MeOH (95: 5) [b] and n-hexane: CHCl3: MeOH (5: 4: 1)

[c] as mobile phase, respectively under 365 nm

205

Appendix 2.7 FT-IR spectra of the standard stigmasterol [black (1)]

and isolated stigmasterol [blue (2)]

205

Appendix 2.8 Ultraviolet spectra of the standard stigmasterol [green

(1) and blue (2)] and isolated stigmasterol [red (3) and

black (4)]

206

Appendix 2.9 Mass spectra of the isolated stigmasterol 206

Appendix 2.10 Mass spectra of standard stigmasterol 207

Appendix 2.11 HPLC chromatograms of standard lupeol (a) and

isolated lupeol (b)

207

xxiii

Appendix 2.12 TLC profiles of the standard lupeol (1) and isolated

lupeol (2) using CHCl3 [a, Rf = 0.24] and CHCl3: MeOH

(95: 5) [b, Rf = 0.89] as mobile phase, respectively under

365 nm

208

Appendix 2.13 3D-HPTLC profiles of the standard lupeol (blue) and

isolated lupeol (red) using CHCl3 [a] and CHCl3: MeOH

(95: 5) [b] as mobile phase, respectively under 365 nm

208

Appendix 2.14 FT-IR spectra of the standard lupeol [blue (1)] and

isolated lupeol [black (2) and green (3)]

209

Appendix 2.15 Ultraviolet spectra of the standard lupeol [(brown (2) and

black (4) and isolated lupeol [green (1) and blue (3)]

209

Appendix 2.16 Mass spectra of isolated lupeol 210

Appendix 2.17 Mass spectra of standard lupeol 210

Appendix 2.18 HPLC chromatograms of standard quercetin (a) and

isolated quercetin (b)

210

Appendix 2.19 TLC profiles of standard quercetin (1) and isolated

quercetin (2) using BAW as mobile phase, before

sprayed with Natural Product reagent (a), after sprayed

using Natural Product reagent (b), under visible (I), 254

nm (II), and 365 nm (III)

211

Appendix 2.20 3D-HPTLC of standard quercetin (green spectra) and

isolated quercetin (violet spectra) using BAW as mobile

phase under 365 nm, before sprayed with Natural

Product reagent (a) and after sprayed with Natural

Product reagent (b)

212

Appendix 2.21 Ultraviolet spectra of standard quercetin [red (2) and

green (3)] and isolated quercetin [blue (1) and black (4)]

212

Appendix 2.22 FT-IR spectra of the standard quercetin [blue (1)] and

isolated quercetin [black (2)]

213

xxiv

Appendix 3.1 Statistical analyses of Inhibition Index of S. crispus to

growth of calcium oxalate crystal

214

Appendix 3.2 Statistical analyses of Inhibition Index of S. arvensis to

growth of calcium oxalate crystal

218

Appendix 3.3 Statistical analyses of free radical scavenging activity of

S. crispus

222

Appendix 3.4 Statistical analyses of free radical scavenging activity of

S. arvensis

226

Appendix 3.5 Statistical analyses of xanthine oxidase inhibitory activity

of 100 ppm S. arvensis extracts to xanthine substrate

232

Appendix 3.6 Statistical analyses of antioxidant activity of extracts and

references using β-carotene linoleic acid method

234

Appendix 3.7 Standard calibration curve of gallic acid 235

Appendix 3.8 Statistical analyses of total phenoliccs contents 236

Appendix 3.9 Statistical analyses the correlation of total phenolic

contents in S. crispus and S. arvensis with their FRSA to

DPPH

237

Appendix 3.10 Standard calibration curve of glucose 238

Appendix 3.11 Standard calibration curve of protein 239

Appendix 4.1 Standard calibration curve of marker compounds 240

xxv

LIST OF ABBREVIATIONS

3D Three of dimension

BAW Buthanol-1: Acetic acid: Water

BHA Butylated Hydroxyl Anisole

BHT Butylated Hydroxyl Toluene

DMSO Dimethylsulfoxide

DPPH 1,1-Diphenyl-2-picrylhydrazyl

E7ac 70% acetone extract

Eac Acetone extract

Em Methanol extract

Em-sox Methanol fraction from soxhlet

Ew Water extract

FTIR Fourier Transform Infra Red

GC Gas Chromatography

GC–MS Gas Chromatography–Mass Spectrometry

GC/TOF-MS Gas Chromatography/Time-of- Flight Mass Spectrometry

HPLC High Performance Liquid Chromatography

HPTLC High Performance Thin Layer Chromatography

MeOH Methanol

NMR Nuclear Magnetic Resonance

Ox-2 anion of oxalate

PCA Principal Component Analysis

Rf Retention factor

RH Relative humidity

Sa Sonchus arvensis

Sc Strobilanthes crispus

SOM Self-Organizing Map

TLC Thin Layer Chromatography

TOF-MS Time-of-flight mass spectrometry

UV Ultraviolet

VIS Visible

XO Xanthine oxidase

xxvi

KAJIAN ANALITIKAL, BIOAKTIVITI DAN STABILITI TERHADAP EKSTRAK STROBILANTHES CRISPUS L. BREMEK DAN SONCHUS ARVENSIS L.

ABSTRAK

Tujuan kajian ini adalah untuk memiawaikan ekstrak-ekstrak (Ew, E7ac, Em,

Eac dan Em-sox) daun Strobilanthes crispus L. Bremek dan Sonchus arvensis

L. untuk tujuan kajian praklinikal. Kaedah pemiawaian dibahagikan kepada tiga

bahagian iaitu profil kimia (kajian analitikal dan fitokimia), profil biokimia (kajian

perencatan pertumbuhan kristal, antioksidan dan kestabilan) dan profil biologi

(kajian antiangiogenik).

