IDENTIFY, ACTIVITY AND STABILITY STUDIES OF by rrn51447

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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
                            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               13



                                        iii
    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 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           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 Crystal         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 Crystal         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   concentration        50%     (EC50)    of    reference       91
            compounds

Table 3.6   Polysaccharide contents in S. crispus and S. arvensis                   100
            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            142
            extracts, respectively




                                         x
                                 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       79
           oxalate in blank, control of sodium citrate and sample of
           water extract from S. crispus (Ew-Sc)




                                         xi
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 β-
            carotene-linoleic acid method

Fig. 3.16   Comparing antioxidant activities of S. arvensis extracts and    97
            quercetin, BHA, and BHT as reference compounds using β-
            carotene-linoleic acid method




                                        xii
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               114
            extracts   (Em)   from   S.     crispus   at   various   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               115
            extracts (Em) from S. arvensis at various storage
            temperatures and time periods

Fig. 4.7    Peak intensities at 330 nm of UV spectra of water extracts         115
            (Ew) from S. arvensis at various storage temperatures and
            time periods




                                          xiii
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.         127
            crispus stored 0 (a), 1 month (b) and 2 months (c) at 25
            0
                C/65% RH




                                        xiv
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
                C/65% RH
Fig. 4.17   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         135
            extracts from S. arvensis stored for 6 months at various
            temperatures




                                        xv
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
                                   0
            room temperature (25 C/60%RH)

Fig. 4.38   PCA of acetone extracts (Eac) from S. crispus stored at            149
            room temperature (25 0C 65% RH) for 0-6 months storage
            period in the spectral region 2000-900 cm-1 (PC2 vs PC1)




                                           xvi
Fig. 4.39   PCA of acetone extracts (Eac) from S. crispus stored at 60    149
            0
                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      153
                   0
            60         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    154
            temperature (25 0C 65% RH) for 0-6 months storage period
            in the spectral region 1800-1200 cm-1 (PC2 vs PC1)



                                         xvii
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
                                                                o
            various temperatures for 1 month storage period (S1- C) 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 (S1-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 (S1-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 (S1-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-1) 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-1) of methanol         160
            extracts (Em) from S. crispus stored at room temperature
            (25°C 65% RH) for 0-6 months storage period




                                       xviii
Fig. 4.59   3D plot of FT-IR spectra (4000–400 cm-1) of methanol                 160
            extracts (Em) from S. crispus stored at 60°C 85% RH for 0-
            6 months storage period

Fig. 4.60   3D plot of FT-IR spectra (4000–400 cm-1) of water extracts           161
            (Ew)   from   S.   crispus     stored   at   room     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-1) 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-1) 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-1) of water extracts           164
            (Ew) 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-1) 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                  165
            extracts   (Eac)   from   S.     crispus     stored   at   various




                                         xix
            temperatures for 1 month storage period

Fig. 4.69   3D plot of FT-IR spectra (4000–400 cm-1) of methanol               165
            extracts   (Em)    from   S.    crispus    stored   at   various
            temperatures for 1 month storage period

Fig. 4.70   3D plot of FT-IR spectra (4000–400 cm-1) 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-1) of acetone                166
            extracts   (Eac)   from   S.    arvensis   stored   at   various
            temperatures for 1 month storage period

Fig. 4.72   3D plot of FT-IR spectra (4000–400 cm-1) of methanol               167
            extracts   (Em)    from   S.    arvensis   stored   at   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)         178




                                           xx
                                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         213
                isolated quercetin [black (2)]




                                         xxiii
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   Standard calibration curve of marker compounds                 240




                                     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-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



                                    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 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.




                                        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 penyakit-

penyakit 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 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




                                        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 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.




                                      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, 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;


                                          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                                 HO
                                       O                  O                    OH
                                           O
                                                    OH
                                                                                                               O
 HO                                                                                   p-hydroxy benzoic acid
                                   O                                           OH
                    H3 C
                    HO
                              HO       OH
                                       verbascoside


 HO                                            HO                                    HO


                                                                              OH                                OH
  O                                    OH                                           H3CO
                                               HO
                                                                                                           O
          p-coumaric acid                                                O
                                                         caffeic acid                       ferulic acid


               OH                                   HO                                                OCH3
      O                                                                  O            HO
                                                                                                           OH
                              OCH3                                                     O
                                                                         OH
                                                                                                      OCH3
                                                               OH                          syringic acid
                         OH
                                                         gentisic acid
            vanillic acid




                                                                                                       C2H5



                                                                        HO
          HO                                                                         β-sitosterol
                           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           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).



