Biological activity & Phytochemical Study of selected Medicinal Plants In

By Musa Khan

A thesis submitted to the Quaid-i-Azam University, Islamabad in partial fulfillment of the requirements for the Degree of

Doctor of Philosophy
in

Plant Sciences
(Plant Taxonomy)

Department of Plant Sciences Quaid-i-Azam University Islamabad 2010

Biological activity & Phytochemical Study of selected Medicinal Plants

By Musa Khan

Department of Plant Sciences Quaid-i-Azam University Islamabad 2010

Certificate CERTIFICATE The theses of Musa Khan is accepted in its present form by the Department of Plant Sciences, Quaid-i-Azam University, Islamabad as satisfying the theses requirement for the degree of Doctor of Philosophy in Plant Taxonomy.

Supervisor ________________________ Pro. Dr. Rizwana Aleem Qureshi

External Examinar._________________________ Dr. Mohammad Khan Laghari (Director PMNH)

External Examinar__________________________

Charperson:____________________________ Prof. Dr. Asghari Bano

Dated:

07/05/2010

Acknowledgements
I have no words to thanks Allah almighty who gives me the opportunity to complete my studies. I feel obliged to my parent department Defense Science & Technology Organization and the Higher Education Commission of Pakistan for providing me financial support during my studies. I heartily appreciate my supervisor Dr Rizwana Aleem Qureshi Prof, Department of Plant Sciences, Quaid-i-Azam University, Islamabad, for her keen interest, kindness and her valuable views and experience. I would like to thanks Chairperson, Department of Plant Sciences, Prof. Dr Asghari Bano for timely providing me all the necessary facilities and administrative support. I also appreciate and thanks my foreign supervisors, Prof. Dr. Dr Brigitte Kopp Department of Pharmacognosy (University of Vienna, Austria) and Dr George Krupitza, Department of Tumor Biology, Medical University of Vienna, Austria, for technical support and guidance during my six months stay in Austria (sponsored by Higher Education Commission of Pakistan). Thanks to all teachers, students and staff members of Department of Pharmacognosy, University of Vienna, Austria, Department of Tumor Biology, Medical University of Vienna and Department of Plant Sciences, Quaid-i-Azam University, Islamabad Pakistan for sharing expertise and for providing a friendly environments. In last I am greatly thankful to my parents who provide me support and put me on this track but my mother could not survive to see me on this stage.

Musa Khan

In memory of my dear mother (July 2008)

ii

ABBREVIATIONS
BuOH C1I C2I Cdc Cdc25A/B/C Cdk Chk1 Chk2 CKI DPPH EtOAc GA HUVEC IC50 IpC50 IR p21 p53 PARP PIC PMSF RB ROS SPE THF TNF UV-light Butanol Chk1 Inhibitor Chk2 Inhibitor Cell division control Cell-devision-cycle 25A/B/C Cyclin-dependent-kinases Checkpoint-kinase 1 Checkpoint-kinase 2 Cyclin dependent kinase inhibitor 1, 1-diphenyl-2-picrylhydrazyl Ethyl acetate Gallic acid Human umbilical vein endothelial cells Concentrations which inhibits by 50 % Concentrations which inhibits proliferation by 50 % Ionizing radiation Protein 21 Protein 53 Poly (ADP-ribose) polymerase Protease inhibitor cocktail Phenylmethylsulfonylfluorid Retinoblastoma protein Reactive oxygen species Solid phase extraction Tetrahydrofurane Tumour necrosis factor Ultraviolet-Light

iii

Table of Contents
Acknowledgments Dedication Abbreviations Summary Introduction 1.1 General introduction 1.2 Pharmacognosy 1.3 Bioassay guided isolation of natural products 1.4 Medicinal plants as a source of important drug 1.5 Secondary metabolites 1.5.1 Small molecules 1.5.1.1 Alkaloids 1.5.1.1 Alkaloids 1.5.1.3 Glycosides 1.5.1.4 Phenols 1.5.1.5 Phenazines 1.5.2 Big small molecules 2.5.2.1 Polyketides 2.5.2.2 Nonribosomal peptides 1.6 Technique used in phytochemistry 1.6.1 Chromatography 1.6.2 Capillary electrophoresis 1.6.3 Spectroscopic Techniques 1.6.3.1 NMR spectroscopy 1.6.3.2 Two-Dimensional Nuclear Magnetic Resonance Spectroscopy (2DNMR) 1.6.3.3 Infrared Spectroscopy 1.6.3.4 Fourier transform infrared spectroscopy 1.6.3.5 Ultraviolet-visible spectroscopy 1.6.4. Liquid chromatography-mass spectrometry 1.6.5. Gas chromatography-mass spectrometry (GC-MS) 1.7 Development of Anticancer agents from Medicinal plants 20 21 21 22 23 23 23 i ii iii 1 4 4 5 5 6 10 10 10 10 12 14 15 15 15 15 16 16 20 20 20

iv

1.8 Development of cancer 1.8.1 Self-sufficiency in growth signals 1.8.2 Insensitivity to antigrowth signals 1.8.3 Evading apoptosis 1.8.4 Limitless replicative potential 1.8.5 Sustained angiogenesis 1.8.6 Tissue invasion and metastasis 1.8.7 The cell cycle 1.8.7.1 Cell cycle phases (short summary) 1.8.7.2 Presence of cyclins and Cdks during single phases 1.8.8 Function and activation of (proto)-oncogenes/oncogenes 1.8.8.1 Oncogenes 1.8.8.2 Cyclin D1 1.8.8 3 Cdc25A (Cell-division-cycle 25A) 1.8.8 4 Function and activation of tumor suppressor genes 1.8.8 5 p53 (protein 53) 1.8.8 6 Activation of p53 1.8.8 7 P21CIP (protein 21) 1.8.8 8 Activation of p21 1.8.8.9 RB 1.8.8.10 Activation of RB 1.8.9 Cell death 1.8.9.1 Apoptosis 1.8.9.2 Autophagy 1.8.9.3 Necroses 1.9 Bioassays Techniques
CIP

24 25 26 26 26 26 27 28 29 29 30 30 30 30 31 31 31 31 32 32 33 33 33 35 35 37

1.9.1 Apoptosis assays (Hoechst 33258 propidium iodide (HOPI) double-staining) 37 1.9.2 Western blot assay 1.9.2.1 Steps in a western blot 1.9.3 Fluorescence Activated Cell Sorting (FACS) assay 1.9.3.1 Flow cytometers 1.9.3.2 Application 1.9.4 Comet assay 37 37 40 41 42 42 v

1.9.4.1 Experimental procedure 1.9.4.2 Principals 1.9.5 Total Phenolics or Folin-Ciocalteau Micro Method 1.9.5.1 Calibration curve 1.9.6 Antioxidant activity 1.9.6.1 (1, 1-diphenyl-2-picrylhydrazyl) (DPPH) 1.10 Selection of Medicinal plants species 1.10.1 Berberis lycium Royle (Berberidaceae) 1.10.2 Mallotus philippensis (Lam.) Muell. Arg. (Euphorbiaceae) 1.10.3 Adhatoda vasica Nees (Acanthaceae) 1.10.4 Albizia lebbeck (L.) Benth. (Mimosaceae) 1.10.5 Bauhinia variegata Linn. (Caesalpinaceae) 1.10.6 Bombax ceiba Linn. (Bombacaceae) 1.10.7 Calotropis procera (Willd.) R. Br. 1. c (Asclepiadaceae) 1.10.8 Carrisa opaca Staff ex Haines (Apocynaceae) 1.10.9 Caryopteris grata Benth. (Verbenaceae) 1.10.10 Cassia fistula Linn (Caesalpinaceae) 1.10.11 Colebrookea oppositifolia Smith (Labiateae) 1.10.12 Debregeasia salicifolia (D.Don) Rendle in Prain (Urticaceae) 1.10.13 Dalbergia sissoo Roxb. (Papilionaceae) 1.10.14 Dodonaea viscosa (L.) Jacq., Enum. Pl. Carib. (Sapindaceae) 1.10.15 Ficus palmata Forssk. (Moraceae) 1.10.16 Ficus racemosa L. (Moraceae) 1.10.17 Jasminum humile Linn. (Oleaceae) 1.10.18 Lantana camara L. (Verbenaceae) 1.10.19 Melia azedarach L. (Meliaceae) 1.10.20 Olea ferruginea Royle (Oleaceae) 1.10.21 Phyllanthus emblica L. (Euphorbiaceae) 1.10.22 Pinus roxburghii Sargent (Pinaceae) 1.10.23 Pyrus pashia Buch. & Ham. (Rosaceae) 1.10.24 Punica granatum L. (Punicaceae) 1.10.25 Rubus ellipticus Smith (Rosacceae) 1.10.26 Viburnum cotinifolium D. Don (Caprifoliaceae) 1.11 Objectives

42 44 44 45 46 47 48 49 49 50 51 51 51 52 53 53 53 54 54 55 55 56 57 57 58 58 59 59 60 60 61 61 62 63 vi

Chapter: 2

Review of Literature

64 64 64 64 65 69 69 69 69 73 72 72 72 73 73 73 73 74 74 74 74 74 74 74 74 75 75 76 76 76 76 76 77 77 vii

2.1 Berberis lycium Royle (Berberidaceae) 2.1.1 Ethnobotanical uses 2.1.2 Chemical constituents 2.1.3 Biological testing 2.2 Mallotus philippensis (Lam.) Muell. Arg. (Euphorbiaceae) 2.2.1 Ethnobotanical uses 2.2.2 Chemical constituents 2.2.3 Biological testing 2.3 Adhatoda vasica Nees in Wall (Acanthaceae) 2.3.1 Ethnobotanical uses 2.3.2 Chemical constituents 2.3.3 Biological testing 2.4 Albizia lebbeck (L.) Benth. (Mimosaceae) 2.4.1 Ethnobotanical uses 2.4.2 Chemical constituents 2.4.3 Biological testing 2.5 Bauhinia variegata Linn. (Caesalpinaceae) 2.5.1 Ethnobotanical uses 2.5.2 Chemical constituents 2.5.3 Biological testing 2.6. Bombax ceiba Linn. (Bombacaceae) 2.6.1 Ethnobotanical uses 2.6.2 Chemical constituents 2.6.3 Biological testing 2.7 Calotropis procera Linn. (Asclepiadaceae) 2.7.1 Ethnobotanical uses 2.7.2 Chemical constituents 2.7.3 Biological testing 2.8 Carissa opaca Stapf ex Haines (Apocynaceae) 2.8.1 Ethnobotanical uses 2.8.2 Chemical constituents 2.9 Cassia fistula Linn. (Caesalpinaceae) 2.9.1 Ethnobotanical uses

2.9.2 Chemical constituents 2.9.3 Biological testing 2.10 Colebrookea oppositifolia Smith (Labiateae) 2.10.1 Ethnobotanical uses 2.10.2 Chemical constituents 2.11 Debregeasia salicifolia (D.Don) (Urticaceae) 2.11.1 Ethnobotanical uses 2.11.2 Chemical constituents 2.11.3 Biological testing 2.12 Dalbergia sissoo Roxb. (Papilionaceae) 2.12.1 Ethnobotanical uses 2.12.2 Chemical constituents 2.12.3 Biological testing 2.13 Dodonaea viscosa Linn. (Sapindaceae) 2.13.1 Ethnobotanical uses 2.13.2 Chemical constituents 2.13.3 Biological testing 2.14 Ficus palmata Forssk. (Moraceae) 2.14.1 Ethnobotanical uses 2.14.2 Chemical constituents 2.15 Ficus racemosa L. (Moraceae) 2.15.1 Ethnobotanical uses 2.15.2 Chemical constituents 2.15.3 Biological testing 2.17 Lantana camara Linn. (Verbenaceae) 2.17.1 Ethnobotanical uses 2.17.2 Chemical constituents 2.17.3 Biological testing 2.18 Melia azedarach Linn. (Meliaceae) 2.18.1 Ethnobotanical uses 2.18.2 Chemical constituents 2.18.3 Biological testing 2.19 Phyllanthus emblica L. (Euphorbiaceae) 2.19.1 Ethnobotanical uses

77 77 77 77 77 78 78 78 78 78 78 79 79 79 79 80 80 81 81 81 81 81 82 82 83 83 83 84 84 84 85 85 86 86 viii

2.19.2 Chemical constituents 2.19.3 Biological testing 2.20 Pinus roxburghii Sargent (Pinaceae) 2.20.1 Ethnobotanical uses 2.21 Punica granatum Linn. (Punicaceae) 2.21.1 Ethnobotanical uses 2.21.2 Chemical constituents 2.21.3 Biological testing 2.22 Rubus ellipticus Smith (Rosaceae) 2.22.1 Ethnobotanical uses 2.22.2 Chemical constituents 2.22.3 Biological testing 2.23 Viburnum cotinifolium D. Don (Caprifoliaceae) 2.23.1 Ethnobotanical uses 2.23.2 Chemical constituents Chapter: 3 Materials & Methods

86 87 87 87 88 88 88 88 89 89 89 90 90 90 90 91 91 91 93 94 95 95 95 95 96 96 96

3.1 Reference Compounds 3.2 Plant Material 3.3 Anti bodies for western blot analyses 3.4 Miscellaneous Chemicals and Reagents 3.5 Cell culture and bacterial strains 3.6 Extraction 3.6.1 Extraction for Antioxidant and Total Phenolics Determination 3.6.2 Extraction of roots powder 3.6.3 Extraction for Flavonoids analyses 3.7 Chromatographic Methods 3.7.1 Thin Layer Chromatography (TLC) 3.7.1.1 Thin Layer Chromatography of Berberis lycium fractions

96 3.7.1.2 Thin Layer Chromatography for Flavonoids analyses 3.7.2 High Performance Liquid Chromatography (HPLC) 3.7.2.1 General HPLC Parameters 3.7.2.2 HPLC Method 3.7.2.3 Sample Preparation 97 97 97 97 98 ix

3.7.3 Gas Chromatography and Mass Spectrometer 3.8 Biological Testing 3.8.1 Antineoplastic Activities 3.8.1.1 Anti-proliferation or Growth inhibition assay 3.8.1.2 Hoechst dye 33258 and propidium iodide double staining (Apoptosis Assay) 3.8.1.3 Western blotting 3.8.1.4 Cell cycle distribution analysis (FACS analyses) 3.8.1.5 Single cell gel electrophoresis (SCGE)/Comet assay 3.8.1.6 Statistical analyses 3.8.2 Total Phenolics determination 3.8.3 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) test 3.8.4 Antibacterial Determination Chapter. 4 Results and Discussion

98 99 99 99

99 99 100 101 102 102 102 103 104 104

4.1 Results

4.1.1 Anti-neoplastic Activities and Phytochemicals studies of Berberis lycium. 104 4.1.1.1 Qualitative Analysis of B. lycium extracts constituents by TLC. 4.1.1.2 Separation and quantification of alkaloids by RP-HPLC 104 4.1.1.3 Inhibition of HL-60 cell proliferation by extracts of B. lycium, Berberine and Palmatine. 112 104

4.1.1.4 Effect of BuOH extract, Berberine and Palmatine on cell cycle distribution 4.1.1.5 Induction of apoptosis by extracts of B. lycium and Berberine 118 115

4.1.1.6 Induction of stress response by extracts of B. lycium and Berberine. 123

4.1.2 Anti-neoplastic Activities and Phytochemicals studies of Mallotus phillipensis. 126

4.1.2.2 Induction of apoptosis by extract of Mallotus phillipensis 126 4.1.2.3 Effect of Hexane fraction on cell cycle distribution. 126 x

4.1.2.4 Induction of stress response by extract of Mallotus phillipensis. 131

4.1.2.5 GC-MS Analysis of Mallotus phillipensis Hexane Fraction. 131 4.1.3 Total Phenolics, Free radical scavenging activity and Flavonoids finger printing of selected Medicinal Plants. 4.1.3.1 Total Phenolics Determination. 4.1.3.2 Determination of Free radical scavenging activity 4.1.3.3 Flavonoids finger printing of selected Plants 4.1.4 Antibacterial and Free radical scavenging activities, Flavonoids finger printing of Mallotus philippensis. 4.1.4.1 Antibacterial activities 4.1.4.2 Free radical scavenging activities 4.1.4.3 Flavonoids finger printing of Mallotus philippensis. 4.2 Discussion 150 150 150 150 155 137 137 137 140

4.2.1 Anti-neoplastic Activities and Phytochemicals studies of Berberis lycium 155 4.2.2 Anti-neoplastic Activities and Phytochemicals studies of Mallotus phillipensis. 4.2.3 Total Phenolics, Free radical scavenging activity and Flavonoids finger printing of selected Medicinal Plants 4.2.4 Antibacterial and Free radical scavenging activities, Flavonoids finger printing of Mallotus Philippensis. Chapter. 5. Conclusion List of Publications Plates Chapter. 6. References 163 166 169 170 182 159 157

List of Figures
Figure 1 Examples of new medicinal plant drugs Figure 2 Acquired capabilities of cancer Figure 3 Cyclin and Cdks distribution during the cell cycle Figure 4 DNA damage induced by UV-light and further the activation of p53 9 25 29 32

xi

Figure 5 Mechanism of Apoptosis Figure 6 Alkaloids of Berberis lycium Figure 7 Compounds of Mallotus philippensis Figure 8 TLC of Berberis lycium extracts Figure 9 RP-HPLC Chromatogram of alkaloids standards

36 65 72 105 106

Figure 10 RP-HPLC chromatogram of n-Butanol fraction of Berberis lycium extract. 107 Figure 11 RP-HPLC chromatogram of water fraction of Berberis lycium extract 108

Figure 12. RP-HPLC chromatogram of Ethyl acetate fraction of Berberis lycium extract 109 Figure 13 Optimum UV spectra of standards compounds Figure 14 Alkaloids percentage in Berberis lycium Figure 15 Anti-proliferative effect of B. lycium extracts and its alkaloids Figure 16 Analysis of cell cycle proteins 110 112 114 115

Figure 17 Cell Cycle Distribution of HL-60 cells upon treatment with of BuOH extract and berberine for 48 h Figure 18 Induction of apoptosis by the B. lycium extracts and berberine Figure 19 Western blot analysis of pro-apoptotic mediators and effectors 117 120 121

Figure 20 The genotoxicity of increasing concentrations of BuOH extract and berberine 122 Figure 21 Comet assay Figure 22 Induction of stress response by the BuOH extract and Berberine Figure 23 Anti-proliferative effect of Mallotus phillipensis extracts Figure 24 Induction of apoptosis by the Mallotus phillipensis Hexane fraction Figure 25 Analysis of cell cycle proteins 123 125 127 128 129

Figure 26 Cell Cycle Distribution of HL-60 cells upon treatment with hexane Fraction of Mallotus phillipensis Figure 27 Induction of stress response by Mallotus phillipensis 130 131

Figure 28 GC/MS chromatogram of hexane soluble fraction of Mallotus phillipensis 136 Figure 29 Gallic acid standard curve Figure 30 Total Phenolics and Extract yield per gram Figure 31 Antioxidant cure of Ascorbic acid Figure 32 Flavonoids finger printing of standard and selected plants Figure 30 Percentage of Flavonoids in Plant samples Figure 34 Types of Flavonoids in each sample 138 139 139 145 146 147 xii

Figure 35 Antibacterial activities of Mallotus philippensis Figure 36 Free radical scavenging activity of Mallotus philippensis Figure 37 Flavonoids finger printing of Mallotus philippensis

151 153 154

List of Tables
Table 1 Reference compounds Table 2 Investigated Plants species Table 3 Anti bodies for western blot analyses Table 4 Miscellaneous Chemicals and Reagents Table 5 Parameters for HPLC-PDA analyses of Alkaloids Table 6 Gradient elution systems used for HPLC separations Table 7 Gas Chromatograph and Mass Spectrometer conditions Table 8 10% Polyacrylamide Gel Preparation Table 9 Linearity study of standard curve for standard compounds Table 10 Percent composition of active alkaloids in Berberis lycium Table 11 Comparative total Phenolic, extract yield per gram and IC50 Values Table 12 Appearance of standards under UV 265nm Table 13 Qualitative analyses of plants samples for Flavonoids types Table 14 Antibacterial activities of roots and flower powder extract 91 92 93 94 97 98 98 101 111 111 140 148 149 152

xiii

Summary

SUMMARY The present study deals with the exploration of some species of medicinal plants found in Pakistan against cancer. Twenty seven plant species were selected from the local flora. Roots of three plants i.e. Berberis lycium (Berberidaceae), Mallotus philippensis (Euphorbiaceae) and Zizyphus nummularia (Rhamnaceae) were studied for antineoplastic activity against p53 deficient human leukemia cell lines (HL-60). Although roots of Zizyphus nummularia possess many complex alkaloids yet its extract was not effective in checking proliferative activity. Berberis lycium extract and its alkaloids berberine and palmatine are known for their beneficial pharmacological properties. In the present study, the anti-neoplastic activities of different B. lycium root extracts and the major constituting alkaloids, berberine and palmatine were investigated in HL-60 cells to elucidate the anti-neoplastic trigger mechanisms of the pure compounds and crude extracts in a p53-deficient background. Growth inhibition, cell cycle distribution, and apoptosis were compared among the ethyl acetate (EtOAc), n-butanol (BuOH) and water (H2O) extracts. The BuOH extract inhibited cell proliferation most efficiently (IC50 < 2.77 g extract weight/ml medium, which corresponded to 250 g dried root/ml). The IC50s for the EtOAc and H2O extracts were 16.65 g/ml and 104.25 g/ml, respectively (corresponding for both extract types to >7.5 mg dried root/ml). The chemical composition of the BuOH extract was analyzed by preparative TLC and quantified by RP-HPLC and it was estimated that it contained 3.73 M Berberine and 1.51M Palmatine per 1 mg dried root. Therefore, HL-60 cells were exposed to the respective concentrations of berberine and palmatine. Berberine showed an IC50 < 1.87M after 72 h of incubation, while palmatine had no significant effect up to 4.68 M. The BuOH extract and berberine induced the intra-S-phase checkpoint causing the accumulation of HL-60 cells in S-phase. In contrast to a very recent report by Liu et al, (2006), It is found that the anti-cancer effects of berberine and the extract are not due to genotoxicity but correlate with -tubulin acetylation, strong activation of Chk2, phosphorylation of Ser177-Cdc25A and its subsequent degradation as well as the consequent inactivation of Cdc2 (CDK1) and furthermore, the down-regulation of the proto-oncogene cyclin D1. The molecular effects were observed at low concentrations (11.1 g BuOH extract/ml; 1.4 g berberine/ml) which inhibited ~ 50 % of the HL-60 cells proliferation after 24 h treatment, hence supporting the mechanistic conjunction. Mallotus philippensis is a well known medicinal plant of Pakistan. It possesses different classes of chemical compounds with unique pharmacological activities. Roots of Mallotus 1

Summary

philippensis was initially extracted and fractionated in organic solvents, n-hexane, ethyl acetate (EtOAc), and n-butanol (BuOH). After evaporating each solvent, 9.23 g dried hexane extract, 4.00 g dried EtOAc extract, and 7.08 g dried BuOH extract was obtained, respectively. The n-hexane fraction showed the highest toxicity against HL-60 cells (IC50 1.5 mg dry roots equivalent /ml medium) after 72h. The hexane fractions regulated protein expression and protein activation in HL-60 cells. The inhibition of HL-60 proliferation that was observed upon treatment with hexane extract was preceded by the down regulation of the proto-oncogene Cdc25A and cyclin D1 after 48 h. All of these effects have not been observed in any p53 deficient cell lines so far by Mallotus phillipensis extracts and its chemical constituents. Valacchi et al (2008) has reported that rottlerin deactivate cyclin D1 in HaCaT cell line. The hexane fraction induced 18% apoptosis after 48h of treatment with 1.5 mg dry roots equivalent /ml medium. The ability of M. phillipensis hexane fraction and the observation indicates that the anti-neoplastic effects have been triggered by induction apoptosis through caspase-2 activation while Brodie et al., 2003 reported that rottlerin activated caspase-3. The chemical composition of the n-hexane fraction of M. phillipensis was analyzed by GC-MS. Different compounds have been detected in the sample. Mass spectrometric data of some compounds have been co-related with already reported compounds from different parts of the same species. Lupeol, Betulin, Kamala Chalcones C like compounds and another unknown compound (GC Rf = 39.9, 45.66, 43.905 and 47.735 minutes respectively) have been detected. Rottlerin that has been reported in M. phillipensis was not detected in the hexane fraction. It has been confirmed from the present anti-neoplastic assay that hexane fraction is active against p53 deficient human leukemia cell lines (HL-60) and the activity was due to compound/compounds other than rottlerin. Kamala or Kamara (a red powder of M. philippensis reported to have different cytotoxic compounds, flavonoids or Phenolic compounds) has compared with the roots of M. philippensis for inhibition of different bacterial strains. Similarly Kamala has compared with the aerial parts (leaves) of M. philippensis in scavenging free radicals. It has been observed that (Kamala or Kamara) extract has shown activities against Gram positive bacteria, Bacillus subtilis and Staphylococcus aureus (MICs 0.7 and 0.6 mg/ml), while it does not shown any response against Salmonella setubal, Staphylococcus epidermidis and Escherichia coli up to maximum concentration of 15 mg/ml. Roots extract was effective against one Gram positive bacteria Bacillus subtilis and one Gram negative bacteria

Summary

Salmonella setubal (MICs 1.00 and 2.00 mg/ml) respectively but it has not shown any activity against Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli up to maximum concentration of 15 mg/ml. It has been observed that both Kamala and leaves extract have free radical scavenging capacity but the leaves extract was more active than Kamala powder in scavenging free radicals. Thin layer chromatography of the leaves has shown the presence of Vitexin, Isovitexin and Rutin. In another set of experiment 24 different plants species were checked to determine total Phenolics, free radical scavenging capacity and flavonoids types. Some plants species were reported medicinally in literature and the others have been selected randomly. The medicinally important plants were Bauhinia variegata, Cassia fistula, Bombax ceiba, Calotropis procera, Carissa opaca, Adhatoda vasica, Albizia lebbeck, Colebrookea oppositifolia, Dalbergia sissoo, Dodonaea viscosa, Ficus palmata, Ficus racemosa, Lantana camara, Melia azedarach, Phyllanthus emblica, Punica granatum, Rubus ellipticus and Viburnum cotinifolium and the non medicinal plats were Jasminum humile, Olea ferruginea, Pinus roxburghii, Caryopteris grata, Debregeasia salicifolia and Pyrus pashia. Total Phenolics were studied by comparing with standard Gallic acid. Phyllanthus emblica has shown highest amount of total Phenolics while comparing with Gallic acid. The extract per gram of Phyllanthus emblica was also greater than others. Phenolic acids, Kaempferol and Vitexin have been detected in the sample of Phyllanthus emblica by thin layer chromatography. Vitexin has been reported for the first time in Phyllanthus emblica. Rubus elepticus has shown comparatively highest capacity in scavenging free radicals. Phenolic acids, Kaempferol, Vitexin, Rutin and Apigenin have been detected in the sample of Rubus ellipticus by thin layer chromatography. All plants species have shown Phenolic acids bands. Vitexin and Isovitexin were present in maximum numbers of plants samples (58.33 and 54.8 % percent respectively); Catechin, Luteolin-7-glucoside, Quercetin and Luteolin were not detected in any sample.

Chapter 1

Introduction

INTRODUCTION 1.1 General introduction The flora of Pakistan due to its diverse climatic and soil conditions and many ecological regions, is very rich in medicinal plants. According to a general survey of Pakistan about 6000 species of flowering plants have been exist, out of 6000 about 400-600 are medicinally important species (Nasir and Ali, 1972; Hamayun et al; 2005). The history of plants to be utilized as medicines is thousands of years old (Samuelsson, 2004). These plant materials initially took the form of crude drugs such as poultices, teas, powders tinctures, and many other herbal formulations (Samuelsson, 2004; Balick and Cox, 1997). From near past it has been discovered that properties of medicinal plants are due to its active chemical compounds and therefore the isolation of active compounds and in the early 19th century morphine has been isolated from opium (Samuelsson, 2004; Kinghorn, 2001). The discovery of drug from medicinal plants has been started from the era when the isolation of primarily drugs such as digitoxin, quinine, cocaine, and codeine has begun. Like morphine some are still in use for different purposes (Butler, 2004; Newman et al., 2000; Samuelsson, 2004). Numbers of scientists have been working in order to isolate and characterize the pharmacologically active compounds from medicinal plants. Drug discovery techniques have been discovered and applying for the standardization of herbal medicines and to obtain analytical marker compounds. Drug discovery from medicinal plants are not simple but it has evolved to include numerous fields of inquiry and take advantages of different analytical procedures. The process initiated with a botanist especially with ethnobotanist, ethnopharmacologist, or plant ecologist that can easily collects and identifies their desired plant(s). Collection may involve those species with known biological activity which need to be study for their active compound(s) and new for isolation (e.g., traditionally used herbal remedies) or may also involve those taxa that have been collected randomly for a large screening purposes. It is also important to take care and respect the intellectual property rights of a given area, country where plant(s) of interest are collected (Baker et al., 1995). Phytochemists are also called natural product chemists. These phytochemists after proper collection, identification and cleaning processes, make crude extracts from the selected parts of the plant materials, subject these crude extracts to biological screening of their desire assays, and commence the process of isolation and characterization of the active chemical compound(s). The whole processes are called bioassay-guided fractionation. Molecular biology is very important and taking essential part in drug discovery from medicinal plant. Molecular biology determines and implements appropriate screening 4

Chapter 1

Introduction

technique science.

that

directed

towards

physiologically

relevant

molecular

targets.

Pharmacognosy encapsulates all of the relevant fields into a distinct interdisciplinary

1.2 Pharmacognosy The term and practice of pharmacognosy have been used since about 200 years ago (Samuelsson, 2004; Kinghorn, 2001), as medicinal plants have progressed to use as drug, the formulation of crude drugs and to isolate the active compounds in drug discovery research. According to the American Society of Pharmacognosy, the pharmacognosy can be stated as the study of the physical, chemical, biochemical and biological properties of drugs, drug substances, or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources. In the present era of research regarding drug discovery from medicinal plants or in broad way from natural origin, pharmacognosy compensate the broad study of natural products from various sources including unicellular and multi cellular organism like bacteria, fungi, plants, and marine organisms. In broad way, Pharmacognosy that study various parameter which includes both botanical dietary supplements, including herbal remedies (Cardellina, 2002; Tyler, 1999), and searching for single chemically and pharmacologically active compound that can be use as drug and may proceed through further development into Food and Drug Administration (FDA)-approved medicines. According to Bruhn and Bohlin the definition of pharmacognosy may proceed as a molecular science that explores naturally occurring structureactivity relationships with a drug potential (Bruhn and Bohlin, 1997). 1.3 Bioassay guided isolation of natural products As natural sources have many useful and important bioactive compounds and many have been discovered using bioactivity directed fractionation and isolation (BDFl). The research of pharmacognosy or isolation of natural products facilitated by newly development of new bioassay methods. It has been found that the bioactive compounds are mostly plant secondary metabolites, which become medicine after processing to pure compounds; some are very useful dietary supplements, and many useful commercial products. Further modification of the active compounds lead to enhance the biological profiles and a large number of such compounds which are approved or undergoing clinical trials for clinical uses against different diseases like pulmonary diseases, cancer, HIV/AIDS, malaria, Alzheimers and other diseases (Butler., 2004; Newman et al., 2003).Crude herbs are used as drugs in different country of the world and therefore it take 5

Chapter 1

Introduction

a basic part of many traditional medicines worldwide. In Asia, traditional Chinese medicine (TCM), Korean Chinese medicine, Japanese Chinese medicine (kampo), ayurvedic medicine (India) and jamu (Indonesia), phytotherapy and hoemeopathy in Europe, Alternative medicines are typically named when herbal therapies use with various other traditional remedies in America. Integrative medicine came into being when the alternative medicine, mainly the aforementioned traditional and folk medicines used worldwide, with conventional medicine (Western medicine). 1.4 Medicinal plants as a source of important drug Different type of isolation methods have been used to obtain pharmacologically active compounds that can use as drug for different diseases. The methods which includes isolation from plants and other natural sources, combinatorial chemistry, synthetic chemistry, and molecular modeling (Geysen et al., 2003; Ley Baxendale, 2002 and Lombardino and Lowe, 2004). Although there is much research in molecular modeling, combinatorial chemistry, and other synthetic chemistry techniques which has been funding by pharmaceutical companies and organizations, natural products which have much complicated structural formulas and particularly medicinal plants, remain an important source of new drugs, new chemical entities (NCEs) and new drug leads, (Butler, 2004; Newman et al., 2000, 2003). According to survey in 2001 and 2002, approximately one quarter of the best-selling drugs in the world were natural products or derived from natural products (Butler, 2004). It has also been reported that approximately 28% of NCEs between 1981 and 2002 were natural products or natural product-derived natural products (Newman et al., 2003) and another survey during this period 20% of NCEs were considered natural product mimics, meaning that the synthetic compound was derived from the study of natural products (Newman et al., 2003). On the bases of this report it has been assumed that research on natural products accounts for approximately 48% of the NCEs reported from 19812002. Further more it has been known that natural products also provide a starting point for laboratory syntheses with diverse structures and often with multiple stereo centers that can be challenging synthetically (Koehn and Carter, 2005; Clardy and Walsh, 2004; Peterson and Overman, 2004; Nicolaou and Snyder, 2004). Natural products shows many structural features in common (e.g., aromatic rings, chiral centers, degree of molecule saturation, complex ring systems, and number ratio of heteroatoms) which have been shown to be very important to drug discovery efforts ( Feher and Schmidt, 2003; Piggott and Karuso, 2004; Clardy and Walsh, 2004; Koehn and Carter, 2005; Lee and Schneider, 2001). Many synthetic and medicinal chemists are working in the creation of natural 6

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product and natural-product like libraries that resembles the structural features of natural products with the compound-generating potential of combinatorial chemistry ( Eldridge et al., 2002; Burke et al., 2004; Hall et al., 2001a; Ganesan, 2004; Tan, 2004). Some natural products that are isolated from medicinal plants can serve not only as new drugs themselves but can also be made useful by further necessary modification by medicinal and synthetic chemists. Sometime new chemical structures are very difficult to found during drug discovery from medicinal plants, in such cases known compounds with new biological activity can provide important drug directions. Molecular target play important rule in drug discovery, since the sequencing of the human genome, a lot new molecular targets have been identified as important and useful in various diseases (Kramer and Cohen, 2004). The developments of high-throughput screening technique may show to the point and more selective activity directed towards these targets, when use the reported compounds from medicinal plants. It has also be known that the compounds isolated from traditionally used medicinal plants shown to act on newly validated molecular targets, one example is indirubin, which targeted and inhibit cyclin dependent kinases (Eisenbrand et al., 2004; Hoessel et al., 1999) and another example is kamebakaurin, which has been shown to target and inhibit NF-nB (Lee et al., 2002; Hwang et al., 2001). There are many known compounds which shown to act on novel molecular targets, this development leads to produce interest in members of these frequently isolated plant compound classes. There are many examples but some are cucurbitacin I, from the National Cancer Institute (NCI) Diversity Set of many known compounds and it is found to be highly selective in inhibiting the JAK/STAT3 pathway in case of tumors with activated STAT3 (Blaskovich et al., 2003), another example is h-lapachone, which also selectively kills cancer cells over normal cells by direct activation of checkpoint during the cell cycle (Li et al., 2003), and betulinic acid is also the same type of compound, with selective melanoma cytotoxicity which control the cell cycle by the activation of p38 (Tan et al., 2003; Cichewicz and Kouzi, 2004; Pisha et al., 1995). According to a review article by (Balunas and Kinghorn, 2005), Four new drugs which have been derived from medicinal plants, and have been introduced recently to the U.S. market (Fig. 1, IIV). The drugs are, Arteether (I, or Artemotil) is an effective antimalarial drug which is derived from artemisinin, which is a sesquiterpene lactone in its class and isolated from Artemisia annua L. (Asteraceae). The plant A. annua are used in traditional Chinese medicine (TCM) (Graul, 2001; van Agtmael et al., 1999;). There are

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many derivatives of artemisinin which are used in Europe in different stages or clinical trials as anti-malarial drugs (Van Agtmael et al., 1999). Galantamine or galanthamine (II, Reminyl) is a also an ethno botanical directed isolated natural product in Russia in the early 1950s, which is first isolated from Galanthus woronowii Losinsk. (Amaryllidaceae) (Pirttila et al., 2004; Heinrich and Teoh, 2004). This compound (Galantamine) is effective in Alzheimers disease and theirfore has been approved for the treatment of Alzheimers disease, it take part in slowing the process of neurological degeneration through inhibiting acetylcholinesterase (AChE) and it also well bind nicotinic acetylcholine receptor (nAChR) and modulating the same. (Pirttila et al., 2004; Heinrich and Teoh, 2004;). An other compound, Nitisinone (III, or Orfadin) is discovered very recently and has been isolated from medicinal plant-derived, it shows a characteristic to control the rare inherited disease, tyrosinaemia, which shows the usefulness of natural products as lead structures (Frantz and Smith, 2003). Nitisinone in actual is the modified form of mesotrione, which is an herbicide based on the natural product leptospermone, isolated from Callistemon citrinus Stapf (Myrtaceae) (Mitchell et al., 2001; Hall et al., 2001b). All these stated three triketones inhibit the same type of enzyme, 4hydroxyphenylpyruvate dehydrogenase (HPPD), while studying in humans and in maize (Mitchell et al., 2001; Hall et al., 2001b). In maize it inhibits the HPPD enzyme which shows an activity as an herbicide by the reduction of tocopherol and plastoquinone biosynthesis. In humans the inhibition of the enzyme HPPD prevents the catabolism of tyrosine and also the toxic byproducts accumulation in the liver and kidneys (Hall et al., 2001b). Tiotropium (IV, Spirival as a trade name\) is another drug which has been released recently to the United States market and has been used for the treatment of chronic obstructive pulmonary disease (COPD) (Frantz, 2005); Mundy and Kirkpatrick, 2004. The drug Tiotroprium which is an inhaled anticholinergic bronchodilator, and ipratropium based, which is a derivative of atropine, isolated from Atropa belladonna L. (Solanaceae)as well as other members of the Solanaceae family (Dewick, 2002; Mundy and Kirkpatrick, 2004; Barnes et al., 1995). Tiotropium is comparatively longer lasting effects while comparing with other available COPD medications (Barnes, 2002; Mundy and Kirkpatrick, 2004).

