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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 Materials and Methods 67
3.2.1 Chemicals 67
vi
3.2.2 Instruments 68
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 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
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 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
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
temperatures and periods
114
Fig. 4.5 Peak intensities at 415 nm of UV spectra of acetone
extracts (Eac) from S. arvensis at various storage
temperatures and periods
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.
crispus stored 0 (a), 1 month (b) and 2 months (c) at 25 0C/65% RH
127
xv
Fig. 4.16 HPLC chromatograms of water extracts (Ew) from S.
crispus stored 0 (a), 1 month (b) and 2 months (c) at 25 0C/65% RH
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.
arvensis stored 0 (a), 1 month (b) and 2 months (c) at 25 0C/65% RH
130
Fig. 4.19 HPLC chromatograms of water extracts (Ew) from S.
arvensis stored 0 (a), 1 month (b) and 2 months (c) at 25 0C/65% RH
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
temperatures and periods
134
Fig. 4.23 Comparing percentage of remaining lupeol concentration in
methanol extracts (Em) from S. arvensis at various storage
temperatures and periods
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
room temperature (25 0C/65% RH) for 0-6 months storage
period in the spectral region 2000-900 cm-1 (PC2 vs PC1)
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
region 2000-900 cm-1 (PC2 vs PC1)
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
region 2000-850 cm-1 (PC3 vs PC1)
151
Fig. 4.44 PCA of acetone extracts (Eac) from S. arvensis stored at
room temperature (25 0C/65% RH) for 0-6 months storage
period in the spectral region 1900-800 cm-1 (PC2 vs PC1)
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
region 1800-1100 cm-1 (PC3 vs PC1)
152
Fig. 4.46 PCA of methanol extracts (Em) from S. arvensis stored at
room temperature (25 0C/65% RH) for 0-6 months storage
period in the spectral region 1800-1100 cm-1 (PC2 vs PC1)
153
Fig. 4.47 PCA of methanol extracts (Em) from S. arvensis stored at
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
region 1800-1200 cm-1 (PC2 vs PC1)
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
various temperatures for 1 month storage period (S1-oC) in
the spectral region 2000-1100 cm-1 (PC2 vs PC1)
155
Fig. 4.52 PCA of water extracts (Ew) from S. crispus stored at
various temperatures for 1 month storage period (S1-oC) in
the spectral region 2000-800 cm-1 (PC2 vs PC1)
156
Fig. 4.53 PCA of acetone extracts (Eac) from S. arvensis stored at
various temperatures for 1 month storage period (S1-oC) in
the spectral region 1900-1200 cm-1 (PC2 vs PC1)
156
Fig. 4.54 PCA of methanol extracts (Em) from S. arvensis stored at
various temperatures for 1 month storage period (S1-oC) in
the spectral region 2000-1000 cm-1 (PC2 vs PC1)
157
Fig. 4.55 PCA of water extracts (Ew) from S. arvensis stored at
various temperatures for 1 month storage period (S1-oC) in
the spectral region 1800-800 cm-1 (PC2 vs PC1)
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
Fig. 4.57 3D plot of FT-IR spectra (4000–400 cm-1) of acetone
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
Fig. 4.59 3D plot of FT-IR spectra (4000–400 cm-1) of methanol
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
(25°C/65% RH) for 0-6 months storage period
161
Fig. 4.61 3D plot of FT-IR spectra (4000–400 cm-1) of water extracts
(Ew) from S. crispus stored at 60°C/85% RH for 0-6
months storage period
161
Fig. 4.62 3D plot of FT-IR spectra (4000–400 cm-1) of acetone
extracts (Eac) from S. arvensis stored at room temperature
(25°C/65% RH) for 0-6 months storage period
162
Fig. 4.63 3D plot of FT-IR spectra (4000–400 cm-1) of acetone
extracts (Eac) from S. arvensis stored at 60°C/85% RH for
0-6 months storage period
162
Fig. 4.64 3D plot of FT-IR spectra (4000–400 cm-1) of methanol
extracts (Em) from S. arvensis stored at room temperature
(25°C/65% RH) for 0-6 months storage period
163
Fig. 4.65 3D plot of FT-IR spectra (4000–400 cm-1) of methanol
extracts (Em) from S. arvensis stored at 60°C/85% RH for
0-6 months storage period
163
Fig. 4.66 3D plot of FT-IR spectra (4000–400 cm-1) of water extracts
(Ew) from S. arvensis stored at room temperature
(25°C/65% RH) for 0-6 months storage period
164
Fig. 4.67 3D plot of FT-IR spectra (4000–400 cm-1) of water extracts
(Ew) from S. arvensis stored at 60°C/85% RH for 0-6
months storage period
164
Fig. 4.68 3D plot of FT-IR spectra (4000–400 cm-1) of acetone
extracts (Eac) from S. crispus stored at various
165
xx
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 various
temperatures for 1 month storage period
165
Fig. 4.70 3D plot of FT-IR spectra (4000–400 cm-1) of water extracts
(Ew) from S. crispus stored at various temperatures for 1
month storage period
166
Fig. 4.71 3D plot of FT-IR spectra (4000–400 cm-1) of acetone
extracts (Eac) from S. arvensis stored at various
temperatures for 1 month storage period
166
Fig. 4.72 3D plot of FT-IR spectra (4000–400 cm-1) of methanol
extracts (Em) from S. arvensis stored at various
temperatures for 1 month storage period
167
Fig. 4.73 3D plot of FT-IR spectra (4000–400 cm-1) of water extracts
(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