Sebatian metabolit sekunder yang dikesan dalam daun S. crispus ialah α-

sitosterol, campesterol, phytol dan stigmasterol, sementara lupeol, phytol dan α-

sitosterol dikesan dalam daun S. arvensis. Sebatian tritriakontana dan

stigmasterol telah diasingkan daripada daun S. crispus, sementara lupeol dan

kuersetin telah diasingkan daripada daun S. arvensis.

Indeks perencatan ekstrak Ew, E7ac, Em, Eac, dan Em-sox S. crispus terhadap

perencatan pertumbuhan kristal kalsium oksalat masing-masing adalah 0.2233

± 0.0875, 0.1861 ± 0.0124, 0.1587 ± 0.0264, 0.1830 ± 0.0335, dan 0.2081 ±

0.0166. Manakala indeks perencatan ekstrak yang sama daripada S. arvensis

masing-masing adalah 0.3375 ± 0.0157, 0.1994 ± 0.0257, 0.1938 ± 0.0662,

0.1347 ± 0.0439, dan 0.3157 ± 0.0457.

Aktiviti antioksidan tertinggi ke atas aktiviti radikal bebas, aktiviti xantina

oksidase dan pelunturan β-karotena oleh asid linoleik daripada ekstrak S.

crispus masing-masing adalah E7ac, Em dan Em-sox. Manakala bagi yang

sama daripada ekstrak S. arvensis masing-masing adalah E7ac, Eac dan Em.

xxvii

Peratusan fenolik daripada ekstrak tumbuh-tumbuhan ini juga ditentukan.

Koefisien penentuan (R2) antara kandungan fenolik dan aktiviti radikalnya

adalah 0.93 (S. arvensis) dan 0.40 (S crispus). Dalam perencatan pelunturan

β-karotena oleh asid linoleik, didapati kesan protein lebih besar berbanding

kesan polisakarida.

Dalam kajian kestabilan dipercepat, kesemua ekstrak (Ew, Em, and Eac) yang

disimpan pada suhu bilik (25 0C, 60% RH) adalah tertinggi berbanding ekstrak

yang disimpan pada suhu 40 0C (75%RH), 50 0C (85% RH) dan 60 0C (85%

RH). Jangka hayat (t90) ekstrak Eac, Em dan Ew daripada S. crispus yang

disimpan pada suhu bilik, masing-masing adalah 2.14, 2.17 dan 1.94 bulan.

Manakala t90 ekstrak yang sama daripada S. arvensis masing-masing adalah

2.10, 7.89 dan 3.50 bulan. t90 stigmasterol dalam Em dan Eac daripada S.

crispus yang disimpan pada suhu bilik, masing-masing adalah 3.60 dan 2.63

bulan. Manakala t90 lupeol dalam ekstrak Em dan Eac daripada S. arvensis,

masing-masing adalah 2.51 dan 2.22 bulan.

Dalam kajian awal antiangiogenik, keputusan menunjukkan bahawa peratus

perencatan Ew dan Em daripada S. crispus, masing-masing adalah 16.67 dan

6.25%, manakala peratus perencatan Ew dan Em daripada S. arvensis,

masing-masing adalah 11.06 dan 8.65%, memperlihatkan bahawa kedua

tumbuhan ini mempunyai kemampuan mencegah atau menyembuh penyakit-

penyakit yang berkaitan dengan angiogenik.

xxviii

ANALYTICAL, BIOACTIVITY AND STABILITY STUDIES ON STROBILANTHES

CRISPUS L. BREMEK AND SONCHUS ARVENSIS L. EXTRACTS

ABSTRACT

The purpose of this study was to standardize the leaf extracts (Ew, E7ac, Em,

Eac and Em-sox) of Strobilanthes crispus L. Bremek and Sonchus arvensis L.

for preclinical studies. The standardization work was divided into three steps:

chemical profiling (analytical and phytochemical studies), biochemical profiling

(crystal growth inhibition, antioxidant and stability studies) and biological

profiling (antiangiogenic studies).

Secondary metabolites detected in S. crispus leaves were α-sitosterol,

campesterol, phytol and stigmasterol, whereas lupeol, phytol and α-sitosterol

were detected in S. arvensis leaves. Tritriacontane and stigmasterol were

isolated from S. crispus leaves whilst lupeol and quercetin were isolated from S.

arvensis leaves.

The inhibition indices of Ew, E7ac, Em, Eac, and Em-sox from S. crispus to

inhibit the growth of calcium oxalate crystals were 0.2233 ± 0.0875, 0.1861 ±

0.0124, 0.1587 ± 0.0264, 0.1830 ± 0.0335, and 0.2081 ± 0.0166, respectively.

The values for similar extracts for S. arvensis were 0.3375 ± 0.0157, 0.1994 ±

0.0257, 0.1938 ± 0.0662, 0.1347 ± 0.0439, and 0.3157 ± 0.0457, respectively.

The highest antioxidant activity on FRSA to DPPH, xanthine oxidase activity

and prevention the bleaching of β-carotene by linoleic acid of S. crispus extracts

are E7ac, Em and Em-sox, respectively whilst those of S. arvensis extracts are

E7ac, Eac and Em, respectively. The percentages of phenolic content from

these plants extract were also determined. Coefficient value (R2) between their

phenolic content and FRSA were 0.93 (S. arvensis) and 0.40 (S crispus). In the

xxix

prevention of bleaching of β-carotene by linoleic acid, effect of protein was more

than that of polysaccharide.