Chapter 1
H
OH

Introduction

O O O O H OCH2CH3 I Arteether
O N II Galatamine O

NO2

O III Nitisinone

CF3

HO
O

O H O S O

O
O O OH VII Calanolide A O

OH S

HO2C HO HO

N H O O H

IV Tiotropium

OH V M6G or morphine-6-glucuronide

NH2

F F H N H OH

N N VII Exatecan

N H H3CO2C

O HO O

H3CO VI Vinflunine

OAc CO2CH3

Figure 1 Examples of some drugs isolated from medicinal plant.

Compounds V-VII (Fig. 1) which are in Phase III clinical trials or registration and are in process of modifications of drugs that currently in clinical use (Butler, 2004). A metabolite of morphine i.e morphine-6-glucuronide (V) , isolated from Papaver somniferum L. (Papaveraceae), which have very little side effect as compared to morphine and will be used as an alternate pain medication (Lotsch and Geisslinger, 2001). A modified vinblastine i.e. Vinflunine (VI), isolated from Catharanthus roseus (L.) G. 9

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Introduction

Don (Apocynaceae) can be use as an anticancer agent with high efficacy (Bonfil et al., 2002; Okouneva et al., 2003). Exatecan (VII) is developed as an anticancer agent and very close similarity with camptothecin that have been isolated from Camptotheca acuminata Decne. (Nyssaceae (Cragg and Newman, 2004; Butler, 2004). The process of modifications of the existing natural products realizes the importance of drugs that have been discovered from medicinal plants as NCEs and consider the possible new drug leads. The drug, Calanolide A (VIII) is isolated from Malaysian rainforest tree (Calophyllum lanigerum var. austrocoriaceum (Whitmore) P.F. Stevens (Clusiaceae), is a dipyranocoumarin natural product, (Yang et al., 2001; Yu et al., 2003; Kashman et al., 1992). It has been investigated that Calanolide A which shows an anti-HIV drug with a very unique and high specific mechanism of action particularly as a non-nucleoside reverse transcriptase inhibitor (NNRTI) of type-1 HIV and is very high effective against AZT-resistant strains of HIV (Yu et al., 2003; Currens et al., 1996; Buckheit et al., 1999;). The drug Calanolide A is in Phase II clinical trials process (Creagh et al., 2001). 1.5 Secondary metabolites All those organic compounds present in plants and in animals that are not working in the normal growth, development or reproduction of organisms but produced in different metabolic processes. Secondary metabolites are not essential for life as compare to primary metabolites, that the absence of secondary metabolites results not in failure of life, but in long-term impairment of the organism's survivability/fecundity or aesthetics, or perhaps in no significant change at all but it is useful for animals ailments and normalizes the physiological abnormalities produced due to different diseases in animal bodies. Secondary metabolites are often very restricted to a particular set of species within a phylogenetic group. In broad sense secondary metabolites may be classify into; small molecules (alkaloids, terpenoids, glycosides, Phenols and Phenazene), big small molecules (Polyketides, Non ribosomal peptides etc), non small molecules (DNA, RNA, ribosome, polysacharides). 1.5.1 Small molecules 1.5.1.1 Alkaloids Alkaloids are natural product that contains basic nitrogen atoms. The name of alkaloids derives from the alkaline and it was used to describe any nitrogen-containing base. Alkaloids are naturally synthesis by a large numbers of organisms, including animals, plants, bacteria and fungi. Alkaloids are a group of natural products (also called secondary metabolites). Alkaloids can be easily purified from various crude extracts by 10

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Introduction

acid-base extraction. There are very many alkaloids which are toxic to other organisms. They often have some pharmacological effects and are used for the treatment of various diseases and recreational drugs. Some alkaloids are used as the local anesthetic and stimulant as cocaine. Some alkaloids have stimulant property as caffeine and nicotine, morphine are used as the analgesic and quinine as the antimalarial drug. Almost all the alkaloids have a bitter taste. Classification Alkaloids may be classified in different groups on the bases of their structure formulas. Pyridine group: Nicotine alkaloid found in tobacco (Nicotiana tabacum) plant and Anabasine alkaloid found in the tree Tobacco (Nicotiana glauca) plant. Pyrrolidine group: Hygrine found in Erythroxylum coca leaves Tropane group: Atropine alkaloid found in Atropa belladonna and Datura stramonium, Cocaine alkaloid found in Erythroxylum coca leaves. Indolizidine group: one example is Swainsonine that was first obtained from a very small plants like pea (e.g. Swainsona sp. and Astragalus sp). Quinoline group: Quinine alkaloids isolated originally from Cinchona succirubra and Strychnine alkaloids was obtained from the seeds of the Strychnos nux vomica tree. Isoquinoline group: The Opium alkaloids like narcotine, papaverine, narceine, morphine, codeine, and heroine, sanguinarine, hydrastine, alkaloids like berberine, emetine, berbamine, oxyacanthine from Berberis species Phenanthrene alkaloids: Opium alkaloids like morphine, codeine, thebaine are included in this group. Phenethylamine group: Alkaloids found in many members of the Cactaceae like Lophophora williamsii and Echinopsis pachanoi i.e. Mescaline alkaloids etc, and some alkaloids found in Ephedra vulgaris i.e. ephedrine alkaloids etc are included in this group. Indole group: Serotonin is found in the enterochromaffin cells in the gut of animals, but also found in mushrooms and plants, including fruits and vegetables, Vinca alkaloids such as vinblastine, vincristine found in Catharanthus roseus etc. Purine group: Caffeine type of alkaloids are abundant in genus Coffea Coffea canephora (also known as Coffea robusta) and Coffea arabica are two speceis which have been grown for this purpose. Terpenoid group: Aconitum alkaloids such as aconitine, Steroid alkaloids such as alkaloids found in Solanum i.e. solanine, solanidine and chaconine etc. 11

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1.5.1.2 Terpenoids The terpenoids sometimes called isoprenoids, are a class of natural products which are very similar to terpenes, that have been derived from five-carbon isoprene units and can be interchanged in thousands of ways. Most of the terpenoids have multi cyclic structures that differ from one another by their functional groups and basic carbon skeletons. These types of natural lipids can be found in every class of living things, and therefore considered as the largest group of natural products

Classification Terpenoids can be thought of as modified terpenes, where terpenes are hydrocarbons resulting from the combination of several isoprene units. The classification of terpenoids can be made according to the number of isoprene units used. Hemiterpenoids: Consist of a single isoprene unit. The only hemiterpene is the Isoprene itself, but oxygen-containing derivatives of isoprene such as isovaleric acid and prenol is classify as hemiterpenoids. Monoterpenoids: Biochemical modifications of monoterpenes such as oxidation or rearrangement produce the related monoterpenoids. Monoterpenoids have two isoprene units. Monoterpenes may be of two types i.e linear (acyclic) or contain rings e.g. Geranyl pyrophosphate, Eucalyptol, Limonene and Pinene. Sesquiterpenes: Sesquiterpenes have three isoprene units e.g. Farnesyl pyrophosphate, Artemisinin, Bisabolol. Diterpenes: It composed for four isoprene units and have the molecular formula C20H32. They derive from geranylgeranyl pyrophosphate. There are some examples of diterpenes such as cembrene, kahweol, taxadiene and cafestol (precursor of taxol). Retinol, retinal, and phytol are the biologically important compounds while using diterpenes as the base. Theses three compounds are known to be antimicrobial and antiinflammatory. Geranylgeranyl pyrophosphate, Retinol, Retinal, Phytol, Taxol, Forskolin Aphidicolin Sesterterpenoids: Terpenoids having 25 carbons and five isoprene units. Triterpenes: It consist of six isoprene units e.g. squalene found in wheat germ, and olives. Tetraterpenoids: It contain eight isoprene units which may be acyclic like lycopene, monocyclic like gamma-carotene, and bicyclic like alpha- and betacarotenes. 12

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Polyterpenoids: It consists of a larger number of isoprene units.

1.5.1.3 Glycosides It is a group of natural product where a sugar group is directly bonded through its anomeric carbon to another group by an O-glycosidic bond or an S-glycosidic bond. The sugar group is then known as the glycone and the non-sugar group as the aglycone or genin part of the glycoside. The glycone can consist of a single sugar group (monosaccharide) or several sugar groups (oligosaccharide).

Classification Glycosides may be classified in three ways i) Type of glycone: If the glycone group of a glycoside is glucose, then the molecule is a glucoside; if it is fructose, then the molecule is a fructoside; if it is glucuronic acid, then the molecule is a glucuronide; etc. In the body, toxic substances are often bonded to glucuronic acid to increase their water solubility; the resulting glucuronides are then excreted. ii) Type of glycosidic bond: It classified as -glycosides or -glycosides which depending on bong geometry that whether the glycosidic bond lies "below" or "above" the plane of the cyclic sugar molecule. On the bases of this particular geometry some enzymes like -amylase can only hydrolyze -linkages; others, like emulsin, can only affect -linkages iii) Type of aglycone. Glycosides are also classified according to the chemical nature of the aglycone e.g. Alcoholic glycoside: salicin is an example of an alcoholic glycoside is which has isolated from the genus Salix. Salicin is converted to salicylic in the body, which is closely related to aspirin and has analgesic, antipyretic and antiinflammatory effects. Anthraquinone glycosides: They are present in senna, rhubarb and aloes; they have a laxative effect.These glycosides contain an aglycone group that is a derivative of anthraquinone. Coumarine glycosides: Psoralin and corylifolin obtained from dried leaves of Psoralea corylifolia and the aglycone is coumarin. Apterin a coumarine glycosides which is reported to dilate the coronary arteries as well as block calcium channels. Cyanogenic glycoside: The aglycone contains a cyanide group, and the glycoside can release the poisonous hydrogen cyanide if acted upon by 13

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some enzyme. They are stored in the vacuole but if the plant is attacked they are released and become activated by enzymes in the cytoplasm. These remove the sugar part of the molecule and release toxic hydrogen cyanide. Storing them in inactive forms in the cytoplasm prevents them from damaging the plant under normal conditions. An example of these is amygdalin from almonds. They can also be found in the fruits (and wilting leaves) of the rose family (including cherries, apples, plums, almonds, peaches, apricots, raspberries, and crabapples). Flavonoid glycosides: In this type of glycosides the aglycone units are flavonoids e.g. Hesperidin (aglycone: Hesperetin, glycone : Rutinose), Rutin (aglycone: Quercetin, glycone: Rutinose), Querctrin (aglycone: Quercetin, glycone: Rhamnose). Phenolic glycosides: The aglycone is a simple phenolic structure e.g. Arbutin found in Arctostaphylos uva-ursi. Saponin glycosides: The characteristic of saponin glycoside that they normally produce soap-like foaming when shaken in aqueous medium, and structurally saponin gycosides composed of one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. Saponin glycosides are found in liquorice (Glycyrrhiza glabra). Steroidal glycosides: The aglycone part is a steroidal nucleus. e.g. the glycosides of Digitalis, Scilla and Strophanthus. These glycosides are more effective in heart diseases. Steviol glycosides: The glycosides found in Stevia rebaudiana bertoni and about 300 times sweetest than sucrose. e.g. stevioside and rebaudioside A, are used as natural sweeteners in many countries. Thioglycosides: These glycosides contain sulfur e.g. sinigrin and sinalbin found in black and white mustard respectively. 1.5.1.4 Phenols Phenols or Phenolic are hydroxyl group (-OH) containing class of chemical compounds where the (-OH) bonded directly to an aromatic hydrocarbon group. Phenol (C6H5OH) is considered the simplest class of this group of natural compounds. Other examples are Resveratrol, Polyphenols (flavonoids and tannins), Gallic acid, Eugenols etc.

14

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1.5.1.5 Phenazines It is also called azophenylene, dibenzo-p-diazine, dibenzopyrazine, and acridizine, is a dibenzo annulated pyrazine and the parent substance of many dyestuffs, such as the eurhodines, toluylene red, indulines and safranines. Pyocyanin is a toxic blue crystalline pigment C13H10N2O that is formed in the metabolism of a bacterium of the genus Pseudomonas (P. aeruginosa), gives a bluish tint to pus infected with this organism, is a quinone imine related to phenazine, and has antibiotic activity especially toward grampositive bacteria 1.5.2 Big small molecules 1.5.2.1 Polyketides Secondary metabolites from bacteria, fungi, plants, and animals. Polyketides are Like a process of fatty acid that are synthesis from fatty acid, the polyketides are also biosynthesized by the polymerization of propionyl and acetyl subunits. They are also the building blocks for variety of natural products or are further derivatized. Examples are Macrolides: It includes Picromycin, the antibiotics of erthromycin A, Clarithromycin and azithromycin, the immunosuppresent tacrolimus (FK506). Polyene antibiotics: It include Amphotercin which was isolated from Streptomyces nodosus, a filamentous type bacterium and use as antifungal drug. Tetracyclines: The tetracycline family broad-spectrum polyketide antibiotic produced by the Streptomyces genus of Actinobacteria, indicated for use against many bacterial infections. Acetogenins: It include Annonacin found in fruits such as the guanabana and Uvaricin is a bis(tetrahydrofuranoid) fatty acid lactone present in the roots of Uvaria accuminata. 1.5.2.2 Nonribosomal peptides It usually produced by microorganisms like bacteria and fungi. Nonribosomal peptides are also found in higher organisms, such as nudibranchs. Nonribosomal peptides are synthesized by nonribosomal peptide synthetases, which, unlike the ribosomes, are independent of messenger RNA. Example are Vancomycin: It produced from the organism Amycolatopsis orientalis. It is a glycopeptide type antibiotic and used for Gram-positive bacteria produced prophylaxis and treatment of infections. It is very important antibiotic and not 15

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always use, but only in cases where the other antibiotics had failed. It is therefore named as a drug of "last resort". Thiostrepton: Cyclic oligopeptide antibiotic, derived from several strains of strepromycetes, such as Streptomyces azureus and Streptomyces laurentii.

1.6 Technique used in phytochemistry 1.6.1 Chromatography Chromatography is a Greek word mean (chroma, color and graphein to write). The term chromatography is used for a set of laboratory techniques which involve the separation of mixtures. The mixture is dissolved in a solvent or a "mobile phase" which pass through a stationary phase, which separates the different constituent of the solution and allows it to be isolated. Chromatography may be classified as Preparative: This type of chromatography is used when the separated components of a mixture is applied for further use (and is thus a form of purification). Analytical: This type of chromatography is use just for measuring the relative proportions of analytes in a mixture and therefore is done normally with smaller amounts of material. Both preparative and analytical are not mutually exclusive. Classification Chromatographic technique can be classified in two ways i) ii) Techniques by difference in bed shape. Techniques by difference physical state of mobile phase

1.6.1.1 Techniques by difference in bed shape It includes Column chromatography and Planar Chromatography. 1.6.1.1.1 Column chromatography Column chromatography is a separating method which is used to purify every chemical compounds from mixtures of different compounds. This type of chromatography is used for from micrograms up to kilograms of separating samples. In this, a glass tube of different diameter and length are used as column. A glass tube with a diameter from 50 mm and a height of 50 cm to 1 m with a tap at the bottom can be used as a classical preparative chromatography column. Slurry of the eluent with the stationary phase powder is prepared and then carefully poured into the column. A special precaution should be taken in order to avoid air bubbles. The slurry is then pipetted on top of the stationary phase. This layer of slurry is usually protected with a small layer of sand or with cotton or glass wool in order to protect the shape of the separating slurry mixture 16

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from the pouring of newly added eluent or solvent. The eluent is slowly passed through the column by opening the tap to move the component of the slurry of organic compounds. It always useful to use a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column. The stationary phase differently retained the individual components from each other and separates them while they are running at different velocities through the column with the eluent and therefore one compound can be elute at the end of the column at a time. A series of fractions is collected during the entire chromatography process. The composition of the eluent flow can be monitored thoroughly and therefore each fraction is analyzed for dissolved compounds. For this purpose analytical chromatography, UV absorption, or fluorescence technique can be used. Colored compounds (or fluorescent compounds with the help of an UV lamp) can be seen through the column glass wall as moving bands. Column chromatography divided into two phases i.e. Stationary phase or adsorbent and mobile phase or eluent. Stationary phase: The stationary phase or adsorbent is a solid material in column chromatography. Mostly silica gel is used as stationary phase for column chromatography and another is alumina which is second used stationary phase. In the past cellulose powder has often been used. Also possible are affinity chromatography or expanded bed adsorption (EBA) and ion exchange chromatography, reversed-phase chromatography (RP). The finely ground powders or gels are used as the stationary phases and/or are microporous for an increased surface, while in EBA a fluidized bed is used. Mobile Phase: It is either a pure solvent or of different solvents mixture. It is very precisely studied so that the retention factor value of the compound of interest is roughly around 0.2 - 0.3, it can be minimizing the time and the amount of eluent to run the chromatography. The chosen of good eluent system is very important so that the different compounds can be separated easily and effectively. The eluent system is optimized in small scale pretests, in each case often using thin layer chromatography (TLC) providing the same stationary phase. The time required to run a column can be minimizes by maximizes the flow rate of the eluent and thereby minimizes diffusion, which results a better separation, see Van Deemter's equation for assistance. Although there are many technique to maximize the column run rate, for example a simple laboratory column can be runs by gravity flow which can be increased by extending the fresh eluent filled column above the top of the stationary phase or negatively controlled with the tap 17

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controls. A pump can also be used for better achievement of flow rates or compressed gas (e.g. air, nitrogen, or argon) can also be used to push the solvent through the column (flash column chromatography) (Still et al, 1978). A spreadsheet that assists in the successful development of flash columns has been developed. The spreadsheet calculate the retention volume as well as the band volume of analytes, the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted (Fair and Kormos, 2008). 1.6.1.1.2 Planar Chromatography Planar chromatography is also a separation technique in which the stationary phase is a plane or present as a plane. A paper can be used as a plane, which may serves as such or impregnated with stationary bed (paper chromatography), Glass plate can also be used on which a layer of solid particles spread (thin layer chromatography). The traveling of different compounds in the sample mixture travel with different velocities according to how strongly they interact with the stationary phase as compared to the mobile phase. The Retardation factor (Rf), which are very specific for each chemical and can be used to aid in the identification of an unknown substance. Planar Chromatography divided into paper chromatographic and thin layer chromatography.

1.6.1.1.2.1 Paper chromatography The technique of paper chromatography is very simple in which a small dot or line of sample solution placed onto a strip of chromatography paper. There is a jar containing a shallow layer of solvent in which the chromatography paper placed and sealed the jar. The solvent rises through the capillary action of the paper, it reach the sample mixture which starts and travel along with the solvent toward the upper side of the paper. As the paper is made of cellulose which is a polar substance, and the compounds within the mixture travel farther in case if they are non-polar. While the polar substances bond with the cellulose paper more strongly and therefore do not travel as far. 1.6.1.1.2.2 Thin layer chromatography Thin layer chromatography (TLC) is very important technique for qualitative study in both small and large scale and therefore widely-employed laboratory technique and it is very closely related with paper chromatography. The only difference between thin layer and paper chromatography is to used a stationary phase of a thin layer of adsorbent like 18

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Introduction

silica gel, alumina, or cellulose on a flat, inert substrate while in the other paper are used as stationary phase. The TLC as compared to paper has the advantage of faster runs rate, better separations of the component, and the choice between different adsorbents. 1.6.1.2 Techniques by physical state of mobile phase 1.6.1.2.1 Gas chromatography The Gas chromatography (GC), or in other words Gas-Liquid chromatography, (GLC), is also a separation technique in which gas is use as the mobile phase. Gas chromatography is always carried out in a particular type of column, which is typically "packed" or "capillary. Stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium) are used in Gas chromatography (GC). Partition equilibrium of analyte is based on both stationary and mobile phase. The material of stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). Such system is always used for in analytical chemistry. GC due to its high temperature unsuitable for high molecular weight biopolymers or proteins (because heat denature protein molecule), frequently encountered in biochemistry. Such type of chromatography is well suited for use in industrial chemical, the petrochemical, environmental monitoring. GC is very important technique and largely used in chemistry research.

1.6.1.2.2 Liquid chromatography Liquid chromatography (LC) is another separation technique for organic compounds in which the mobile phase is always a liquid. Liquid chromatography can be performed both in a column or a plane. In the recent research liquid chromatography that generally utilizes very small packing particles along with a relatively high pressure, such technique is named as high performance liquid chromatography (HPLC). In order to use the HPLC technique, the sample is accelerated by a liquid (mobile phase) at high pressure through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase). HPLC is further divided into two different sub-classes which are based on both the polarity of the mobile and stationary phases. Such GC technique in which the mobile phase is less polar than stationary phase (e.g. toluene use as the mobile phase, and silica use as the stationary phase) is known as normal phase liquid chromatography (NPLC), while in cases where the mobile phase is polar than stationary phase (e.g. water-methanol mixture use as the mobile phase and C18 = octadecylsilyl use as the stationary phase) is known as reversed phase liquid 19

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chromatography (RPLC). It has been known that the "normal phase" has very few applications as compared to RPLC which has been used considerably more. Such technique in which no pressure is used to accelerate the mobile phase through the stationary phase are named as fast protein liquid chromatography which come under the broad heading of chromatography. The above mentioned chromatographic techniques are always used in phytochemistry research. There are different other chromatographic techniques are also used e.g., Supercritical fluid chromatography, Affinity chromatography, Size exclusion chromatography, Chiral chromatography, Ion exchange chromatography, Countercurrent chromatography etc. 1.6.2 Capillary electrophoresis Capillary electrophoresis (CE) introduced in the 1960s. As shown by its name the Capillary electrophoresis (CE) or capillary zone electrophoresis (CZE), very small and thin capillary tube can be used to separate ionic species by their charge and frictional forces. In ordinary electrophoresis, electrically charged analytes move under the influence of an electric field while using a conductive liquid medium. The technique of capillary electrophoresis (CE) was designed under the principal of separating species that are based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. 1.6.3 Spectroscopic Techniques 1.6.3.1 NMR spectroscopy Nuclear magnetic resonance spectroscopy or which is also known as NMR spectroscopy, which has been named due to which the magnetic properties of certain nuclei used in this technique. The principal and its origins of NMR spectroscopy are detailed in a separate section. Both proton NMR and carbon-13 NMR spectroscopy are important applications for the organic chemist. In principle, NMR is applicable to that entire nucleus which possessing spin. NMR spectrum gives us many types of information. Functional groups can be determined by using infrared spectroscopy similarly analysis of a 1D NMR spectrum gives information on the type and number of chemical entities which is present in a molecule. However, NMR is much useful as compared to IR because a lot of information obtained from NMR. NMR can be applied to a wide variety of samples, both in the solution and the solid state. Therefore its impact on the natural sciences has been substantial. NMR is also used to the mixtures of analytes. It can also be used to understand the dynamic effects like 20

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temperature and reaction mechanism and can also provide useful information regarding protein and nucleic acid structure and function. 1.6.3.2 Two-Dimensional Nuclear Magnetic Resonance Spectroscopy (2DNMR) Two-dimensional NMR is useful as compared to one-dimensional NMR because the two dimensional spectra provide more information than one dimensional spectra about a molecule and are gives a detail information regarding the structure of a molecule, particularly in case of molecules that are too complicated to work with using onedimensional NMR. It has also known that Jean Jeener first proposed the first twodimensional experiment, COSY, in 1971, who was a professor at Universit Libre de Bruxelle. This work of Jean Jeener was further studied by Walter P. Aue, Enrico Bartholdi and Richard R. Ernst, who published their work in 1976 (Martin and Zekter, 1988). There are other types of two-dimensional NMR such as exchange spectroscopy (EXSY), J-spectroscopy, Nuclear Overhauser effect spectroscopy (NOESY), total correlation spectroscopy (TOCSY) and heteronuclear correlation experiments, such as HMBC, HMQC, and HSQC. 1.6.3.3 Infrared Spectroscopy Infrared spectroscopy (IR spectroscopy) is also a part of spectroscopy that studies the infrared region of the electromagnetic spectrum. There are different techniques which are related with IR spectroscopy, the most common one is absorption spectroscopy. As with all other spectroscopic techniques, it can also be useful in identifying compounds or examination of sample composition. Infrared spectroscopy related tables are easily available in literature. Uses and applications Applications of infrared spectroscopy for both organic and inorganic chemistry have been highly successful (Lau, 1999). The applications of IR spectroscopy in the field of semiconductor microelectronics are much beneficial. IR spectroscopy is useful in both research and industry as very reliable and simple technique for dynamic measurement, quality control and measurement. IR spectroscopy is useful technique in forensic analysis for both criminal and civil cases and also useful to find out the degree of polymerization in polymer synthesis. Due to the development in the instruments the infrared measurements became easy across the whole range of interest as fast as 32 times a second. IR spectroscopy techniques have been developed to analyze the quality of tealeaves. It has been understood that a well trained manpower can be used more sparingly, at a significant cost saving (Luypaert et al., 2003). 21

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1.6.3.4 Fourier transform infrared spectroscopy Fourier transform infrared (FTIR) spectroscopy is form of IR spectroscopy and it is measurement technique for collecting infrared spectra. Instead of recording the intensity of energy absorbed when the frequency of the infra-red light is non constant (monochromator), the infra red light is guided through an interferometer. After passing through the sample under investigation, the measured signal is the interferogram. Performing a mathematical Fourier transform on this signal results in a spectrum identical to that from conventional (dispersive) infrared spectroscopy. FTIR spectrometers are very cheaper than other conventional spectrometers because building of interferometers is very easier as compared to the fabrication of a monochromator. It has been noted that that the measurement of a single spectrum is much faster for the FTIR technique due to simultaneous collection of the information at all frequencies. These are the usefulness of the multiple samples to be collected and calculated the averaged together which results an improvement in sensitivity. Due to the various advantages of FTIR, virtually all latest infrared spectrometers are FTIR instruments 1.6.3.5 Ultraviolet-visible spectroscopy UV-visible spectroscopy or in other words ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) related to the spectroscopy of photons in the UV-visible region. UV-visible spectroscopy uses light in the visible ranges or its adjacent ranges i.e. near ultraviolet (UV) and near infrared (NIR) ranges. The color of the chemicals involved is directly affects the absorption in the visible ranges. Molecules undergo electronic transitions in these ranges of the electromagnetic spectrum. This technique apposite the fluorescence spectroscopy, in that fluorescence involved with transitions of molecule from the excited state to the ground state, while in UV-visible spectroscopy the absorption measures transitions from the ground state to the excited state.( Skoog, et al., 2007). Application UV/Visible spectroscopy is widely used in the quantitative analysis of transition metal ions and highly conjugated organic compounds solutions. It has also been used for the detector for HPLC. The presence and absence of an analyte gives an indication which can be considered to be proportional to the concentration. For perfect results, the instrument's indication about an analyte in the unknown should be compared with the indication of a standard; this is identical to the use of calibration curves. The response or indications

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(e.g., peak height) for a particular amount of concentration is known as the response factor. 1.6.4. Liquid chromatography-mass spectrometry Both liquid chromatography-mass spectrometry (LC-MS), or alternatively HPLC-MS) is one of the technique that extensively used in analytical chemistry. It combines both the physical separation capabilities of liquid chromatography and HPLC with the mass analysis capabilities of mass spectrometry. There are many applications of LC-MS which is much sensitive and specific. In the presence of other chemicals, one can determine the specific one because its application is oriented towards the specific detection and potential identification. Applications LC-MS is widely used in the field of drug development at many different stages including Glycoprotein Mapping, Natural Products Dereplication, Peptide Mapping, Bioaffinity Screening, In Vivo Drug Screening, Metabolic Stability determination, Metabolite Identification, Impurity Identification and quantification, Degradant Identification, Quantitative Bioanalysis, and in field of Quality Control. LC-MS also used in pharmacokinetic studies of pharmaceuticals. On the basis of these studies one can understand how quickly a drug will be cleared from the hepatic blood flow, and other organs of the body. Due to high sensitivity and short analysis time MS is used for this and exceptional specificity compared to UV (as long as the analyte can be suitably ionised). 1.6.5. Gas chromatography-mass spectrometry (GC-MS) The combines features of gas-liquid chromatography and mass spectrometry are combined in Gas chromatography-mass spectrometry (GC-MS to identify different substances within a test sample. GC-MS have different application which includes drug detection, fire investigation, environmental studies, explosives detection, and determination of unknown samples. Airport security can also be used the GC/MS to identify substances in both luggage and human beings. GC/MS can also identify trace elements in materials that were far away of investigation previously and thought to have disintegrated beyond identification. The GC-MS is used to perform a specific test, it is therefore considered as a "gold standard" for forensic substance identification. It has also been used to identify a particular substance in a given sample. A non-specific test only shows that a substance falls into a category of substances. Although a non-specific test could statistically recommend the identity of the substance, this could lead to false positive identification. 23

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1.7 Development of Anticancer agents from Medicinal plants In order to develop new and clinically useful anticancer agents, both the sample sources and bioassay screening systems are highly important. There are tow methods which have been regarding screening methods i.e. mechanism of action (MOA)-based and cell-based method. There are different cell cultures which are use in preliminary screening for anticancer activity. Different screening techniques against a panel of human cancer cell lines are implemented in order to develop active cancer agents against different types of cancer. All those compounds that are successful in the in vitro studies are then further tested for efficacy through in vivo xenograft studies. In the present scenario of research in the field of anticancer drugs new MOA-based bioassay systems which are aimed at particular molecular targets have also revolutionized the discovery of potential drug candidates. There are different cell proteins which have been targeted by the anticancer drugs; the protein includes DNA topoisomerases I and II, cyclin dependent kinases (CDKs), growth and transcription factors, etc. In order to consider sample sources, many effective, clinically useful anticancer drugs are obtained from the higher plants. Some examples are the compounds such as Vinca alkaloids, diterpenes from Taxus, alkaloids of Camptotheca, and lignans of Podophyllum. There are also some modified related compounds. There a number of extensive reviews on research in anticancer drugs (Suffness & Douros, 1982; Itokawa, 1988; Lee, 1993; Itokawa et al., 1999, 2000, 2006; Tang et al., 2003a, 2003b; Lee., 2004, Mukherjee et al., 2001, Cragg & Newman., 2004). The reviews that describing the influential discoveries and development of taxol, which is a tubulin-interactive and camptothecin, which is topo I-interactive, by Wall and Wani illustrate that how natural products have influenced the further development of natural product-derived and synthetic entities (Cragg & Newman., 2004, Wall & Wani., 1996, Oberlies & Kroll., 2004). The terminology of cancer has often varied (Suffness and Douros, 1982) and they recommended the following definitions to avoid confusion. The word cytotoxicity is used when extracts or compounds contain activity against tumor cell lines and the word antitumor or antineoplastic are used when the materials shows activity in vivo in experimental systems, and the word anticancer used to extracts or compounds that are clinically active against human cancer. 1.8 Development of cancer The people of developing countries are more killed by cancer each year than AIDS, tuberculosis or malaria and it has been confirmed in 2008 that more than 12 million new cases of cancer were diagnosed world wide. Out of 12 millions 7.6 million deaths have 24

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been occurred. The percentage is more in developing countries i.e. 60 percent and it has been calculated that more than half of all new cases and fatalities occurred in developing countries. Due to poverty in development countries the poor medical infrastructure often means that cancer is a sure-fire death sentence. The rates of survival from cancer in developing countries are exceptionally poor. Most people do not seek medical help until their disease is advanced and incurable; it is due to lack of awareness, stigma and reliance on traditional healers mean. Cancer diseases are after cardiovascular diseases the second common cause of death. Because of the dramatic development, cancer research has give rise to a rich and complex body of knowledge. The primarily step was set in the discovery of mutations in proto-oncogenes that produce oncogenes with dominant gain of function (Cyclin D1 and Cdc25A described below and tumour suppressor genes with recessive loss of function (p53 and RB describe below) (Bishop and Weinberg, 1996). This first mutation of these degenerated cells helps them to get an advantage in proliferation and progression compared to normal cells. Hanahan and Weinberg published few years ago The Hallmarks of Cancer (figure 2). In this review they described six different capabilities which each cell needs to degenerate in a malignant cancer cell. 1.8.1 Self-sufficiency in growth signals Self-sufficiency in growth signals was the first step which was clearly defined by cancer researchers. Normally cells required growth signals to move from G0/G1 state of cell cycle into an active proliferation state. These signals are found for example in the extracellular matrix and are transmitted into the cell by transmembrane receptors. In absence of these signals a normal cell and their receptors cannot start the proliferation machine, but many oncogenes mimic these growth signals and initiate cell cycle on their own. For example the epidermal growth factor receptor (EGFR) is upregulated in stomach, brain and breast tumours. This liberation from dependence on exogenously derived signals disrupts a critically important homeostatic mechanism that normally operates to ensure a proper behaviour of various cell types within a tissue (Hanahan and Weinberg 2000).

