ANALYTICAL,_BIOACTIVITY_AND_STABILITY_STUDIES_2

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, parents, Itam St. St. Pamenan and Tinun Tinun My wife, Hartati My children, children, Indah Permata, Permata, Ridho Sahary Adha and Nadya Chantika

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.

ii

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

iii

13

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

19

Agents 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

24

Product 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

28

Reactions Kinetic 1.8.6 Determination of the Reaction Order and Calculation of the

28

Rate Constants 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

31

STROBILANTHES

CRISPUS 

AND

SONCHUS 

ARVENSIS EXTRACTS

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

36

Maceration Method

iv

2.2.5 Analysis by Gas Chromatography/Time of Flight Mass

37

Spectrometry (GC/TOF-MS) 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 H-NMR and C-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

41

Maceration Method 2.3.2 Analysis by Gas Chromatography/Time of Flight Mass

42

Spectrometry (GC/TOF-MS) 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.

62

arvensis  2.4 Conclusion

63

CHAPTER 3 CRYSTAL GROWTH INHIBITION AND ANTIOXIDANT

64

ACTIVITY STUDIES ON STROBILANTHES CRISPUS  AND SONCHUS ARVENSIS EXTRACTS 3.1 Introduction

64

3.2 Materials and Methods

67

3.2.1 Chemicals

67

v

3.2.2 Instruments

68

3.2.3 Determination of Inhibition on Calcium Oxalate Cr ystal

68

Growth 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

71

Model System 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 Cr ystal

75

Growth 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

95

System 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

vi

CHAPTER 4 STABILITY STUDIES ON

STROBILANTHES CRISPUS 

105

AND SONCHUS ARVENSIS EXTRACTS 4.1 Introduction

105

4.2 Materials and Methods

106

4.2.1 Chemicals

106

4.2.2 Instruments

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 Results and Discussion

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

131

reaction for marker compounds 4.3.5.1 Kinetic of Stigmasterol and Lupeol Degradation

132

4.3.5.2 Determination of the Order of the Degradation

135

Reaction

vii

4.3.5.3 Determination of the Rate Constant for Marker 

138

Compound Degradation 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 

170

CRISPUS AND SONCHUS ARVENSIS EXTRACTS

5.1 Introduction

170

5.2 Materials and Methods

172

5.2.1 Chemicals

172

5.2.2 Instruments

172

5.2.3 Rat Aorta Assay

172

5.3 Results and Discussion

173

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

viii

LIST OF TABLES Page Table 1.1

Plants used for treatment of kidney stone and related

13

diseases Table 1.2

Storage conditions of the stability studies of drug

25

substance and drug product Table 2.1

Comparison

of

compounds

having

1%

or

greater 

43

percentage in one of the leaf  S. crispus  extracts detected by GC/TOF-MS Table 2.2

Comparison

of

compounds

having

1%

or

greater 

46

percentage in one of the leaf  S. arvensis  extracts detected by GC/TOF-MS Table 2.3

Selected compounds having the highest percentage in S.

53

crispus extracts Table 2.4

Selected compounds having the highest percentage in S.

54

arvensis extracts Table 2.5

Comparison of the chemical shift of the isolated compound

58

and tridecane, CH3(CH2)11CH3 (Breitmaier, 1979) Table 3.1

The ability to inhibit the calcium oxalate crystal growth of 

78

the water extracts compared to that of 25 mM sodium citrate Table 3.2

Crystal numbers of blank, positive control, and sample at

83

various incubation times 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

reference

91

Polysaccharide contents in S. crispus  and S. arvensis 

100

concentration

50%

(EC50)

of

compounds Table 3.6

water extracts

ix

Table 3.7

Protein and nitrogen compounds content in S. crispus  and

102

S. arvensis water extracts Table 4.1

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

107

arvensis  extracts with controlled relative humidity (RH) using saturated salt solution Table 4.2

Calibration data of marker compounds using Agilent HPLC

124

system Table 4.3

Analytical precision of stigmasterol and lupeol as marker 

124

and in samples Table 4.4

Recovery test for marker in S crispus  and S. arvensis 

125

extract Table 4.5

Activation Energy (Ea) of stigmasterol and lupeol in extracts, respectively

x

142

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

21

exhibiting antiangiogenic activity Fig. 2.1

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

38

arvensis leaves Fig. 2.2

Comparing percentages of total extracts from S. crispus 

42

and S. arvensis leaves using different macerating solvents 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 

57

and S. arvensis  leaves with n-hexane, chloroform and methanol solvents, using continuous soxhlet extractor. 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

77

sodium citrate on calcium oxalate crystal growth at various incubation times Fig. 3.3