In accelerated stability studies, the extracts (Ew, Em, and Eac) stored at room

temperature (25 0C, 60% RH) was highest when compared to stored at 40 0C

(75%RH), 50 0C (85% RH) and 60 0C (85% RH). Shelf life (t90) of Eac, Em and

Ew from S. crispus stored at room temperature was 2.14, 2.17 and 1.94

months, respectively. Meanwhile the t90 of similar extracts from S. arvensis was

2.10, 7.89 and 3.50 months, respectively. The t90 of stigmasterol in Em and Eac

from S. crispus stored at room temperature was 3.60 and 2.63 months

respectively, whilst lupeol in Em and Eac from S. arvensis was 2.51 and 2.22

months respectively.

In preliminary antiangiogenic studies, the results showed that inhibition

percentages of Ew and Em from S. crispus are 16.67 and 6.25% respectively,

whilst those of Ew and Em from S. arvensis are 11.06 and 8.65% respectively,

exhibiting that these plants possess the potential to prevent or cure

angiogenesis related diseases.

1

CHAPTER 1

INTRODUCTION

1. 1 The Usage of Medicinal Plants

Plants have been used as source of medicines for thousands of years in

maintaining health as an alternative to or in conjunction with modern medicines.

The majority of the world's population in developing countries used herbal

medicines to meet their health needs, following traditional beliefs and practices

adopted from their elders and ancestors. The World Health Organization (WHO)

estimated about 70% of the world population uses medicinal plants for

medicines, and they are highly used mainly in Asia, South America and Africa

(Chapman, K. and Chomchalow, N., 2005). Mamedov et al. (2005) reported the

flora of Russia and Central Asia contains approximately 300 species of plants

that have been used in prescription and non-prescription pharmaceutical

preparations, while nearly 2500 plants are known to have been used in

traditional medicine. A study from Kenya showed that patients had a clear

sense of which diseases when they visit a traditional healer although previously

they would go to a western clinic. In South Africa, traditional healers are

flourishing in urban areas where western health care is also available (Van der

Geest, 1997; Mander et al., 1997 cited in Jäger, A. K., 2005). Another study

reported that the rate of having used an alternative treatment method is 42.1%

in the U.S, 48% in Australia, 70% in Canada, 38% in Belgium, 90% in Germany,

75% in France and 75.9% in Turkey (Recai et al., 2006). Meanwhile, Lai et al.

(2007) reported that over two-thirds of the older Chinese immigrants in Canada

use traditional Chinese medicine in combination with Western health services.

About half (50.3%) of the older Chinese immigrants used Chinese herbs, 48.7%

2

used Chinese herbal formulas, and 23.8% consulted a Chinese herbalist. In

Indonesia, Sulaksana et al. (2004) reported that at least 1,845 of medicinal

plants have been identified and inventoried, and at least 400 ethnic

communities have experiences in use of medicinal plants.

The use of herbal medicine is extensive, increasing and complex. In England,

from a survey of the use of complementary and alternative medicine (CAM)

reported that purchasing of herbal medicine product (HMPs) have increased

almost 20% per year (Heinrich et al., 2004). In 2002, the global trade in herbal

product to have a value of US$12 billion, with trades in crude medicinal plants

exceeding US $800 M., herbal extracts and semi-finished raw materials

exceeding US $8 billion and herbal cosmetics about US$1.5 billion (Parke and

Tikasingh, 2002). The demand for medicinal plants is increasing everyday and

the World Health Organization (WHO) has projected that the global herbal

market will grow to $5 trillion by 2050 from the current level of $62 billion with

growth rate of 7 to 30 per cent annually (Reddy, 2003). According to Malaysian

Deputy Minister of Natural Resources and Environment, currently the value of

the local herbal market in Malaysia is estimated to be around 3.8 billion Ringgit

(1.03 billion U.S. dollars) and this amount is expected to reach 8 billion Ringgit

(2.16 billion U.S. dollars) by 2010, a handsome annual growth rate of 15 to 20

percent (http://english.people.com.cn/ 200609/13/eng20060913_302302.html).

In addition, combination of traditional and modern medicine has an important

role in promoting health care system. In many countries, herbal medicine is

3

making a strong comeback and the world of medicine today embraces both

single pure chemical entities and herbal medicine side by side (WHO, 2001).

There are many medicinal herbs used in health care, such as Eurycoma

longifolia, Orthosiphon stamineus, Phyllanthus niruri, Andrographis paniculata,

and Catharanthus roseus (Wiart, 2002; Zakaria and Ali, 1994; Dalimartha,

1999). To enable medicinal plants to be use in modern medicine, researches

and development are important for the advancement of traditional medicines.

My research work will focus on two widely used medicine plant species,

Strobilanthes crispus and Sonchus arvensis. Both of the latter has their origins

from Padang Sumatera.

1.2 Strobilanthes crispus Plant

1.2.1 Botanical Description

Strobilanthes crispus L. Bremek is an annual plant, which grows easily in the

forest, riverbanks and abandoned fields. It is commonly used as fence hedges.

The plant is native to countries from Madagascar to Indonesia, which can be

grown 50 to 1200 meters above sea level. This bush-like plant can attain a

height between 1 to 2 m. The circular bark can be divided into segments and

similar to its branches, they are hairy and green. The leaf is oblong-lanceolate,

rather obtuse, and shallowly crenate-crispate. The top surface of the leaf is

darker green in color and less rough compared to the under surface. The leaves

are very scabrous on both surfaces and covered with short hairs, whereas the

flower is short, dense, and consists of penciled spikes. The leaf is 9-18 cm in

length and 3-8 cm in width. The plant can be propagated using cut steams.

4

The classification for S. crispus is as follows, Division is Spermatophyta, Sub

division is Angiospermae, Class is Dicotyledonae, Sub class is Solanales,

Family is Acanthaceae, Genus is Strobilanthes and Species is Strobilanthes

crispus.