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Figure 2. Acquired capabilities of cancer

Legend figure 2: Acquired capabilities of cancer. Most of cancer types have acquired the same or near the same set of functional capabilities during their development (Hanahan and Weinberg, 2000). 1.8.2 Insensitivity to antigrowth signals To assure the tissue homeostasis, many signals are known, which stop the proliferation of cells. These antigrowth signals are like their antagonists localised in the extracellular matrix and on the surface of nearby cells. The growth inhibitory signals (p53 and RB) can stop proliferation via two different ways. First the cells may be forced out of the active cell cycle into the G0 state, or second they may be induced to permanently dismiss the possibility to proliferate (neurons as example). Loss of these growth inhibitory signals results in hyper proliferation of cells, especially degenerated cells, and further on cancer development (Hanahan and Weinberg, 2000). 2.8.3 Evading apoptosis Normal cells have a limited rate of cell cycles and afterwards these cells start the programmed cell death (apoptosis). The apoptotic machinery can be divided into sensors and effectors, the two classes of components. The sensors are proper for monitoring the extracellular and intracellular room for conditions of normality and abnormality, which influence the future of the cell, to stay alive or to die. Therefore, intracellular sensors monitor the cells well-being and activate the death pathway in response to detecting abnormalities, including DNA damage, signalling imbalance induced by oncogene action, 26

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survival factor insufficiency, or hypoxia (Evan et al., 1998). The effectors are regulated by these sensors and could start, if necessary, the apoptotic machine. This hallmark has a profound consequence, because until this step, degenerated cells could be disposed and eliminated via the programmed cell death and the homeostasis is assured, but loss of this function is another step for cancer development. 1.8.4 Limitless replicative potential All hitherto described capabilities together lead to an uncoupling of a cells growth program from signals in its environment. After completing of a certain number of doublings, they stop growing. Cancer cells have the ability to overcome this. They get immortalized and thats a big advantage compared to normal cells (Hanahan and Weinberg, 2000) 1.8.5 Sustained angiogenesis Nutrients and other important substances are supplied by vasculature and they are crucial for the function and survival of cells and tissue. Cells which are within 100 m of a capillary blood vessel get nourish by this vessel. Because of this dependence on nearby capillaries, cells within a tissue would have an intrinsic ability to encourage blood vessel growth. To growth exuberantly and progress a lager size of tumor, cancer cells need this ability to sustain angiogenesis. The two different types of angiogenesis initiating signals are vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Each of these factors binds to transmembrane tyrosine kinase receptors displayed by endothelial cells (Fedi et al., 1997). The ability to induce and sustain angiogenesis seems to be acquired in a discrete step during tumour development, via an angiogenic switch from vascular quiescence (Hanahan and Weinberg, 2000). 1.8.6 Tissue invasion and metastasis Last but not least, the primary tumor masses start to spawn pioneer cells that move out of the tumor, invade in other normal tissues and develop new colonies. This type of pioneer cells is called metastase. This is the last step of tumor development; the metastasis enables cancer cells to escape the primary tumor mass and colonize new terrain in the body, where initially, nutrients and space are not limiting. For this step the degenerated cells need a mutation and further an over-expression of cell-cell adhesion molecules (CAMs); mostly an increasing level of E-cadherin was found in cancer, which is ubiquitously expressed in endothelial cells. The function of E - cadherin is lost in a majority of epithelial cancer. Only three of these described mutations are enough to develop a tumor. But normally the 27

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development of cancer is a long term disease. In the last years the increasing awareness of patients help to detect a lot of early stage cancer by the preventive medical backup, and treatment with drugs facilitates in many cases a longer life. But there is one big problem, after long term treatment cancer cells start to get resistant against the drugs and research aims to develop new pharmaceuticals to combat chemo resistant cancer cells. 1.8.7 The cell cycle The Cell cycle in most of the cells includes four coordinated and controlled steps, G0/G1phase, S-phase, G2-phase and M-phase, which is classified in mitosis and cytokinesis. Between this steps are three checkpoints, which protect cells of uncontrolled cell cycle or replication of damaged DNA. The checkpoints are between G1/S-phase, G2/M-phase and last but not least the spindle checkpoint during the M - phase. Only if each step is duly completed the cell could transcend checkpoints. This time between transcend the next phase will be used to repair the mismatch in the cell. The progression of the cell cycle is catalyzed by cyclin and cyclin-dependentkinases (Cdk) in mammalian and cell division control (Cdc) in yeast. In humans, we find six different cyclins, cyclin A, B1, B2, D1-D3, and E. Only D-cyclins are represented in all phases, all others are fixed in one or two cell cycle steps. The degradation of cyclins is assured by proteolysis. Cdks are divided in Cdk1, Cdk2, Cdk4, Cdk5 and Cdk6. The catalytic unit of Cdks is activated when they are associated with the regulatory unit of cyclins. Increasing activity of Cdks is given when a conservative threonine rest (position 160 in Cdk1) is phosphorylated. On the other site decreases the activity of Cdks when tyrosine- and threonine rests (Thr15 in Cdk1) are phosphorylated. The Cyclin/Cdk complexes can be inhibited by cyclin dependent kinase inhibitors (CKI). In mammalian cells are two different families of CKIs, Cdk interacting protein (CIP) and polypeptide inhibitors of Cdk4 and Cdk6 (INK4). CIP family inhibits complexes between Cdk2, Cdk4 and Cdk6 with cyclins respectively. The most important member of the CIP family is p21CIP. The expression of p21CIP is stimulated by the tumor suppressor gene p53 and p21C I P is also present during S-phase and has the possibility to inhibit the DNA replication. The members of the second family are p16INK4a, p15INK4b, p18INK4c and p19INK4d. These inhibitors are tissue dependent and control only Cdk4 and Cdk6. In summary, there are three different ways to control cyclin/Cdk complexes. a) proteolysis of cyclins, b) phosphorylation of tyrosine and threonine rests and c) CKI. Cdks, cyclins and CKIs are mediators between extracellular signals and the state of phosphorylation of the tumor suppressor gene retinoblastoma protein (RB). The activation of Cdks in the G1- and S-phase of cell cycle involves phosphorylation of RB. 28

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This phosphorylation releases the inactivation of RB. The state of RB phosphorylation determines the future of the cell: proliferation, differentiation, or cell death via apoptosis. 1.8.7.1 Cell cycle phases (short summary) 1. G1-phase is called the phase between interphase and S-phase. This step of cell cycle is marked by synthesis of various enzymes that are required in S-phase, mainly those needed for DNA replication (RNAs, proteins). In this step the DNA is not duplicated yet (2n DNA). 2. S-phase involves synthesis and replication of DNA (2n DNA 4n DNA) 3. G2-phase is responsible for significant protein synthesis, mainly involving the production of microtubules, which are required during the process of mitosis. Inhibition of protein synthesis during G2-phase prevents the cell from undergoing mitosis. 4. M-phase is classified in mitosis and cytokinesis. a. Mitosis: division of duplicated chromosomes to the pole regions b. Cytokinesis: the cells cytoplasm divides forming distinct cells 1.8.7.2 Presence of cyclins and Cdks during single phases The first cyclins, which are detectable after mitosis, are D-type-cyclins. The regulatory relevance of cyclin D1 is limited to the G1-phase of cell cycle, but if cyclin D1 is upregulated, cell cycle starts autonomously and cannot be stopped. This capability defines cyclin D1 as a proto-oncogene. The preferred binding partner of cyclin D1 is Cdk4 and 6. Subsequently, in the late G1- and early S-phase cyclin E associates with Cdk2. During the S-phase cyclin E will be replaced by cyclin A. In G2- and M-phase, Cdk1 in combination with cyclin A or B is predominant (figure 3).

Figure 3 Cyclin and Cdks distribution during the cell cycle 29

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1.8.8 Function and activation of (proto)-oncogenes/oncogenes 1.8.8.1 Oncogenes Genes, which can potentially induce neoplastic transformation. They include genes coding for growth factors, growth factor receptors, protein kinases, phosphatases, nuclear phosphoproteins, and transcription factors. When these genes are constitutively expressed after structural and or regulatory changes, uncontrolled cell proliferation may result I just want to refer to two proto-oncogenes, which are crucial in this work, the cyclin D1 and a member of the Cdc25 family, Cdc25A.

1.8.8.2 Cyclin D1 Cyclin D1 is known as an important proto-oncogene. It regulates the transition between G1- to S-phase (as described above). The binding of cyclin D1 to its kinase partners (Cdk4/6) results in the active complexes, which phosphorylate RB. Hyperphosphorylation of RB causes the release of E2F transcription factor and furthermore the expression of genes, which are required for entry into S-phase (Alao et al., 2006). Normally, protein levels of cyclin D1 begin to increase in the early G1-phase of cell cycle and after transition into S-phase cyclin D1 is translocated from nucleus into the cytoplasm and degraded. Continuous over expression of cyclin D1 results in uncontrolled cell proliferation and may cause cancer. Cyclin D1 is over-expressed in many types of cancer, mantle cell lymphoma, prostate and breast cancer respectively. Inostamycin is an effective cytostatic drug against cyclin D1 over expression (Baba et al., 2004). 1.8.8 3 Cdc25A (Cell-division-cycle 25A) Cdc25A is a member of the Cdc25 family. Cdc25 was first described in Schizosaccharomyces pombe fission yeast. In mammals the Cdc25 family includes three types of Cdc25 homologs, Cdc25A, Cdc25B and Cdc25C. All members are essential and specific for cell cycle. The mRNA of Cdc25C is predominantly expressed in G2/M-phase, Cdc25B is expressed throughout the cell cycle and is elevated in G2-phase. In addition, the mRNA of Cdc25A is expressed throughout the cell cycle, however with peak expression during late G1- and S-phases (Ducruet et al., 1997). The role of Cdc25A in initiation of DNA replication is also consistent with the ubiquitin proteasome-mediated destruction of Cdc25A in G1/S-phase checkpoint responses to DNA damage and replication stress. This requires Chk1 or Chk2 (Checkpoint kinase 1/2) mediated phosphorylation of Cdc25A, and phosphorylation of Ser123, Ser75 and Ser177 are a prerequisite for such accelerated ubiquitylation and degradation. Consequently, the 30

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absence of Cdc25A in damaged cells precludes dephosphorylation of Thr14 and Tyr15 of Cdk2, and locks this essential S-phase promoting kinase in its inactive form (Mailand et al., 2000; Falck et al., 2001). New studies showed that Cdc25A is also stabilized in G2/M-phase of cell cycle and could abrogate the G2 checkpoint (Mailand et al. 2002). These findings mark Cdc25A as a potential proto-oncogene, because when Cdc25A is over expressed and phosphorylated on the activating Ser17, all checkpoints in the cell cycle is crossed. 1.8.8 4 Function and activation of tumor suppressor genes A gene that encodes a product that negatively regulates the cell cycle and must be inactivated or degraded before a cell can proceed division. Tumor suppressor genes encode proteins, which normally suppress cell proliferation. Mutations, which decrease their activity, may cause cancer. Examples for tumor suppressor genes are differentiation factors, signal transductions proteins and negative cell cycle regulators (Leisser, 2004). 1.8.8 5 p53 (protein 53) p53 a nuclear protein is the most relevant tumor suppressor gene in mammalian with mutation in 50 % of all cancers. The name of p53 is based on the molecular weight. The loss of p53 causes a proliferation advantage. Normally, cells have low p53 levels. The level of p53 in cells increases when the cells are exposed to UV-light or other DNA damaging treatments (Fig. 4). 1.8.8 6 Activation of p53 Activation of p53 has two different consequences, stop of proliferation or apoptosis. The consequence depends on the state of cell cycle. If the activation of p53 is in the late G1phase, p21CIP will be induced by p53 and blocks the cycle, until the DNA damage is repaired, but if the cell already passes the S-phase, p53 induces programmed cell death apoptosis. 1.8.8 7 P21CIP (protein 21) P21CIP as described in before is known as a Cdk-inhibitor and is induced by both p53dependent and -independent mechanisms following stress. Induction of p21CIP may cause cell cycle arrest (Cayrol et al., 1998). Increased expression of p21CIP inhibits the activity of Cdk2-cyclin E complexes, as well as other Cdk/cyclin complexes, while p16INK4a specifically sequesters and inactivates Cdk4/6 complexes. Inactivation of these cyclin-Cdk complexes prevents phosphorylation of RB protein, which is necessary for progression from G1- to S-phase of the cell cycle (Taylor et al., 2004). 31

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Cell cycle blocked by activation of p21 via p53, and re-entry cell cycle after repairing DNA damage.

Induced programmed cell death apoptosis, cell died.

Figure 4. DNA damage induced by UV-light and further the activation of p53. 1.8.8 8 Activation of p21CIP After DNA damage caused by UV-light or chemical agents (Doxorubicin) p 2 1
C I P

is

activated via p53 (Wood et al., 2006). p21CIP inhibits Cdk2-cyclin E complexes which are necessary to cross the G1 block. Hence p21 C I P has the potential to stop the cell cycle in G1 until the DNA damage is repaired. 1.8.8.9 RB RB protein was first found in a retina tumor, which occurs at early age. The onset of this disease could be heritable or spontaneous due to a mutation in the RB gene (position 13q14). To manifest a retinal tumor both alleles must be mutated for breakout. The loss of functional RB could cause many other types of cancer (osteosarcomas and small platelet lung carcinomas respectively). 1.8.8.10 Activation of RB RB is like p53 a nuclear protein which negatively regulates the cell cycle. In non-active or arrested cells (G0/G1) RB is hypo-phosphorylated. At the end of G1-phase RB becomes hyper-phosphorylated by Cdk/cyclin complex and returns to a hypophosphorylated state during mitosis. Only in a hyper-phosphorylated state RB binds to specific proteins. This interaction happens during the S-phase of the cell cycle. Target genes of RB are the E2F family (transcription factor). Because of this binding to RB the 32

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E2F genes are blocked and cannot activate transcription. An over-expression of RB inhibits cell proliferation, but this effect could be abolished by over-expression of D cyclins. They build complexes with Cdks and this combination is responsible for phosphorylation of RB. In summary, dephosphorylated RB blocks cell proliferation and its activation must be abolished to assure transit through the cell cycle. This is ensured by cyclic phosphorylation (Levin, 1998) 1.8.9 Cell death Cell death is one of the fundamental cell regulatory system, which graduates the tissue integrity, and in series the homeostasis of tissues. In general we distinguish between two different types of cell death - the programmed cell death that includes apoptosis (type I) and autophagy (type II) in contrast to necrosis which introduce inflammation of the tissue (figure 18). 1.8.9.1 Apoptosis Apoptosis is the main type of programmed cell death and is mediated by an intracellular program. It was first described by John Kerr in the late 1960s (O' Rourke and Ellem 2000). A series of metabolic events lead to morphological differentiation of cells, including blebbing, changes to cell membrane, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation. The cells which are eliminated by apoptosis can be classified in different categories (Wagner,1999). I. Cells without function; in fact apoptosis is essential for human embryonic development. In example, the differentiation of fingers and toes in a developing human embryo occurs because cells between the fingers undergo programmed cell death - apoptosis. II. Cells which are produced in excess; in this category are for example blood cells and male gametes. III. Cells which have already fulfil their functions; for example cells of the endometrium. The cells will be eliminated by apoptosis during the premenstrual and menstrual cycles. Cells which could impair the cell integrity with their negative potential; it is qualitative and quantitative the most important function of apoptosis. One example are the auto reactive T-lymphocytes. They are negatively selected and eliminated in the thymus. Last but not least, apoptosis is an important mechanism to innoxious tumour cells Each cell 33

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has the potential to start the biochemical machine of apoptosis. This program is classified in 4 different steps: I. Determination and activation of apoptosis: The first step of the program is dependent on two different categories of factors. One category of factor releases (positive signals), and the second one inhibits (negative signals) the apoptosis program. Positive signals are Fas-Fas-ligand and the TNF-induced (tumour necrosis factor) model and negative signals are all growth factors, human growth hormones and cell adhesion. If there is an imbalance between these antagonists, the activation or inactivation of apoptosis starts. II. Execution of cell death Four genes are necessary in this step. Three of this group are cell death genes (ced) and the fourth is called egg-laying defective 1 (egl-1). Egl-1, ced-3 and ced4 are essential to perform the cell death program. If one of these genes is deactivated the cell death program stops. The last ced gene is ced-9. This four genes interact with themselves (elg-1 inhibits ced-9, ced-9 inhibits ced-4, ced-4 activates ced-3). III. Phagocytosis When cells undergo final stages of apoptosis they display phagocytotic molecules on their cell surface (phosphatidylserine). These molecules mark the apoptotic bodies of cells for phagocytosis. The removal of the apoptotic bodies by phagocytes release no inflammation of the tissue. IV. Degradation After phagocytosis cell remnants will be removed by proteases and Ca++ dependent endonucleases. 1.8.9.2 Autophagy Autophagy is a catabolic process where the cells start degradation of their own components through the lysosomal machinery. It was first described in 1960. This kind of programmed cell death is strictly regulated and is important for developing, cell growth and homeostasis. Autophagy plays a role in some diseases to protect the organism against infections by intracellular pathogens. In higher eukaryotes autophagic dysfunction has 34

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been associated with heart disease, neurodegenerative disorders and tumour progression (Leisser, 2004). 1.8.9.3 Necroses Necrosis is definitely a non-programmed cell death. Necrotive cells start swelling, chromatin becomes digested at random, plasma membranes and organelle membranes become disrupted and finally cells lyse. This type of cell death effects injury and provokes an inflammatory response.

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2. Cell shrinkage, and nucleus condensation

2. Cell starts swelling and theorganellesare damaged.

3. Nucleus fragmentation and apoptotic bodies.

3. Cell lysis, the organelles and building the chromatin are destroyed.

Phagocytosis and no inflammation

Phagocytosis and inflammation

Figure 5. Mechanism of Apoptosis

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1.9 Bioassays Techniques 1.9.1 Apoptosis assays (Hoechst 33258 propidium iodide (HOPI) double-staining) Grusch et al, (2002) first described this method of apoptosis, in which propidium iodide (PI) and Hoechst 33258 are directly added to the culture medium to final concentrations of 2 mg/ml and 5 mg/ml, respectively. After one h incubation period at 37C, the cells are then examined under a Zeiss Axiovert 35 fluorescence microscope with DAPI filters. The cells were then photographed on Kodak Ektachrome P1600 film (Eastman Kodak Company, Rochester, NY, USA) and the three type of cells i.e. viable, apoptotic, and necrotic, were counted manually. The Hoechst dye will stains the nuclei of all cells and therefore it became clear and easily monitor nuclear changes associated with apoptosis, such as nuclear fragmentation and chromatin condensation. On the other hand PI is excluded from live and early apoptotic cells and the uptake of PI by necrotic and late apoptotic cells indicates loss of its membrane integrity characteristic. The selective uptake of the two dyes along with the combination of fluorescence microscopy, help the monitoring of the induction of apoptosis in intact cultures and also to distinguish it from non-apoptotic cell death (necrosis). Necrosis is therefore characterized by nuclear PI uptake without nuclear fragmentation or chromatin condensation.

1.9.2 Western blot assay W. Neal Burnette (Burnette) first gave the name of western blot to the technique. The western blot is also name as protein immunoblot. It is an analytical technique which is widely used to identify specific proteins both qualitatively and quantitatively in a given sample of extract or tissue homogenate. Gel electrophoresis is first use to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3D structure of the protein (native/ non-denaturing conditions). After separation of the proteins, it then transferred to nitrocellulose or PVDF membrane. The membrane are then probed (detected) with specific antibodies in order to identify the target protein (Towbin et al., 1979; Renart et al., 1979). The method of Western blotting is widely used in different biological fields such as molecular biology, biochemistry, immunogenetics and other molecular biology disciplines

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1.9.2.1 Steps in a western blot Tissue preparation: whole tissue or cell culture is used for extraction. In case of tissue, blender is used in case of larger sample volume in order to broken down the solid tissues and a homogenizer or sonication is used in case of smaller volume for breaking the tissue. The above mention method can be used in case of cell culture. Lyses of cells and solubilization of proteins can be encouraged by employing of assorted detergents, salts, and buffers. In order to prevent the protein from the digestion by its own enzymes, Protease and phosphatase inhibitors are often used. At the time of protein and tissue preparation the temperature should be very low or work should be completed in ice in order to avoid protein denaturing. After completion the lyses process centrifugation can be made in order to separate different organelles and cell compartments. Gel electrophoresis: Gel electrophoresis is a separation technique which used in biology for protein separation. Proteins separation is based on isoelectric point (pI), electric charge, molecular weight, or a combination of all these factors. Both treatment of the sample and the nature of the gel are responsible for the nature of the separation. Polyacrylamide gels along with buffers loaded with sodium dodecyl sulfate (SDS) are used in common type of gel electrophoresis. Once the protein have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]), it has the characteristic of SDS-PAGE (SDS polyacrylamide gel electrophoresis) is to maintains polypeptides in its denatured state and thus it became easy in separation of proteins by their molecular weight. The protein of sampled become covered in the negatively charged SDS and move through the acrylamide mesh of the gel to the positively charged electrode. The protein thus migrate through this mesh and hence smaller proteins migrate faster are thus separated according to size (usually measured in kilo Daltons, kDa). The resolution of the gel depends on the concentration of acrylamide- better resolution of lower molecular weight proteins obtain with greater acrylamide concentration. The resolution of higher molecular weight proteins will be better when the acrylamide concentration lower. In most blots proteins travel only in one dimension along the gel. Different samples are loaded one by one into wells in the gel. One lane of the gel is always reserved for a marker or ladder. The marker is a commercially available mixture of proteins and each having specific molecular weights which are typically stained 38

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so as to form visible, colored bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane. Twodimensional (2-D) gel can also be used, which spreads the proteins from a single sample out in two dimensions. Transfer of separated protein to a membrane: A membrane made of nitrocellulose or polyvinylidene difluoride (PVDF) is used in order to transfer proteins from the gel onto the membrane. The protein transfers up the paper by capillary action, bringing the proteins with it. Electro blotting is another method for transferring the proteins which uses an electric current to pull proteins from the gel into membrane. The proteins which are present in the gel transfer with the same organization as they had with in the gel. Coomassie or Ponceau S dyes are used in order to check the uniformity and overall effectiveness of transfer of protein from the gel to the membrane. Ponceau S is the more common of the two, due to Ponceau S's higher sensitivity and its water solubility makes it easier to subsequently destain and probe the membrane. Blocking of membrane: Both bovine serum albumin (BSA) and non-fat dry milk are used a dilute solution of protein for the blocking of non-specific binding. The blocking is achieved by placing the membrane in the dilute solution of the protein and Tween 20 also used as a detergent in a minute percentage. The membrane is covered with the protein in the dilute solution accept the places where the target proteins have not attached. After addition of antibody, there is no space on the membrane for it to attach other than on the binding sites of the specific target protein. This confusion in the final product of the Western blot is reduces in this way, which leads to clearer results, and eliminates false positives. Detection of specific protein: The following two steps are involved in detection. 1. Primary antibodies: After the process of blocking the membrane, the membrane is incubated under gentle agitation along with a very dilute solution of the primary antibody (0.5 and 5 micrograms/ml). A small percentage of detergent, buffered saline solution and sometimes with powdered milk or BSA are present in the solution. The membrane can be sealed along with the antibody solution and incubated anywhere from 30 minutes to overnight. The incubation temperature can be varied. The warmer temperatures being associated with more binding, both specific (to the target protein, the "signal") and non-specific ("noise"). 39

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2. Secondary antibodies: After removing unbound primary antibody by rinsing the membrane, the membrane is treated with another antibody, directed at a species-specific portion of the primary antibody. Due to its targeting properties it is known as a secondary antibody which tends to be referred to as "anti-mouse," "anti-goat," etc. All these antibodies isolated from animal sources (or animal sourced hybridoma cultures); it therefore very specific because an anti-mouse secondary will bind to just about any mouse-sourced primary antibody. This allows some cost savings by allowing an entire lab to share a single source of mass-produced antibody, and obtain far more similar results. The secondary antibody has the property that it is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. The secondary antibodies will bind to one primary antibody and therefore enhance the signal. Analysis: After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is indexed to the structural protein to control between groups. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers. Detection of protein intensity: Different types can be used for the detection of protein intensity like Radioactive detection, Fluorescent detection, Chemiluminescent detection, Colorimetric detection. Radioactive detection: Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest. The importance of radioactive detections methods is declining because it is very expensive, health and safety risks are high and ECL provides a useful alternative. 1.9.3 Fluorescence Activated Cell Sorting (FACS) assay The Fluorescence-activated cell sorting (FACS) is a unique type of flow cytometry. Heterogeneous mixtures of biological cells (one cell at a time) are sorted into two or more 40

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containers which is run on a principal of the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, the fluorescent signals from individual cells and the physical separation of cells of particular interest as well, are performed by it through its objective and quantitative recording. The acronym FACS is trademarked and owned by Becton Dickinson (Loken, 1990). While many immunologists use this term frequently for all types of sorting and non-sorting applications, it is not a generic term for flow cytometry. The first cell sorter was invented by Mack Fulwyler in 1965, using the principle of Coulter volume a relatively difficult technique to use for sorting and one no longer used in modern instruments. The technique was expanded by Len Herzenberg who was responsible for coining the term FACS. Herzenberg won the Kyoto Prize in 2006 for his work in flow cytometry. The suspension of cell which is analyzed is injected in the center of a narrow, rapidly flowing stream of liquid. The system flow is arranged in such a way that there is a large separation between the cells as compare to their diameter. A vibrating mechanism causes the stream of the cells divide into individual droplets. The system is set in such a way that there is a low probability of more than one cell's being in a droplet. Just before the stream divide into droplets, the flow passes through a fluorescence intensity measuring station where the fluorescent character of interest of each cell is measured. Just at the point where the stream divides into droplets, an electrical charging ring is placed. A charge is placed on the ring based on the immediately-prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off. 1.9.3.1 Flow cytometer Modern flow cytometer are able to analyze several thousand particles every second, in "real time," and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers "high-throughput" (for a large number of cells) automated quantification of set parameters. To analyze solid tissues, single-cell suspension must first be prepared. A flow cytometer has 5 main components: Flow cell: liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing. 41

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Optical system: commonly used are lamps (mercury, xenon); high-power watercooled lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon (488nm), red-HeNe (633nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals.

Detector and Analogue-to-Digital Conversion (ADC) system: which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer

Amplification system: linear or logarithmic Computer: for analysis of the signals

1.9.3.2 Application The technology can be applied in various fields such as pathology, molecular biology, immunology, plant biology and marine biology. It became very useful when used with fluorescence tagged antibodies in the field of molecular biology. These specific antibodies bind to antigens on the target cells and help to give information on specific characteristics of the cells being studied in the cytometer. It has many applications in medicine (especially in transplantation, tumor immunology, hematology, and chemotherapy, genetics and sperm sorting for sex preselection). In marine biology, the auto-fluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterize abundance and community structure. In protein engineering, flow cytometry is used in conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with desired properties. 1.9.4 Comet assay Comet assay also known as the Single Cell Gel Electrophoresis (SCGE) assay is a simple and much sensitive technique for the study of DNA damage at the level of the individual eukaryotic cell. The technique was first described by Singh et al. in 1988. Now a days it is much popular and standard technique for the study of DNA damage/repair, bio monitoring and genotoxicity testing. The cells have encapsulated in a low-melting-point agarose suspension, the cells are then lysis in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software. 1.9.4.1 Experimental procedure Experimental are involved the following steps. 42

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Encapsulation: A sample of cells either derived from an in vitro cell culture or from an in vivo test subject is dispersed into individual cells and suspended in molten low-melting-point agarose at 37C. This mono-suspension is cast on a microscope slide. A glass cover slip is held at an angle and the mono-suspension applied to the point of contact between the coverslip and the slide. As the cover slip is lowered onto the slide the molten agarose spreads to form a thin layer. The agarose is gelled at 4C and the coverslip removed. The agarose forms a matrix of carbohydrate fibers that encapsulate the cells, anchoring them in place. The agarose is considered to be osmotic-neutral, therefore solutions can penetrate the gel and affect the cells without cells shifting position. In an in vitro study the cells would be exposed to a test agent - typically UV light, ionizing radiation, or a genotoxic chemical - to induce DNA damage in the encapsulated cells. For calibration, hydrogen peroxide is usually used to provide a standardized level of DNA damage

Lysis: The slides are then immersed in a solution that causes the cells to lyse. The lysis solution often used in the comet assay consists of a highly concentrated aqueous salt (often, common table salt can be used) and a detergent (such as Triton X-100 or sarcosinate). The pH of the lyses solution can be adjusted (usually between neutral and alkaline pH) depending upon the type of damage the researcher is investigating. The aqueous salt disrupts proteins and their bonding patterns within the cell as well as disrupting the RNA content of the cell. The detergent dissolves the cellular membranes. Through the action of the lysis solution the cells are destroyed. All proteins, RNA, membranes and cytoplasmic and nucleoplasmic constituents are disrupted and diffuse into the agarose matrix. Only the DNA of the cell remains, and unravels to fill the cavity in the agarose that the whole cell formerly filled. This structure is called nucleoid (a general term for a structure in which DNA is concentrated).

Electrophoresis: After lysis of the cells (typically 1 to 2 hours at 4C) the slides are washed in distilled water to remove all salts and immersed in a second solution (an electrophoresis solution). Again this solution can have its pH adjusted depending upon the type of damage that is being investigated. The slides are left for ~20 minutes in the electrophoresis solution prior to an electric field being applied. In alkaline conditions the DNA double helix is denatured and the nucleoid becomes single stranded. An electric field is applied (typically 1 V/cm) for ~20 minutes. The slides are then neutralized to pH 7, stained with a DNA43

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specific fluorescent stain and analyzed using a microscope with an attached CCD (charge-coupled device - essentially a digital camera) that is connected to a computer with image analysis software e.g. Comet IV, Perceptive Instruments Ltd., Haverhill, UK. 1.9.4.2 Principles The concept underlying the SCGE assay is that undamaged DNA retains a highly organized association with matrix proteins in the nucleus. When damaged, this organization is disrupted. The individual strands of DNA lose their compact structure and relax, expanding out of the cavity into the agarose. When the electric field is applied the DNA, which has an overall negative charge is drawn towards the anode. Undamaged DNA strands are too large and do not leave the cavity, whereas the smaller the fragments, the farther they are free to move in a given period of time. Therefore, the amount of DNA that leaves the cavity is a measure of the amount of DNA damage in the cell. The image analysis measures the overall intensity of the fluorescence for the whole nucleoid and the fluorescence of the migrated DNA and compares the two signals. The stronger the signal from the migrated DNA the more damage there is present. The overall structure resembles a comet (hence "comet assay") with a circular head corresponding to the undamaged DNA that remains in the cavity and a tail of damaged DNA. The brighter and longer the tail, the higher the level of damage.

1.9.5 Total Phenolics or Folin-Ciocalteau Micro Method This method is used routinely in our lab to measure total phenol. The procedure is also used for analysis of total phenol in various plants and fruits. It uses the minimum volume of reagents and almost eliminates wasted reagent. Good micro pipets must be used for reproducibility. Plastic or glass cuvettes can be used. It is based on the method reported by Slinkard and Singleton, (1977), only the volumes have been reduced. If you cannot reproducibly measure such small volumes, try to reduce the volumes to the smallest you can. This reduces waste and disposal volume. The following reagents are used in this assay. Folin Ciocalteu Reagent: It is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric assay of phenolic and polyphenolic antioxidants (Singleton et al., 1999). It works by measuring the amount of the substance being tested needed to inhibit the oxidation of the reagent (Vinson et al., 2005). However, this reagent does not only measure total phenols and will 44

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react with any reducing substance. The reagent therefore measures the total reducing capacity of a sample, not just the level of phenolic compounds. This reagent forms part of the Lowry protein assay and will also react with some nitrogen-containing compounds such as hydroxylamine and guanidine (Ikawa et al., 2003). This is usually purchased as the 2N reagent available from Sigma (F9252) or from Fisher Scientific (ICN19518690), and presumably others. Singleton and Rossi, (1965) describe the preparation of the reagent from sodium tungstate, sodium molybdate, lithium sulfate, bromine, and some acids. Gallic Acid Stock Solution: In a 100-mL volumetric flask, dissolve 0.500 g of dry gallic acid in10 mL of ethanol and dilute to volume with water. Can be opened daily, but to store, keep closed in a refrigerator up to two weeks. Sodium Carbonate Solution: Dissolve 200 g of anhydrous sodium carbonate in 800 mL of water and bring to a boil. After cooling, add a few crystals of sodium carbonate, and after 24 hr, filter and add water to 1 L.

1.9.5.1 Calibration curve To prepare a calibration curve, add 0, 1, 2, 3, 5, and 10 mL of the above phenol stock solution into 100 mL volumetric flasks, and then dilute to volume with water. These solutions will have phenol concentrations of 0, 50, 100, 150, 250, and 500 mg/L gallic acid, the effective range of the assay. From each calibration solution, sample, or blank, pipet 20 L into separate cuvettes, and to each add 1.58 mL water, and then add 100 L of the Folin-Ciocalteu reagent, and mix well. Wait for between 30 sec and 8 min, and then add 300 L of the sodium carbonate solution, and shake to mix. Leave the solutions at 20C for 2 hour and determine the absorbance of each solution at 765 nm against the blank (the "0 mL" solution) and plot absorbance vs. concentration. Alternatively, they can be left at 40C for 30 min before reading the absorbance. A calibration curve is created with the standards and determined the levels in the samples. Do not neglect to multiply the observed concentrations by any dilution factor of the original sample. Results are reported at Gallic Acid Equivalent, GAE, because the phenols in sample contain mostly other phenols, and only small amounts of Gallic acid. Since the assay measures all phenolics, the choice of Gallic acid as standard is based on the availability of a stable and pure substance, and Gallic acid is both, and it is less expensive than other options. In addition, the response to Gallic acid has been shown to be equivalent to most other phenolics in extract on a mass basis. It has also tested the 45

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stability of Gallic acid standard solutions and it can say they lose less than 5% of their value over two weeks when refrigerated and kept tightly closed.

1.9.6 Antioxidant activity The body uses oxygen and nutrients to make energy. Oxygen also helps the immune system fight disease and harmful substances. Oxidation is a process that uses by products formed from oxygen fighting disease to create molecular agents that react with body tissues. Unfortunately, this process can form free radicals that cause cell damage. Antioxidants help reduce the number of free radicals that form in the body, lower the energy levels of existing free radicals, and stop oxidation chain reactions to lower the amount of damage caused by free radicals. The antioxidants of food are thought to prevent diseases caused by oxidative stress (Cutler, 1984; Frankel et al., 1993). Free radicals are believed to be one of the causes of over sixty health problems, according to various scientific and medical groups. These problems include cancer, aging, and atherosclerosis. By increasing antioxidant intake and reducing exposure to free radicals can help lower health risks and problems. Antioxidant enzymes are also produced by our bodies and include catalase, superoxide dismutase, and glutathione peroxidase. These enzymes also fight against free radicals. The enzymes are available in supplemental forms, but it is believed that taking the building blocks of the enzymes in supplemental form is more effective. Zinc, selenium, copper, and manganese are some of the building blocks. Minerals and vitamins are also often antioxidants. Vitamins including lutein, cysteine, beta-carotene, vitamin B2, vitamin C, vitamin E, and coenzyme Q10 and herbs. Among the natural secondary metabolites, flavonoids play a key role in the antioxidant mechanism by scavenging free radical produced during oxidation process. Flavonoids are widely distributed in plant foods such as vegetables and fruits. They possess a unique C6C3C6 structure (diphenylpropane structure) with phenolic hydroxy groups; more than 4000 different functional group substitution patterns have been identified as natural flavonoids. The average intake of flavonoids in Western diets was estimated to be 1 g/day (Kuhnan, 1976). Flavonoids in citrus fruits are known as bioflavonoids or vitamin P, which exhibit beneficial effects on capillary permeability and fragility (Rusznyak and Szent-Gyorgyi, 1936). These compounds have been investigated regarding their physiological functions such as anti-inflammatory, anticarcinogenic, and antitumor activities (Bracke et al., 1994; Middleton and Kandaswami, 1994; Attaway, 1994). The antioxidant activity of flavonoids has attracted much attention in relation to their physiological functions. Dietary flavonoids are considered to aid in the prevention of 46

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coronary heart disease because epidemiological studies have shown an inverse relationship between the intake of dietary flavonoids and coronary heart disease (Hertog et al., 1993). The so-called French paradox is at least partially related to the consumption of red wine rich in flavonoids and other phenolic compounds. It is, however, necessary to know the biodynamics of flavonoids after intake for estimation of in vivo antioxidant activity. Various techniques are use to determine the antioxidant activity of a testing sample. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radicals are also use for the same assay.