Comparing inhibition indices of  S. arvensis  extracts and

77

sodium citrate on calcium oxalate crystal growth at various incubation times 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)

xi

79

Fig. 3.5

The histogram of the crystal size distribution of calcium

80

oxalate in blank, control of sodium citrate and sample of  water extract from S. arvensis (Ew-Sa) Fig. 3.6

The growth profile of calcium oxalate crystal for blank,

82

positive control and S. crispus  extract (Ew-Sc) at 2, 4, 8, and 24 hours of incubation time Fig. 3.7

The growth profile of calcium oxalate crystal for blank,

82

positive control and S. arvensis  extract (Ew-Sa) at 2, 4, 8, and 24 hours of incubation time 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

86

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

crispus (violet) Fig. 3.10

Comparing FRSA of S. crispus extracts using DPPH

89

method Fig. 3.11

Comparing FRSA of S. arvensis extracts using DPPH

90

method Fig. 3.12

Comparing FRSA of reference compounds using DPPH

90

method Fig. 3.13

Comparing xanthine oxidase inhibitory activities of 100 ppm

94

S. crispus extracts to xanthine substrate Fig. 3.14

Comparing xanthine oxidase inhibitory activities of 100 ppm

94

S. arvensis extracts to xanthine substrate Fig. 3.15

Comparing antioxidant activities of  S. crispus  extracts and

97

quercetin, BHA, and BHT as reference compounds using βcarotene-linoleic acid method Fig. 3.16

Comparing antioxidant activities of S. arvensis  extracts and quercetin, BHA, and BHT as reference compounds using βcarotene-linoleic acid method

xii

97

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)

112

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). Fig. 4.2

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

113

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) Fig. 4.3

Peak intensities at 415 nm of UV spectra of acetone

114

extracts (Eac) from S. crispus at various storage temperatures and periods Fig. 4.4

Peak intensities at 415 nm of UV spectra of methanol extracts

(Em)

from

S.

crispus  at

various

114

storage

temperatures and periods Fig. 4.5

Peak intensities at 415 nm of UV spectra of acetone

114

extracts (Eac) from S. arvensis at various storage temperatures and periods Fig. 4.6

Peak intensities at 415 nm of UV spectra of methanol extracts

(Em)

from

S.

arvensis  at

various

115

storage

temperatures and time periods 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

xiii

115

Fig. 4.8

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

117

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) Fig. 4.9

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

118

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) Fig. 4.10

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

120

extracts from S. crispus ; methanol, Em (a), acetone, Eac (b), and water, Ew (c) before storage Fig. 4.11

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

120

extracts from S. arvensis ; methanol, Em (a); acetone, Eac (b), and water, Ew (c) before storage Fig. 4.12

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

121

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) Fig. 4.13

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

122

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) Fig. 4.14

HPLC chromatograms of acetone extracts (Eac) from S.

126

crispus  stored 0 (a), 1 month (b) and 2 months (c) at 25 0

C/65% RH

Fig. 4.15

HPLC chromatograms of methanol extracts (Em) from S.

crispus  stored 0 (a), 1 month (b) and 2 months (c) at 25 0

C/65% RH

xiv

127

Fig. 4.16

HPLC chromatograms of water extracts (Ew) from S.

128

crispus  stored 0 (a), 1 month (b) and 2 months (c) at 25 0

Fig. 4.17

C/65% RH

HPLC chromatograms of acetone extracts (Eac) from S.

129

arvensis  stored 0 (a), 1 month (b) and 2 months (c) at 25 0

C/65% RH

Fig. 4.18

HPLC chromatograms of methanol extracts (Em) from S.

130

arvensis  stored 0 (a), 1 month (b) and 2 months (c) at 25 0

C/65% RH

Fig. 4.19

HPLC chromatograms of water extracts (Ew) from S.

131

arvensis  stored 0 (a), 1 month (b) and 2 months (c) at 25 0

C/65% RH

Fig. 4.20

Comparing

percentage

of

remaining

stigmasterol

133

concentration in acetone extracts (Eac) from S. crispus  at various storage temperatures and periods Fig. 4.21

Comparing

percentage

of

remaining

stigmasterol

133

concentration in methanol extracts (Em) from S. crispus  at various storage temperatures and periods Fig. 4.22

Comparing percentage of remaining lupeol concentration in

134

acetone extracts (Eac) from S. arvensis  at various storage temperatures and periods Fig. 4.23

Comparing percentage of remaining lupeol concentration in

134

methanol extracts (Em) from S. arvensis at various storage temperatures and periods Fig. 4.24