                              OH                                OH                                                        OH
                                   OH                                    OH                                                    OH

                                            HO         O                                    O        O             O
HO                O                                                           HO

                                                                                  HO                 OH
                                                               OH
                                                                                            OH             OH      O
                                                  OH   O
           OH     O                                                                     luteolin-7-O-glucoside
                luteolin                          quercetin

                                             OH                                                                            OCH3
                                                  OH
                                                                                       O        O              O
                                                                              O
                  O     O          O                            H3C      O
      HO
                                                                              HO                OH
          HO            OH                                          HO        OH       OH                      O
                  OH               O                                     OH
                       isocinaroside                                                        linarin
                                       OH                                          OH                                          OCH3
                                             OH
                                                   HO               O                       HO                     O
            O      O           O
 HO
                                       OH
     HO            OH
            OH           OH    O                           OH       O                                     OH       O
                 sonchoside                                    apigenin                                        acacetin


Fig. 1.2 Chemical structures of S. arvensis constituents


                                                               9
                          OH

   HO         O                                        H
                                                   H


                   OH
                                               H
         OH   O
                                   HO
          kaempferol                                              HO
                                         taraxasterol
                                                                            lupeol



                          H




        HO                                                  HO

                  β-amyrin                                       α-amyrin

                     OH                  HO         OH                 HO         OH HO
                          OCH3

   HO         O                     HO                     OH     HO

                                                                             HO           OH
                   OH                    HO         OH                  mannitol
         OH   O                            inositol
          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.




                                       12
Table 1.1 Plants used for treatment of kidney stone and related diseases

 No   Plant Name                     Constituent                        Reference

 1    Plantago      Glycoside aucubin, plantagin, plantenolic,     Perry and Metzger,
      major         succinic acid, adenine, cholin, and aucubin.   1980; Samuelsen,
                    Polysaccharides, lipids, cafeic acid           2000.
                    derivatives, flavonoids, iridoid glycosides,
                    terpenoids, alkaloid, organic acid.
 2    Zea mays      Galactan, xylan, dextrose, sugar,              Perry and Metzger,
                    zeaxanthin, protein, inosite,                  1980; Pedreschi
                    hexaphosphoric acid, maizenic acid, resins,    and Cisneros,
                    potassium and calcium salt. Anthocyanins       2007; Pozo-Insfran
                    cyanidin-3-glucoside, pelargonidin-3-          et al., 2006
                    glucoside etc. Phenolic acid p-coumaric
                    acid, vanillic acid etc.
 3    Raphanus      Acylated anthocyanin (as pelargonidin),        Otsuki et al., 2002 ;
      sativus       alkaloids pyrolidine, isoquinoline,            Vargas et al., 1999;
                    phenethylamine, sulphuric compounds
                    glucoparin, sinigrin, allylisothiocyanate.     Basile et al., 2003.
                    Flavonoids apigenin, apigenin-7-O-
                    triglycoside etc.
 4    Phylanthus    Potassium, phyllanthin, hypophyllanthin,       Perry and Metzger,
      niruri        triacontanal, triacontanol, lignans,           1980;
                    glycosides, flavonoids, alkaloids, tannins,    Syamasundar et
                    phenylpropanoids, saponins                     al., 1985
 5    Orthosiphon   High potassium salt, glucoside, diterpenes,    Perry and Metzger,
      stamineus     orthosiphols, rosmarinic acid, salvigenin,     1980; Takeda et
                    orthosiphols, flavonoids (eupatorin,           al., 1993; Akowuah
                    sinensitin, 3′-hydroxy-5,6,7,4′-               et al., 2005
                    tetramethoxyflavone,TMF)




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 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



                                       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 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


                                        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 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.


                                        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, 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.



                                                                                      OH                                       O
                      HH                                                                                                  OH
O                                                                               HO                       O
                                                                          HO          O
        O
                                                 O                                                               H                 OH
                                                                           F
                           H
        O
                                                                                                             H       H
medroxyprogesterone acetate                                                                  O
                                                              O
                                                                  dexamethasone                          cortisone

                                                                  OSO3H                                                        OH
                                                                                                 O       COOH
                                                                               HO
                                                                                                     O                         OH
                                                                                                         H
                                                                               HO
H2N
                      N             N
                                             H
                                                     OH                                    rosmarinic acid
                      H             H
                               squalamine
                                        OH
                                               OH
                                                                  O
                                                                                                         O
                                                          H
        HO                O+
                                             OH H O    O          C   C   C          OH
                                                   H3C                H   H
                                                  H H
                                                                                                                 OH
                                          H2C O
        CH2OH                                          H                                                 O
                                O            H
    H      O
                  O                     O
                                        H HO
                                                  OH OH                                          3-hydroxyflavone
        OH H
                                    H          OH
HO                H
                                        OH H
        H    OH
                                             nasunin

                               OH                                              OH                                    OH
                                    OH                                HO                                                  OH
                  O                                                   O                    HO            O

                                                                                                                 OH
            O                                                                                          O
                                                                      O
    3′,4′-dihydroxyflavone                                                                           fisetin
                                                     2′,3′-dihydroxyflavone

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



                                                                          21
                          OH                       OH             HO          O
                                                        OH
 HO          O
                                 HO        O
                                                                        OH O
                                                                                             OH
      OH     O                                                              genistein
                                      OH   O
         apigenin
                                        luteolin

HO         O                                       OH                                   OH
                                                        OH                                   OH

                                HO         O                      HO          O
           O                                            OH
                          OH
           daidzein                             OH                                  OH
                                      OH O                              OH    O
                                       myricetin                         quercetin


                                 OH

        HO            O                                      HO         O


                           OH                                                  OH
                 OH   O                                            OH   O
                  kaempferol                                       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
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



                                         24

								
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