1.9.6.1 (1, 1-diphenyl-2-picrylhydrazyl) (DPPH) It is one of such method that is currently popular and is based upon the use of the stable free radical diphenylpicrylhydrazyl (DPPH). The molecule of 1,1-diphenyl-2picrylhydrazyl (,-diphenyl--picrylhydrazyl; DPPH: (1) is characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that the molecules do not dimerise, as would be the case with most other free radicals. The delocalization also gives rise to the deep violet color, characterized by an absorption band in ethanol solution centered at about 520 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form (2) with the loss of this violet color (although there would be expected to be a residual pale yellow color from the picryl group still present). Representing the DPPH radical by X and the donor molecule by YZ, the primary reaction is X + Y Z = X Y + Z [1]

NO2 O2N . . N NO2 1. DiPhenylicrylhydrazyle (free radical) N

O2N

NO2 H N NO2 2. Diphenylepicrylhydrazine (nonradical) N

where X Y is the reduced form and Z is free radical produced in this first step. This latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorized) by one molecule of the reductant. The reaction [1] is therefore intended to provide the link with the reactions 47

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taking place in an oxidising system, such as the autoxidation of a lipid or other unsaturated substance; the DPPH molecule X is thus intended to represent the free radicals formed in the system whose activity is to be suppressed by the substance Y Z. The DPPH method as summarized above was evidently introduced nearly 50 years ago by Marsden Blois, working at Stanford University (Blois, 1958). It was noted in the original paper that among other compounds active in this reaction are glutathione, aromatic amines (such as p-phenylene diamine and p-aminophenol), and -tocopherol (Vitamin E 2:1 stoichiometry) and polyhydroxy aromatic compounds (such as hydroquinone and pyrogallol). On the other hand, monohydric phenols (such as tyrosine), simple sugars (such as glucose), purines and pyrimidines, do not react, while proteins are precipitated. It was also noted that inorganic ions in lower valence states may of course interfere and must be eliminated or determined separately which presumably applies most importantly to ferrous iron (Blois, 1958).

1.10 Selection of Medicinal plants species Twenty seven plants were selected and collected from Margalla Hills Islamabad Pakistan. Some species are reported as medicinal while some are not reported medicinal plants species. Three plants species Berberis lycium Royle (Berberidaceae), Zizyphus nummularia (Rhamnaceae) and Mallotus philippensis (Euphorbiaceae) were analyzed for antineoplastic activities, other twenty four plants species were analyzed for free radical scavenging activity, total Phenolic content and Flavonoids types. The plant samples used in this study were Albizia lebbeck (Mimosaceae), Bauhinia variegata and Cassia fistula (Caesalpinaceae), Bombax ceiba (Bombacaceae), Calotropis procera (Asclepiadaceae), Carissa opaca (Apocynaceae), Colebrookea oppositifolia (Labiateae), Dalbergia sissoo (Papilionaceae), Dodonaea viscosa (Sapindaceae), Ficus palmata and Ficus racemosa (Moraceae), Jasminum humile and Olea ferruginea (Oleaceae), Adhadoda vasica (Acanthaceae), Lantana camara Linn. (Verbenaceae), Melia azedarach L. (Meliaceae), Phyllanthus emblica. L. (Euphorbiaceae), Pinus roxburghii Sargent (Pinaceae), Punica granatum L. (Punicaceae), Rubus ellipticus Smith, Pyrus pashia Buch. & Ham. (Rosacceae), Viburnum cotinifolium D. Don (Caprifoliaceae), Dabregeasia salicifolia (Urticaceae) and Caryopteris grata (Verbenaceae).

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2.10.1 Berberis lycium Royle (Berberidaceae) Berberis lycium is a widely used medicinal plant in Pakistan, known by the common name Zyarh larghai or Kashmal, whereas its English name is Barberry (Anwar, 1979) Al-Biruni describes the plant under the name of Ambaribis and also mentioned its Persian names as Zirkash (Said, 1996 ). Berberis taxa are important plants, with various medicinal properties. Berberis is also included in Indian and British pharmacopoeias.

Description Shrub, 2-3(-4) m tall, erect or suberect, semideciduous; stem and branches pale, whitish to greyish, terete to subsulcate, glabrescent, younger ones obscurely to distinctly puberulous; yellowish to straw-coloured. Leaves oblanceolate to oblong-obovate, subsessile, usually conspicuously papillose, grey or white below, Racemes (6-)10-25flowered, 3-6(-7) cm long, rarely shorter and subfascicled. Flowers usually pale-yellow; Outer sepals much smaller than the middle and inner sepals; inner sepals 4.5-5 mm long, 3 mm broad, obovate. Petals slightly shorter than the inner sepals, obovate, emarginate, with lanceolate basal glands. Stamens slightly shorter than petals, connectives produced or anthers apiculate. Ovules usually 4, shortly stipitate. Berries ovoid or obovoidsubglobose, excluding 1 mm long style, blackish with heavy grey-white bloom.

Distribution Native in the whole Himalaya Mountains range and widely distributed in temperate and semi-temperate areas of India, Nepal, Afghanistan, Bangladesh and Pakistan.

1.10.2 Mallotus philippensis (Lam.) Muell. Arg. (Euphorbiaceae)

Description Shrubs or small trees, 2-15 m tall. Branchlets, petiole, and inflorescences yellowbrownish stellate-tomentose. Leaves ovate to lanceolate, leathery, margin subentire, apex acuminate; basal veins 3. Male flowers 1-5-fascicled; calyx lobes 3 or 4, oblong, ca. 2 mm, tomentulose; stamens 15-30. Female flowers: calyx lobes 3-5, subovate, ca. 3 mm, tomentose; ovary tomentose and red glandular-scaly; styles 3, 3-4 mm, plumose. Capsule

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subglobose, 8-10 mm in diam., (2 or)3-locular, covered with a red glandular-scaly layer. Seeds subglobose, ca. 4 mm in diam., black. Fl. Mar-May, fr. Jun-Aug.

Distribution Mountain slopes or valleys, limestone hills or river valleys, forests; 300-1600 m. Anhui, Fujian, Guangdong, Guangxi, Guizhou, Hainan, Hubei, Hunan, Jiangsu, Jiangxi, Sichuan, Taiwan, Xizang, Yunnan, Zhejiang, Bangladesh, Bhutan, India, Laos, Malaysia, Myanmar, Nepal, New Guinea, Pakistan, Philippines, Sri Lanka, Thailand, Vietnam; N Australia.

1.10.3 Adhatoda vasica Nees (Acanthaceae). Description An erect much branched, gregarious, evergreen shrub, up to 2 (-2.5) m. Stem quadrangular to nearly terete, young shoots greyish-pubescent Leaves elliptic-lanceolate, glabrous above, pubescent on nerves beneath, basally attenuate, entire, acuinate. Flowers white, c. 3 cm long, nearly sessile, in terminal and axillary spikes, up to 10 cm long, 2.5-3 cm broad; bracts leafy, broadly-elliptic; Calyx 5-lobed, lobes linear-lanceolate, 6-10 x c. 2 mm, acute, puberulous, imbricate. Corolla pale-white, tube 1.2-1.5 cm long, pubescent outside, throat villous, limb 2-lipped, upper lip erect, shortly bifid, galeate, lower lip with 3 elliptic, obtuse lobes. Ovary oblong, c. 3 mm long, style 2-2.5 cm long. Capsule stipitate, broadly clavate, pubescent. Seeds orbicular, 2-3 mm across, glabrous. Fl. Per.: November-April (plains); July-October (hills). Distribution: Panama (probably) introduced), Indonesia, Malaya, S.E. Asia, India and Pakistan. In Pakistan, it does well on waste lands up to 1300 m; it is also cultivated as an ornamental.

1.10.4 Albizia lebbeck (L.) Benth. (Mimosaceae) Description 50

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A large deciduous tree with dark grey bark, usually cracked, young parts usually hairy. Leaves bipinnate, rachis glabrous or tomentose, with a large gland 1.2-3.7 cm from the base; Pinnae 1-4 pairs, often with glands between the upper pairs of leaflets or between all the pairs. Leaflets 3-9 pairs, petiolule c. 1 mm long, the lateral leaflets oblong, terminal obovate, obtuse or retuse, glabrous or hairy. Inflorescence pedunculate heads, solitary or fasciculated; Flowers whitish, very fragrant, pedicel hairy, bracteate; Calyx campanulate long, hairy, short toothed, teeth deltoid-acute. Corolla funnel shaped, lobes c. 2 mm long, ovate, acute, hairy externally. Pod thin, pale straw coloured. Fl. Per. April May.

Distribution W. Pakistan, widely cultivated; Tropical Asia; N. Australia and Tropical Africa. Commonly planted as a roadside tree. mills and wheels

1.10.5 Bauhinia variegata Linn. (Caesalpinaceae) Description A medium sized tree with dark brown nearly smooth bark; young shoots pubescent. Leaves with a medium cleft reaching from 1 /4 to 1/3 the way down, lobes obtuse, the base is deeply heart shaped, 9-15 nerved, pubescent beneath when young. Inflorescence few flowered pubescent raceme tomentose, 5 toothed at the apex. Petals obovate, with long rather broad claw, all white or 4 petals pale purple and fifth darker with purple veins. Stamens 5, fertile, no staminodes. Ovary hairy, stipe 10-17 mm long; style long, stigma capitate. Pods hard, flat; Fl. Per.: Feb.-April.

Distribution Kashmir; W. Pakistan; India (Punjab, Uttar Pradesh, Bengal, Assam, Central India, Madras; Sikkim); Nepal; Burma; China; widely cultivated in tropics.

1.10.6 Bombax ceiba Linn. (Bombacaceae) Description Tall trees, trunk usually unbranched up to considerable height. Bark grey, covered with hard small conical prickles. usually disappearing with age. Petiole 10-30 cm long, pulvinate at the base; stipules triangular, 5-10 mm x 4 mm with hairy margin, caducous. 51

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Leaflets 5-7, glabrous, entire, elliptic-lanceolate, acuminate, attenuate at base, more or less leathery, Inflorescence many fascicles of 1-4 flowers borne, at or near the end of branches. Flowers large, showy, red (occasionally yellow or white); pedicel thick, Calyx 3-lobed (rarely 2-lobed), cup-shaped, Petals twisted in bud, elliptic-oblong. Ovary conical, green, covered with silky hairs, 0.5-1.2 cm long; style simple, 5.9-6.5 cm long; stigmas 5, filiform. 5-6 mm long. Capsule 10-12.5 cm long; oblong, woody, 5 valved, profusely to finely tomentose. Fl.Per.: December-March. Distribution: Commonly cultivated as a roadside and garden tree in Pakistan. Wild in subhimalayan tract from Hazara to eastword, up to 3500 ft., India, Ceylon, S,E.Asia, China, Australia (Queenslands North Australia) and China (Yunnan).

1.10.7 Calotropis procera (Willd.) R. Br. (Asclepiadaceae) Description Erect shrub or small tree up to 3 m tall, much branched from the base, latex milky; young branches covered with white cottony tomentum. Bark soft, corky. Leaves 5-15 x 1.8-10 cm, broadly ovate, ovate-oblong, elliptic or obovate, entire, base cordate, apex acute, subsessile, young leaves covered with white cottony tomentum, becoming subglabrous. Flowers white outside, purplish within, tips darker. Sepals c. 5 mm long. Corolla divided c. 2/3 the way down, glabrous, lobes acute. Fruit recurved, tip not invaginated in the tissue of the fruit. Fl. Per.: All the year round. Distribution: Pakistan, India, Afghanistan, Iran , Iraq

1.10.8 Carissa opaca Stapf ex Haines (Apocynaceae) Description Shrub, up to 3.5 meter, evergreen, branches glabrous, or puberulous, spines arising between the petiole, straight or bifurcate, sharp, hard, 2.5-3.5 cm long, young shoots with milky juice. Leaves glabrous, opposite, elliptic, ovate or rounded, shining green above, paler, puberulous on nerves beneath; Flowers white or light rose, sweet scented; Calyx c. 2 mm long, lobes lanceolate, acuminate, puberulous or ciliate, reaching almost to the base 52

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of the calyx tube. Corolla tube slender, 8-12 mm long, lanceolate, acute overlapping to the right, in bud, spreading. Ovary one ovuled; stigma slightly bifid. Berry somewhat ellipsoid or subglobose, 6-8 mm long, dark purple when ripe, with milky juice, edible. Fl. Per.: April-June. Distribution: Drier parts of India and Pakistan (from Punjab-Himalayas upto 6000 ft, in Murree), Burma and Sri Lanka.

1.10.9 Caryopteris grata Benth. (Verbenaceae).

Description A straggling or rambling shrub, often purplish or brownish in colour. Leaves lanceolate or elliptic, crenate-serrate to entire or subentire, acuminate, petiolate, pubescent. Cymes short, axillary. Flowers small, white or purplish; bracts 2-3 mm long, somewhat subulate, pubescent. Calyx with spreading lobes in fruit, divided half-way down but scarcely enlarged in fruit. Corolla-tube , lobes 3.5-5 mm long, lower larger. Stamens exserted. Capsules 2.5-4 mm long, subglobose, glabrous, slightly 4-lobed, red when ripe. Fl.Per.: Feb.-May.

Distribution Outer and sub-Himalayan tracts.

1.10.10 Cassia fistula Linn (Caesalpinaceae) Description Tree, up to 20 m tall. Rachis 12-25 cm long, terete, glabrous. Leaves compound with 3-8 pairs of opposite leaflets, smooth above, hairy below. Flowers arranged in drooping racemes, each raceme c. 10-45 cm long; Calyx 5, green, folded backward on the stalk, hairy, ovate, 9 mm long. Petals 5, obovate, blunt, distinctly veined. Ovary slender, thinly

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appressed hairy, style sturdy, stigma punctiform. Pods terete, glabrous, indehiscent, 40-60 cm long, 1.5-2 cm broad, black glossy brown, 40-100 seeded. Fl. Per.: April June.

Distribution W. Pakistan, Swat and Hazara eastwards, ascending to 4000 ft. and commonly planted in gardens; common in deciduous forests throughout the greater part of India, Burma and Ceylon.

1.10.11 Colebrookea oppositifolia Smith (Labiatae). Description Plants 1-3 m, much branched. Base broadly cuneate to rounded, margin crenulateserrulate, apex long acuminate, adaxially rugulose and puberulent, abaxially denselytomentose to lanate-tomentose. Panicles 10-15 cm, branches 4-7 cm; verticillasters 10-18flowered, globose; bracteoles ca. 1 mm, densely tomentose outside, glabrous inside. Flowers ca. 2 mm., calyx minute, ca. 0.6 mm. Corolla to 3 mm; upper lip ovate-oblong, ca. 0.5 mm, straight, emarginate; lower lip elongated, spreading, ca. 1.5 mm, middle lobe ovate-oblong, 2 as long as ovate lateral lobes. Style erect, slightly longer than corolla. Nutlets obovoid, ca. 1 mm, yellow-brown, with a small basal white scar. Fl. Jan-Mar, fr. Mar-Apr.

Distribution Savanna forests, thickets in hot, dry regions; 200-2200 m. Yunnan [India, Myanmar, Nepal, Thailand].

1.10.12 Debregeasia salicifolia (D.Don) Rendle in Prain (Urticaceae) Description A dioecious, evergreen tall shrub or small tree. Stem with dark brown fibrous bark scabrous, young shoots whitish tomentose. Leaves with up to 2.5 cm long, densely tomentose petiole; lamina oblong - lanceolate 2-15 cm long, 0.6-3 cm broad, silvery tomentose beneath, scabrous and rugose above, serrate, acute; stipules linear-lanceolate up to c. 1 cm long, brown, deciduous. Male flower clusters larger than female flower heads. Calyx of male flowers campanulate, streaked orange-red and white, tomentose 54

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outside, 4-lobed, shorter than brown bracteoles; tubular-ovoid with narrowed mouth in female flowers. Stamens 3-5, exserted, anthers pale purple. Achenes fleshy, yellow, c. 1.5 mm long, pointed. Fl.Per.: March-June. Distribution: India, Pakistan (Punjab, N.W.F.Province, Kashmir) Afghanistan and Tropical Africa. Common in moist places in the Northern Himalayas and Salt range, up to 2000 m.

1.10.13 Dalbergia sissoo Roxb. (Papilionaceae) Description Tree with rough bark and mainly longitudinal furrows, young branch pubescent. Leaf imparipinnate, rachis c. 3.7-7.5 cm long; leaflets 3-5, c. 3.5-6.5 cm long, broadly ovate or suborbicular, acuminate, glabrescent, petiolule c. 5-8 mm long; stipules c. 5 mm long. Inflorescence an axillary panicle, composed of several short spikes with sessile to subsessile flowers. Bract small, pubescent, caducous. Calyx c. 5 mm long, teeth ciliate, unequal, shorter than the tube. Corolla yellowish white. Stamens 9, monadelphous, tube slit on the upper side only, anthers uniform. Ovary pubescent, 2-4-ovulate, style glabrous, stigma capitate. Fruit c. 3.7-10 cm long, c. 7.0-13 mm broad, strap-shaped, glabrous, 1-4seeded. Seed flattened. Fl. Per.: March-May.

Distribution Pakistan; India; Sikkim; Afghanistan; Persia; Iraq; Very widely planted in the plains along the roadsides, canals and fields and in the forest plantations.

1.10.14 Dodonaea viscosa (L.) Jacq., (Sapindaceae) Description An evergreen shrub up to 5 m tall; young parts covered with a yellow, viscid resin. Leaves sub-sessile, oblanceolate to spathulate, glabrous, entire, sub-acute to apiculate. Sepals 3-5, connate at the base, ovate, 3 mm long, puberulous; persistent. Stamens 6-8, free, rudimentary in the female flower; anthers subsessile, oblong, 2-5 mm long, sparsely hairy at the tip. Disc annular, cushion-shaped. Ovary triquetrous, 3-locular, sparsely 55

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hairy, rudimentary in the male flower; style 3 mm long, minutely papillose; stigma 3-fid. Capsule 2-4 valved; valves membranous, light brown, green or maroon, winged at the back. Seed sub-globose, c. 4 mm long, black. Fl. Per: Jan-March.

Distribution Australia, S. Africa, N. America, China, India, Ceylon and W. Pakistan. A component of the scrub vegetation of low hilly areas.

1.10.15 Ficus palmata Forssk. (Moraceae) Description A large deciduous shrub or small tree, up to 10 m tall. Truck and branches. without aerial roots, bark smooth, brownish-grey, young twigs densely hairy. Leaves broadly ovate to suborbicular or orbicular upper surface scabrid, soft hairy on lower side to glabrate, Hypanthodia solitary or sometimes paired, axillary, on c. 1-2.5 an long, tomentose peduncles, subglobose to pear-shaped, tomentose, subtended by 3, deltoid, acute basal bracts, apical orifice umbonate. Male flowers: numerous in the upper half, pedicellate; sepals 4-5, free, lanceolate, hairy; stamens 3-6. Female flowers: basal, numerous; sepals 5, basally united, hairy; ovary ovoid with subterminal, long hairy style. Figs constricted or gradually narrowed at base, 1.5-2.5 cm long, yellow or purple, hairy. Fl. & Fr. Per.: May-November Distribution: Nepal, N. & N.W. India, Pakistan, Afghanistan, Iran, Arabian Peninsula, Somalia, Sudan, Ethiopia and S. Egypt. This is a highly variable and common wild fig occurring in N.W. Hills up to 2500 m on hot dry slopes in clay-loam soils in Baluchistan, Punjab and North Western Frontier Province and Kashmir. Two subspecies are recoginzed. The type subspecies from E. Africa and Saudi Arabia has more elongate, distinctly acute or acuminate leaves with slight pubescence.

1.10.16 Ficus racemosa L. (Moraceae) Description 56

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A small to large, 10-20 (- 30) m tall, evergreen or occasionally deciduous tree. Leaves with 2.6 (-7.5) cm long, grooved minutely hairy, brownish-scurfy petiole; lamina ovatelanceolate to elliptic-lanceolate, margin entire to used obtuse or subacute to occasionally acuminate at apex, glabrous on both sides; lateral nerves 4-7 (-8) pairs, bulging beneath, intercostals present; Male flowers: sessile, ostiolar in 23-whorls; 3(-4), united, lobes dentate-lacerate, red; stamens usually 2, pistillode present. Female flowers: sessile or subsessile. sepals as in male; ovary substipitate, with lateral, 2.3 long, glabrous style, stigma simple. Gall flowers pedicellate, dispersed among female. Figs depressed subglobose or pyriform, 2.54 cm in, diameter red, usually streaked. Seeds lenticular, c. 1 mm long. Fl. & Fr. Per.: March-May & September-November. Distribution: Pakistan, India, Sri Lanaka, Bangle Dish, S. Chins, Burma, Thailand, Malayasia, Indonesia to N. Australia.

1.10.17 Jasminum humile Linn. (Oleaceae) Description Shrub erect, 1 (-2) m tall, deciduous or evergreen, glabrous. Branches green, angular. Leaves alternate, very variable in size, sometimes revolute; leaflets coriaceous, dark green above, paler beneath, variable in shape, elliptic, ovate, or lanceolate, acute or obtuse, terminal sometimes larger than lateral. Flowers in terminal corymbose cymes; Calyx tube c. 3 mm long, teeth very short. Corolla yellow, tube 1-2.5 cm long, lobes 5, broadly ovate-obtuse or round, reflexed when the flower is open. Berry simple or didymous, globular-ellipsoid, 4-6 mm long, black when ripe, full of crimson juice. Fl. Per: AprilJune. Fruit: September-December.

Distribution Himalaya and Hindukush, from Afghanistan to Western China. Northern regions of Pakistan, South Waziristan, Baluchistan, in temperate forests, 1000-3000 m, common. Sometimes cultivated with Jasminum officinale.

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1.10.18 Lantana camara L. (Verbenaceae) Description Evergreen shrub with rambling or straggling branches, 1-2(-4) m, tall; branches usually minutely or inconspicuously pubescent, unarmed to conspicuously prickly with hooked spines. Leaves opposite, decussate, ovate to ovate-oblong crenate-serrate, acute to shortly acuminate, rugose, scabrid; Flowering heads axillary, peduncled, umbellate in flower, shorter to exceeding the subtending leaves, 2-3 cm across. Bracts lanceolate to linear, acute to subulate, rarely a few larger ones also present. Flowers mostly orange or yellow, turning to red or scarlet later. Calyx thin, pubescent. Corolla-tube pubescent, slightly enlarged and curved above the middle; limb 4 lobed with spreading, rounded lobes. Drupe 3-5 mm in diameter, globose; fleshy, black, shining, 2-seeded. Fl. Per.: Throughout the year.

Distribution A native of trop. America widely introduced and naturalized in many tropical and subtropical regions.

1.10.19 Melia azedarach L. (Meliaceae) Description Tree, up to 12 m tall; young shoots tomentose. Leaves 2-(3)-pinnate, up to 60 cm long; leaflets opposite, elliptic, 2.5-5 cm long, 5-19 mm broad, serrate to sub-serrate, acuminate, often oblique, sub-sessile. Flowers lilac, sweet-scented, in axillary panicles; pedicel 2-3 mm long, puberulous. Calyx 5-6-lobed; lobes c. 2 mm long, acute, pubescent. Petals 7-9 mm long, spathulate to lanceolate, ciliate, imbricate in bud. Staminal tube 6-7 mm long, cylindrical, expanded at the base and apex, 10-striate, with 20 teeth at the apex; anthers sessile, 1 between each pair of teeth. Disc glabrous, fused with the ovary base. Ovary usually 5-locular; style 4-5 mm long; stigma capitate. Drupe 1.5-2 cm long, globose, 3-6-seeded, yellow when ripe. Fl. Per. March-April.

Distribution

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Wild in W. Himalaya, up to 1700m. Cultivated and naturalized in parts of Iran, China, Burma, Turkey, India & W. Pakistan.

1.10.20 Olea ferruginea Royle (Oleaceae) Description Trees or shrubs, up to 10 m high, greyish green. Bark smooth when young, peeling off in narrow strips when old. Leaves oblong-lanceolate to ovate, 3-10 cm long, often cuspidate, very coriaceous, dark green and shining above, with a dense film of minute scales beneath which turn reddish brown on older leaves, margins recurved, midrib prominent; petiole short. Flowers whitish, in trichotomous axillary 2-4 cm long cymes. Calyx truncate or with 4 short teeth. Corolla tube very short, lobes 4, 1-2 mm long, elliptic, obtuse or acute, with a ridge along the middle. Drupe c. 8 mm long, 5 mm in diameter, ovoid, black when ripe; pulp scanty, oily. Fl. Per.: April-May, sometimes September. Fruit: AugustNovember.

Distribution Afghanistan, Pakistan, Kashmir. Very common in the lower hills, 500-2000 m, gregarious, usually with Acacia modesta. Frequently planted in graveyards. the complex.

1.10.21 Phyllanthus emblica L. (Euphorbiaceae) Description Monoecious, deciduous tree; bark brownish; main stems terete, Leaves distichous; stipules triangular-ovate, margins entire or denticulate, ciliate; leaf blade oblong or linearoblong, paler abaxially, green adaxially, drying reddish or brownish, base shallowly cordate and slightly oblique, margin narrowly revolute, apex truncate, rounded or obtuse, mucronate or retuse at tip; Fascicles with many male flowers and sometimes 1 or 2 larger female flowers. Male flowers: sepals 6, membranous, yellow, obovate or spatulate, subequal, apex obtuse or rounded, margin entire or shallowly denticulate; disk glands 6, subtriangular; stamens 3; filaments coherent into column, 0.3-0.7 mm; anthers erect, 59

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oblong, 0.5-0.9 mm, longitudinally dehiscent, apex mucronate. Female flowers: sepals 6, oblong or spatulate, apex obtuse or rounded, thicker, margin membranous, lobate; ovary ovoid, ca. 1.5 mm, 3-celled; styles , connate at base, deeply bifid, lobes divided at tip. Fruit a drupe, globose, 1-1.3 cm in diam., exocarp fleshy, pale green or yellowish white, endocarp crustaceous. Fl. Apr-Jun, fr. Jul-Sep.

Distribution Dry open sparse forests or scrub, village groves; 200-2300 m. Fujian, Guangdong, Guangxi, Guizhou, Hainan, Jiangxi, Sichuan, Taiwan, Yunnan [Bhutan, Cambodia, India, Indonesia, Laos, Malaysia, Myanmar, Nepal, Philippines, Sri Lanka, Thailand; South America (cultivated)].

1.10.22 Pinus roxburghii Sargent (Pinaceae) Description Trees up to 30 m tall with a soft flaky bark 2-5 cm thick. Leaves in clusters of 3,20-30 cm long. Male cones c. 1.5 cm long, yellowish, in dense terminal clusters. Female cones solitary or 2-3 at the tips of branches, mature ones woody; bract and scale distinct, umbo prominently beaked. Wing 2-3 times longer than seed.

Distribution Afghanistan, the Himalaya from Chitral eastward to Bhutan, Sikkim.

1.10.23 Pyrus pashia Buch. & Ham. (Rosaceae) Description Trees to 12 m tall, with branches often armed. Branchlets purplish brown or dark brown; buds ovoid, apex obtuse; scales puberulous along margin. Stipules caducous, linearlanceolate, membranous, adaxially pubescent, margin entire, apex acuminate; petiole initially pilose, soon glabrescent; leaf blade ovate or narrowly ovate, glabrescent, base rounded, rarely broadly cuneate, margin obtusely serrate, apex acuminate or acute. Raceme umbel-like, 713-flowered; peduncle initially tomentose, glabrescent; bracts caducous, linear, membranous, both surfaces tomentose, margin entire, apex acuminate. Pedicel initially tomentose, glabrescent. Petals white, obovate, base shortly clawed, apex 60

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rounded. Stamens slightly shorter than petals. Pome brown, with pale dots, sepals caducous. Fl. MarApr, fr. AugSep.

Destribution Valleys, among shrubs; 600--3000 m. Guizhou, Sichuan, Xizang, Yunnan, Bhutan, India, Kashmir, Laos, Myanmar, Nepal, W Pakistan, Sikkim, Thailand, Vietnam. This tree is cultivated in Yunnan, and is often used as stock for grafting pear cultivars.

1.10.24 Punica granatum L. (Punicaceae)

Description Tree or shrub, Branches terete, opposite, branchlets usually ending in spines. Leaves glabrous, oblong-lanceolate to obovate or elliptic, subpetiolate, entire, apex sub-actue to obtuse. Flowers scarlet red or white, conspicuous, 3 cm or more in length. Calyx indented slightly above the middle, reddish, somewhat succulent; lobes triangular. Petals broadly obovate, wrinkled, alternating with the sepal lobes. Ovary subglobose; style thick reddish; stigma simple; slightly bilobed. Fruit globose, 2-8 cm in diameter, sometimes persistent, pale red to scarlet, or brownish, partitioned by thin leathery yellow septa; the rind thick and coriaceous. Fl.Per.: April July. Fr. Per.: Sept.-Dec.

Distribution: Mediterranean Europe, Africa, and Asia. In Pakistan it grows wild from 1000-2000 m, throughout the western range, (Baluchistan, N. & S. Waziristan, NWFP, Kurram, Dir, Chitral); grows gregariously on dry limestone soils in the salt range and in the Hazara, Also found in the Kashmir and Himalayan areas. 1.10.25 Rubus ellipticus Smith (Rosacceae) Description Shrubs 13 m tall. Branchlets purplish brown or brownish, pubescent, with sparse, curved prickles and dense, purplish brown bristles or glandular hairs. Leaves imparipinnate, 3foliolate; petiole 26 cm, petiolule of terminal leaflet 23 cm, lateral leaflets subsessile, petiolule and rachis purplish red bristly, pubescent, with minute prickles; stipules linear, 61

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711 mm, pubescent, with intermixed glandular hairs; blade of leaflets elliptic or obovate, terminal leaflet much larger than lateral leaflets, abaxially densely tomentose, with purplish red bristles along prominent veins, adaxially veins impressed, pubescent along midvein, base rounded, margin unevenly minute sharply serrate, apex acute, abruptly pointed, shallowly cordate, or subtruncate. Inflorescences terminal, dense glomerate racemes, Flowers: Calyx abaxially pubescent, intermixed yellowish tomentose, sparsely bristly; sepals erect, ovate, abaxially densely yellowish gray tomentose, apex acute and abruptly pointed. Petals white or pink, spatulate, longer than sepals, margin premorse, densely pubescent, base clawed. Ovary pubescent; styles glabrous, slightly longer than stamens. Aggregate fruit golden yellow, subglobose, glabrous or drupelets pubescent at apex; pyrenes triangular-ovoid, densely rugulose. Fl. MarApr, fr. AprMay. 2n = 14.

Distribution Slopes, montane valleys, sparse forests, thickets, roadsides; 300--2600 m. Guangxi, Guizhou, Sichuan, S Xizang, Yunnan Bhutan, India, Laos, Myanmar, Nepal, Pakistan, Philippines, Sikkim, Sri Lanka, Thailand, Vietnam.

1.10.26 Viburnum cotinifolium D. Don (Caprifoliaceae) Description A large shrub up to 3 m tall. Young branches and undersurface of leaves stellately tomentose. Leaves ovate or orbicular, entire, crenate or wavy, lateral nerves 5-6 pairs, obliquely bifurcating halfway between midrib and edge of leaf, prominent beneath. Flower: 6-8 mm long, in peduncled, corymbose cymes; branches of inflorescence woody. Bracts linear, narrow. Corolla white, shortly campanulate; Stigma subsessile. Drupe 8-9 mm long, oblong, compressed, red-black. Seed dorsally 2-grooved, ventrally 3-grooved. Fl. Per.: March-May. Distribution: Afghanistan & Pakistan Himalaya. A common shrub with leaves white cottony below and small white flowers which appear in early spring before the leaves. Found in open sunny places N. W. Himalaya from 900-3500 m.

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1.11 Objectives The study was initiated with following objectives To explore the national flora for medicinally importance species. To identify the effect of Berberis and Mallotus scientifically against different diseases. To study the active chemical constituents of medicinal plants. To help the national scientist in the field of drug discovery from medicinal plants.