Decrease of stigmasterol percentage in methanol and

135

acetone extracts from S. crispus  stored for 6 months at various temperatures Fig. 4.25

Decrease of lupeol percentage in methanol and acetone extracts from S. arvensis  stored for 6 months at various temperatures

xv

135

Fig. 4.26

Plot of ln C of remaining stigmasterol in acetone extracts

136

(Eac) from S. crispus  against time at various storage conditions Fig. 4.27

Plot of ln C of remaining stigmasterol in methanol extracts

137

(Em) from S. crispus  against time at various storage conditions Fig. 4.28

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

137

from S. arvensis against time at various storage conditions Fig. 4.29

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

138

from S. arvensis against time at various storage conditions Fig. 4.30

Comparing degradation rate constant of stigmasterol in S.

139

crispus extracts at various storage conditions Fig. 4.31

Comparing degradation rate constant of lupeol in S.

140

arvensis extracts at various storage conditions Fig. 4.32

Arrhenius plot for stigmasterol in acetone (a) and methanol

141

extracts (b) from S. crispus  Fig. 4.33

Arrhenius plot for lupeol in acetone (a) and methanol

141

extracts (b) from S. arvensis  Fig. 4.34

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

143

extracts at various storage conditions Fig. 4.35

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

144

at various storage conditions Fig. 4.36

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

144

room temperature (250C/60%RH) Fig. 4.37

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

144

room temperature (250C/60%RH) 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)

xvi

149

Fig. 4.39

PCA of acetone extracts (Eac) from S. crispus stored at 60 0

149

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

region 2000-900 cm-1 (PC2 vs PC1) Fig. 4.40

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

150

room temperature (25 0C/65% RH) for 0-6 months storage period in the spectral region 2000-900 cm-1 (PC2 vs PC1) Fig. 4.41

PCA of methanol extracts (Em) from S. crispus stored at 60

150

0

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

region 2000-900 cm-1 (PC2 vs PC1) Fig. 4.42

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

151

temperature (25 0C/65% RH) for 0-6 months storage period in the spectral region 2000-850 cm-1 (PC3 vs PC1) Fig. 4.43

PCA of water extracts (Ew) from S. crispus  stored at 60

151

0

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

region 2000-850 cm-1 (PC3 vs PC1) Fig. 4.44

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

152

room temperature (25 0C/65% RH) for 0-6 months storage period in the spectral region 1900-800 cm-1 (PC2 vs PC1) Fig. 4.45

PCA of acetone extracts (Eac) from S. arvensis stored at 60

152

0

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

region 1800-1100 cm-1 (PC3 vs PC1) Fig. 4.46

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

153

room temperature (25 0C/65% RH) for 0-6 months storage period in the spectral region 1800-1100 cm-1 (PC2 vs PC1) Fig. 4.47

PCA of methanol extracts (Em) from S. arvensis  stored at 60

153

0

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

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

xvii

154

Fig. 4.49

PCA of water extracts (Ew) from S. arvensis  stored at 60

154

0

C/85% RH for 0-6 month storage period in the spectral

region 1800-1200 cm-1 (PC2 vs PC1) Fig. 4.50

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

155

various temperatures for 1 month storage period (S 1-oC) in the spectral region 2000-1200 cm-1 (PC2 vs PC1) Fig. 4.51

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

155

various temperatures for 1 month storage period (S1-oC) in the spectral region 2000-1100 cm-1 (PC2 vs PC1) Fig. 4.52

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

156

various temperatures for 1 month storage period (S1-oC) in the spectral region 2000-800 cm-1 (PC2 vs PC1) Fig. 4.53

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

156

various temperatures for 1 month storage period (S 1-oC) in the spectral region 1900-1200 cm-1 (PC2 vs PC1) Fig. 4.54

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

157

various temperatures for 1 month storage period (S 1-oC) in the spectral region 2000-1000 cm-1 (PC2 vs PC1) Fig. 4.55

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

157

various temperatures for 1 month storage period (S 1-oC) in the spectral region 1800-800 cm-1 (PC2 vs PC1) Fig. 4.56

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

159

extracts (Eac) from S. crispus  stored at room temperature (25°C/65% RH) for 0-6 month storage period Fig. 4.57

3D plot of FT-IR spectra (4000–400 cm- ) of acetone

159

extracts (Eac) from S. crispus  stored at 60°C/85% RH for  0-6 month storage period Fig. 4.58

3D plot of FT-IR spectra (4000–400 cm- ) of methanol extracts (Em) from S. crispus  stored at room temperature (25°C/65% RH) for 0-6 months storage period

xviii

160

Fig. 4.59

3D plot of FT-IR spectra (4000–400 cm- ) of methanol

160

extracts (Em) from S. crispus stored at 60°C/85% RH for 06 months storage period Fig. 4.60