The local name is daun picah beling (Jakarta), enyoh kelo, kecibeling,

kejibeling, ngokilo (Java), pecah kaca or jin batu (Malay). The Latin synonym is

Sericocalyx crispus L. Bremek (Departemen Kesehatan Republik Indonesia,

19771; Syamsuhidayat and Hutapea, 19911; Wijayakusuma et al., 2000; Heyne,

1987; Fadzelly et al., 2006). Picture of S. crispus is presented in Plate 1.1.

Plate 1.1 Picture of S. crispus plant

5

1.2.2 Biological Activity

Studies in Indonesia have found that infusion of the dried leaves has been used

as antidiabetic, diuretic, antilithic, and laxative (Perry and Metzger, 1980;

Syamsuhidayat and Hutapea, 19911; Wijayakusuma et al., 2000). They

suggested boiling 25 – 50g of fresh leaves in 200 ml boiling water, and then

drinking the infusion after filtration. For external use, poultice of the fresh leaves

can be directly applied on to wounds caused by the bite of poisonous snakes or

other animals (Wijayakusuma et al., 2000). Ismail et al., (2000) reported that the

extract showed antioxidant activity using ferric thiocyanate (FTC) and

thiobarbituric acid (TBA) methods. Jaksa et al. (2004) reported that the extract

showed anti hepatocarcinogenesis effect on rats. The hot water-extract of

fermented and unfermented leaves was found to reduce blood glucose in

hyperglycemic rats, while unfermented leaves also reduced glucose level in

normal rats. Both fermented and unfermented leaves also exhibited improved

lipid profiles (Fadzelly et al., 2006). Rahmat et al. (2006) reported that the

methanolic extract displayed strong cytotoxic effect on colon cancer (Caco-2),

human breast cancer hormone non-dependent (MDA-MB-231) and liver cancer

(HepG-2). The chloroform extract of this plant was also shown to have cytotoxic

effect against Caco-2 and HepG-2.

1.2.3 Phytochemistry

Soediro et al. (1983, 1988) isolated and identified verbacoside, glycosidic ester

of caffeic acid and seven phenolic acids; namely p-hydroxy benzoic, p-

coumaric, caffeic, vanilic, gentinic, ferulic, and syryngic acids in the leaves.

Besides, the leaves also contained tannin, saponin, salt of potassium, sodium

and silicate (Departemen Kesehatan Republik Indonesia, 19771, 1980;

6

Syamsuhidayat and Hutapea, 19911; Wijayakusuma et al., 2000). Rahmat et al.

(2006) reported the presence of β-sitosterol, and stigmasterol in the leaves. The

chemical structures of the constituents are presented in Fig. 1.1.

HO

HOO

O

OHOH2C

OHO

O

OHHOHOH3C

O

OH

OH

verbascoside

O

OH

HO

p-hydroxy benzoic acid

OHO

HO

p-coumaric acid

HO

HO

O

OH

caffeic acid

H3CO

HO

O

OH

ferulic acid

OCH3

OH

O

OH

vanillic acid

OH

HO

O

OH

gentisic acid

OCH3

OH

OCH3

O

HO

syringic acid

HO stigmasterol

HO

C2H5

β-sitosterol

Fig. 1.1 Chemical structures of S. crispus constituents

7

1.3 Sonchus arvensis Plant

1.3.1 Botanical Description

S. arvensis L. is an annual plant that is easy to grow in rainy and sunshine

areas, such as on riverbanks, ridges of rice field and abandoned fields 50 –

1650 meters above sea level. The plant is a native of Eurasia with a tapered

root and produces bitter latex. The stem is hollow inside. The leaves are single,

6 – 48 cm in length and 3 – 12 cm in width, elliptical, and lanceolate in shape,

highly variable, entire to deeply pinnate-lobed, clasping the stem at the base

with rounded basal lobes (auricles), sharp-pointed at end side, while at the base

is like heart, and green in color.

Flowers are humped shape and having a long stalk, light yellow in color and

turns brownish red on maturity. The fruits are thin box shape with five sides, 4

mm in length, hairy and yellowish brown in color. The plants can be propagated

using the seeds.

The classification for Sonchus arvensis is as follows: Division is Spermatophyta,

Sub division is Angiospermae, Class is Dicotyledonae, Sub class is Asterales,

Family is Asteraceae, Genus is Sonchus, and Species: Sonchus arvensis.

The local name is lempung, rayana, jombang and galibug, lalakina (Sunda),

tempuyung (Jawa). Other names are Niu she tou (China), Laitron des champs

(France), Sow thistle (British) (Dalimartha, 2001; Departemen Kesehatan

Republik Indonesia, 19772; Sulaksana et al., 2004; Syamsuhidayat and

Hutapea, 19912; Wijayakusuma et al., 2001). Picture of S. arvensis is presented

in Plate 1.2.

8

1.3.2 Biological Activity

S. arvensis L. is one of the medicinal herbs used in traditional medicines, in

which the leaf extract was used as a diuretic, lithotriptic and antiurolithiasis

agent; also indicated for fever, poisoning and swelling or abscess (Dalimartha,

2001; Syamsuhidayat and Hutapea, 19912). Dalimartha (2001) recommended

using 15 – 60 g fresh leaves, boiled in water, and the filtered infusion taken as

medicine. For external use, the ground fresh leaves were applied directly on the

wounds or the pressed liquid can be used as a compress for abscess, injured

skin and wasir (Dalimartha, 2001).

Plate 1.2 Picture of S. arvensis plant.