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2.1 Berberis lycium Royle (Berberidaceae) 2.1.1 Ethnobotanical uses The roots of B. lycium known as Darhald which are used for diaphoretic, as astringent, as well as bleeding piles (Nadkarni, 1980). In Pakistan folk medicine, the roots powdered the plant are recommended for the treatment of rheumatism and muscular pain and it is to be taken with milk probably to protect the gastric mucosa from damage, (Ikram et al., 1966). The roots of Berberis species are used for the treatment of a number of ailments which includes rheumatism, eye and ear diseases, malarial fever, diabetics, jaundice, stomach disorder, fever, skin disease, and also used as a tonic (Watt, 1889; Kirtikar and Basu, 1933 a; Chopra et al., 1958 a; Ambastha, 1988). Several other Berberis species were found to be used in the treatment of various inflammatory conditions, including rheumatism, fever and Pyrexia (Yesilada and Kpeli, 2002). 2.1.2 Chemical constituents The active constituents are alkaloids. The major alkaloids of B. lycium are berberine and umbellatine (Ali and Khan, 1978), chelidonic acid and oxyacanthine (Karnick, 1994). Heterocyclic constituents, named berberisterol, berberifuranol and berberilycine, have been isolated from the roots of Berberis lycium (Ali and Sharma, 1996). Berberine (I), berbericine and berbericinine hydriodide were also reported in the roots of B. lycium (Ikram et al., 1966). Palmatinechloroform a tertiary dihydroprotoberberine alkaloid (Miana, 1973) and compounds such as the alkaloids sindamine (III), punjabine, gilgitine, chenabine (IV) and jhelumine are also reported (Leet et al., 1982, 1983). Besides these, Ali and Khan (1978), reported berbamine (V) but in the present study the main constituent was berberine and palmatine (II) (Fig. 6), while berbamine was not detected in Thin Layer Chromatography (TLC), High Pressure Liquid Chromatography (HPLC) and Capillary Electrophoreses (CE).
O O

O O

CH3O OCH3

CH3O
I Berberine

N OCH3

II Palmatine

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OMe MeN OMe

MeO NMe O O CHO

MeN

OMe MeO MeO O CHO NMe

O OH

OH

III Sindamine

IV Chenabine

O O O

O N

O V Berbamine O H

Figure 1 Alkaloids of Berberis lycium 2.1.3 Biological testing Berberis lycium shows antimicrobial activities (Harsh and Nag, 1988; Sing et al., 2007).The wound-healing activity has recently investigated in rats, the reports shows an increase in epithelialization and wound contraction(Asif et al., 2007). A significant reduction in both blood glucose levels and glycosylated haemoglobin has reported while treated the Alloxane- induced diabetic Rates with Berberis lycium roots extract and berberine (Gulfaraz et al., 2008). Among the reported chemical constituents of B. lycium, berberine shows different pharmacological activities According to literature berberine is a benzylisoquinoline alkaloid mainly distributed in the genus Berberis and some other medicinal plants. Berberine is to be considered the major active principle of B. lycium. There are different pharmacologic activities of berberine which include metabolic inhibition of certain organisms, inhibit the bacterial enterotoxin formation, inhibit the intestinal fluid accumulation and ion secretion as well, inhibit the smooth muscle contraction, control 65

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and minimizes the inflammation, inhibit the aggregation of platelet, elevate platelet count in certain types of hrombocytopenia, stimulate the secretion of bile and bilirubin, and also inhibit the of ventricular tachyarrhythmias (Birdsall et al., 1997; Akhter et al., 1979). Diarrhea caused by Vibrio cholera and Escherichia coli has been the focus of numerous berberine studies, and results indicate several mechanisms which may explain its ability to inhibit bacterial diarrhea. An animal study found berberine reduced the intestinal secretion of water and electrolytes induced by cholera toxin (Swabb et al., 1981). Other studies have shown berberine directly inhibits some V. cholera and E. coli enterotoxins (Sack and Froelich, 1982). It significantly reduces smooth muscle contraction and intestinal motility (Akhter et al., 1979) and delays intestinal transit time in humans (Yuan et al., 1994). Berberine sulfate has also been found to be directly bacteriocidal to V. cholera (Amin et al., 1969). In a report about it affect on E. coli, berberine sulfate was used in vitro research which shows the bacterial inhibition of adherence to epithelial or mucosal surfaces, the first step in the infective process. The over all effect may be due to the berberines inhibitory activity on fimbrial structure formation on the surface of the treated bacteria (Sun et al., 1988). Growth of some organism like Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis and Leishmania donovani were positively inhibited by berberine extracts and its salts (Kaneda et al., 1991; (Ghosh et al., 1985). It has also be studied that the crude extracts of berberine are more effective than the salts of berberine (Kaneda et al., 1990). In tropical climates Giardia lamblia infestation (giardiasis) is a common occurrence, particularly in pediatric populations (Nair, 1970). In India, it has been concluded after various clinical trials that berberine administration positively improved gastrointestinal symptoms and reduction is occur in Giardia-positive stools. In comparison to metronidazole (Flagyl), another popular giardiasis medication, Berberine was nearly as effective at half the dose (Choudhry et al., 1979). The in vitro and in vivo studies of berberines effects on Entamoeba histolytica indicated berberine sulfate was rapidly amoebicidal and caused encystation, degeneration, and eventual lyses of the trophozoite forms (Subbaiah and Amin, 1967). Berberine sulfate rapidly inhibited the growth of Trichomonas vaginalis via formation of large autophagic vacuoles that eventually result in lysis of the trophozoite forms (Kaneda et al., 1991). Studies have shown berberine markedly decreased parasitic load and rapidly improved hematologic parameters in infected animals. In vitro results indicated berberine inhibited multiplication, respiration, and macromolecular biosynthesis of amastigote forms of the 66

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parasite, interfered with the nuclear DNA of the promastigote form, and inhibited organism maturation (Ghosh et al., 1985). Aqueous berberine and sulfacetamide were both studied in a clinical trial against Chlamydia trachomatis infection which was conducted on 51 subjects in an outpatient eye clinic. It was concluded that while sulfacetamide eye drops gave some better clinical results, while conjunctival scrapings of the patient under investigation were remained positive for the infective agent and relapses occurred. While in case of, the conjunctival scrapings of patients that intake the berberine chloride eye drops were found negative for C. trachomatis and the relapses were also negative up to one year after treatment. It was further studied that the berberine chloride had no direct anti-chlamydial properties, but it is possible that it treated the infection by stimulating some protective mechanism in the host (Babbar et al., 1982). In another clinical study it was found that berberine chloride is better than sulfacetamide in both the clinical course of trachoma and in achieving drop inserum antibody titers against C. trachomatis (Khosla et al., 1992). Berberine administration were studied in both clinical trials and animal research, it was found that it prevented ischemiainduced ventricular tachyarrhythmia, stimulated cardiac contractility, and lowered both blood pressure and peripheral vascular resistance (Chun et al., 1978; Marin-Neto et al., 1988). The mechanism for berberines antiarrhythmic effect is unclear, but an animal study indicated it may be due to suppression of delayed afterdepolarization in the ventricular muscle (Wang et al., 1994). An animal study suggested, in addition to affecting several other parameters of cardiac performance, berberine may have a vasodilatory / hypotensive effect attributable to its potentiation of acetylcholine (Chun et al., 1978). In vitro studies utilizing human cell lines demonstrated that berberine inhibited activator protein 1 (AP-1), a key transcription factor in inflammation and carcinogenesis (Fukuda et al., 1999). Another study, utilizing human peripheral lymphocytes, showed berberine to exert a significant inhibitory effect on lymphocyte transformation, concluding that its anti-inflammatory action may be due to inhibition of DNA synthesis in activated lymphocytes (Ckless et al., 1995). A third study concluded that during platelet activation in response to tissue injury, berberine had a direct affect on several aspects of the inflammatory process. It exhibited dose-dependent inhibition of arachidonic acid release from cell membrane phospholipids, inhibition of thromboxane A2 from platelets (Huang et al., 1991) and inhibition of thrombus formation (Wu and Liu, 1995).

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Berberine has demonstrated a number of other beneficial effects, including immunostimulation because berberine increased blood flow to the spleen, activated of macrophage, rising of platelet numbers in cases of primary and secondary thrombocytopenia, and the excretion of conjugated bilirubin are increased in experimental hyperbilirubinemia (Birdsall et al., 1997). The anticancer properties of berberine against cancer cells established from cervical, esophageal, oral, colonic, prostate cancers, leukemia melanoma and glioblastoma are known by different studies (Iizika et al., 2000; Jantova et al., 2003, 2006; Kuo et al., 1995; Letasiova et al., 2006; Li et al., 2000; Lin et al., 2006a, 2006b, 2007; Mantena et al., 2006a, 2006b; Piyanuch et al., 2007., Sanders et al., 1998; Serafim et al., 2007; Zhang et al., 1990; Katiyar et al., 2008). Berberine was studied in different assays it is concluded that it inhibits tumor cell growth via inducing cell cycle arrest and/or apoptosis, and the expression pattern of genes which is responsible for the regulation of cell cycle progression and apoptosis was correlated to the inhibition of cellular proliferation. The activity of berberine against tumor cells may vary depending on the duration of treatment , amount of dose and type of cancer cells, (Iizika et al., 2000; Jantova et al., 2003, 2006; Kuo et al., 1995; Letasiova et al., 2006; Li et al., 2000a; Lin et al., 2006a, 2006b, 2007; Mantena et al., 2006a, 2006b; Piyanuch et al., 2007; Sanders et al., 1998; Serafim et al., 2007; Zhang et al., 1990). It has been studied the effect of berberine on non-small cell lung cancer cells and concluded that the growth inhibition of the cells were mediated by p53 (Zhang et al., 1990). But still it is under investigation that how berberine initiates the cascade that eventually leads to cell cycle arrest and/or apoptosis and it suggested in some studies that berberine may interfere with DNA replication as a topoisomerase I inhibitor (Gatto et al., 1996; Kobayashi et al., 1995), while in some others experiment it showed that berberine may cause directly DNA damage (Krey and Hahn, 1969; Davidson et al., 1977; Li et al., 2000b; Ihmels et al., 2005; Letasiova et al., 2006). A very recent study addressed the molecular mechanisms of Berbeine-induced cell cycle arrest and apoptosis in osteosarcoma cells. The authors concluded that Berberine inhibited osteosarcoma cell proliferation through its genotoxicity causing p53-dependent G1 arrest and apoptosis, whereas G2 arrest was p53independent (Liu et al., 2009). In the present investigation studying extracts of B. lycium and its main constituent, Berberine, we discovered another growth inhibitory mechanism that did not involve genotoxicity, but the inactivation of Cdc25A and the acetylation of atubulin reminiscent to the anti-neoplastic mechanism of taxol.The doses usage of berberine in clinical situations is not considered toxic and cytotoxic or mutagenic. High 68

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dosages of berberine can result some side-effects which may include dyspnea, lowered blood pressure, gastrointestinal discomfort, flu-like symptoms, and cardiac damage. In pregnancy care should be taken while using berberine, because berberine can cause uterine contractions and miscarriage. Berberine may be avoided in jaundiced neonates because of its bilirubin displacement properties. The berberine can be use in most clinical situations for various therapeutic purposes is 200 mg orally two to four times daily. 2.2 Mallotus philippensis (Lam.) Muell. Arg. (Euphorbiaceae) 2.2.1 Ethnobotanical uses. Kamala, a red powder consisting of glandular hairs from the capsules of the plant, It has been used as a drug and dye and has long been used as an anthelminticum and cathartic in traditional medicine (Satyavati et al., 1987; Srivastava et al., 1967; Gupta et al., 1984 and an orange dye for silk (Lounasmaa et al., 1975). Fruit is purgative for animal (Zabihullah et al., 2006) .the red powder (Local name; Kamela) coating the fruit is commonly administered in curd for the elimination of intestinal worms and also for skin irritation, ringworm, and freckles (Usmanghani et al., 1997). 2.2.2 Chemical constituents Its many chemical constituent of Mallotus philippensis include -sitosterol, stigmasterol, bergenin, and alphaamyrin. (Bandopandhyay et al., 1972; Zaidi et al., 2009). Flavonoids such as Kamalachalcones A and B have been isolated by Toshiyuk et al (1998) from kamala. A new flavanone, 4-hydroxy isorottlerin (I), and two new chalcone derivatives, kamalachalcones C (II) and D (III) and 5,7-dihydroxy-8-methyl-6-prenylflavanone (VI) (Furusawa et al., 2005), were isolated from the red powder of glandular hairs kamala. Phloroglucinol derivatives, Mallotophilippens A (IV) and B(V); Mallotophilippens C, D and E (Daikonya et al., 2002, 2004), Friedelin (Tanaka et al., 1988), 3-hydroxy-D:Afriedoolean-3-en-2-one (Kikuchi and Toyoda, 1967), 2 -hydroxy-D:A-friedooleanan-3one and 3 -hydroxy-D:A-friedoolean-an-2-one (Talapatra et al., 1978), lupane-type triterpenoids, lupeol and betulin (Tanaka et al., 1988). 3'-prenylrubranine (VII) (Zaidi et al., 2009), red compound (VIII) (Lounasmaa et al., 1975), isorottlerin (IX) and rottlerin (X) (Furusawa et al., 2005). (Fig.7) 2.2.3 Biological testings Biological studies such as cytotoxic (Arisawa et al., 1986, 1990; Fujita et al., 1980), antitumor (Arisawa et al., 1990), and human immunodeficiency virus (HIV) reverse transcriptase inhibitory activities have been described (Nakane et al., 1991) Anthelmintic, 69

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antibacterial and antiallergic activities of Mallotus philippensis has been justified, especially, its ethnomedical use against intestinal worms.( Jabbar et al., 2006; Kumar et al., 2006; Daikonya et al., 2002). In recent report Zaidi et al (2009) has describe the bactericidal potential of the chemical compounds isolated from Mallotus philippensiss and concluded that rottlerin was inhibit Helicobacter pylori most potently. Rottlerin (5,7dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1 ,2chromine), also called mallotoxin, is one of the major constituents of Mallotus philippensis exhibiting various pharmacological activities including mitochondrial uncoupler effects. (Zaidi et al., 2009). Rottlerin was considered as a specific inhibitor of the novel protein kinase C (PKC) isoform, PKC d, and was shown to have anticarcinogenic properties (Soltoff,, 2007). PKC d translocation and activation are induced by different apoptotic stimuli in different cellular systems (Brodie et al., 2003). It has been studied that activation of specific pathways in the plasma membrane in mitochondria and nucleus and translocation by PKC d that eventually converge to the activation of caspase-3 and subsequent apoptosis (Brodie et al., 2003). However, there are a large number of studies that have concluded that rottlerin might not act directly on PKC d, but can activate some cellular changes that is very similar those produced by the direct inhibition of PKC d (Soltoff, 2001;Tapia et al., 2006). In one latest experiment, In which colon carcinoma cells and glioma cells are sensitized by rottlerin to TRAIL-mediated apoptosis by uncoupling of the mitochondria and inhibition of Cdc2, respectively (Tillman et al., 2003; Kim et al., 2005), However the mechanisms was not clear that how rottlerin-induced apoptosis and rottlerin sensitizes cancer cells to TRAIL-mediated apoptosis . It has also found that the apoptosis induced by rottlerin is mediated through DR5 up regulation (Lim et al., 2009). Rottlerin also sensitized different type of cancer cells, but has no effect on normal cells. In case of TRAIL-mediated apoptosis, it has been suggested that the combined treatment with rottlerin and TRAIL may offer a safe and effective cancer therapy and it has also found in the same experiment that CHOP-mediated DR5 up regulation, which is independent of PKC d activity, also take a significant rule in rottlerin-induced apoptosis. Tanaka et al, (2008) has isolated known friedelane-type triterpenoids compounds from the stem bark of M. phillipensis and described the anti-tumor promoting activity of 3-hydroxy-D:Afriedoolean-3-en-2-one ( IC50 = 292 mol ratio/ 32 pmol/TPA); 3-hydroxy-D:Afriedoolean -2-one (IC50 = 288); positive control, curcumin (IC50 = 343); Epstein-

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Barrvirus early antigen (EBV-EA) activation induced by 12-O-tetradecanoyl phorbol 13acetate (TPA) used in the experiment.

Me HO Me Me Me

O OH OH OH O
Me HO

O Me O Me H OH Me OH O

H OH

O Me

OH

I 4'-Hydroxyisorottlerin

II Kamalachalcones C

Me O O HO OH

Me Me Me O O HO O Me O HO Me Me Me O O OH

Me CO Pr i HO OH HO O O Me Me Me

Me OH OH

HO Me

OH III Kamalachalcone D

IV Mallotophilippen A

Me CO Me HO OH HO O O Me Me Me

HO

Me OH OH

OH

V Mallotophilippen B

VI 5,7-dihydroxy-8 methyl-6-prenylflavanone

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O HO OH

OH

HO
O O

O
OH O O

OH
VII 3 -prenylrubranine

O
OH O IX Isorottlerin

VIII Red compound

Figure 2 Compounds of Mallotus philippensis

2.3 Adhatoda vasica Nees (Acanthaceae) 2.3.1 Ethnobotanical uses Adhatoda vasica is widely used in the Ayurvedic and Unani system of medicine for treating bronchitis, asthma, fever and jaundice on account of the antispasmodic properties of roots and leaves. A. vasica has also been used for the treatment of bronchial obstruction which is created by allergen (Sharma et al., 1999; Amin and Mehta, 1959). It has been used for asthma and tuberculosis (Dorsch and Wagner, 1991; Paliwa et al., 2000; Barry et al., 1955; Grang et el., 1996; Gupta et al., 1954). 2.3.2 Chemical constituents The chemical examination of Adhatoda vasica revealed to contain different types of alkaloids, glycosides, different phenolic compounds and sterols components (Lateef et al., 2003). The major chemically active components identified are two alkaloids: vasicinone and vasicine (Das et al., 2005). Some minor alkaloids viz. Vasicol, adhatodinine and vasicinol also present. The leaves contain an alkaloid vasicine and an essential oil. 2.3.3 Biological testing Adhatoda vasica possesses hepatoprotective activity (Bhattacharyya et al., 2005). It possesses antioxidant, chemopreventive agent and antibacterial (Karthikeyan et al.,2009). It has the tendency to restore the hematological changes produced by irradiation in Swiss albino mice (Kumar et al., 2005). The activities of Glutathione S-transferase are enhanced in the liver of mice. A. vasica reduced glutathione (GSH) levels in liver, and also reduced lipid peroxidation(LPO). It also reduced the acid and alkaline phosphatases in testis of 72

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normal and irradiated mice (Singh et al., 2000). Leaves of Adhatoda vasica possess anticestodal efficacy (Yadav and Tangpu, 2008). Due to alkaloids such as vasicine and vasicinone it possesses the biological activities such as expectorant and mild bronchial antispasmodic. (Lahiri and Pradhan, 1964; Gupta et al., 1971).

2.4 Albizia lebbeck (L.) Benth. (Mimosaceae) 2.4.1 Ethnobotanical uses The wood of Albizia lebbeck is very similar to walnut and therefore very good for canoes, furniture, house building, and picture frames, etc. the wood of A. lebbeck is also used for cane crushers, oil. In the Ayurveda both the leaves and the bark of A. lebbeck have been in clinical use for centuries. Spongy and ulcerative gums have been strengthening with the powder of the bark of the roots. The leaves Juice are very useful in ophthalmia. The leaves decoction are useful for night-blindness and therefore given internally. Bark is astringent and is employed in diarrhea, dysentery, and hemorrhoids. Powdered bark is useful for ulcers, and especially for snake wounds flowers possess the power of causing retention of the seminal fluid. Seeds are astringent and are employed in diarrhea, dysentery and hemorrhoids. The seeds are also used for ophthalmic diseases. The oil from the seeds is considered useful in leprosy. The seeds are crushed and made into a paste, which is applied to reduce enlarged cervical glands. Bronchial asthma is being treated with the decoction of stem bark. Bark of A. lebbeck used as antifertility drugs (Shah et al., 2009). 2.4.2 Chemical constituents The leaves of Albizia lebbeck are good source of saponins (Pal et al., 1995). 2.4.3 Biological testing The leaves of A. lebbeck, which contain different type of saponins, and possessed nootropic activity (Chintawar et al., 2002), anticonvulsant activity (Kasture et al., 2000). Antiasthmatic and antianaphylactic activity of A. lebbeck have been reported (Tripathi and Das, 1977). Tripathi et al (1979) have conclude that A. lebbeck is not like disodium chromoglycate, it exerts antianaphylactic activity in guinea pigs. A. lebbeck also enhance the concentration of plasma cortisol level in patients of bronchial asthma (Tripathi et al., 1978). Albizia lebbeck showed antioxidant activities in alloxan-induced diabetic rats (Resmi et al., 2006).

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2.5 Bauhinia variegata Linn. (Caesalpinaceae) 2.5.1 Ethnobotanical uses Bauhinia variegata generally cultivated as an ornamental plant. The leaves are given to cattle as fodder, flowers are used as pot herb and also made into pickles; wood is useful in buildings and for making agricultural goods. The plant yields gum; the bark is useful for tanning and dyeing. The plant is reputed to have medicinal properties also. The root is tonic and carminative, the flowers laxative and the bark is astringent; various parts of the plant are reputed to have healing properties also. In the traditional medicines of South East Asia the same is used for skin diseases, as an astringent, bronchitis, tonic, leprosy, anti inflammatory and for ulcers (Kirtikar and Basu, 1993). The roots decoction is useful in dyspepsia and also used as an antidote to snake poison (Thammanna et al., 1990). 2.5.2 Chemical constituents Several flavonoids have been isolated during the phytochemical studies on the stems, bark, flowers and seeds (Gupta et al., 1979, 1980, 1984; Rahman and Begum, 1966; Wahab et al., 1987;Yadava and Reddy, 2001). The non-woody aerial parts of B. variegata were studied and isolated six flavonoids, and a triterpene caffeate, ( Rao et al., 2008). Phenanthraquinone, named bauhinione has been isolated from Bauhinia variegate (Zhao et al., 2005). 2.5.3 Biological testing The non-woody aerial parts of B. variegate yield anti-inflammatory agents. It shows insuline secretion activity from INS-1 cells. The ethanolic extract of B. variegate at the rate of 250 mg/kg positively suppressed liver tumor (Rajkapoor et al., 2006). It has been determined the anthelmintic activity of the leaves. (Sing et al., 2005).

2.6. Bombax ceiba Linn. (Bombacaceae) 2.6.1 Ethnobotanical uses The cotton inside the fruits was used a substitute of cotton. Various parts of plant are used in small pox, bleeding gums, toothache, carries, sores in mouth, pain in leg, fever, enlarged spleen, atrophy, emaciation, rheumatism, spermatorrhoea, cholera, pneumonia, pleurisy, intestinal neuralgia, leprosy and skin diseases (Ansari, 2004). The flower was a common ingredient in Chinese herb tea. The gum has aphrodisiac, astringent, demulcent, haemoptysis of pulmonary tuberculosis and influenza, malaena and menorrhagia and 74

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acute dysentery with beneficial results. Flowers are used for haemorrhoids. Root has stimulant, tonic and aphrodisiac properties. Plants are used for making light packing boxes and in fisherman floats. In Punjab it is used for making water conduits, troughs and bridges, the timber is also utilized in match industry. Buds are used as vegetables. 2.6.2 Chemical constituents Preliminary tests show the presence of glycosides and tannins from root, stem and leaf. In the stem some alkaloids and in root proteins are identified (Mehra, 1968). The stem bark contains lupeol and b-sitostrol (Mukherjee, 1971). The root bark has 3 naphthalene derivatives related to gossypol (toxic principle of cotton seed) and called as 'semigossypol' (Seshadri, 1973). Flowers contain b-sitosterol, traces of essential oil, kaempherol and Quercetin (Gopal, 1972). On hydrolysis gum yield arabinose, galactose, galacturonic acid and rhamnose. 2.6.3 Biological testing Aqueous extract has moderate oxytoxic activity on gravid and non-gravid isolated rat uteri and guinea pig and rabbit uterine strips. It has musculotrapic action in guinea pig ileum and cardiac stimulant action on frog's heart (Misra, 1968). It has a negligible bloodpressure elevating action in anaesthetized dog (Misra, 1966).

2.7 Calotropis procera Linn. (Asclepiadaceae) 2.7.1 Ethnobotanical uses Calotropis procera is a medicinal plant. The latex is irritant to the skin and mucous membrane and said to cause blindness. It is also used as a purgative and said to be specific for Guinea worms. The seed floss is used for stuffing mattresses, pillows etc. It is sometimes used to adulterate Indian Kapok but it is inferior to it in resilience and water repellent properties. In Indian traditional system of medicine the different parts of the C. procerat have been used for the treatment of various diseases such as ulcers, leprosy, piles, tumors, and certain disease of abdomen, liver and spleen (Kirtikar and Basu, 1935). C. procera (root) are useful carminative drug in the treatment of dyspepsia (Kumar and Arya, 2006). Various tribes of central India use C. procera (root bark and leaves) as a useful drug for jaundice, a curative agent and as antidote for snake poisoning (Samvatsar, 2000; Nandkarni,1976).

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2.7.2 Chemical constituents The latexes of C. procera are a rich source of many biologically active constituents that include some glucosides, different tannins and proteins (Wititsuwannakul et al., 2002; Dubey and Jagannadham, 2003). Alkaloids, cardiac glycosides, tannins, flavonoids, sterols and triterpenes has been reported (Mossa et al., 1991). Singh and Rastogi reported Calactin, Calotropin and Uscharidin were formed by substitution of glycosides at C-1 of 4, 6 deoxy sugar (Singh and Rastogi, 1970), Calotropin isolated from leaves and stalkes (Perry and Metzger, 1980), toxic flavonoids have also been reported (Salunke et al., 2005). 2.7.3 Biological testing According to the study of Choedon et al, the aqueous extract of C. procera (latex) has inhibit cellular infiltration and concluded that it afford protection against development of neoplastic changes while using the transgenic mouse model of hepatocellular carcinoma (Choedon et al., 2006). The roots of C. procera have been extracted with chloroform and studied the protective activity against carbon tetrachloride induced liver damage which shows a significance protective result. (Basu et al., 1992). Methanol extract possess antioxidant activity in Trema orientalis (Uddin et al 2008) and Senna tora (Uddin et al 2008a). C. procera latex is also reported to possess very interesting unrelated activities such as the ability to combat diarrhea or retard insect larval development and (Kumar et al., 2001, 1994; Morsy et al., 2001). Chloroform extract of roots has been reported to possess anti-inflammatory activity (Kumar and Basu, 1994; Basu and Chaudhuri, 1991). Aqueous extract of the flowers has been found to exhibit analgesic, antipyretic and antiinflammatory activity (Mascolo et al., 1988).The alcoholic extract from different parts has been found to possess antimicrobial and spermicidal activity (Kishore et al., 1997; Qureshi et al., 1991). Alcoholic roots extract possesses a very strong antiimplantation activity 100%.which may be due to its estrogenic activity (Jagadish et al., 2002). Laticifer proteins (LP) recovered from the latex of the medicinal plant. Calotropis procera, is a target for DNA topoisomerase I triggering apoptosis in cancer cell lines. (Oliveira et al., 2007). Calotropis procera flowers possess hepatoprotective activity (Ramachandra et al., 2007).

2.8 Carissa opaca Stapf ex Haines (Apocynaceae)

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2.8.1 Ethnobotanical uses Fresh leaves of Carissa opaca and roots of Sageretia brandrethiana are boiled in water and used in case of Jaundice and Hepatitis, the decoction is taken orally, approximately one cup twice a day for two to three weeks (Abbasi et al., 2009). Fruit is edible. 2.8.2 Chemical constituent Carissone, palmatic acid, benzyl salicylate, benzyl benzoate, farnesene (Rai et al., 2005)

2.9 Cassia fistula Linn. (Caesalpinaceae) 2.9.1 Ethnobotanical uses Cassia fistula is an ornamental tree, the bark is used as tanning material and wood ash is used as mordant in dyeing. The pulp of pods is used in Bengal to flavour tobacco. The durable wood is used for various purposes. The different parts of the plant are also reported to have medicinal properties. It is also useful in the treatment of different skin diseases, rheumatism, inflammatory diseases, anorexia and jaundice (Anonymous,1992; Kirtikar and Basu 1991). 2.9.2 Chemical constituents A flavone glycoside 5,3',4'-tri-hydroxy-6-methoxy-7-O-alpha-L-rhamnopyranosyl-(1 --> 2)-O-beta-D-galactopyranoside with antimicrobial activity was reported by (Yadava and Verma, 2003). Compounds, 5-(2-hydroxyphenoxymethyl) furfural, (2'S)-7-hydroxy-5hydroxymethyl-2(2'-hydroxypropyl)chromone,benzyl-2-hydroxy-3,6-dimethoxybenzoate, and benzyl 2beta-O-D-glucopyranosyl-3,6-dimethoxybenzoate, 5-hydroxymethylfurfural, (2'S)-7-hydroxy-2-(2'-hydroxypropyl)-5-methylchromone, and two oxyanthraquinones, chrysophanol and chrysophanein, were isolated from the seeds of Cassia fistula. (Kuo et al., 2002) 2.9.3 Biological Testing Luximon-Ramma et al (2002) has reported that antioxidant activities correlated to the total Phenolic compounds. The hepatoprotective activity (Bhakta et al., 1999; Bhakta et al., 2001) and the hypoglycemic activity (Esposito Avella et al., 1991) have been reported.

2.10 Colebrookea oppositifolia Smith (Labiateae)

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2.10.1 Ethnobotanical uses In traditional medicine, the epilepsy diseases is treated with the roots of C. oppositifolia and the leaves of the same plants are used for the healing of wounds and bruises (Chopra et al., 1956) 2.10.2 Chemical constituents Several flavone and flavone glycosides have isolated from the bark, stems, leaves, and flowers of C. oppositifolia. (Ahmed et al., 1974; Patwardhan et al., 1981; Yang et al., 1996).

2.11 Debregeasia salicifolia (D.Don) (Urticaceae) 2.11.1 Ethnobotanical uses A strong fiber used to make ropes, is obtained from the bark. 2.11.2 Chemical constituent Akber et al. (2001) reported quercetin, hisperidine, 3b-19alpha-dihydroxy-30-norurs-12ene,b-sitosterol, stigmasterol, oleanolic acid and lupeol in extracts of Debregeasia salicifolia 2.11.3 Biological Testing Ahmed et al. (2006) has identified that leaves extracts has IC50 values more than 100 g/ml of DPPH radical scavenging activity while comparing with Ascorbic acid IC50 = 1.75

2.12 Dalbergia sissoo Roxb. (Papilionaceae) 2.12.1 Ethnobotanical uses Plants of the genus Dalbergia are medicinally important and have been used for the treatment of gonorrhoea, arthritis, and rheumatic pains (Anonymous. 1950; Nadkarni, 1982; Singh and Chaturvedi, 1966). It has been reported in folk medicine and is used mainly as aphrodisiac, abortifacient, expectorant, anthelmintic and antipyretic. It is also used in conditions like emesis, ulcers, leucoderma, dysentery, stomach troubles and skin diseases. (Kirtikar and Basu, 1933 b; Nadkarni. 1954; Chopra et al., 1956 b). In Arabic countries the aqueous leaves extract of D. sissoo has been used for the treatment of

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gonorrhea (El-Dagwy, 1996).The hard wood D. sissoo which is very heavy and durable, widely used for the manufacturing of boats furniture, wheels and carts, etc. 2.12.2 Chemical constituents Phytochemical examination of genus Dalbergia has provided a large number of compounds, which include flavonoids, furans, benzophenones, styrenes, and terpenoids (Chawla and Chibber, 1981; Khan and Javed, 1997). Chemical constituent of D. Sissoo have also been studied before (Ahluwalia et al., 1965; Ahluwalia et al., 1963; Banerji et al., 1965, 1966; Banerji et al., 1963; Dhingra et al., 1974; Farag et al., 2001; Mukerjee et al., 1971; Ramakrishna et al., 2001;Sharma et al., 1979a, 1979b, 1980a, 1980b). Two new isoflavones glycosides along with other five known isoflavone glycosides have been isolated from the leaves and stem bark by D. sissoo Salwa et al (2001). It has been isolated several type of chemicals constituent from the green branches and aerial parts of D. sissoo Roxb, such as biochanin-A, tectorigenin, isoflavones irisolidone, prunetin, muningin, genestein, the flavone nor-artocarpotin, sissotrin and prunetin-4- O galactoside stigmasterol, -sitosterol and -amyrin (Kinjo et al., 1987; Ishikura et al., 1989; Rao et al., 1989; Ramesh and Yuvarajan, 1995; Mathias et al., 1998). Sarg et al (1999) and Labreque, (1983) reported the composition of the fatty acids in the fixed oil which are myristic 5.56%, palmitic 21.70%, stearic 24.33%, arachidic 19.37%, linoleic 10.81% and oleic 9.4% (Labreque, 1983; The Wealth of India, 1988) 2.12.3 Biological testing Hajare et al (2000) reported marked antipyretic and moderate analgesic activities by Dalbergia sissoo leaves. Ethanolic extract of D. sissoo leaves possesses antiinflammatory activity (Hajare, 2001).The oil also showed strong repellent action (Ansari et al., 2000). A dose-dependent inhibitory effect have been shown by the alcohol extract of the green aerial parts on the motility of isolated rabbit duodenum, pronounced bronchodilation and also shows a significant antipyretic, anti-inflammatory, analgesic, and estrogen-like activities. The plant extract was studied with out any side effects in rats. (Sarg et al.,1999). 2.13 Dodonaea viscosa Linn. (Sapindaceae) 2.13.1 Ethnobotanical uses Leaves D. viscosa are useful for wounds healing, burns and swellings. It is also used as febrifuge and is useful in rheumatism. The fruit is used as a fish poison. The decoction should be used as mouthwash only and should not be swallowed (Qureshi et al., 2008). It 79

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is useful remedy for the treatment of diarrhea, skin infections and rheumatism. The roots of D. viscose are used for the treatment of inflammation and spasmodic in traditional medicine. D. viscosa is used for malaria, wounds and burns (Al-Dubai and Al-khulaidi, 1996). It is also used as an antipuritic in skin rashes and for the treatment of sore throat, dermatitis and hemorrhoids (Chhabra et al., 1991; Hedberg et al., 1983). In India, the infusion of leaves were used to treat rheumatism, gout, hemorrhoids, fractures and snake bites (Kirtikar and Basu, 1995; Nadkarni and Nadkarni, 1982) .The quick growth and gregarious habit of this shrub makes it an excellent hedge plant. The branches are used as fire-wood and as a support for the flat mud roofs in village houses. The wood can be used for making walking sticks and tool-handles. 2.13.2 Chemical constituents Aliarin, dodonic acid, viscosol (Sachdev and Kulshreshtha, 1986), stigmosterol, isorhamnetin (Rao, 1962; Ramachandra et al., 1975), penduletin, quercetin, doviscogenin (Khan et al., 1988), dodonosides A and B (Wagner et al., 1987) have been isolated from D. viscose. Flavonoids, terpenes, coumarins and steroids are also reported by AbdelMogib et al (2001) and Ahmad et al (1987). 2.13.3 Biological testing Literature survey reveals that D. viscosa has an antimicrobial effect (Rojas et al., 1992; Getie et al., 2004), this plant collected in different countries demonstrates variable biological activity. The methanol extract of the entire plant collected in Saudi Arabia possesses no activity against Escherichia coli, Proteus vulgaris, Staphylococcus aureus and Pseudomonas aeruginosa and Candida albicans (Getie et al., 2003). On the other hand, a similar extract of the leaves of the Mexican species showed weak activity against E. coli, P. aeruginosa, S. aureus, Bacillus subtilis and C. albicans (Rojas et al., 1982).The 50% methanol of the flowers and leaves of the Nigerian species demonstrated antibacterial activity against B. subtilis, E. coli, Proteus species, P. aeruginosa and S. aureus (Ogunlana and Ramstad, 1975). An antipyretic and an antimicrobial agent has been reported (Rojas et al., 1992, 1995, 1996; Getie et al., 2003; Ahmad et al., 1994) The leaves were reported to possess local anesthetic, smooth muscle relaxant (Rojas et al, 1996), antifungal (Al-Yahya et al., 1983; Naovi et al., 1991) anti-inflammatory (Mahadevan et al., 1998; Getie et al., 2003) and anti-ulcerogenic activity (Veerapur et al., 2004). Sukkawala and Desai (1962) have reported that 95% ethanol extract of D. viscose leaves has shown anti-scariasis, anthelmintic, cardiac depressant, hypotensive, uterine 80

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relaxation and asoconstrictor activity in different experimental models. However the pharmacopoeial standards of D. viscosa leaves have not been reported. Khalil et al., (2006) reported that alcoholic extract of D. viscose possess anti-inflamatory activity without toxic effect. Ramzi et al (2008) reported that the diterpenoid and flavonoids derivatives (Sachdev and Kulshreshtha, 1984; Abdel-Mogib et al., 2001; Getie et al., 2002) are mainly responsible for the remarkable antioxidant and antimicrobial effect of this plant.

2.14 Ficus palmata Forssk. (Moraceae) 2.14.1 Ethnobotanical uses The fruit is demulcent, emollient, laxative and poultice (Parmar and Kaushal, 1982; Chopra et al., 1986). It is used as a part of the diet in the treatment of constipation and diseases of the lungs and bladder (Chopra et al., 1986). The sap is used in the treatment of warts. Fruit in raw is sweet and succulent (Hedrick, 1972). A very tasty fruit (Parmar. and Kaushal, 1982), it is often dried for later use. The fruit is about 2.5cm in diameter and annual yields from wild trees are about 25kg (Parmar. and Kaushal, 1982). The unripe fruits and young growth are cooked and eaten as a vegetable. They are boiled, the water is removed by squeezing and they are then fried. Used as a nice green vegetable (Parmar and Kaushal, 1982). The pliable wood is of little value but has been used for making hoops, garlands, ornaments. 2.14.2 Chemical constituents The fruit contains about 6% sugars, 1.7% protein, 0.9% ash and 0.2% pectin (Parmar and Kaushal, 1982 ). Low in vitamin C, about 3.3mg per 100g (Parmar. and Kaushal, 1982).

2.15 Ficus racemosa L. (Moraceae) 2.15.1 Ethnobotanical uses The mature fruits are astringent, stomachic and carminative. Traditionally the fruit extract is used in diabetes, leucoderma and menorrhagia. It is also used locally to relive inflammation of skin wounds, lymphadenitis. They are eaten by locals people. The wood is often employed in making cart frames, ploughs, box, fittings, match boxes and cheap furniture. A decoction of the bark is used as a wash for wounds. The tree is planted for shade in gardens.