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

from

S.

crispus  stored

at

room

161

temperature

(25°C/65% RH) for 0-6 months storage period Fig. 4.61

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

161

(Ew) from S. crispus  stored at 60°C/85% RH for 0-6 months storage period Fig. 4.62

3D plot of FT-IR spectra (4000–400 cm- ) of acetone

162

extracts (Eac) from S. arvensis  stored at room temperature (25°C/65% RH) for 0-6 months storage period Fig. 4.63

3D plot of FT-IR spectra (4000–400 cm- ) of acetone

162

extracts (Eac) from S. arvensis  stored at 60°C/85% RH for  0-6 months storage period Fig. 4.64

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

163

extracts (Em) from S. arvensis  stored at room temperature (25°C/65% RH) for 0-6 months storage period Fig. 4.65

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

163

extracts (Em) from S. arvensis  stored at 60°C/85% RH for  0-6 months storage period Fig. 4.66

3D plot of FT-IR spectra (4000–400 cm- ) of water extracts (Ew)

164

from S. arvensis  stored at room temperature

(25°C/65% RH) for 0-6 months storage period Fig. 4.67

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

164

(Ew) from S. arvensis  stored at 60°C/85% RH for 0-6 months storage period Fig. 4.68

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

(Eac)

from

S.

crispus  stored

xix

at

various

165

temperatures for 1 month storage period Fig. 4.69

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

(Em)

from

S.

crispus  stored

at

165

various

temperatures for 1 month storage period Fig. 4.70

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

166

(Ew) from S. crispus  stored at various temperatures for 1 month storage period Fig. 4.71

3D plot of FT-IR spectra (4000–400 cm- ) of acetone extracts

(Eac)

from

S.

arvensis  stored

at

166

various

temperatures for 1 month storage period Fig. 4.72

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

(Em)

from

S.

arvensis  stored

at

167

various

temperatures for 1 month storage period Fig. 4.73

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

167

(Ew) from S. arvensis  stored at various temperatures for 1 month storage period Fig. 5.1

Inhibition percentages of S. crispus and S. arvensis extracts

175

on angiogenic using rat aorta ring assay. Fig. 5.2

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

176

from S. crispus as angiogenesis inhibitor (b) Fig. 5.3

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

177

from S. arvensis as angiogenesis inhibitor (b) Fig. 5.4

Images of rat aorta and control (a) and betulinic acid (b)

xx

178

LIST OF PLATES Page Plate 1.1

Picture of S . crispus plant

4

Plate 1.2

Picture of S . arvensis plant

8

xxi

LIST OF APPENDICES Page Appendix 2.1

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

200

70% acetone, E7ac (b), methanol, Em (c), water, Ew (d), and n-hexane, Eh (e) extracts of S. crispus L leaves Appendix 2.2

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

201

70% acetone, E7ac (b), methanol, Em (c), water, Ew (d), and n-hexane, Eh (e) extracts of S. arvensis L leaves Appendix 2.3

Spectroscopic data of isolated tritriacontane

202

Appendix 2.4

HPLC chromatograms of standard stigmasterol (a) and

204

isolated stigmasterol (b) Appendix 2.5

TLC profiles of the standard stigmasterol (1) and isolated

204

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 Appendix 2.6

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

205

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 Appendix 2.7

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

205

and isolated stigmasterol [blue (2)] Appendix 2.8

Ultraviolet spectra of the standard stigmasterol [green

206

(1) and blue (2)] and isolated stigmasterol [red (3) and black (4)] 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

207

isolated lupeol (b)

xxii

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

208

lupeol (2) using CHCl3 [a, Rf = 0.24] and CHCl3: MeOH (95: 5) [b, Rf = 0.89] as mobile phase, respectively under  365 nm Appendix 2.13 3D-HPTLC profiles of the standard lupeol (blue) and

208

isolated lupeol (red) using CHCl3 [a] and CHCl3: MeOH (95: 5) [b] as mobile phase, respectively under 365 nm Appendix 2.14 FT-IR spectra of the standard lupeol [blue (1)] and

209

isolated lupeol [black (2) and green (3)] Appendix 2.15 Ultraviolet spectra of the standard lupeol [(brown (2) and