9

1.3.3 Phytochemistry

From the leaves of S. arvensis several compounds have been isolated and

identified, including luteolin, luteolin-7-O-glucoside (Bondarenko et al., 1973),

isocinaroside (Bondarenko et al., 1974), luteolin-7-O-glucoside, linarin

(Bondarenko et al., 1975), quercetin, isorhamnetin, chrysoeriol, isorhamnetin-7-

β-D-glucoside, quercetin-7-β-D-glucopyranoside (Bondarenko et al., 1976),

sonchoside (Bondarenko et al., 1978), and apigenin, luteolin-7-O-glucoside (Qu

Guirong et al.,1993), acacetin, kaempferol, chrysoeriol, luteolin, isorhamnetin

(Qu Guirong et al., 1995), quercetin-3-O-α-L-rhamnoside, kaempferol-3,7-α-L-

dirhamnoside (Qu, Guirong et al., 1996), α-amyrin, β-amyrin, lupeol,

taraxasterol (lactuserol), pseudo-taraxasterol (Hooper et al., 1982). The leaves

also contain manitol, inositol, silica, potassium and saponin (Fig. 1.2).

OOH

HO O

OHOH

luteolin

OHOOH

HO O

OHOH

quercetin

OOH

O

OHOH

O

OHOH

HO

OHO

luteolin-7-O-glucoside

O

O

OHOH

O

OHOH

HO

OHO

isocinaroside

O

O

OCH3

O

OHOH

HO

OO

OH3C

OHOHHO

linarin

OOH

O

OHOH

O

OHOH

HO

OHO

OH

sonchoside

OOH

HO O

OH

apigenin OOH

HO O

OCH3

acacetin

Fig. 1.2 Chemical structures of S. arvensis constituents

10

OHOOH

HO O

OH

kaempferol HO

H

HH

taraxasterol

HO lupeol

HO

H

β-amyrin

HO

α-amyrin

OHOOH

HO O

OHOCH3

isorhamnetin

OH

OHHO

HO

HO OH

inositol

HO

HO OH

HO OH

HO

mannitol

Fig. 1.2 (continued)

1.4. Analysis and Isolation of the Chemical Constituents

The purpose of this analysis is to determine the presence of substances in a

sample, qualitative or quantitatively. In this study, the aim of the isolation is to

obtain compound/s useful as chemical marker.

“Markers are constituents of medicinal plant material that are chemically defined

and are of interest for control purpose. Markers are generally used when

constituents with known therapeutic activity are not found or are uncertain and

may serve to calculate the quantity of plant material or preparation in the

finished product. However, the marker has to be quantitatively determined in the

11

plant material or preparation when the starting materials are tested” (WHO,

1993).

“Chemical constituents in plant vary depending on the genetic heterogeneity of

plant species, part of plant, differences in conditions of growth, the age of the

plant, the time and manner of collection or harvest, method of processing and

storage, shelf life and interaction with the other plant constituents. Furthermore,

identification and characterization of the structure of unknown substances are

an important part of natural product drug analysis” (Cannell, 1998).

1.5 Kidney Stone Disease

1.5.1 Aspects of Kidney Stone Disease

Kidney stones are not a product of modern life, but the Scientists have found

evidence of kidney stones in a 7,000-year-old Egyptian mummy. In year 2000,

2.7 million of patients visited health care centers and of these more than

600,000 patients were found to suffer from kidney stone. Men tend to be

affected more frequently than women. Prevalence of kidney stone rises for men

in their 40s and this continues until they are in their 70s, whilst women tend to

suffer disease during their 50s (Coe, 2004).

A kidney stone is a hard mass developed from crystals that separate from the

urine and build up on the inner surfaces of the kidney. Urine normally contains

chemicals that can inhibit the crystal formation. These chemicals may not

function effectively in certain cases leading to stone formation. Fine stone may

pass out of the body through the urinary tract. The chemical composition of

kidney stones depends on the chemical imbalance in the urine. There are four

12

types of kidney stones i.e. calcium, uric acid, struvite and cystine stones.

Calcium type is predominantly stone, approximately 80%, and the most

common type of stone in combination with either oxalate or phosphate. Struvite

called infection stone is a less common followed by uric acid stone which is the

least common of all. Cystine stones are also very rare (Coe, 2004; Hesse et al.,

1976)

1.5.2 Natural Product Inhibitor of Urinary Calculi

Traditionally, some plants were found to be acceptable in treating kidney stone

and related kidney disorders, for example, Orthosiphon stamineus Benth,

Strobilanthes crispus L. Bremek, Soncus arvensis L., Malpighia coccigera and

genus Phyllanthus were used (Perry and Metzger, 1980). The plants used

traditionally in kidney stone diseases are presented in Table 1.1.

13

Table 1.1 Plants used for treatment of kidney stone and related diseases

No Plant Name Constituent Reference

1 Plantago major

Glycoside aucubin, plantagin, plantenolic, succinic acid, adenine, cholin, and aucubin. Polysaccharides, lipids, cafeic acid derivatives, flavonoids, iridoid glycosides, terpenoids, alkaloid, organic acid.

Perry and Metzger, 1980; Samuelsen, 2000.

2 Zea mays Galactan, xylan, dextrose, sugar, zeaxanthin, protein, inosite, hexaphosphoric acid, maizenic acid, resins, potassium and calcium salt. Anthocyanins cyanidin-3-glucoside, pelargonidin-3-glucoside etc. Phenolic acid p-coumaric acid, vanillic acid etc.

Perry and Metzger, 1980; Pedreschi and Cisneros, 2007; Pozo-Insfran et al., 2006

3 Raphanus sativus

Acylated anthocyanin (as pelargonidin), alkaloids pyrolidine, isoquinoline, phenethylamine, sulphuric compounds glucoparin, sinigrin, allylisothiocyanate. Flavonoids apigenin, apigenin-7-O-triglycoside etc.

Otsuki et al., 2002 ; Vargas et al., 1999; Basile et al., 2003.