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2.16.2 Chemical constituents Ficus racemosa is a chemically rich plant and possess glycosides, beta-sitosterol, lupeol, dumurin, tiglic acid ester, taraxasterol and a new compound racemosic acid (Ghani, 1998; Li et al., 2004). Jahan et al., (2008) has reported an antioxidant compound 3-O-(E)caffeoyl quinate through bio assay guided isolation. A tetra cyclic triterpene, Gluanol acetate was isolated from bark acetone extract of F. racemosa and identified as new mosquito larvicidal compound. A new anti-inflammatory glucoside, Racemosic acid along with Bergenin isolated from Ficus racemosa. Racemosic acid possesses antioxidant activity (Li et al., 2004). 2.16.3 Biological testing The F. racemosa fruits are hypoglycaemic and antioxidant activities (Jahan et al, (2008). The leaves and stem bark also contain hypoglycaemic activity (Baslas & Agha, 1985). The aqueous bark extract possesses wormicidal activity and useful anthelmintic (Chandrashekhar et al., 2008). The leaves of F. racemosa also contain antifilarial activity, antidiuretic, antihepatotoxic, anti-pyretic, anti-inflammatory, Antifungal, analgesic, antipyretic and hepatoprotective activities (Deranjyagala et al., 1988; Forestieri et al., 1996; Mandal et al., 1998, 1999, 2000; Mishra et al., 2005; Rao et al., 2003, 2002). The ethanolic extract of the bark is hypoglycemic and antiprotozoal activity. Decoction of the bark is used as wash for wounds, in asthma, piles and menorrhagia (Yusuf et al., 1994; Ghani, 1998). The bark and leaf of F. racemosa were also reported to have significant antidiuretic activity, wound healing activity, antitussive activity, antinociceptive activity, anti-pyretic activity, hypoglycemic activity, anti-bacterial activity, hepatoprotective activity and anti-diarrhoeal activity (Mukherjee et al., 1998; Mandal et al., 1999, 2000; Rao et al., 2002; Bhaskara et al., 2003; Ratnasooriya et al., 2003, Ferdous et al., 2008). Khan and Sultana (2005) have reported in renal carcinogenesis induced with ferric nitrilotriaceatate (Fe-NTA) treated rats and evaluate the chemomodulatory effect by F. racemosa. KBrO3-mediated nephrotoxicity in rats is significantly suppresses by Ficus racemosa extract and therefore considered a potent chemo preventive agent (Khan and Sultana, 2005).

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2.17 Lantana camara Linn. (Verbenaceae) 2.17.1 Ethnobotanical uses A tea prepared from the leaves and flowers is useful against influenza and stomachache. The leaves of L. camara are useful for different diseases like swelling or inflammation, ulcers, catarrhal affection, itches, cuts, bilious fever, rheumatism, eczema eruptions, and also useful for the treatment of snake-bite. The oil isolated from the leaves is useful antiseptic for wound healing. The roots and flower are useful for toothache and chest complaints of children respectively. Diaphoretic, Vulnerary, and carminative properties are also present in L. camara. Several other activities are present in L. camara such as treatment of high blood pressure, fistulae, asthma, pustules, tumours and cancers, atoxy of abdominal viscera, malaria, in tetanus, leprosy, scabies, and bronchitis (Kirtikar and Basu, 1918; Ghisalberti, 2000; Pullaiah, 2006; Mahathir, 2002; Johns et al., 1983; Begum et al., 2000; Barua, 1969). The roots of L. camara are useful for the treatment of gonorrhea. The plant is also useful for Alzheimcr's disease as well. It is a tonic to the nervous system and used to treat insomnia and epilepsy. It relaxes the muscles, quickens the senses and strengthens the memory (Siddiqui et al., 1995; Begum et al., 2003). 2.17.2 Chemical constituents
Lantanin by P. G. J. Louw (1943), lantadene B (Barton et al ., 54), lantanolic acid (Barua

et al., 1969). Various steroids, terpenoids, and flavonoids have isolated by different groups from the different parts of the plant (Pullaiah, 2006; Johns et al., 1983; Begum et al., 2000). lantanoic acid and camaranoic acid, lantic acid (Begum et al., 2008), camarinic acid (Siddiqui et al., 1995), camangeloyl acid, camarinin (Begum et al., 2003, 2006), oleanonic acid, and ursonic acid (Siddiqui et al., 2000) lantanolic acid (Begum et al., 2008, lantanilic acid (Barua et al., 1976, 1985), -amyrin , -sitosterol and lantadene B (Ahmed et al., 1972), Iantoic acid (Roy and Barua, 1985), lantadene D (Sharma et al., 1990), lantadenes.( Sastry and Mahadevan, 1963; Sharma et al., 2000), lantanolic acid, oleanolic acid, 22/.-O-angeloyl-oleanolic acid, 22 -O-senecioyl-oleanolic acid, 22 hydroxyl-oleanonic acid , 19-hydroxy ursolic acid and a new triterpenoid 3isovaleroyl-l9-hydroxy-ursolic acid (lantaiursolic acid) (Pan et al., 1993), camarinic acid, camaric acid, camarilic acid and camaracinic acid (Siddiqui et al., 1995; Begum et al., 1995), 25-hydroxy-3-oxolean-12-en-28-oic acid, hederagenin and 19-hydroxyursolic acid (Singh, et al., 1996), novel trans lactone containing euphane triterpenes A, B and C (O'Neill et al., 1998), phcnylpropanoid glycosides verbascoside, isoverbascoside,

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isonuomioside A, calceolarioside E and derhamnosylverbascoside (Taoubi et al., 1997), martynoside and verbascoside (Syah et al., 1998), theveside (Ford and Bcndall, 1980),

2.17.3 Biological testing Wound-healing property and antihyperglycaemic activities have been reported in the aqueous extract of the leaves and the shoot of L. camara exhibit antibacterial properties. A steroid Lancamarone, isolated from the leaves of L. camara, possesses cardiotonicproperty. The lantamine alkaloid isolated from the bark of stems and roots, possesses effective antispasmodic and antipyretic properties as such as those of quinine (Kirtikar and Basu, 1918; Ghisalberti, 2000; Pullaiah, 2006; Mahathir, 2002).

2.18 Melia azedarach Linn. (Meliaceae) 2.18.1 Ethnobotanical uses Persian Lilac is a fast growing tree of the plains and foot-hills, cultivated along roadsides and in villages. The fruit is eaten by goats and sheep, and the stony endocarps are used as beads. The exuded gum obtained from its trunk is considered useful in spleen enlargement, its wood extract is prescribed internally in asthma (Dhiman, 2003), decoction of bark is used in paroxysmal fever to relieve thirst, nausea, vomiting and general debility, loss of appetite and skin diseases (Sharma et al., 2001). Leaves are applied in the form of poultice to relieve nerves headach and to cure the eruption on the scalp. Leaf juice is anthelmintic, diuretic and emmenagouge, expectorant, vermifuge and their decoction is astringent, stomachic (Warrier et al., 1995; Dhiman, 2003; Sharma et al., 2001), employed in hysteria, they are used internally and externally in leprosy, scrofula and other skin diseases (Nadkarni, 1954). Flowers are astringent, anodyne, refrigerent, emmenagouge, diuretic, resolvent, deobstruent and alexipharmic (Warrier et al., 1995; Sharma et al., 2001). They are applied as a poultice to relieve nervous headache (Dhiman, 2003 ). They are stomachic (Zhou et al., 2005), vermicide and valuable in eruptive skin diseases (Nadkarni, 1954) and for killing lice. Fruits are anthelmintic, emmolient and purgative (Rani et al., 1999). Fruits are considered tonic. Sushruta prescribed mahanimb fruits internally in indigestion, colic and intestinal catarrh. Seeds: seeds are bitter, expectorent, anthelmintic and aphrodiasic, and are useful in helminthiasis, typhoid fever, pain in the pelvic region, uropathy, vitiated conditions of vata and scrofula (Warrier et al., 1995). They are prescribed in rheumatism; oil obtained from seeds is applied locally in skin diseases 84

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(Dhiman, 2003). They are taken with adjuvants like rice water and clarified butter; ramyak Ghrita of sushurta was a specific remedy for gout. Sharangadhara prescribed seeds for urinary disorders. Ashroghna vati, a classical compound of 16th centuary, was prescribed for piles (Khare). Roots are bitter, astringent, mildly thermogenic, anodyne, depurative, vulnerary, antiseptic, constipating, expectorant, febrifuge, antiperiodic, urinary astringent, anthelmintic, emmenagogue and bitter tonic in low doses. They are useful in headache, sciatica, lumbago, leprosy, leucoderma, skin diseases, wounds, ulcers, piles, worm infestation, cough, asthma, ammenorrhoea, dysmenorrhoea, diabetes, abnormal urethral discharge, chronic and intermittent fevers, vomiting, post labour pain in uterus (Warrier et al., 1995; Sharma et al., 2001). 2.18.2 Chemical constituents Besides the chemical constituent of stem, fruits and bark, the leaves has been shown to contain nimbinene, meliacin, quercetrin, quercetin-3-0-b-rutinoside, kaempferol- 3-0-b rutinoside, rutin and kaempferol-3-L-rhamno-Dglucoside (Sharma et al., 2001). Hot methanolic extract of Melia azedarach leaves contain dipentadecyl ketone, glycerol 1, 3bis-undec-9- enoate 2-dodec-9-enoate and glycerol tris-tridec-9-enoate.( Suhag et al., 2003). Ethyl acetate extract of leaves of M. azedarach led to the isolation of the limonoid 1-cinnamoyl-3,11- dihydroxymeliacarpin (Alche et al., 2003). 2.18.3 Biological testing Melia azedarach possesses

haematological

activity

(Benencia

et

al.,

1992),

Immunomodulatory activity (Benencia et al., 1997), Insecticidal activity (Rani et al., 1999; Mahla et al., Pandey and Verma, 2002; Gajmer et al., 2003), Antiviral activity (Wachsman et al., 1998; Alche et al., 2002, 2000), Antifungal activity (Carpinella et al., 1999), Antibacterial activity (Carpinella et al., 1999; Khan et al., 2001), Cytotoxic activity (Itokawa and. Qiao, 1995; Nam and Lee; 2004; Zhou et al., 2004; Petrera and Coto., 2003) , Antimalarial activity: (Ofulla et al., 1995), Anthelmintic activity: (Pervez et al., 1994). Antilithic activity: (Christina et al., 2006). Antifertility activity Choudhary et al., 1990; Keshri et al., 2003; Roop, 2005; Keshri et al., 2005; Sharanabasappa and Saraswati, 2004), Analgesic activity (Vohra and Dandia., 1992), Antifeedant activity (ElLakwah et al., 1995).

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2.19 Phyllanthus emblica L. (Euphorbiaceae) 2.19.1 Ethnobotanical uses The mature fruits are very sour and contain 1%-1.8% Vitamin C. They are eaten raw or sweetened or preserved. The seeds, roots, and leaves are used as medicine. The dried leaves are sometimes used as fillings in pillows. Different parts of P. emblica has been used in traditional way of treatment for various purposes and diseases such as bleeding piles, vomiting, gout, asthma, heart and bladder diseases, sore throat, hiccough, diarrhea, (Kirtikar et al., 1935). Due to its special taste Emblica fruit is well accepted by the people. It has both superoxide dismutase and vitamin C in large amount (Verma and Gupta, 2004). The fruit is very popular and therefore used in various traditional medicinal systems, such Ayurvedic medicine, Chinese herbal medicine, and Tibetan medicine (Zhang et al., 2000). 2.19.2 Chemical constituents Many new sesquiterpenoids has been isolated from the roots of P. emblica, (Zhang et al., 2000, 2001a), the fruit juice contain many polyphenols and organic acid gallates (Zhang et al., 2001b, 2001c), the leaves and branches contain flavonoids and ellagitannins i.e naringenin, eriodictyol, kaempferol, dihydrokaempferol, quercetin, naringenin 7-Oglucoside (prunin), naringenin 7-O-(60-O-galloyl)-glucoside, naringenin 7-O-(60-Otrans-p-coumaroyl)-glucoside, kaempferol 3-O-rhamnoside, quercetin 3-O-rhamnoside, myricetin 3-O-rhamnoside, 2-(2-methylbutyryl)-phloroglucinol 1-O-b-D-glucopyranoside (multifidol glucoside) v,epigallocatechin 3-O-gallate, 1,2,3,6-tetra-O-, 1,2,4,6-tetra-O,15) and 1,2,3,4,5-penta-O-galloyl-b -Dglucose, and decarboxyellagic acid (Zhang et al., 2001c, 2002), phyllaemblic acid and its glycosides phyllaemblicins AC sesquiterpenoids from the roots, organic acid gallates, L-malic acid 2-O-gallate , and mucic acid 2-O-gallate together with hydrolysable tannins, 1-O-galloyl-bDglucose, corilagin, and chebulagic acid, Elaeocarpusin and putranjivain A were the other two main ellagitannins obtained from the fruit juice. Moreover, seven other tannins and flavonoids, geraniin, phyllanemblinins C and E , prodelphinidin B1, prodelphinidin B2, epigallocatechin 3-O-gallate , and (S)-eriodictyol 7-[6-O-(E)-p-coumaroyl]-b-D-glucoside (were the main phenolic compounds isolated from the branches and leaves of the plant 2.19.3 Biological testing The fruit of Emblica have hypolipidemic activity (Thakur et al., 1988; Jacob et al., 1988; Mathur et al., 1996; Anila and Vijayalakshmi, 2000), contain hypoglycemic activities 86

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(Anila and Vijayalakshmi, 2000; Abesundara et al., 2004), it is also one of the important constituent of many prescription available for hepatoprotective (Antarkar et al., 1980; De et al., 1993; Panda and Kar, 2003). Emblica is useful antimicrobial agent (Dutta et al., 1998; Godbole and Pendse, 1960; Rani and Khullar, 2004), anticancer (Jeena et al., 2001; Zhang et al., 2004), and anti-inflammatory agent (Asmawi et al., 1993; Lampronti et al., 2004; Perianayagam et al., 2004). The clastogenic effects induced with metal are highly improved with Emblica. (Biswas et al., 1999; Dhir et al., 1990).

2.20 Pinus roxburghii Sargent (Pinaceae) 2.20.1 Ethnobotanical uses The resin extracted from chir pine and other pines have been in use traditionally for various purposes across the world. The resin used to repair broken ceramic pottery by Hopi Indians, of American southwest, (Lanner, 1981). The resin of Pinus roxburghii, known locally as Ahule sallo, used to relieve the symptoms of a cough in Nepal. About two grams of resin and an equal amount of common salt are boiled in 250 -300 ml of water and drunk warm before bedtime for 2-4 days. In addition, the resin from Pinus wallichiana is used as a plaster for bone fractures. The resin is also mixed with an equal amount of butter and is warmed to make a paste. This ointment is applied to the affected parts regularly before bedtime to soften scar tissue (Bhattarai, 1992). In Uttaranchal, the resin of chir pine was applied to boils, heel cracks and on either side of the eye to reduce swelling (Singh et al., 1990). As the cones of chir pine is used for decoration, which can be a flourishing business for indigenous communities. Besides United States, Europe is becoming a strong market for decorative cones. For cones and most botanical products, entrepreneurs have noted that the German market is about ten times that of the United States' market (Coppen and Hone, 1995). There are opportunities in developing countries with extensive conifer forests (e.g. Mexico and Central America or Eastern Europe) to help meet the demand for decorative cones.

2.21 Punica granatum Linn. (Punicaceae) 2.21.1 Ethnobotanical uses Pomegranate is grown for its edible fruit and as an ornamental plant. It exhibits many varieties distinguished by the size of flower and fruit and taste of the fruit. Cultivated in

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Baluchistan and NWFP (Pakistan) areas is Kandahari, originally from Kandahar, Afghanistan, for its large, deep red, mostly acid-sweet pomegranates. The fruit is delicious to eat; the juice is a useful tonic in fevers. The dried seeds of Pomegranate are used for adding taste to certain foods. Bark of the root and wood is used as a vermifuge for tapeworms; also used for diarrhea and dysentery. A number of dyes can be obtained from it; black writing ink is also made from it. In Ayurvedic system of treatment the pomegranate is considered a pharmacy unto itself and consider useful antiparasitic agent (Naqvi et al., 1991), a blood tonic, (Lad et al., 1986), heal aphthae, diarrhea, and ulcers (Caceres et al., 1987). In the Unani system of medicine the Pomegranate is useful prescription for the treatment of diabetes and therefore much popular in the Middle East and India ( Saxena and Vikram, 2004). 2.21.2 Chemical constituents Quercetin, luteolin, and kaempferol were analyzed from pomegranate extracts, Hydroquinone pyridinium alkaloid isolated from the leaves of Punica granatum L (Schmidt et al., 2005). Various chemical constituents such as flavone glycosides i.e. apigenin and luteolin (Nawwar et al., 1994) and tannins i.e. punicafolin and punicalin, are reported from the leaves of Pomegranate. 2.21.3 Biological Testing The fruit extracts of Pomegranate possesses different therapeutic properties (Lansky and Newman, 2007) and other parts of the plant i.e. bark, roots, and leaves reported to have various medicinal properties (Naqvi et al., 1991). Pomegranate leaves can inhibit the development of obesity and hyperlipidemia in high-fat diet induced obese mice (Lei et al., 2007).), antibacterial activity.(Melndez et al., 2006; Mathabe et al., 2007). Jimnez Misas et al., (1979) has reported that Punica granatum inhibit 50% inhibition while studying plants of different plant families. Punica granatum showed moderate anthelmintic action against human Ascaris lumbricoides (Raj, 1975). Parts of P. granatum other than leaves were investigated for antioxidants activities (Gil et al., 2000; Rosenblat et al., 2006; Guo et al., 2008). Different parts of P.granatum shows in vitro anticancer activity (Lansky et al., 2005a, 2005b; Seeram et al., 2004, 2006; Cerda et al., 2004; Mertens-Talcott et al., 2006; Gil et al., 2000; Rosenblat et al., 2006; Guo et al., 2006; Guo et al., 2006; Rosenblat et al., 2006; Guo et al., 2008; Chidambara Murthy et al., 2002; Albrecht et al., 2004; Malik and Mukhtar, 2006; Malik et al., 2005). It has been tested for Alzheimers diseases (Hartman et al., 2006). 88

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2.22 Rubus ellipticus Smith (Rosaceae) 2.22.1 Ethnobotanical uses Due to useful medicinal properties Rubus species, it has been used in folk medicine (Patel et al., 2004). Roots and young shoots of Rubus ellipticus are used for colic pain and (Bhakumi, 1987). The leaves of (Rubus) blackberry are useful for the treatment of various ailments such as hypoglycemic activities, antidiarrhoeic, astringent, and also used for inflammation in mucous membrane of the oral cavity and throat (Borkowski et al., 1994; Ozarowski and Jaroniewski, 1989). Various diseases such as heart and the cardiovascular system, colic pain, diabetes, treating fever, influenza, alimentary canal, diarrhea, menstrual pain, air-passage are treated traditionally with the leaves of Raspberry leaves (R. idaeus L.). Externally the leaves of raspberry may also be applied as choleretic agents sudorific, antibacterial, anti-inflammatory, diuretic (Ozarowski and Jaroniewski, 1989; Czygan, 1995). Relaxant effects, particularly on uterine muscles have been reported in Raspberry leaf extract (Burn and Withell, 1941; Robbers and Tyler, 1999; Rojas-Vera et al., 2002). It has been noticed excellent supporting effects during pregnancy and labor in the leaves of raspberry (Simpson et al., 2001). The inner bark of the Rubus ellipticus plant is valued as a medicinal herb in traditional Tibetan medicine, including its use as a renal tonic and antidiuretic. Its fruits are edible and can also be used to produce a purplish blue dye (Plants For A Future, 2002). The juice of Rubus ellipticus Smith, which has an attractive color and rich flavor, can be preserved as such and can also be used for squashmaking. A very good jam can also be prepared from this fruit. This fruit has also been successfully introduced into Florida in the United States as a fruit and ornamental plant (Anonymous, 1948). The fruits are juicy and contain 64.00 per cent extractable juice, which comes out with a slight pressure. 2.22.2 Chemical constituents Ursolic acid and Acuminatic acid has been reported in the roots of R. ellipticus (Talapatra et al., 1989). New Pentacyclic Triterpene Acid elliptic acid from the leaves of Rubus ellipticus has been isolated (Dutta et al., 1997). Leaves of Rubus species contains tannins (Marczal, 1963; Okuda et al.,1992), derivatives of kaempferol and quercetin, phenolic acids, triterpenes, mineral salts as well as vitamin C are reported in Rubus species (Gudej and Rychlinska, 1996; Krzaczek, 1984; Wojcik, 1989). The leaves of raspberry contain some derivatives of ellagic acid, quercetin and kaempferol (Gudej, 2003). Methyl gallate 89

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and Methyl brevifolincarboxylate.is also reported with another known compound from Rubus speceis (Gudej et al., 1998). 1-Octacosanol was isolated previously from roots of Rubus ellipticus (Bhakuni et al., 1987) 2.22.3 Biological testing Rubus ellipticus leaves were found to have anticonvulsant activity against electrically induced convulsions, it potentiated the hypnotic effect of pentobarbitone sodium, it also possessed positive inotropic and chronotropic effects (Rana et al., 1990). The extract of R. ellipticus is active against hypothermia (Bhakumi et al., 1971). The roots of R. ellipticus possess antiprotozoal activty against Entamoeba histolitica, and hypoglycemic activity (Abraham et al., 1986). Antifertility activity of R. ellepticus has been reported in Ayurvedic and Unani literature (Casey, 1960). Sharma et al (1981) reported antiimplantation activity in roots and aerial parts of R. ellipticus. Some closely related species of Rubus such as R. fruticosus contain hypoglycaemic activity, (Newall, 1996), R. brasiliensis possesses anxiolysis activities (Nogueira et al., 1998). It has been studied several times the effect of total extracts of the leaves of R. idaeus on the uterus in vitro and studied the pharmacological effect on other smooth muscles preparation.(Burn et al., 1941; Beckett et al., 1954; Patel et al., 1995). The constituent of R. pinfaensis such as triterpenoids and phenol spossesses antibacterial activities (Richards et al., 1994) and the constituent of Rubus imperialis such as triterpenes possesses and antinociceptive properties (Niero et al., 1999). Methanolic extract of the leaves of Rubus idaeus possesses more than 80% relaxant acticity in Guianea-pig ( Rojas-Vera et al., 2002).

2.23 Viburnum cotinifolium D. Don (Caprifoliaceae) 2.23.1 Ethnobotanical uses The fruit is sweetish and edible. Fruit is considered to be laxative and blood purifiers. Leaves extract is applied in menorrhagia. (Saghir et al., 2001; Qureshi et al., 2007) 2.23.2 Chemical constituents A new biflavonoid named as 1-5, 11-5, 1-7, 11-7, 1-4', II-4'-hexahydroxy [6-0-8] biflavone along with seven known flavonoids have been isolated from leaves of Viburnum cotinifolium (Muhaisen Hasan et al., 2001).

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3.1 Reference Compounds Standard reference compounds (Alkaloids and Phenolic) for quantitative and qualitative analyses RP-HPLC and TLC are listed in Table 1. Table 1 Reference compounds Reference compound Berberine chloride dihydrate Palmatine chloride Berbamine dihydrochloride Codeine hydrochloride Kampferol Myricetin Catechin Vitexin Orientine Isoquercetin Hyperosid isovitexin Luteolin-7-glucoside Rutin Kampferol-7neohesperidoside Quercetin Luteolin Apigenin Purity factor 98.92% 96.98% >95% >99% >99% >99% >98% 96.30% 97.70% >99% >99% >99% 97.70% 96.80% 97.30% >99% 98.10% 93.80% Company Phyto Lab GmbH & Co, Germany Phyto Lab GmbH & Co, Germany Sigma-Aldrich (Schnelldorf, Germany) Heilmittelwerk Wien, Austria Roth, Karisruhe, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Extrasynthese, Geney, France Merk, Darmstadt, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Roth, Karisruhe, Germany Extrasynthese, Geney, France

3.2 Plant Material Roots and aerial parts of selected plants species were collected from Margalla Hills and Quaid-i-Azam University campus, Islamabad during flowering periods 2006-2009. The investigated species are listed in Table 2. Identification of the plants material based on morphological criteria was carried out by a plant taxonomist Professor Dr Rizwana Aleem Qureshi, Faculty of Biological Sciences and Department of Plant Sciences Quaidi-Azam University Islamabad. The voucher specimens of the collected plant materials are deposited at the Herbarium of Pakistan, of the same department.

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Table 2 Investigated Plants species S/No Name 1 Berberis lycium 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Mallotus phillipensis Rubus ellipticus Bauhinia variegata Caryopteris grata Colebrookea oppositifolia Phyllanthus emblica. Melia azedarach Ficus racemosa Dodonaea viscosa Jasminum humile Albizia lebbeck Pinus roxburghii Olea ferruginea Bombax ceiba Cassia fistula Lantana camara Punica granatum Pyrus pashia Dalbergia sissoo Debregeasia salicifolia Adhadoda vasica Carissa opaca Viburnum cotinifolium Ficus palmata Calotropis procera Family name Berberidacea Locality Margalla Hills Euphorbiaceae Margalla Hills Rosaceae Margalla Hills Caesalpinaceae Margalla Hills Verbenaceae Margalla Hills Labiateae Margalla Hills Euphorbiaceae Campus Meliaceae Moraceae Sapindaceae Oleaceae Campus Parts Roots Roots Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial Aerial 1252282 125266 125270 125284 22265 125311 125274 125280 125371 125281 125264 125265 125370 125277 125271 125276 125279 125312 125269 125283 Acc. No. 125174 125263 125272 125275 125262

Campus Campus Margalla Hills Mimosaceae Campus Pinaceae Margalla Hills Oleaceae Margalla Hills Bombacaceae Margalla Hills Caesalpinaceae Campus Verbenaceae Campus Punicaceae Campus Rosaceae Papilionaceae Urticaceae Acanthaceae Apocynaceae Margalla Hills Campus Margalla Hills Campus

Margalla Hills Caprifoliaceae Margalla Hills Moraceae Margalla Hills Asclepiadaceae Campus

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3.3 Anti bodies for western blot analyses Twenty three different antibodies were purchased from different companies. Details of the antibodies are listed in Table 3.

Table 3 Anti bodies for western blot analyses Anti-bodies Cdc2-p34 (17) Cdc25A (M-191) phospho-Cdc25A-(phSer17) -tubulin PARP -tubulin cleaved Caspase-3(Asp17) pospho-p38-MAPK (Thr180/Tyr182) p38-MAPK cyclin D1 p21 phospho-Cdc2(phTyr15) Chk2 phospho-Chk2 (Thr68) H2AX (phSer139) phoshpho-Cdc25A-(phSer177) Cdc25A phospho Ser17 Caspase-2 (H-19) Cdc25A (F-6) -Actine Acetylated -tubulin Company Santa Cruz (Santa Cruz, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Signaling (Danvers, MA, USA) Signaling (Danvers, MA, USA) Signaling (Danvers, MA, USA) Signaling (Danvers, MA, USA) Signaling (Danvers, MA, USA) Signaling (Danvers, MA, USA) Signaling (Danvers, MA, USA) Signaling (Danvers, MA, USA) Calbiochem, (San Diego, CA, USA) Abgent (San Diego, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Santa Cruz (Santa Cruz, CA, USA) Sigma (St. Louis, MO Sigma (St. Louis, MO

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3.4 Miscellaneous Chemicals and Reagents Chemicals reagents along with its company names and its use are listed in Table 4. Table 4. Miscellaneous Chemicals and Reagents Chemicals/reagents 1, 1-Diphenyl-2-picrylhydrazyl Gallic acid Ascorbic acid Hoechst 33258 propidium iodide Trice buffer Triton X-100 Phenyl methyl sulfonyl fluoride (PMSF) Protease inhibitor cocktail (PIC) Sodium dodecyl sulfate (SDS) Amonium per sulfate (APES) N, N, N', N'-tetra methyle ethylene diamine Acrylamide/Bis solution RPMI 1640 medium Heat inactivated fetal calf serum Glutamine Pencillin-streptomycin Agarose Ethidium bromide Folin-Ciocalteus phenol reagent 2-Aminoethyle diphenyl borinate Nutreint Broth medium Nutreint Agar medium Company Sigma-Aldrich (Schnelldorf, Germany) BDH, Poole, England Sigma-Aldrich (Schnelldorf, Germany) HO, Sigma, St Louis, MO, USA PI, both Sigma, St Louis, MO Sigma-Aldrich (Schnelldorf, Germany) Sigma-Aldrich (Schnelldorf, Germany) Sigma-Aldrich (Schnelldorf, Germany) Sigma-Aldrich (Schnelldorf, Germany) Sigma-Aldrich (Schnelldorf, Germany) Sigma-Aldrich (Schnelldorf, Germany) Bio-Red laboratories, India Bio-Red laboratories, India Life Technologies (Paisley, Scotland, UK) Life Technologies (Paisley, Scotland, UK) Life Technologies (Paisley, Scotland, UK) Life Technologies (Paisley, Scotland, UK) Gibco, Paisley, Scotland Sigma-Aldrich, Munich, Germany MERK, Darmastadt Germany Sigma-Aldrich (Germany) MERK, Darmastadt Germany MERK, Darmastadt Germany Uses Free radical Total phenolics Free radical Apoptoses Apoptoses Western blotting Western blotting Western blotting Western blotting Western blotting Western blotting Western blotting Western blotting Cell Prolifiration Cell Prolifiration Cell Prolifiration Cell Prolifiration Comet assay Comet assay Total phenolics Flavonoids detection Antibacterial assay Antibacterial assay

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3.5 Cell culture and bacterial strains The ATCC (American Type Culture Collection, Manassas, VA, USA) brand culture HL60 human promyelocytic cells were purchased. Three gram positive bacterial strains, staphylococcus aureus (ATCC 65438), Bacillus subtilis (ATCC 6633) and Staphylococcus epidermidis (ATCC 12228); three gram negative bacterial strains Escherichia coli (ATCC 15224), and Salmonella setubal (ATCC 19196)

3.6 Extraction Three different methods were used for extraction one for analyses of free radical scavenging activity (antioxidant activity) and total Phenolics determination in the aerial parts of selected plants (Chapter 4.6.1), the second methods was to extracts the roots for antibacterial and antineoplastic (anticancer) activities (Chapter 4.6.2), the third method was to extract and prepare the aerial parts for flavonoids finger printing of the selected medicinal plants (Chapter 4.6.3).

3.6.1 Extraction for Antioxidant and Total Phenolics Determination Four grams of the aerial part of each plant species powder were weighed into a flask and mixed in 40 ml (80% aqueous) methanol, and slightly stirred the suspension. Tubes were sonicated for twenty minutes and centrifugated for ten min at (1500g), and collected supernatants. Re-extracted the plant materials two times and the combined supernatants were evaporated by Rota vapor to a volume of about 10 ml. These concentrated extracts were lyophilized and weighed. Extracts obtained per gram dry powder are listed in (Table 11).

3.6.2 Extraction of roots powder Roots of the plants were carefully washed, dried under shed and grounded. 200 g of powdered B. lycium root and Mallotus phillipensis were extracted four times with methanol (MeOH). These extracts were collected and concentrated with a Rotavapor at 40 C. The concentrated MeOH extract of B. lycium was dissolved in distilled water and extracted three times each with organic solvents in sequence of increasing polarity such as ethyl acetate (EtOAc), and n-butanol (BuOH). After evaporating each solvent from B. lycium 0.44 g dried EtOAc extract, and 2.22 g dried BuOH extract was obtained,

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respectively 2.78 g dried material was recovered from the aqueous phase and considered as H2O extract. The concentrated MeOH extract of Mallotus phillipensis was dissolved in distilled water and extracted three times each with organic solvents in sequence of increasing polarity such as hexane, ethyl acetate (EtOAc), and n-butanol (BuOH). After evaporating each solvent 9.23 g dried hexane extract, 4.00 g dried EtOAc extract, and 7.08 g dried BuOH extract was obtained, respectively.

3.6.3 Extraction for Flavonoids analyses One grams of the aerial part of each plant species powder were weighed and mixed in 10 ml of 70% aqueous methanol in a test tube. The suspension was prepared and stirred slightly. After sonication of the tubes for twenty minutes, the tubes were centrifuged for ten min at (1500g), and collected the supernatants. The plant materials were re-extracted twice. The supernatants were combined and evaporated by Rota vapor to a volume of about 2 ml. These concentrated extracts were lyophilized. The concentrated extracts redissolved in 5 mL distilled water and extracted two times with hexane. The water soluble fractions were acidified and reflux for twenty minutes. The extracts were extracted with ethyl acetate. The ethyl acetate fractions were washed two times with distilled water.

3.7 Chromatographic Methods 3.7.1 Thin Layer Chromatography (TLC) Thin layer chromatography was used for qualitative studies of alkaloids in Berberis lycium and flavonoids analyses in the aerial parts of selected plants. 3.7.1.1 Thin Layer Chromatography of Berberis lycium fractions The constituents of the Berberis lycium fractions were qualitatively studied by TLC. Toluene-isopropanol-ethyl acetate-methanol and water (12:6:3:3:0.6) were used as a mobile phase, TLC aluminum sheets 20 x 20 cm Silica gel 60 F254 (Merck, Darmstadt, Germany) were used as stationary Phase. Two tank TLC chambers were used and one of the tanks filled with the mobile phase and the other with liquid ammonia. The atmosphere of the chamber was saturated 20 minutes prior chromatographic separation. Detection was made by Dragendorff Reagent.

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3.7.1.2 Thin Layer Chromatography for Flavonoids analyses Diluted samples (Chapter 3.6.3) of selected medicinal plant were qualitatively studied by TLC. Butanol-Acetic acid-Water (4:1:5) upper layer were used as a mobile phase, TLC aluminum sheets 20 x 20 cm Silica gel 60 F254 (Merck, Darmstadt, Germany) were used as stationary Phase. Detections were made by Natural Product-Polyethylene glycol reagent. The plates were sprayed with a solution of 1% ethanolic 2-Aminoethyle diphenyl borinate followed by a 5% ethanolic solution of polyethylene glycol-400. Flavonoids appear in different color zone under UV 365 nm. Standard Flavonoids (Chapter 3.2) were used for identification.

3.7.2 High Performance Liquid Chromatography (HPLC) 3.7.2.1 General HPLC Parameters For the qualitative and quantitative determination of Alkaloids in Berberis lycium, high performance liquid chromatography (HPLC) on reverse phase material was employed as analytical method. Both quantitative and qualitative determinations were done on a HPLC instruments equipped with a photo diode array detector (see Table 3.5). Table 5 Parameters for HPLC-PDA analyses of Alkaloids Controller Degasser Autosampler Pump Flow Stationar Phase Detector Software Shimadzu SCL-10AD VP system controller Shimadzu DGU-14A Degasser Shimadzu SIL-10AD VP Auto Injector Shimadzu SPD 10AD VP Liquid Chromatograph 1.3 ml/min Guard Column: Hypersil ODS C18, 5, 4 x 4 mm. Column: Hypersil ODS C18, 5, 125 x4 mm. Shimadzu SPD 10AD VP Diode Array Detector LC Solution

3.7.2.2 HPLC Method Separations of the alkaloids were achieved by gradient elution with A mixture of acetonitrile and buffer solution (consisting of 0.0116 molar solution of sodium 1heptansulfonate monohydrate in water, adjusted to pH 2.7 with Phosphoric acid). Mobile phase A consisted of buffer and Acetonitrile (HPLC grade) served as mobile phase B (see Table 6)

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Table 6 Gradient elution systems used for HPLC separations S/No 1 2 3 4 5 6 Time (min) 0 12 13 15 17 25 Flow rate (ml/min) 1.3 1.3 1.3 1.3 1.3 1.3
A= buffer, B=Acetonitril

Eluent A% 75 30 10 10 75 75

Eluent B% 25 70 90 90 25 25

3.7.2.3 Sample Preparation For preparation of stock solutions, dissolved 30.03 mg/mL of Butanol soluble fraction, 29.97 mg/mL of ethyl acetate soluble fraction and 30.08 mg/mL water remaining, in methanol (HPLC grade) and centrifuged (14x103 rpm) for 10 minutes in order to sediments the insoluble particle. The supernatants were removed for analyses. For calibration curve, standard stock solutions were prepared in the same way and dilutions were made (0.1-100 g/mL) using methanol as solvent. 3.7.3 Gas Chromatography and Mass Spectrometer Active hexane soluble fraction of Mallotus phillipensis root was qualitatively determine with conducted using Gas chromatography system that was interfaced with an Agilent 5973 inert mass selective detector. See Table 7 for analytical conditions. Table 7 Gas Chromatograph and Mass Spectrometer conditions MSD Agilent 5973 inert mass selective detector (MSD) system (Wilmingto, USA) Column DB-5 MS; 30 m x 0.25 mm x 0.5 m (Agilent J&W DB5ms Ultra Inert) Mode Electron ionization (EI) Scan Mode Mass range Scanned 25-800 amu Source tempratue 230 C Scan time 0 - 60 min Transfer line temperature 280 C Mass data processed soft Agilent Chemstation ware GC Agilent 5890N GC system Injection mode Split mode 10:1 Injection temperature 250C Injection volume 1 l Carrier gas Helium; Flow rate: 1.5 mL/min Oven temperature 120 C - 300 C 98

Chapter 3

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3.8 Biological Testing 3.8.1 Antineoplastic Activities 3.8.1.1 Anti-proliferation or Growth inhibition assay The cells culture of HL-60 (human promyelocytic cells) were seeded in T-25 tissue culture flasks at a concentration of 1x105 per ml and incubated with increasing concentrations of the different extracts of Berberis lycium and Mallotus phillipensis or with solutions of berberine and palmatine. Cell counts and IC50 values were determined in the different fractions after 48 and 72 h, using a KX 21 N microcell counter (Sysmex, Kobe, Japan). The media and other supplements were purchased from Life Technologies (Paisley, Scotland, UK). The percent of cell divisions compared to the untreated control were calculated as follows: [(C48 h + drug - C24 h + drug) / (C48 h - drug - C24 h - drug)] x 100 = % cell division C48 h + drug = cell number after 48 hours of drug treatment C24 h + drug = cell number after 24 hours of drug treatment C48 h - drug = cell number after 48 hours without drug treatment C = cell number after 24 hours without drug treatment

3.8.1.2 Hoechst dye 33258 and propidium iodide double staining (Apoptosis Assay) Hoechst staining was performed according to the method described by Grusch et al (2002). HL-60 cells (0.1x106 per ml) were seeded in T25 cell culture flasks and exposed to increasing concentrations of B.lycium, M. phillipensis fractions and standard berberine for 48 h. Hoechst 33258 (HO) and propidium iodide (PI, both Sigma, St Louis, MO) were added directly to the cells to final concentrations of 5 and 2 mg/ml, respectively. After 60 min of incubation at 37 Co, the cells were examined under a fluorescence microscope (Axiovert, Zeiss) equipped with a DAPI filter and a camera. This method allows discriminating between early apoptosis, late apoptosis, and necrosis. Cells were judged according to their morphology and the integrity of their cell membranes, which can easily be seen after propidium iodide staining.