209

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

210

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

211

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

212

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

212

green (3)] and isolated quercetin [blue (1) and black (4)] Appendix 2.22 FT-IR spectra of the standard quercetin [blue (1)] and isolated quercetin [black (2)]

xxiii

213

Appendix 3.1

Statistical analyses of Inhibition Index of  S. crispus  to

214

growth of calcium oxalate crystal Appendix 3.2

Statistical analyses of Inhibition Index of  S. arvensis  to

218

growth of calcium oxalate crystal Appendix 3.3

Statistical analyses of free radical scavenging activity of 

222

S. crispus  Appendix 3.4

Statistical analyses of free radical scavenging activity of  226

S. arvensis  Appendix 3.5

Statistical analyses of xanthine oxidase inhibitory activity

232

of 100 ppm S. arvensis extracts to xanthine substrate Appendix 3.6

Statistical analyses of antioxidant activity of extracts and

234

references using β-carotene linoleic acid method 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

237

contents in S. crispus and S. arvensis with their FRSA to DPPH Appendix 3.10 Standard calibration curve of glucose

238

Appendix 3.11 Standard calibration curve of protein

239

Appendix 4.1

240

Standard calibration curve of marker compounds

xxiv

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-

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

xxv

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 αsitosterol 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.

xxvi

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 penyakitpenyakit yang berkaitan dengan angiogenik.

xxvii

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 campesterol, phytol and stigmasterol, whereas lupeol, phytol and

-sitosterol,

α

-sitosterol

α

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 (R 2) between their  phenolic content and FRSA were 0.93 ( S . arvensis ) and 0.40 (S crispus ). In the

xxviii

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 t 90 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.

xxix

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%

1

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

2

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.

3

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

4

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, pcoumaric, 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;

5

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.

O

OH

HOH2C HO

O O

O

OH

O

O

OH HO

O

H3C

HO

OH

p-hydroxy benzoic acid

HO HO

OH

verbascoside HO

HO

HO

O

OH

OH

OH H3CO

HO

p-coumaric acid

O

O

ferulic acid

caffeic acid OH

OCH3

HO

O

O

HO OH

OCH3

OH

vanillic acid

OH

O OCH3

OH

gentisic acid

syringic acid

C2H5

HO

β-sitosterol

HO

stigmasterol

Fig. 1.1 Chemical structures of S . crispus constituents

6

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.

7

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.

8

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

al.,

et

1974),

luteolin-7-O-glucoside,

linarin

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

OH

HO

O

OH

OH

OH HO

OH

OH

O

OH

O

O

O

OH OH

OH

O

luteolin-7-O-glucoside

quercetin

luteolin

O

HO

OH OH

O

HO

OCH3

OH OH O

O

HO

O

HO

O

H3C

OH O

O

O

O HO

HO

OH

O

OH

OH OH

O

OH

isocinaroside

linarin

OH

OH

OCH3

OH

HO HO

O

HO

O

HO

O

OH

OH OH

O

O

OH

O

sonchoside

OH

O

apigenin

Fig. 1.2 Chemical structures of S. arvensis constituents

9

OH

O

acacetin

OH HO

O

H

H

OH OH

H

O HO

kaempferol

HO

taraxasterol

lupeol

H

HO

HO

α-amyrin

β-amyrin OH

HO

HO

OH

OH HO

OCH3 HO

HO

O

OH

HO HO

OH OH

O

HO

OH

inositol

OH

mannitol

isorhamnetin

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 10

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 

11

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.

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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-3glucoside 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-Otriglycoside etc.

Otsuki et al., 2002 ;

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

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

4

Phylanthus  niruri 

5

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

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

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

13

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.

14

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 t erminal 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 

15

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

16

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 βcarotene-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

17

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

18

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 antioxidative, 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.

19

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

20

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

OH

O OH

H H O

HO

O

HO

O

O

O

H

F

OH

H

O

H

medroxyprogesterone acetate

H

O O

cortisone

dexamethasone

OH

OSO3H

O

COOH

HO O

OH H

HO

H2N

N H

N H

H

rosmarinic acid

OH

squalamine OH OH H HO

O

CH2OH H

O

O

O OH H O H3C H H H2C O H H O OH OH H HO

O

OH H HO

H

H H

O

O

+

C

C H

C H

OH

OH O

3-hydroxyflavone

OH OH H

OH

nasunin OH

OH OH O

OH OH

HO

HO

O

O OH

O

3′ ,4′ -dihydroxyflavone

O

O

2′ ,3′ -dihydroxyflavone

fisetin

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

21

OH

OH

HO

O

OH

HO

O HO

O

OH OH

O

OH

apigenin HO

O

OH

genistein

O

luteolin

O

OH

OH OH

OH HO

O

HO

O

OH

daidzein

O

OH OH

OH OH

OH

O

O

quercetin

myricetin OH HO

HO

O

O

OH OH

OH

O

OH

kaempferol

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

22

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).

23