4 Phylanthus niruri

Potassium, phyllanthin, hypophyllanthin, triacontanal, triacontanol, lignans, glycosides, flavonoids, alkaloids, tannins, phenylpropanoids, saponins

Perry and Metzger, 1980; Syamasundar et al., 1985

5 Orthosiphon stamineus

High potassium salt, glucoside, diterpenes, orthosiphols, rosmarinic acid, salvigenin, orthosiphols, flavonoids (eupatorin, sinensitin, 3′-hydroxy-5,6,7,4′-tetramethoxyflavone,TMF)

Perry and Metzger, 1980; Takeda et al., 1993; Akowuah et al., 2005

1.6 Antioxidant

1.6.1 Aspect and Process of Antioxidation

An antioxidant is a chemical or any substance or any enzyme that prevents or

reduces the oxidation or oxidative damage due to oxygen or other chemicals.

All living organisms contain complex systems of antioxidant enzymes and

chemicals. Antioxidants in biological systems have multiple roles and these

include deterring oxidative damage and participating in the major signaling

pathways of the cells. One major action of antioxidants in cells is to prevent

14

damage due to the action of reactive oxygen species (ROS) involved hydrogen

peroxide (H2O2), the superoxide anion (O2•−), and free radicals such as the

hydroxyl radical (•OH). These molecules are unstable and highly reactive, and

can damage cells by chemical chain reactions such as lipid peroxidation, or

formation of DNA adducts that could cause cancer-promoting mutations or cell

death (Ames et al., 1993; Finkel and Holbrook, 2000). Oxidative stress, induced

by oxygen radicals, is believed to be a primary factor in various degenerative

diseases, such as cancer (Muramatsu et al., 1995), atherosclerosis (Steinberg

et al., 1989), gastric ulcer (Das et al., 1997).

The classification of antioxidants is one of two ways, i.e. chain-breaking and

preventive. In the chain-breaking event, a free radical releases or steals an

electron leading to the formation of a second radical. This molecule in turn

follows the same path that leads to the formation of a third molecule. This

process repeats itself leading to the generation of more unstable products. The

process continues until the radical is stabilized by a chain-breaking antioxidant

or it simply decays into a harmless product. In preventive, antioxidant enzymes

like superoxide dismutase, catalase and glutathione peroxidase prevent

oxidation by reducing the rate of chain initiation (Parnes, 1998). In the works,

antioxidants reduce the free radical energy, stop the free radical from forming in

the first place, or interrupt an oxidizing chain reaction to minimize the damage

caused by free radicals (Ames et al., 1993).

1.6.2 Systems of Antioxidation

These systems can be divided into enzymatic and non enzymatic. The

enzymatic involved superoxide dismutase (SOD), which catalyses such as the

conversion of O2●⎯ to H2O2 and H2O, and then convert H2O2 to H2O and O2.

15

Meanwhile non enzymatic involved the lipid-soluble vitamin for example

vitamins E and A or provitamin A (β-carotene) (Fouad, 2007).

The example of enzymatic antioxidant is xanthine oxidase (XO), which is a very

important enzyme in the purine metabolism involved in the formation of uric acid

in the body, i.e. catalyzes the oxidation of hypoxanthine to xanthine and can

further catalyze the oxidation of xanthine to uric acid. XO is responsible for the

medical condition known as gout. Gout is caused by deposition of uric acid in

the joints leading to painful inflammation, with inhibition of XO leading to a

remission in gout.

The active site of XO is composed of a molybdopterin unit with the molybdenum

atom also coordinated by terminal oxygen (oxo) and sulfur atoms and a terminal

hydroxide. In the reaction with xanthine to form uric acid, an oxygen atom is

tranferred from molybdenum to xanthine. The reformation of the active

molybdenum center occurs by the addition of water. Like other known

molybdenum-containing oxidoreductases, the oxygen atom introduced to the

substrate by XO originates from water rather than from dioxygen (O2) (Chiang

et al., 1994; Hille, 2005; Harrison, 2002; Rastelli et al., 1997; Parnes, 2006).

Xanthinuria, hypouricemia, hypercalcinuria, and decreased bone density are the

diseases caused by insufficient function of xanthine oxidase. Similar symptoms

are increased xanthine excretion, decreased uric acid excretion, and mental

retardation. One proposed hypothesis says that drinking tea decreases the risk

of cancer because of the presence polyphenols which are know as inhibitors of

xanthine oxidase (Xu et al., 1994). Some dietary phenolic compounds might

function as natural biological response modifier (BRM) by protecting cells or

16

tissues against injuries especially those caused by lipid peroxidation and/or

enzyme mediated oxidation (Nakagami et al., 1995).

The followings are example of non enzymatic antioxidant:

a. Free Radicals

Free radicals are believed to play a role in different health conditions, including

the aging process, cancer, and atherosclerosis. Reducing exposure to free

radicals and increasing intake of antioxidant nutrient has the potential to reduce

the risk of free radical-related health problem.

In the free radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl

(DPPH) assay, the purple colored DPPH constitute the stable free radical,

which is reduced to 1,1- diphenyl-2-picrylhydrazine (yellow colored) by reacting

with an antioxidant The antioxidant donates hydrogen from the hydroxyl group

to free radical (DPPH) to inhibit the chain oxidation by the free radical. The

product is a stable molecule, which will not initiate or propagate further oxidation

of lipids (Sherwin, 1978; Blois, 1958).

b. β-Carotene

β-Carotene is a member of a class of substance called carotenoids is a vitamin

that acts as an antioxidant, protecting cells against oxidation damage. Some

studies have showed differences in the in vitro activities of the β-carotene

isomers. One study showed that 9-cis β-carotene that isolated from Dunaliella

bardawil has higher potency to protect methyl linoleate from oxidation than that

of the all-trans β-carotene isomer (Levin and Mokady, 1994). Another study

demonstrated that 9-cis β-carotene and all-trans β-carotene had equal

17

antioxidant activities when assessed by enhanced human neutrophil

chemiluminescence (Liu et al., 2000).