3.8.1.3 Western blotting HL-60 cells were preincubated for increasing time periods (from 2 to 48 h) with 11.1 g BuOH extract /ml and 1.4 g berberine/ml medium. In another set of experiment HL -60

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cells were preincubated for increasing time periods (from 2 to 48 h) with 1.5 mg/mL hexane fraction of M. phillipensis. Then, cells were placed on ice, washed with ice-cold PBS (pH 7.2), centrifuged (1000 rpm, 4 C, 4 min) and the pellets lysed in 150 l buffer containing 150 mM NaCl, 50 mM Tris ph 8.0, 1 % Triton X-100, 2.5 % 0.5 mM PMSF and PIC (Sigma, Schnelldorf, Germany). Debris was removed by centrifugation (12,000 rpm, 4 C, 20 min) and the supernatant collected. Then, equal amounts of protein were loaded onto 10 % polyacrylamide gels (See Table 8) Proteins were electrophoresed for 2 h and then electro-blotted onto PVDF membranes (Hybond P, Amersham, Buckinghamshire, UK) at 4 C for 1 h. To confirm equal sample loading, membranes were stained with Poinceau S. After washing with TBS, the membranes were blocked for 1 h in Blotto (blocking solution: 5 % skimmed milk in TBS and 0.5 % Tween 20), washed 3 times in TBS/T, and incubated by gentle rocking with primary anti-bodies in Blotto (0.20.3 : 1000), at 4 C overnight. Then, the membranes were washed in TBS/T (3 x for 5 min) and further incubated with the second antibody (peroxidase-conjugated anti-rabbit IgG, or anti-mouse IgG dilution 1:2000 in Blotto, for 1 h at room temperature. The membranes were washed with TBS/T and the chemo luminescence (ECL detection kit, Amersham, Buckinghamshire, UK) was detected by exposure of the membranes to Amersham HyperfilmTM ECL. The antibodies against Cdc2-p34 (17), Cdc25A (M-191), phospho-Cdc25A-(phSer17), -tubulin, PARP and -tubulin were from Santa Cruz (Santa Cruz, CA, USA), against cleaved Caspase-3(Asp17), pospho-p38-MAPK (Thr180/Tyr182), p38-MAPK, cyclin D1, p21, phospho-Cdc2(phTyr15), Chk2, and phospho-Chk2 (Thr68) were from Cell Signaling (Danvers, MA, USA), against H2AX (phSer139) from Calbiochem, (San Diego, CA, USA), and actin were from Sigma (St. Louis, MO). phoshpho-Cdc25A(phSer177) from Abgent (San Diego, CA, USA), and against acetylated -tubulin and -

3.8.1.4 Cell cycle distribution analysis (FACS analyses) The cells culture of HL-60 (0.5x106 per ml) were seeded in T-25 tissue culture flasks and incubated with 5.6 g/ml BuOH extract, 0.7 g/ml (1.86 M) berberine, or 0.28g/ml (0.75 M) palmatine (which were equivalent to 0.5 mg/ml dried root powder, respectively) and 1.5 mg/mL hexane fraction of M. phillipensis according to cell culture parameters. After 24 h of incubation, the cells were separated from the medium and resuspended in 5 ml cold PBS, after centrifugation (600 rpm, 5 min) the culture were again

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suspended and fixed in 3 ml cold ethanol (70 %) for 30 min at 4 Co. After washing two time with cold PBS, propidium iodide and RNAse A were mixed to a final concentration of 50 mg/ml each and incubated at 4 C for 60 min before analyses. Cells were analyzed with a FACS Caliber flow cytometer (BD Biosciences, San Jose, CA, USA) and ModFit LT software were used for calculating the cell cycle distribution (Verity Software House, Topsham, ME, USA). Table 8. 10% Polyacrylamide Gel Preparation Stalking gel Chemical Distilled water Tris buffer 0.6 (pH 6.8) Sodium dodecyl sulfate (SDS) 10% Acrylamide/Bis solution 30% Ammonium per sulfate (APES) N, N, N', N'-tetra methyl ethylene diamine Tris buffer 1.6 M (pH 8.8) Distilled water Sodium dodecyl sulfate (SDS) 10% Acrylamide/Bis solution 30% Ammonium per sulfate (APES) N, N, N', N'-tetra methyl ethylene diamine quantity 6 mL 2.52 mL 100 L 1.32 mL 50 L 10 L 2.5 mL 4.04 mL 100 L 3.3 mL 50 L 10 L

Seperating gel

3.8.1.5 Single cell gel electrophoresis (SCGE)/Comet assay The experiments were conducted according to the guidelines of Tice et al, (2000). After treatment of the cells with BuOH extract or berberine, the cells were centrifuged (400 x g, 5 min, 23 C, Sigma-Aldrich, 4K 15C, Germany) and the pellet resuspended with 200 l PBS. The cytotoxicity was determined with trypan blue (Lindl et al, 1994), which is a measure for the integrity of the cell membrane. Only cultures with survival rates 80 % were analyzed for comet formation. To monitor DNA migration 0.05 x 10-6 cells were mixed with 80 L low melting agarose (0.5 %, Gibco, Paisley, Scotland) and transferred to agarose-coated slides. The slides were immersed in lyses solution (1 % Triton X, 10 % DMSO, 2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, pH 10.0) at 4C for 1 h. After unwinding and electrophoresis (300 mA, 25 V, 20 min) under alkaline conditions (pH > 13), which allows the determination of single and double strand breaks, DNA-protein crosslinks and apurinic sites, the DNA was stained with 40 L ethidium bromide (20 g/ml, Sigma-Aldrich, Munich, Germany) and the percentage DNA in tail was analyzed

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with a computer aided image analysis system (Comet IV, Perceptive Instruments Ltd., Haverhill, UK). From each experimental point one slide was prepared and 50 cells were scored per slide.

3.8.1.6 Statistical analyses The results of the SCGE (single cell gel electrophoresis) experiments were analysed with one-way ANOVA followed by Dunnetts multiple comparison test, and the apoptosis and proliferation experiments with t-test using GraphPad Prism version 4 (GraphPad Prism Software, Inc., San Diego, CA, USA). 3.8.2 Total Phenolics determination Phenolic compounds were determined with the method of FolinCiocalteu (Singleton and Rossi, 1965). For the preparation of Gallic acid stock solution, took a 100-mL volumetric flask, dissolved 0.5g of Gallic acid powder in 10 ml of ethanol, after complete dissolving the volume were made up to 100 mL. To prepared Sodium carbonate solution, anhydrous sodium carbonate (200g) was dissolved in 0.8 L of distill water and was boiled. After cooling the solution, few crystals of sodium carbonate (Na2CO3) were added. After a period of 24 hr, filtered the solution and add water to 1L. In order to draw a calibration curve (Fig. 4.15), add 0, 100, 200, 500, 750, 1250, 2000 L and extended to 4.00 mL of the above Gallic acid stock solution into 100 ml volumetric flasks, and then dilute to volume with water (100ml). These solutions will have phenol concentrations of 0, 5, 10, 25, 37.5, 62.5,100 and 4000 mg/L Gallic acid equivalent. Took 20 L into separate cuvettes from each calibration solution, sample or blank and to each add 1.58 ml distill water, and then pipet 100 L of the Folin-Ciocalteu reagent. After mixing the reagent and sample well, kept for 30 sec and 8 min. Add 300 L of the sodium carbonate solution, and shacked well to mix. Left the solutions at 40C for 30 min before reading the absorbance and determine the absorbance of each solution at 765 nm against the blank (the "0 mL" solution) and plot absorbance vs. concentration.

3.8.3 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) test The free radical scavenging activities of the leaves powder extracts were assessed by using the DPPH radical (Brand-Williams, et al., 1995). The experiment was slightly

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modified by making 0.5 m. mole /L solution of DPPH in methanol and again diluted four times (4X) in order to showed absorbance below 1. The solution of DPPH (1000l) was mixed with different concentration of each test compound (2.5-200g/ml, 500l) and the absorbance change at 517 nm was measured 30 min later (Blois, et al., 1958). The reaction solution without DPPH was used as a blank test and ascorbic acid (2.5-15g/ml, 500l) was used a positive control. The experiment conducted on Shimadzu spectrophotometer (UV-120-01), by using low lens. Mean value of triplicate was plotted in graph in order to calculate the concentration required for 50% reduction (50% inhibition concentration, IC50) of DPPH radical (Yen, et al., 1994 and Kubo, et al., 1984). GraphPad Prism version 4 (GraphPad Prim Software, Inc., San Diego, CA, USA) was used for calculating the concentration.

3.8.4 Antibacterial Determination The test was performed by simple Agar diffusion assay (Kavanagh., 1963; Leven et al., 1979). Eight dilutions were tested (dilution were 1, 2, 3, 5, 7.5, 10, 12.5 and 15mg/ml) against five strains of bacteria. Three gram positive bacterial strains, staphylococcus aureus (ATCC 65438), Bacillus subtilis (ATCC 6633) and Staphylococcus epidermidis (ATCC 12228); two gram negative bacterial strains Escherichia coli (ATCC 15224), and Salmonella setubal (ATCC 19196).

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4.1 Results 4.1.1 Anti-neoplastic Activities and Phytochemicals studies of Berberis lycium 4.1.1.1 Qualitative Analysis of B. lycium extracts constituents by TLC The EtOAc, BuOH, and H2O extracts of B.lycium roots were loaded on TLC plates which were immersed in a saturated chromatography chamber containing toluene-EtOAcisopropanol-methanol-H2O (12:6:3:3:0.6) as running phase, and compared standards berberine, berbamine and palmatine, which are known constituents of various Berberis taxa with distinct anti-neoplastic properties. All extracts contained berberine. The BuOH extract showed comparatively the highest concentration (retention factor, Rf = 0.151). Also palmatine was detected in all extracts but only traces were found in the H2O and EtOAc extracts (Rf = 0.088). Berbamine was not found in any extract (the standard was occurring as a black spot under 254 nm UV light, Rf = 0.405). Besides berberine and palmatine another unknown bands were present in all extracts. (see figure 8).

4.1.1.2 Separation and quantification of alkaloids by RP-HPLC The alkaloids seen in the different fractions during TLC analyses have been successfully separated and determined by HP-HPLC. In order to obtained good resolutions and minimize peaks tailing. A series of experiments using different solvent systems, column types and running parameters (results not shown) were performed. A mixture of acetonitrile and buffer solution (consisting of sodium 1-heptansulfonate monohydrate in water, adjusted to pH 2.7 with Phosphoric acid) was considered best. Solvent gradients are shown in table. 6 (chapter 4; page. 98). Chromatograms that shows separation in different fractions of Berberis lycium and standards compounds are shown in Fig. 9. Average retention times for internal standard codeine, berberine, palmatine and berbamine were 4.52, 9.753, 9.190 and 8.056 minutes respectively. An unknown peak (Rf = 8.062) was found in the UV range of palmatine (Fig. 10). Calibration curves have been developed separately for Berberine and Palmatine. The linearity studies of standard curve for standard compounds have been calculated (see Table. 9). Estimated concentrations of berberine and palmatine are listed in Table 10.and Fig.14 page 112.

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Figure 8 TLC of Berberis lycium extracts 1. H2O Fraction (1 mg/ mL) 6 l 2. Ethyl acetate.fraction (1 mg/ mL) 6l 3. Ethyl acetate.Fraction (1 mg/ mL) 8 l, 4. Standard palmatine chloride (0.125 mg/mL) 4l 5. Standard berberine chloride dihydrate (0.25 mg/mL) 4 l 6. Standard berbamine dihydrochloride (1 mg/mL) 2l 7. BuOH Fraction (1mg/mL) 4l 8. BuOH Fraction

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280nm,4nm (1.00)

4.483/1071318

mAU

bar A.Press.(Status)

150 125 100 75 50 25 0 -25 0.0

1 3
9.209/1068637

0.95

9A

0.90 0.85 0.80 0.75 0.70

0.65 0.60 0.55 0.50 0.45

6.917/1954 7.465/13985 8.098/140361 8.585/10536

9.659/850449

0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

5.0

5.524/2921 6.121/10704

10.0

15.0

20.0

min

Minutes Figure 9 RP-HPLC Chromatogram of alkaloids standards. RP-HPLC chromatogram of standard compounds: 1. Codeine hydrochloride, 2. Berbamine dihydrochloride, 3. Palmatine chloride, 4. Berberine chloride dehydrate.

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mAU
280nm,4nm (1.00)

bar

4.524/971034

A.Press.(Status) 0.95 0.90 0.85 0.80 0.75 0.70

125

9B
100
9.693/933662

0.65 0.60 0.55 0.50 0.45 0.40

75
5.570/52834 6.157/12530 6.568/4705 6.979/3879 7.558/3920 7.754/2751 8.066/348850

50

9.337/136112

0.35 0.30 0.25 0.20 0.15 0.10 0.05

25

-25 0.0

5.0

10.0

15.0

20.0

0.00

min

Minutes

Figure 10. RP-HPLC chromatogram of n-Butanol fraction of Berberis lycium extract.

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280nm,4nm (1.00)

4.524/1090246

mAU

bar A.Press.(Status) 0.95

150

9C

0.90 0.85 0.80 0.75 0.70 0.65 0.60

125

100

75
5.554/193428 6.153/23925 6.564/9520 6.447/7921 7.209/60156.983/18401 7.546/49385 8.062/415770 9.350/154142 9.734/474556

0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05

50

25

-25 0.0

5.0

10.0

15.0

20.0

0.00

min

Minutes Figure 11. RP-HPLC chromatogram of water fraction of Berberis lycium extract.

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280nm4nm (1.00)

4.501/1094990

mAU

bar A.Press.(Status) 0.95 0.90

150 125 100 75 50 25 0 -25 0.0

9D

0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

5.0

8.077/42709 8.350/5232 8.517/26538.681/36302 8.875/4745 9.128/11131 9.408/10378 9.782/152207

10.0

15.0

20.0

min

Minutes

Figure 12. RP-HPLC chromatogram of Ethyl acetate fraction of Berberis lycium extract

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mAU(x100) 9.54 2.0 229 264 209 248 347 302

Results & Discussion

1. Berberine

1.0

0.0 200.0 mAU(x100) 2.0 9.13 1.5 226 265 346 1.0 202 206 212 0.5 0.0 200.0
mAU(x100) 8.05 2.0

225.0

250.0

275.0

300.0

325.0

350.0

375.0

380

nm

2. Palmatine
249

302

225.0

250.0

275.0

300.0

325.0

350.0

375.0

378

nm

3. Berbamine

1.0 256 279 304 344 379 375.0

0.0 200.0 225.0 250.0 275.0

300.0

325.0

350.0

nm

mAU(x1,000) 1.5 4.54 201 206

1.0

4. Codeine

0.5 262 285 0.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 397 nm

Figure 13 Optimum UV spectra of standards compounds: Optimum UV spectra of standards compounds: 1. Berberine chloride dihydrate, 2 Palmatine Chloride., 3. Berbamine dihydrochloride, 4. Codeine hydrochloride

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Table 9 Linearity study of standard curve for standard compounds Compounds Calibration curve Slope Berberine Palmatine Berbamine y= 34.59x+16.59 y=34.24x+44.368 y=3.0872x+22.946 34.59 34.24 3.0872 Intercept R2 16.59 44.37 22.95 Range of injection amount (g/mL) 0.9994 10-100 0.9974 1-50.00 0.9948 1-100

Calculation: The concentrations/percentage of Berberine and Palmatine were calculated with the following equations Factor of correction (FC) = Area (internal standard) x Mass (sample) Area (sample) x Mass (internal standard)

Sample (%) = Mass (internal standard) x Area (Sample) x FC (Factor of correction x 100 M (sample) x Area (internal standard)

Table 10 Percent composition of active alkaloids in Berberis lycium Fractions BuOH Fraction Ethyl acetate Fraction H2O Fraction Berberine % 18.04 0.01 0.54 0.0015 2.76 0.026 Palmatine % 2.80 0.04 0.00255 0.93 0.065

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Percent composition of active alkaloids

20

H2O Fraction
15

BuOH Fraction EtOAc Fraction

% age

10 5 0 Berberine Palmatine

Figure 14 Alkaloids percentage in Berberis lycium:

4.1.1.3 Inhibition of HL-60 cell proliferation by extracts of B. lycium, Berberine and Palmatine. Logarithmically growing cells were incubated with increasing concentrations of EtOAc, BuOH and H2O extract, or the purified alkaloids berberine and palmatine for 72 h. Then, cells were counted and the inhibition of proliferation was calculated. The BuOH extract showed the highest toxicity against HL-60 cells (IC50 2.8 g extract/ml medium, corresponding to <0.25 mg dried root/ml) after 72h (Fig. 15). Similar extract concentrations were used to facilitate comparability and therefore, the measured differences in the extract activities (see table 10) were due to different chemical compositions of the extracts. To evaluate which of the major constituents of the BuOH extract may have caused growth inhibition, HL-60 cells were treated with the measured equivalent concentrations of berberine (0.7-2.1 g/ml, corresponding to 0.51.5 mg dried root weight/ml medium) and palmatine (0.28-0.83 g/ml, corresponding to 0.51.5 mg dried root weight/ml 112

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medium). The IC50 for berberine was less than 3.7 M (= 1.4 g/ml, corresponding to an equivalent of ~ 1 mg dried roots of B. lycium/ml medium) after 48 h and < 1.9 M after 72 h of incubation. Palmatine did not inhibit growth after 48 h. The inhibition of HL-60 proliferation that was observed upon treatment with BuOH extract or berberine was preceded by the induction of p21, which has been also observed by Liu et al. (2009) and by a dramatic down regulation of the proto-oncogene cyclin D1 after 48 h (Fig. 16). Both, the up regulation of p21 and the suppression of cyclin D1 are potent mechanisms to block cancer cell growth.

Treatment with EtOAc extract % cell proliferation

Treatment with BuOH extract % cell proliferation

100

100

*
75 50 25 0

* * * * * * * *

* *
75

*
50 25 0

* * * * * *

Co nt r 17ol 35.5 4 .0 Co 6.6 nt r 17ol 35.5 4 .0 Co 6.6 nt r 17ol 35.5 46.0 .6

extract concentration (g/ml)

Co nt ro 2. l 8 5. 11 6 Co .1 nt ro 2. l 8 5. 11 6 Co .1 nt ro 2. l 8 5. 11 6 .1

extract concentration (g/ml)

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Treatment with H2O extract % cell proliferation

100 75 50 25 0

Treatment with Palmatine % cell proliferation

100 75 50 25 0

* * * * * * * * * * **

Co nt r 6o 109.5l 134.3 209.0 Co 8.5 nt r 6o 109.5l 134.3 209.0 Co 8.5 nt r 6o 109.5l 134.3 209.0 8. 5

extract concentration (g/ml)

Treatment with Berberine % cell proliferation

100

*
75 50 25 0

* * * * * *

Figure 15 Anti-proliferative effect of B. lycium extracts and its alkaloids: (a) EtOAc extract (17.5, 35.0 and 46.6 g/ml medium); (b) BuOH extract (2.8, 5.6 and 11.1 g/ml); (c) H2O extract, (69.5, 104.3, 139.0 and 208.5 g/ml); (d) Berberine (0.7, 1.4, and 2.1 g/ml); and (e) Palmatine (0.28, 0.55 and 0.83 g/ml). Cells were counted after 24, 48 and 72 h of treatment (white, light gray and dark gray columns, respectively) and the percentage of proliferation was calculated and compared to DMSO-controls (Control). Controls were considered as cells with a maximal proliferation rate (100%). Experiments were done in triplicate. Error bars indicate SEM, asterisks significance (p< 0.05).

Co nt ro l 0. 7 1. 4 2 Co .1 nt ro l 0. 7 1. 4 2 Co .1 nt ro l 0. 7 1. 4 2. 1

drug concentration (g/ml)

Co nt ro 0. l 2 0. 8 5 0 5 Co .83 nt ro 0. l 2 0. 8 5 0 5 Co .83 nt ro 0. l 2 0. 8 5 0. 5 83

drug concentration (g/ml)

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Figure 16 Analysis of cell cycle proteins: HL-60 cells (1x106 cells) were seeded into T25 tissue culture flasks and allowed to grow for 48 h when cells were incubated with 11.1g BuOH extract/ml medium (left side panels) and 1.4 g berberine/ml medium (right side panels) for 2, 4, 8, 24 and 48 h. Then, isolated protein samples were subjected to 10 % SDS-PAGE separation and subsequent Western blot analysis using antibodies against p21 and cyclin D1. Equal sample loading was controlled by Poinceau S staining and -actin analysis.

4.1.1.4 Effect of BuOH extract, Berberine and Palmatine on cell cycle distribution. HL-60 cells were exposed to 5.5 g BuOH extract/ml (corresponding 0.5 mg dried root /ml), and 0.7 g berberine/ml (which is contained in 5.5 g BuOH extract) for 48 h to investigate the cell cycle distribution. Both, the extract and the pure compound caused a reduction of G1 cells and accumulation of cells in the S phase (Fig. 16), which was most likely due to activation of intra S-phase checkpoint, because checkpoint kinase 2 (Chk2) became highly activated (Luo et al., 2008)(Fig.22). Palmatine had no effect on cell cycle distribution (data not shown) which was consistent with the observation that it did not have an effect on growth inhibition.

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Control

BuOH (5.6 g /mL)

116

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Berberine (0.7 g/ mL equ.)

Cell cycle distribution after BuOH extract treatment (48 h)

50

Cell cycle distribution after Berberine treatment (48 h)

60 50

* *

Control 5.6 g

* *

Control 0.7 g

30 20 10 0 G0-G1 S G2-M

% cells

% cells

40

40 30 20 10 0

*
G0-G1 S G2-M

Figure 17 Cell Cycle Distribution of HL-60 cells upon treatment with of BuOH extract and berberine for 48 h: Cell Cycle Distribution of HL-60 cells upon treatment with of BuOH extract and berberine for 48 h. Logarithmically growing HL-60 cells were incubated with 5.6 g/ml BuOH extract and 0.7 g/ml berberine and then subjected to FACS analysis. Experiments were done in triplicate. Error bars indicate SEM, and asterisks significance (p<0.05).

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4.1.1.5 Induction of apoptosis by extracts of Berberis lycium and Berberine HL-60 cells were treated with the three extracts (EtOAc, BuOH and H2O) and berberine for 48h and the induction of cell death was analyzed. The three extract types induced apoptosis and the BuOH extract was the most active followed by the EtOAc- and the H2O extracts (see table 10; page.112) for comparison). Berberine was used at a comparable concentration as contained in the BuOH extract and this concentration caused a similar pro-apoptotic effect as the extract (Fig. 18). High concentrations of berberine (10 50 g/ml) were shown earlier to induce H2AX phosphorylation (H2AX) in osteosarcoma cells indicating genotoxicity (Liu et al., 2009). In the present study we demonstrate that 0.7 and 1.4 g/ml berberine and the corresponding concentration of BuOH extract specifically induced apoptosis in HL-60 cells without concomitant induction of H2AX (Fig. 19). This observation indicates that the anti-neoplastic effects have not been triggered by berberine-caused genotoxicity. Comet assay detecting DNA single strand breaks provided no evidence that berberine or the BuOH extract cause DNA damage (Fig. 20). Thus, other mechanisms must be responsible for cell cycle inhibition and apoptosis. Interestingly, berberine and the BuOH extract caused acetylation of -tubulin (Fig. 19), which is indicative for tubulin polymerization reminiscent of the mechanism of taxol. Tilting the fine-tuned equilibrium of polymerized/de-polymerized microtubule is incompatible with normal cell division and this causes not only cell cycle arrest but also apoptosis.

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119

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EtOAc extract treatment (48 h)

30

BuOH extract treatment (48 h)

30

*
% apoptotic cells

% apoptotic cells

* *

20

20

*
10

10

*
0

*
0

C on tr ol

Co nt ro l

11 .7

extract concentration (g/ml)

17 .5

extract concentration (g/ml)

H2O extract treatment (48 h)

30

Berberine treatment (48 h)

30

% apoptotic cells

% apoptotic cells

20

* *

20

*
10

10

13 9. 2

Co nt ro l

20 8. 5

69 .5

C on tr ol

1. 4

0. 7

extract concentration (g/ ml)

extract concentration (g/ml)

Figure 18 Induction of apoptosis by the Berberis lycium extracts and berberine: (1) Control (2) BuOH extract 11.1 g/ml (3) EtOAc extract 17.5 g/ml (4) H2O extract 208.5 g /ml. Error bars indicate SEM, asterisks significance (p<0.05).

2. 1

11 .1

2. 8

5. 9

5. 6

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Figure 19 Western blot analyses of pro-apoptotic mediators and effectors

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Figure 20 The genotoxicity of increasing concentrations of BuOH extract and berberine: The genotoxicity of increasing concentrations of BuOH extract and berberine was investigated in logarithmically growing HL-60 cells. 50 M H2O2 was used as positive control and solvent-treated cells were used as negative control. The cytotoxicity was determined with trypan blue (Lindl et al, 1994), which is a measure for the integrity of the cell membrane. Only cultures with survival rates 80 % were analyzed for comet formation. To monitor DNA migration 0.05 x 10-6 cells were mixed with 80 L low melting agarose (0.5 %, Gibco, Paisley, Scotland) and transferred to agarose-coated slides. The slides were immersed in lyses solution (1 % Triton X, 10 % DMSO, 2.5 M

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NaCl, 10 mM Tris, 100 mM Na2EDTA, pH 10.0) at 4C for 1 h. After unwinding and electrophoresis (300 mA, 25 V, 20 min) under alkaline conditions (pH > 13), which allows the determination of single and double strand breaks, DNA-protein crosslinks and apurinic sites, the DNA was stained with 40 L ethidium bromide (20 g/ml, SigmaAldrich, Munich, Germany) and the percentage DNA in tail was analyzed with a computer aided image analysis system (Comet IV, Perceptive Instruments Ltd., Haverhill, UK). From each experimental point one slide was prepared and 50 cells were scored per slide. 1. Control; 2. Berberine 1.4g/ mL; 3. B. lycium (BuOH fraction 11.14g/ ml) 4. Positive control (H2O2); Statistical analysis: Dunnetts test.

C om et assay a ft e r c e ll tr e a tm e n t fo r 4 8 h
30

% tail DNA

25 20 15 10 5 0

BuOH B e r b e r in e

ol

7 0.

2O

.1

2.

5.

50

c o n c e n tr a t io n ( g /m l)

Figure 21 Comet assay: The genotoxicity of increasing concentrations of BuOH extract and berberine was investigated in logarithmically growing HL-60 cells. 50 M H2O2 was used as positive control and solvent-treated cells were used as negative control. Bars indicate means SD of results obtained with three independent cultures (from each culture 50 cells were evaluated).

4.1.1.6 Induction of stress response by extracts of Berberis lycium and Berberine Cellular stress is a prominent inducer of apoptosis and cell cycle arrest. Therefore, I analyzed the activation of p38-MAPK and found that this stress-induced kinase became transiently phosphorylated (at Thr180/Tyr182) and hence activated after 8 h of treatment with 11.1 g BuOH extract /ml and 1.4 g berberine/ml medium (Fig. 22). Also Chk2

on

11

1.

0.

tr

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became activated within 4 h treatment (Fig. 22). This activation pattern was consistent with that of intra-S-phase arrest reported by Luo et al, (2008). Chk1 was not induced (data not shown). Cdc25A became phosphorylated at Ser177 and therefore, Cdc25A became inactivated (within 2 h, Fig. 22) leading finally to its degradation (Madlener et al., 2009). This resulted in the accumulation of Tyr15 phosphorylation of Cdc2, which is a specific target site of the Cdc25A phosphatase, Ray and Kiyokawa, (2008). Tyr15-Cdc2 phosphorylation inactivates this cell cycle specific kinase. The treatment with BuOH extract and berberine changed also the phosphorylation pattern at Ser17 of Cdc25A. The inactivation of the Cdc25A proto-oncogene was the most immediate event elicited by the BuOH extract and berberine (Fig.22). This was followed by the acetylation of -tubulin (Fig. 19), the activation of Chk2 and p38, and the down regulation of cyclin D1. All of these effects have not been observed so far in berberine or in Berberis-extract treated cells.

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Figure 22 Induction of stress response by the BuOH extract of Berberis lycium and Berberine:

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4.1.2 Anti-neoplastic Activities and Phytochemicals studies of Mallotus phillipensis 5.1.2.1 Inhibition of HL-60 cell proliferation by Mallotus phillipensis extracts Logarithmically growing HL-60 cells were incubated with increasing concentrations of nHexane, EtOAc and BuOH extract for 72 h. Then, cells were counted and the inhibition of proliferation was calculated. The Hexane extract showed the highest toxicity against HL-60 cells (IC50 1.5 mg dry roots equivalent /ml medium) after 72h (Fig. 23; page. 127). The inhibition of HL-60 proliferation that was observed upon treatment with hexane extract was preceded by the down regulation of the proto-oncogene cyclin D1 after 48 h (Fig. 27; page. 131). Suppression of cyclin D1 is potent mechanisms to block cancer cell growth.

4.1.2.2 Induction of apoptosis by extract of Mallotus phillipensis Logarithmically growing cells were incubated with increasing concentrations of n-hexane fraction of Mallotus phillipensis for 48h and the induction of cell death was analyzed. The extract induced apoptosis 18% after 48h of treatment with 1.5 mg dry roots equivalent /ml medium (Fig. 24; page. 128). I monitored the ability of Mallotus phillipensis hexane fraction and the observation indicates that the anti-neoplastic effects have been triggered by induction apoptosis through caspase-2 activation (Fig. 22; page. 129).

4.1.2.3 Effect of Hexane fraction on cell cycle distribution. HL-60 cells were exposed to 1.5 mg dry roots equivalent /ml medium hexane fraction 48 h to investigate the cell cycle distribution. There were observed a slight alteration in cell distribution but the results were not significant (Fig. 26; page 130).

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Treatment with Hexan extract % cell proliferation

Treatment with EtOAc extract % cell proliferation

100

100

* *
75 50 25 0

* *

* * *

*
75 50 25 0

* * *

C on tr ol 1 1. 5 2 C on tr ol 1 1. 5 2 C on tr ol 1 1. 5 2

extract concentration (mg/ml)

Treatment with BuOH extract % cell proliferation

100 75 50 25 0

* * *

* * *

l 1 5 2

on t

extract concentration (mg/ml)

Figure 23 Anti-proliferative effect of Mallotus phillipensis extracts: HL-60 cells were seeded into T-25 tissue culture flasks (1x105 cells/ml), grown for 24 h to enter logarithmic growth phase, and incubated with increasing concentrations (a) Hexane extract (1.00, 1.50, and 2.00 mg dry roots equivalent /ml medium); (b) EtOAc extract (1.00, 1.50 and 2.0 mg dry roots equivalent /ml medium); (c)BuOH extract (1.00, 1.5 and 2.0 mg dry roots equivalent /ml) Cells were counted after 24, 48 and 72 h of treatment (white, light gray and dark gray columns, respectively) and the percentage of proliferation was calculated and compared to DMSO-controls (Control). Controls were considered as cells with a maximal proliferation rate (100%). Experiments were done in triplicate. Error bars indicate SEM, asterisks significance (p< 0.05).

on t

on t

l 1 1. 5 2

ro

l 1 5 2

ro

1.

1.

ro

C on tr ol 1 1. 5 2 C on tr ol 1 1. 5 2 C on tr ol 1 1. 5 2

extract concentration (mg/ml)

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2
30

Hexan extract treatment (48 h) % apoptotic cells

20

10

Figure 24 Induction of apoptosis by the Mallotus phillipensis Hexane fraction: HL-60 cells were incubated with increasing extract concentrations (1 and 1.5 mg/ml dry powder equivalent) for 48 h. Then, cells were double stained with Hoechst 33258 and propidium iodide and examined under a fluorescence microscope and a DAPI filter. Nuclei with morphological changes indicating apoptosis (Methods) were counted. C. control; 1. 1 mg dry roots equivalent /ml medium and 2. 1.5 mg dry roots equivalent /ml medium. The percentages of vital and apoptotic cells calculated. Experiments were done in triplicate. Error bars indicate SEM, asterisks significance (p<0.05).

on tr

extract concentration (mg/ml)

1. 5

ol

128

Chapter 4

Results & Discussion

Figure 22 Analysis of cell cycle proteins: HL-60 cells (1x106 cells) were seeded into T25 tissue culture flasks and allowed to grow for 48 h when cells were incubated with 1.5 mg dry roots equivalent /ml medium) for 2, 4, 8, 24 and 48 h.

Control

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Results & Discussion

1.5 mg/ml dry roots equivalent /ml medium

Cell cycle distribution after BuOH extract treatment (48 h)

50

Control 1.5 mg/ml

% cells

40 30 20 10 0 G0-G1 S G2-M

Figure 26 Cell Cycle Distribution of HL-60 cells upon treatment with hexane Fraction of Mallotus phillipensis:

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Results & Discussion

4.1.2.4 Induction of stress response by extract of Mallotus phillipensis Cellular stress is a prominent inducer of apoptosis and cell cycle arrest. Therefore, I analyzed the down regulation of Cyclin D1, both Cdc 25A (F-6) and Cdc 25A (M-191). The inactivation of the Cdc25A proto-oncogene and the down regulation of cyclin D1 were the most immediate event elicited by the hexane Fraction of Mallotus phillipensis. All of these effects have not been observed in any p27 deficient cell lines so far by Mallotus phillipensis extracts and its chemical constituents (Fig. 27 page. 131).