1.6.3 Nutritional Antioxidants

The following substances are example of nutritional antioxidant:

a. Vitamins: Vitamin A, C (ascorbic acid), E.

The example of food containing high levels of these antioxidants is fruits,

vegetables and vegetable oils. Vitamins are believed to play a role in preventing

the development of such chronic diseases as cancer, heart disease, stroke,

memory loss, rheumatoid arthritis, and cataracts (Parnes, 1998). Low dietary

intake of antioxidant vitamins and minerals increase the incidence rate of

cardiovascular disease and cancer (Hercberg et al., 2004)

b. Carotenoid terpenoids (α-carotene, β-carotene).

Carrot is the example of food containing carotenoids.

c. Flavonoid and polyphenolics.

Food containing of these antioxidant are tea, coffee, chocolate, fruits and

soy. Flavonoids have a variety of biological effects in numerous mammalian cell

systems, in vitro as well as in vivo. Recently much attention has been paid to

their antioxidant properties and to their inhibitory role in various stage of tumor

development in animal studies (Hollman et al., 1996; Miller, 1996).

In addition, Yu et al. (2006) reported that in a β-carotene-linoleate system,

crude protein showed antioxidant activity and Li et al. (2007) and Kishk et al.

(2007) reported that polysaccharides showed also inhibitory activity in β-

carotene-linoleate model system. Chuanguang et al. (2002) reported that

Misgurnus anguillicaudatus polysaccharides have ability to remove O2●⎯, HO●,

H2O2 and other oxygen active compounds. Polysaccharides, which are widely

18

distributed in animals, plants, and microorganisms, have been demonstrated to

play an important role as dietary free-radical scavenger for the prevention of

oxidative damage (Blander et al., 2003; Harman, 1993; Liu et al., 1997).

As we known, both, S. crispus and S. arvensis contain such as phenolic and

flavonoids, thereby, the purpose of this work is to evaluate the antioxidant

activity of the extracts on the oxidative potential.

1.7 Angiogenesis

1.7.1 Definition and Process of Angiogenesis

Angiogenesis can be defined as the process by which new blood vessel form

from pre-existing vessel, which is controlled by certain chemicals produced in

the body. The other chemicals stopped the process called angiogenesis

inhibitors. The angiogenesis process consists of the following steps, beginning

with activation of endothelial cells by growth factors, followed by enzymatic

degradation of basement membrane, detachment of endothelial cells from

adhesion proteins, endothelial cell migration into the perivascular spaces and

proliferation, and final new vessels formation. The process is regulated by

various growth factors and cytokines. Vascular endothelial growth factor

(VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor alpha

(TNF-a) and interleukin-8 (IL-8) are the potent angiogenic growth factors

(Brooks et al., 1999; Huang and Zheng, 2006; Mochizuki et al., 2007).

Angiogenesis plays an important role in the growth and metastasis of tumor and

several chronic inflammatory diseases including rheumatoid arthritis and

proliferative diabetic retinopathy. Meanwhile many ischemic diseases for

examples ischemic coronary artery disease, critical limb ischemia and brain

19

infarction may benefit from the induction of angiogenesis. Inhibition of

angiogenesis has been recognized as a promising therapeutic approach for the

control of tumor or cancer growth and metastasis and chronic inflammatory

diseases. Tumor or chronic inflammatory diseases cannot grow or spread

without the formation of the new blood vessels, the oxygen and nutrients be

brought into cells via blood vessels, allowing the cells to grow, invade nearby

tissue, spread to other part of the body, and form new cells colonies (Huang

and Zheng, 2006, Sheeja et al., 2007, Sylvia et al., 2003, Tsuneki et al., 2005).

1.7.2 The Relationship between Antioxidants and Antiangiogenic Agents

A number of antiangiogenesis compounds have been recognized and many

have antioxidative properties. Matsubara et al. (2005) reported that nasunin; an

antioxidant anthocyanin isolated from eggplant peels was demonstrated as an

angiogenesis inhibitor. They also implied that nasunin may also be useful to

prevent angiogenesis related diseases. Huang and Zheng (2006) reported that

rosmarinic acid inhibited angiogenesis of human umbilical vein endothelial cells.

Rosmarinic acid, a water soluble polyphenolic compound which is isolated from

medicinal plants has been reported to have biological activities such as anti-

oxidative, anti-inflammatory and anti-depressive activities.

Several flavonoids that are more widely distributed in the plant kingdom,

including 3-hydroxyflavone, 3′,4′-dihydroxyflavone, 2′,3′-dihydroxyflavone,

fisetin, apigenin and luteolin have ability to inhibit the in vitro angiogenesis

process (Fotsis et al., 1997 cited in Mukherjee et al., 1999; Engelmann et al.,

2002). Mukherjee et al. (1999) reported other flavonoids including genistein and

daidzein, an isoflavone have ability in inhibiting of angiogenesis process.

20

Meanwhile Kim et al. (2006) reported flavonol of myricetin, quercetin,

kaempferol and galangin can also inhibit angiogenesis process. Previously, Tan

et al. (2003) reported that quercetin which is found in many fruits and

vegetables, as well as olive oil, red wine, and tea, possesses antiangiogenic

potential. Various pharmacological activities of quercetin have been

demonstrated including antioxidation by scavenging free radicals, prevention of

atherosclerosis, and chronic inflammation. Some of the earlier antiangiogenic

compounds identified were steroids, including progestin, medroxyprogesterone

acetate (MPA), the glucocorticoids, dexamethasone and cortisone (Williams et

al., 1999). The other antiangiogenesis compound is squalamine, a natural

amino sterol purified and characterized from tissues of the dogfish shark

(Williams et al., 1999).

Antiangiogenic activity of the herb extracts was recently reported by Song et al.

(2003). In this study it was reported that Phellinus linteus extract showed strong

antiangiogenic and antioxidant activity. The researchers suggested that

antioxidant and anti-angiogenic activities of Phellinus linteus would be partly

responsible for its anti-tumor effect. In vitro assay using human endothelial cells

of edible berry extracts showed that the extracts impaired angiogenesis (Bagchi

et al., 2004). Berries are rich in anthocyanins, compounds that provide

pigmentation to fruits and serve as natural antioxidants. Anthocyanins also

serve as anti-inflammatory, anti-mutagenic agents and natural antioxidant.

Extracts of Gastrodia elata rhizome demonstrated potent anti-angiogenic

activity in the CAM assay (Ahn et al., 2007). Rhizome of Gastrodia elata Blume

is a traditional herbal medicine in Oriental countries. Several phenolic

21

compounds, such as 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-

hydroxy-3-methoxybenzaldehyde and gastrodin [4-(β-D-glucopyranosyl)benzyl

alcohol] have been identified from this plant.

Chemical structures of a variety of phytochemicals exhibiting antiangiogenic

activity are presented in Fig. 1.3.

O

O

O

O H

HH

medroxyprogesterone acetate

O

HO

FO

OH

HO

dexamethasone

O

O

OH

O

H

H

H

OH

cortisone

OSO3H

NH H OHN

HH2N

squalamine

O OHHO

HO

OOH

COOH

H

rosmarinic acid

OO

HO O+

OH

OH

OH

O

CH2OH

H OH

HOHH

HHO

O

H2C O

O

OH HOHH

HHOH

H

OH OH

HH

O

H

HH3C

C

O

CH

CH

OH

nasunin

O

O

OH

3-hydroxyflavone

O

O

OHOH

3′,4′-dihydroxyflavone O

O

OHHO

2′,3′-dihydroxyflavone

O

OH

HO

O

OHOH

fisetin

Fig. 1.3 Chemical structures of a variety of phytochemicals exhibiting antiangiogenic activity

22

OOH

HO O

OH

apigenin OOH

HO O

OHOH

luteolin

OOH

HO O

OH genistein

O

HO O

OH daidzein OH

OOH

HO O

OHOH

OH

myricetin

OHOOH

HO O

OHOH

quercetin

OHOOH

HO O

OH

kaempferol

OHOOH

HO O

galangin

Fig. 1.3 (continued).

1.8 Drug Stability

1.8.1 General Concept in Drug Stability

“The purpose of stability tests is to provide evidences on how the quality of a

drug substance or drug product varies with time under the influence of a variety

of environmental factors such as temperature, humidity, and light, as well as to

establish a re-test period for the drug substance or a shelf life for the drug

product and recommended storage conditions” (ICH, 2003).

“Stability is one of the most important factors, which determine whether a

compound or mixture of compounds can be developed into a therapeutically

useful pharmaceutical product. The recognition of this concept, along with ability

23

to optimize drug stability and product shelf life has been among the most

significant achievements in drug research and development. The stability of a

pharmaceutical preparation may be defined as its degree of resistance to

chemical and physical changes. The efficacy of the preparation must remain

constant (or change only within the limits specified by legal provision) until the

date of expiration” (Racz, 1989).

Since the herbal drug or herbal drug preparation in its entirety is regarded as

the active substance, a mere determination of the stability of the constituents

with known therapeutic activity will not suffice. It must also be shown, as far as

possible e.g. by means of appropriate fingerprint chromatogram, that other

substances present in the herbal drug or in the herbal drug preparation are

likewise stable and that their proportional content remains constant. If herbal

medicinal product contains several herbal drugs or preparation of several herbal

drugs and if it is not possible to determine the stability of each active substance,

the stability of the medicinal product should be determined by appropriate

fingerprint chromatograms, appropriate overall methods of assay and physical

and sensory tests or other appropriate tests.

The variation in content during the proposed shelf-life of herbal medicinal

product containing a herbal drug or herbal drug preparation whose constituents

of known therapeutic activity should not more than 5% of the initial assay value

whilst those whose constituents of unknown therapeutic activity should not

exceed 10 % of the initial assay value (CPMP, 2001).

24

1.8.2 Guideline for Stability Testing of Drug Substance and Drug Product

Stress tests of the drug substance can help to identify the likely degradation

products, which in turn can help to establish the degradation pathway and the

intrinsic stability of the molecule and validate the stability indicating power of the

analytical procedure used. The nature of the stress tests will depend on the

individual drug substance and the type of drug product involved. Stress tests

are likely to be carried out on a single batch of the drug substance. It should

include the effect of temperature in 10 0C increment above that for accelerated

testing (e.g. 50°C, 60°C etc.) and humidity at 75 % or greater. The design of the

formal stability studies for the product should be based on knowledge of the

behavior and properties of the drug substance and from stability studies on the

drug substance and on experience gained from clinical formulation studies. The

likely changes on storage and the rationale for the selection of attributes to be

tested in the formal stability studies should be stated. For long term studies,

frequency of testing should be sufficient to establish the stability profile of the

drug substance or drug product. Either for drug substances with a proposed re-

test period or for drug product with a proposed shelf life of at least 12 months,

the frequency of testing at the long term storage condition should normally be

every 3 months over the first year, every 6 months over the second year, and

annually thereafter through the proposed re-test period for the drug substance

or the proposed shelf life for the drug product. At the accelerated storage

condition, a minimum of three time points, including the initial and final time

points (e.g., 0, 3, and 6 months), from a 6-month study is recommended. In

general, a drug substance should be evaluated under storage conditions (with

appropriate tolerances) that test its thermal stability and, if applicable, its

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