Figure 27 Induction of stress response by Mallotus phillipensis

4.1.2.5 GC-MS Analysis of Mallotus phillipensis Hexane Fraction. GC-MS analyses of Mallotus phillipensis hexane fraction were performed to identify the volatile and semi volatile components. Analyses of the extract confirmed with the help of mass spectrometric characteristics and compared with the already reported compounds from the same species and other. Although the ion ratio of some compounds were the same but they were completely different compounds. Unknown compound (GC Rf = 39.9, 45.66, 43.905 and 47.735 minutes respectively) are detected while comparing its Mass spectrum with literature (See Fig. 28). Compound (V) EI-MS m/z, 530 [M+], 515, 219, 147, 57, 43 and 28; (VI) EI-MS m/z 426 [M+], 411, 218, 207, 189, 135, 95 and 28; (VII)

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EI-MS m/z 442 [M+], 424, 355, 281, 207and 28; (VIII) EI-MS m/z 484[M+], 466, 406, 257, 189, 109, 43 and 28. Compounds (IX) EI-MS m/z, 351 [M+], 314, 286, 256, 197 and 97; (X) EI-MS m/z, 396 [M+], 337, 320, 294, 240, 154, 126, 83, 59 and 41 were present in abundant (Rf = 17.917 and 31.125 minutes respectively).

O Me HO O Me H OH Me OH O

H H H H
Me H OH

O H I Lupeol

Me

II Kamalachalcones C

H H H H O H

O H III Betulin

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(IV)

(V)

133

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Results & Discussion

(VI)

(VII)

134

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Results & Discussion

(VIII)

(IX)

135

Chapter 4

Results & Discussion

(X) Figure 28 GC/MS chromatogram of hexane soluble fraction of Mallotus phillipensis.

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4.1.3 Total Phenolics, Free radical scavenging activity and Flavonoids finger printing of selected Medicinal Plants.

4.1.3.1 Total Phenolics Determination Aerial parts of twenty four Plants species were studied for total Phenolics. Gallic acid standard curve was established prior to identification of total Phenolics (see Fig 29 page. 138) The plants include Albizia lebbeck, Bauhinia variegata and Cassia fistula, Bombax ceiba, Calotropis procera, Carissa opaca, Colebrookea oppositifolia, Dalbergia sissoo, Dodonaea viscosa, Ficus palmata, Ficus racemosa, Jasminum humile and Olea ferruginea, Adhadoda vasica, Lantana camara, Melia azedarach, Phyllanthus emblica, Pinus roxburghii, Punica granatum., Rubus ellipticus, Pyrus pashia, Viburnum cotinifolium, Debregeasia salicifolia and Caryopteris grata. A range of 167-2160 (mg/gram) Gallic acid equivalent is formed in which Phyllanthus emblica, Rubus ellipticus., Bauhinia variegata, and Caryopteris grata shows comparatively better total Phenolic, and percentage yield while Debregeasia salicifolia showed minimum total Phenolic and percentage yield ( see Fig. 30; page. 139 and Table 11; page. 141)

4.1.3.2 Determination of Free radical scavenging activity Aerial parts of twenty four Plants species were studied for Free radical scavenging activities. Ascorbic acid was used as a standard antioxidant compound (IC50= 5.75 g/ml) (see Fig. 31; page. 139). Separate antioxidant cure was established for each sample. Results showed that Rubus ellipticus was more active in scavenging 1, 1-Diphenyl-2picrylhydrazyl (DPPH) Free radical (I/C50= 2.5) and Calotropis procera with minimum activity (I/C50=125). The results are shown in Table 11; page. 141. I/C50,s were calculated by using GraphPad Prism Software, Inc., San Diego, CA, USA).

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Galic acid Curve

0.8 Absorbance 0.6 0.4 0.2 0 0 500 1000 1500 2000 2500 Concentration (mg/l) y = 0.0004x + 0.046 R2 = 0.9991

Figure 29 Gallic acid standard curve

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Results & Discussion

( extract yeild (mg/g)

Gallic acid equavalent (mg/g)

2500

2000

1500 (mg/g) 1000 500 0 P. pashia D. sisso M. azadereca. F. recemosa Bombex ceiba P. roxburgii O. ferruginea P. granatum A. labbeck V. cotinifolium. F. Palmata R. ellipticus. B. variegata C. oppositifolia D. Salicifolium P. emblica. L. camara C. procera C. grata D. viscosa J. humile A. Vasica C. fistula C. opaca

Figure 30 Total Phenolics and Extract yield per gram:

Antioxidant curve of Ascorbic acid

100 80

% inhibition

60 40 20 0 0 5 10 15 20 25

Concentration in ug IC50= 5.75 ug

Figure 31 Antioxidant cure of Ascorbic acid

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Table 11 Comparative total Phenolic, extract yield per gram and IC50 Values. S/No Plants name 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Amount of Total Phenolics a extract/g (mg of GAE/g dw) Rubus ellipticus 0.137 0.001 1558 Bauhinia variegata 0.13 0.01 1775 Caryopteris grata 0.197 0.001 1450 Colebrookea oppositifolia 0.07 0.01 383 Phyllanthus emblica 0.255 0.001 2160 Melia azedarach 0.21 0.01 2120 Ficus racemosa 0.115 0.001 1775 Dodonaea viscosa 0.19 0.01 1600 Jasminum humile 0.137 0.001 1050 Albizia lebbeck 0.25 0.001 1850 Pinus roxburghii 0.085 0.001 308 Olea ferruginea 0.11 0.01 450 Bombax ceiba 0.09 0.01 725 Cassia fistula 0.21 0.01 1383 Lantana camara 0.185 0.001 1358 Punica granatum. 0.165 0.001 1817 Pyrus pashia. 0.225 0.001 2027 Dalbergia sissoo 0.16 0.01 1025 Debregeasia salicifolia 0.077 0.001 167 Adhadoda vasica 0.117 0.001 426 Carissa opaca 0.15 0.01 800 Viburnum cotinifolium 0.137 0.001 500 Ficus palmata 0.1150.001 883 Calotropis procera 0.130.001 500 dw dry weight of the original sample.

IC50 2.54 3.26 3.29 3.36 3.58 7.32 9.14 10.26 11.32 12.1 15.13 16.26 24.56 25.03 30.65 32.38 32.50 32.56 33.37 39.27 40.26 41.33 64.26 125

4.1.3.3 Flavonoids finger printing of selected Plants Selected medicinal plants have been studied for flavonoids types. Fourteen different standards flavonoids have been run to determine the samples qualitatively. The standards which have been used are Rutin, Quercetin, Kaempferol, Myricetin, Catechin, Vitexin, Orientin, Isoquercitrin, Isovitexin, Hyperoside, Luteolin-7-glucoside, Kampferol-7neohesperidoside, Luteolin and Apigenin. Each sample and standard flavonoids have been run two times on thin layer chromatographic plates as describe in TLC method (4.7.1.2) (without standard and with standard). Different colours zone of flavonoids have been appeared after spraying of reagent A (1% ethanolic 2-Aminoethyle diphenyl borinate solution) and reagent B (5% ethanolic solution of polyethylene glycol-400) under 365 nm UV light (Fig. 32). Retention time (R t) and colors after spraying reagent A 140

Chapter 4

Results & Discussion

and B have been thoroughly studied under UV 365 nm (Table 12; page. 150).) The results of possible flavonoids types in the plants sample are listed in table 13; page. 151. The ratio of flavonoids types in plant specimens are shown in Fig. 30.
OH HO HO O HO O OH HO O O OH

OH HO

O OH OH

HO HO

OH

O 2F Myricetin

OH

1F Kaempferol-7-neohesperidoside

OH

O OH

OH HO O OH
HO HO HO H O OH OH 4F Vitexin O OH

HO OH 3F Catechin

OH O
OH O

OH

O
HO HO HO OH OH H O OH O OH

HO H H O

O H

OH

HO

H HO

H OH

5F Orientin

F6 Isoquercitrin

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Results & Discussion

OH HO O OH
OH

O HO O
HO

OH

HO

OH

HO

O OH

HO HO OH

O HO O 8F Isovitexin OH

7F Hyperoside

OH HO

OH HO O O HO O OH OH O OH
HO O OH HO

OH O OH

OH

HO O O O

OH

9F Luteolin 7-O-glucoside

11F Kaempferol-7-neohesperidoside

OH HO O OH

O HO O H HO HO O O
HO O OH

OH
OH

HO OH

OH

HO O OH

10F Rutin

12F Quercetin

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Results & Discussion

OH HO O OH
HO O OH

OH

OH

13F Luteolin

14F Apigenin

B 143

Chapter 4

Results & Discussion

144

Chapter 4

Results & Discussion

Figure 32. Flavonoids finger printing.of standard and selected plants (1: R. ellipticus; 2. B. variegata; 3. C. grata; 4. C. oppositifolia; 5. P. emblica; 6. M. azedarach; 7. F. racemosa; 8. D. viscosa; 9. J. humile; 10. A. lebbeck; 11. P. roxburghii; 12. O. ferruginea; 13. B. ceiba; 14. C. fistula; 15. L. camara; 16. P. granatum; 17. P. pashia 18. D. sissoo; 19. D. salicifolia; 20. A. Vasica; 21. C. opaca; 22. V. cotinifolium; 23. F. 145

Chapter 4

Results & Discussion

palmata; 24. C. procera) have been collected. The plants materials are extracted as described in extraction procedure (see extraction procedure 3.6.1). Standard flavonoids. F1. Kaempferol; F2. Myricetin; F3. Catechin; F4 Vitexin; F5 Orientin; F6 Isoquercitrin; F7. Hyperoside; F8. Isovitexin; F9. Luteolin-7-glucoside; F10. Rutin; F11 Kaempferol-7neohesperidoside; F12 Quercetin; F13 Luteolin and F14 Apigenin)

PERCENTAGE OF FLAVONOIDS
120 100 100 80 58.33 54.16 60 33.33 29.16 29.16 25 40 16.66 8.33 4.16 0 0 0 20 0
F2 F3 F6 F7 Ph en ol ic A ci ds F1 F4 F5 F8

%age

12.5

Flavonoids

Figure 33 Percentage of Flavonoids in Plant samples: Percentage of standards flavonoids (F1. Kaempferol; F2. Myricetin; F3. Catechin; F4 Vitexin; F5 Orientin; F6 Isoquercitrin; F7. Hyperoside; F8. Isovitexin; F9. Luteolin-7-glucoside; F10. Rutin; F11 Kaempferol-7-neohesperidoside; F12 Quercetin; F13 Luteolin and F14 Apigenin)

F9 F1 0 F1 1 F1 2 F1 3 F1 4

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Chapter 4

R. ellipticus. B. variegata C. grata

F10 F5

F11 F7 F2

F14 F8 F4

C. oppositifolia P. emblica M. azedarach F. racemosa D. viscosa J. humile A. lebbeck P. roxburghii O. ferruginea B. ceiba C. fistula

F1

Figure 34. Types of Flavonoids in each sample

L. camara P. granatum P. pashia . D. sissoo . D. Salicifolia A. Vasica C. opaca V. cotinifolium F. Palmata C. procera

Phenolic Acid

Results & Discussion

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Results & Discussion

Table 12 Appearance of standards under UV 265nm

S/No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Standard Kaempferol Myricetin Catechin Vitexin Orientin Isoquercitrin Hyperoside Isovitexin Luteolin 7-glucoside Rutin Kamferol-7neohesperidoside Quercetin Luteolin Apigenin R /time 0.81 0.73 0.74 0.65 0.48 0.52 0.54 0.57 0.57 0.42 0.55 0.87 0.9 0.86 Colour (Reagent A) light green orange dark green light green light green orange orange light green Fluorescent yellow Fluorescent yellow light olive green yellow Fluorescent yellow light green Colour (Reagent B) light green dark green dark brown light green light green orange dark brown light green orange orange light olive green orange fluorescent yellow light green

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Table 13 Qualitative analyses of plants samples for Flavonoids types

Plant R. ellipticus. B. variegata C. grata C. oppositifolia P. emblica M. azedarach F. racemosa D. viscosa J. humile A. lebbeck P. roxburghii O. ferruginea B. ceiba C. fistula L. camara P. granatum P. pashia D. sissoo D. salicifolia A. vasica C. opaca V. cotinifolium F. palmata C. procera Flavonoids detected Five Five Two Four Three Four Eight Six Five Six Four Three Three Four Five Five Five Three Five Seven Ten Five Seven Five Phenolic acid + + + + + + + + + + + + + + + + + + + + + + + + F1 + + + + + + + F2 + F4 + + + + + + + + + + + + + + F5 + + + + + + + + F6 F7 + + F8 + + + + + + + + + + + + + F9 F10 + + + + + + + F11 + + + F14 + + + -

(+) = present; (-) = absent; F1. Kaempferol; F2. Myricetin; F3. Catechin; F4 Vitexin; F5 Orientin; F6 Isoquercitrin; F7. Hyperoside; F8. Isovitexin; F9. Luteolin-7-glucoside; F10. Rutin; F11 Kaempferol-7-neohesperidoside; F12 Quercetin; F13 Luteolin and F14 Apigenin

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4.1.4 Antibacterial and Free radical scavenging activities, Flavonoids finger printing of Mallotus philippensis. 4.1.4.1 Antibacterial activities Methanolic extracts of (roots and flower powder) M. philippensis were tested against five strains of bacteria. Flower powder (Kamala or Kamara) extract was effective against Gram positive bacteria, Bacillus subtilis and Staphylococcus aureus (MICs 0.7 and 0.6 mg/ml), while it could not showed any response against the remaining bacterial strains up to maximum concentration of 15mg/ml. Roots extract was effective against one Gram positive bacteria Bacillus subtilis and one Gram negative bacteria Salmonella setubal (MICs 1.00 and 2.00 mg/ml) respectively, while it did not showed any response against the remaining bacterial strains up to maximum concentration of 15mg/ml. the results shown in Figure 35 and table 14 below.

4.1.4.2 Free radical scavenging activities Methanolic extract of (flower powder Kamala and leaves) M. philippensis were investigated for its free radical scavenging capacity. DPPH (1, 1-Diphenyl-2picrylhydrazyl) was used in the experiment. The bleaching of DPPH colour in the experiment by Mallotus philippensis extracts represents the scavenging capacity of its extracts. Results shows that the leaves extracts was more reactive than the kamala extracts (IC50 33 and 47 g/ml respectively) see Figure 36, Ascorbic acid was used as standard antioxidant (IC50 = 5.75 g /ml).

4.1.4.3 Flavonoids finger printing of Mallotus philippensis Aerial parts of Mallotus philippensis have been studied for flavonoids types. Fourteen different standards flavonoids have been run parallel to sample in order to identify flavonoids in the samples qualitatively. The standards which have been used are Rutin, Quercetin, Kaempferol, Myricetin, Catechin, Vitexin, Orientin, Isoquercitrin, Isovitexin, Hyperoside, Luteolin-7-glucoside, Kaempferol-7-neohesperidoside, Luteolin and Apigenin. The samples and standard flavonoids have been run on thin layer chromatographic plates as describe in TLC method (4.7.1.2). Different colors zone of flavonoids have been appeared after spraying of reagent A (1% ethanolic 2-Aminoethyle

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diphenyl borinate solution) and reagent B (5% ethanolic solution of polyethylene glycol400) under 365 nm UV light (Fig. 37). Retention time (Rt ) and colours after spraying reagent A and B have been thoroughly studied under UV 365 nm. Vitexin, Isovitexin and Rutin have been detected in the extract.

roots extract kamala extract 2 1.8 1.6 1.4 1.2

mg/ml

1 0.8 0.6 0.4 0.2 0 Bacillus subtilis Staph. aureus Salmonella setubal

Figure 35 Antibacterial activities of Mallotus philippensis.

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Table 14. Antibacterial activities of roots and flower powder extract

Concentration (mg/ml) 15.00

Inhibition zone (mm) (roots extract) B1 B2 B3 B4 B5 24.0 6.0

Inhibition zone (mm) (flower powder or kamala) B1 B2 B3 B4 B5 24 20 -

12.50 10.00 7.50 5.00 3.00 2.00 1.00 Ciprofloxacine 35.0 (2 mg/ml) DMSO -

22.0 21.5 21.5 20.0 19.5 19.0 17.0 39.0 22.0 -

5.5 5.0 4.0 4.0 4.0 4.0 0.0 36.0 35.0 -

22 20 21.5 20 21.5 18 20.0 17 19.5 17 19.0 15 17 13 39.0 22.0 29.0 36. -

B1 = Escherichia coli, B2 = Bacillus subtilis, B3= Staphylococcus aureus, B4 = Staphylococcus epidermidis, B5 = Salmonella setubal

0.6

(Flower powder) (Leaves)

0.5

Absorbance at (517 nm)

0.4

0.3

0.2

0.1

0.0 20 40 60 80 100 120 140 160 180 200 220

Concentration (ug/ml)

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100 90 80 70

Flower powder (Leaves)

% inhibition

60 50 40 30 20 20 40 60 80 100 120 140 160 180 200 220

Concentration(ug/ml)

50 45 40 35 30 g /ml 25 20 15 10 5 0 Ascorbic acid Leaves Kamala

Figure 36 Free radical scavenging activity of Mallotus philippensis

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A B

Results & Discussion

Figure 37 Flavonoids finger printing of Mallotus philippensis: (MP) Sample and standard flavonoids (1F. Kaempferol; 4F Vitexin; 6F Isoquercetin; 7F. Hyperoside; 8F. Isovitexin; 9F. Luteolin-7-glucoside; 10F. Rutin and S3 Quercetin and Rutin Mix)

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4.2 Discussion 4.2.1 Anti-neoplastic Activities and Phytochemicals studies of Berberis lycium The roots of B. lycium were extracted with organic solvent methanol, evaporated methanol and the extract was again dissolved in distilled water. The water solution was again extracted with organic solvent hexane, ethyl acetate, butanol and the remaining of water. The fractions were analyzed qualitatively by thin layer chromatography (TLC). Berberine, palmatine and berbamine were used as reference compounds. Berbamine was reported to be a constituent of B. lycium (Ali and Khan, 1978), while there was no evidence of its presence in the here performed TLC and RP-HPLC analyses. All extracts contained berberine (retention factor, Rf = 0.151) and palmatine (Rf = 0.088), whereas the highest concentration of both compounds was detected in the BuOH extract. For quantification RP-HPLC was used. Therefore berberine and palmatine were selected to study as reference compounds in ordered to identify the active compound in B. lycium. The effects of root extracts of B. lycium were studied against HL-60 human leukemia cells and compared them with the purified constituent alkaloids, Berberine and Palmatine. B. lycium is an erect small rigid shrub about 1.0-2.5 meters tall, with a thick woody shoot covered with a thin brittle bark (Hooker, 1882) and is native in the whole Himalaya Mountains range and widely distributed in temperate and semi-temperate areas of India, Nepal, Afghanistan, Bangladesh and Pakistan. The active constituents of B. lycium are alkaloids. The major alkaloids are Umbellatine, Berberine (Ali and Khan, 1978), Oxyacanthine and Chelidonic acid (Karnick, 1994), Heterocyclic constituents named Berberisterol, Berberifuranol and Berberilycine (Ali and Sharma, 1996), the alkaloids Sindamine, Punjabine and Gilgitine (Leet et al., 1982, 1983), Palmatinechloroform a tertiary dihydroprotoberberine alkaloid (Miana, 1973), Berbericine and Berbericinine hydriodide (Ikram et al., 1966) were also found in the roots of B. lycium. Besides these, Berbamine, starch grains and tannins are also present in small quantities (Ali and Khan, 1978). In the present investigation berberine and the crude BuOH extract regulated protein expression and protein activation in HL-60 cells similarly. Also the growth inhibitingand apoptosis-inducing potential was similar and FACS- and Comet data were almost identical. This is a strong indication that BuOH-mediated cell cycle arrest was due to berberine. I shows that the growth inhibitory properties of berberine and BuOH extract correlated directly with the inactivation and down-regulation of the proto-oncogene

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Cdc25A resulting in the inactivation of Cdc2. Also the inhibition of human nasopharyngeal carcinoma CNE-2 cell growth by berberine was associated with suppression of cyclin B1, CDK1 (Cdc2), and Cdc25C proteins (Cai et al, 2006). In human glioblastoma T98G cells berberine induced cell cycle retardation in G1-phase through increased expression of p27 and suppression of CDK2, CDK4, cyclin D, and cyclin E proteins (Eom et al., 2008), and HL-60 cell growth was significantly inhibited by berberine in G0/G1 phase with a decrease in S-phase cells (Wang and Lin, 2004). In another study FACS analyses indicated that berberine induced G2/M-phase arrest in HL60 cells and murine myelomonocytic leukemia WEHI-3 cells that was accompanied by increased levels of Wee1 and 14-3-3sigma, and decreased levels of Cdc25C, CDK1 and cyclin B1 (Lin et al., 2006). This is in contradiction to the reported G0/G1 arrest (Eom et al., 2008) and to the intra-S-phase arrest observed in this study, but the differences were most likely due to the different berberine concentrations used in these investigations. Notably, intra-S-phase arrest correlated with the activation of Chk2 and this was also demonstrated in the context of ionizing radiation (Luo et al., 2008). In addition, the extract and the purified compound caused the down-regulation of the proto-oncogene cyclin D1 after 48 h and this certainly added up to the cell division arrest. Therefore, berberine and the BuOH extract down-regulated two potent oncogenes, Cdc25A and cyclin D1. The proliferation of human umbilical vein endothelial cells (HUVECs) was inhibited upon incubation with 20 g/ml berberine. This phenomenon was accompanied by a significant decrease of PCNA, and a typical apoptotic appearance correlated with a marked decline in the mitochondrial membrane potential. Berberine-mediated inhibition of vascular endothelial cell proliferation suppressed neo-vascularization, and this might be one of the mechanisms attenuating growth and metastasis of tumors (Hao et al., 2005) Berberine and the BuOH extract in a 3-D metastasis model. This model utilizes singlelayers of lymphendothelial cells onto which MCF-7 cell spheroids are placed that repulse the endothelial cells thereby generating gaps in the lyphendothelium underneath that function as gates through which cancer cell bulks can penetrate. However, 10 - 100 M Berberine did not prevent lymphendothelial gap formation induced by MCF-7 spheroids (data not shown). It was further reported that an ethanol extract of Coptis teeta, which contains berberine and other components, as well as purified berberine induced apoptosis of MCF-7 breast cancer cells (Kang et al., 2005). Berberine triggered cell death was reported also in 156

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several other human cancer cell lines [Lin et al., 2006; Mantena et al., 2006; Hwang et al., 2006), such as in human glioblastoma T98G cells that was concomitant with an increased Bax/Bcl-2 ratio, disruption of the mitochondrial membrane potential, and the activation of caspase-9 and caspase-3 (Eom et al., 2008). Berberine-induced apoptosis of human leukemia HL-60 cells was shown to be associated with down-regulation of nucleophosmin/B23 and telomerase activity (Wu et al., 1999). Furthermore, Liu et al (2009) reported a cell cycle inhibitory effect of Berberine in a high concentration range (between 10 - 50 M), which correlated with DNA damage. In this study, the authors hypothesize that Berberine inhibited osteosarcoma cell proliferation and induced apoptosis through genotoxicity. In contrast, we found that the inhibition of proliferation and the induction of apoptosis occurred with berberine doses and extract concentrations that were devoid of genotoxic activity, although we agree that high Berberine concentrations could cause DNA strand breaks. Our data suggest that another molecular/cellular mechanism transduces the pro-apoptotic properties of Berberine and BuOH extract and this correlated with -tubulin acetylation, which is indicative for microfilament polymerization (Marcus et al., 2005) Tilting the fine-tuned balance of polymerized/de-polymerized microtubule is incompatible with proper cell division as well as cell survival. Therefore, the anticancer properties of berberine and the BuOH extract are reminiscent of that of taxol (Wilson and Forer, 1997) and independent of genotoxicity.

4.2.2 Anti-neoplastic Activities and Phytochemicals studies of Mallotus phillipensis The roots of M. phillipensis were extracted with organic solvent methanol. The concentrated MeOH extract of M phillipensis was dissolved in distilled water and extracted three times each with organic solvents in sequence of increasing polarity such as hexane, ethyl acetate (EtOAc), and n-butanol (BuOH). After evaporating each solvent, 9.23 g dred hexane extract, 4.00 g dried EtOAc extract, and 7.08 g dried BuOH extract was obtained, respectively. M. phillipensis is a deciduous tree widely distributed throughout tropical Asia, Mountain slopes or valleys, limestone hills or river valleys, forests; 300-1600 m. Fujian, Anhui, Guangdong, Guizhou, Guangxi, Hainan, Hunan, Jiangsu, Taiwan, Jiangxi, Sichuan, Xizang, Yunnan, Hubei, Zhejiang, Bhutan, Philippines, India, Laos, Thailand, Myanmar, Malaysia, Bangladesh, Nepal, Pakistan, New Guinea, Sri Lanka, Vietnam and N Australia. Different type of chemical compounds

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(big and small) have been found in the different parts of the plant. Rottlerin (5, 7dihydroxy-2, 2-dimethyl-6-(2, 4, 6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1 ,2-chromine), which is also called mallotoxin, is one of the major constituents that have been confirmed in different parts of M. phillipensis, exhibiting various pharmacological activities including mitochondrial uncoupler effects. (Zaidi et al., 2009). Rottlerin was evaluated and considered that it is a specific inhibitor of the novel protein kinase C (PKC) isoform, PKC d, and identified as an anticarcinogenic chemical compound (Soltoff,, 2007). The activation and translocation of PKC d are induced by different apoptotic stimuli in various cellular systems (Brodie et al., 2003). It has been demonstrated in various studies that PKC d might not directly inhibited by rottlerin, but it can produce some cellular changes that is very closely resembles to those produced by the direct inhibition of PKC d (Soltoff, 2001;Tapia et al., 2006). In one recent study, It has been found that rottlerin sensitized both glioma cells and colon carcinoma cells to TRAIL-mediated apoptosis through inhibition of Cdc2 and uncoupling of the mitochondria, respectively (Tillman et al., 2003; Kim et al., 2005). However, both the mentioned mechanisms by which rottlerin sensitizes cancer cells to TRAIL-mediated apoptosis and rottlerin-induced apoptosis are not completely known. In very recent study it is demonstrated that apoptosis induced by rottlerin was due to its up regulation property of DR5 (Lim et al., 2009). Furthermore, it has been noticed that rottlerin does not sensitized normal cell but potentially sensitized various cancer cells, to TRAILmediated apoptosis. Therefore, it is strongly recommended that the combined treatment with both TRAIL and rottlerin can be use as a safe and effective cancer therapy. It also noticed that the up regulation of DR5 mediated by CHOP, that is independent of PKC d activity, contributes to rottlerin-induced apoptosis. Tanaka et al, (2008) has isolated known friedelane-type triterpenoids compounds from the stem bark of M. phillipensis and described the anti-tumor promoting activity, and they found for 3-hydroxy-D:Afriedoolean-3-en-2-one ( IC50 = 292 mol ratio/ 32 pmol/TPA); 3-hydroxy-D:Afriedoolean -2-one (IC50 = 288); curcumin was used as positive control, (IC50 = 343); Epstein-Barrvirus was used as early antigen (EBV-EA) and activation induced by 12-Otetradecanoyl phorbol 13-acetate (TPA) used in the experiment. In the present investigation the hexane fractions regulated protein expression and protein activation in HL-60 cells. The extract showed the highest toxicity against HL-60 cells (IC50 1.5 mg dry roots equivalent /ml medium) after 72h. The inhibition of HL-60 proliferation that was observed upon treatment with hexane extract was preceded by the 158

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down regulation of the proto-oncogene Cdc25A and cyclin D1 after 48 h. All of these effects have not been observed in any p53 deficient cell lines so far by Mallotus phillipensis extracts and its chemical constituents. Valacchi et al, 2008 has reported that rottlerin deactivate cyclin D1 in HaCaT cell line. The hexane fraction induced 18% apoptosis after 48h of treatment with 1.5 mg dry roots equivalent /ml medium. I monitored the ability of M. phillipensis hexane fraction and the observation indicates that the anti-neoplastic effects have been triggered by induction apoptosis through caspase-2 activation while Brodie et al., 2003 reported that rottlerin activated caspase-3. Caspase-2, maybe even more versatile as previously thought by mediating such opposing functions as either killing (Sidi et al ., 2008; Olsson et al ., 2009) or saving a cell after DNA damage (Shi et al., 2009) and, subsequently, even more useful in protecting the whole organism from developing cancer (Ho et al ., 2009). It was therefore concluded that , caspase-2 may represent the archetype member of this protease family that still unifies many of the above-mentioned functions in a single enzyme. The hexane extract was analyzed with GC/MS and different compounds were detected in the fraction. Mass spectrometric data of some compounds have been co-related with already reported compounds from different parts of the same species. Some unknown compounds which have the same m/z ratio as of Lupeol, Betulin, Kamala Chalcones C (GC Rf = 39.9, 45.66, 43.905 and 47.735 minutes respectively) have been detected. Rottlerin and friedeline types compound that has been reported cytotoxic from M. phillipensis was not detected in the fraction. It has been confirmed from the present antineoplastic assay that hexane fraction is active against p53 deficient human leukemia cell lines (HL-60) and the activity was due to other than rottlerin and friedeline types compound.

4.2.3 Total Phenolics, Free radical scavenging activity and Flavonoids finger printing of selected Medicinal Plants. I had selected twenty four different plants species were selected. Some plants species were reported medicinally in literature and the others have been selected randomly. The medicinally important plants were Bauhinia variegata, Cassia fistula, Bombax ceiba, Calotropis procera, Carissa opaca, Adhatoda vasica, Albizia lebbeck, Colebrookea oppositifolia, Dalbergia sissoo, Dodonaea viscosa, Ficus palmata, Ficus racemosa,

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Lantana camara, Melia azedarach, Phyllanthus emblica, Punica granatum, Rubus ellipticus and Viburnum cotinifolium and the non medicinal plats were Jasminum humile, Olea ferruginea, Pinus roxburghii, Pyrus pashia., Caryopteris grata and Debregeasia salicifolia. Total Phenolics were studied by comparing with Gallic acid. A calibration curve was established for Gallic acid. Phyllanthus emblica has shown highest amount of total Phenolics while comparing with Gallic acid. The extract per gram of Phyllanthus emblica was also greater than others; it is assumed that the highest amount of extract yield by Phyllanthus emblica responsible for its highest total Phenolic contents. The compounds isolated so far from the aerial parts of Phyllanthus emblica are ellagitannins and flavonoids naringenin, eriodictyol, Kaempferol, dihydro Kaempferol, quercetin, naringenin 7-O-glucoside (prunin), naringenin 7-O-(60-O-galloyl)-glucoside, naringenin 7-O-(60-O-trans-p-coumaroyl)-glucoside, Kaempferol 3-O-rhamnoside, quercetin 3-Orhamnoside, myricetin 3-O-rhamnoside, 2-(2-methylbutyryl)-phloroglucinol 1-O-b-Dglucopyranoside (multifidol glucoside) v,epigallocatechin 3-O-gallate, 1,2,3,6-tetra-O-, 1,2,4,6-tetra-O-,15) and 1,2,3,4,5-penta-O-galloyl-b -Dglucose, and decarboxyellagic acid (Zhang et al., 2001c, 2002). Seven other tannins and flavonoids, geraniin, phyllanemblinins C and E , prodelphinidin B1, prodelphinidin B2, -epigallocatechin 3-Ogallate , and (S)-eriodictyol 7-[6-O-(E)-p-coumaroyl]-b-D-glucoside were the main Phenolic compounds isolated from the branches and leaves of the plants. Phenolic acid, Kaempferol and Vitexin were detected in Phyllanthus emblica by thin layer chromatography. Vitexin was reported for the first time in Phyllanthus emblica. Melia
azadereca contain second highest amount of total Phenolics. The compounds that have

been isolated from the aerial parts of Melia azedarach are nimbinene, meliacin, quercetin, quercetin-3-0-b-rutinoside, Kaempferol- 3-0-b rutinoside, rutin and kaempferol-3-Lrhamno-Dglucoside (Sharma et al., 2001). Dipentadecyl ketone, glycerol 1, 3-bis-undec9- enoate 2-dodec-9-enoate and glycerol tris-tridec-9-enoate were isolated from the hot methanolic extract of Melia azedarach leaves (Suhag et al., 2003). Limonoid 1cinnamoyl-3,11- dihydroxy meliacarpin have been isolated from the ethyl acetate extract of M. azedarach leaves (Alche et al., 2003). Phenolic compounds, isovitexin and rutin were detected in Melia azedarach by thin layer chromatography. Isovitexin was not reported in Melia azedarach before. Among the randomly selected plant species, Pyrus pashia. showed highest yield per gram of dried powder and total Phenolics as well. It has delicious fruits but no ethno botanical and phytochemical data in literature. Phenolic acid, orientin and isovitexin were detected in Pyrus pashia. 160

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Rubus ellipticus has shown comparatively highest capacity in scavenging free radicals. Its activity was strong than standards ascorbic acid. The compounds isolated from the aerial parts of Rubus ellipticus are elliptic acid (Dutta et al., 1997), tannins (Marczal, 1963; Okuda et al., 1992), derivatives of Kaempferol and Quercetin, Phenolic acids, triterpenes, mineral salts as well as vitamin C are reported in Rubus species (Gudej and Rychlinska, 1996; Krzaczek, 1984; Wojcik, 1989). Gudej (2003) has reported derivatives in raspberry leaves i.e. Kaempferol quercetin, ellagic acid and Methyl gallate. Methyl brevifolin carboxylate is also reported with another known compound from Rubus species (Gudej et al., 1998). Phenolic acids, Kaempferol, Vitexin, Rutin and Apigenin were detected from the aerial parts of Rubus ellipticus by thin layer chromatography. Vitexin, Rutin and Apigenin reported for the first time from the species. Rutin (quercetin-3-rhamnosyl glucoside) is a kind of flavonoid glycoside found in buckwheat, many vegetables, fruits, tea and wine, which are the plant-derived beverages (Manach et al., 1997). Rutin or Vitamin P has antihypertensive, antiviral and antiplatelet properties, as well as strengthen the capillaries, which is the result of its high radical scavenging activity and antioxidant capacity (Guo et al., 2007). In addition, hypolipidaemic, cytoprotective antispasmodic and anticarcinogenic activities have also been reported. All these properties are highly useful in preventing different type of diseases and also help in protecting the stability of the genetic material (Yang et al., 2008). Diagnosing genome instability in the cell are performed by the micronucleus (MN) assay which is an efficient biomarker for such diagnosing (Bonassi et al., 2001). Fenech, (2008) has suggested that the supplementation with specific micronutrients such as rutin, a-tocopherol, ascorbic acid can normalized or reduced the MN frequency , and that the genome damage rate can be minimizing with optimal level of micronutrient intake. Furthermore, La Casa et al (2000) clearly indicate that the gastric mucosal damage produced by intragastric instillation of the necrotizing agent are significantly reduced by rutin, and also increased GPx activity. Robak and Gryglewski (1988) have also observed that SOD-sensitive free radicals also scavenged by rutin , which are produced during the activity of xanthine-oxidase. Dugas et al (2000) measured the antioxidant activity of a series of flavonoids against peroxyl radicals generated. In their study, the most active compound was the quercetin, followed by rutin. They suggest that potential role for dietary intake of rutin and quercetin containing foods in lowering the risk of certain pathophysiologies that have been associated with free radical-mediated disease. It has also been studied that showed a dose-response effect in inhibiting low density lipoprotein (LDL) per oxidation of rutin (Jiang et al., 2007; 161

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Ne`gre-Salvayre et al 1995. Moreover, Milde et al (2000) suggested that rutin is a promising flavonoid for reducing the risk of atherosclerosis due to its inhibiting on LDL oxidation. Bauhinia variegata, Colebrookea oppositifolia and Phyllanthus emblica also shows comparatively strong free radical scavenging activity. Six flavonoids, and a triterpene caffeate, were also obtained from the non-woody aerial parts of Bauhinia variegata, (Rao et al., 2008) Phenanthraquinone, named bauhinione has been isolated from Bauhinia variegata. (Zhao et al., 2005). Several flavone and flavone glycosides are reported from Colebrookea oppositifolia (Ahmed et al., 1974; Patwardhan et al., 1981; Yang et al., 1996). Phenolic acid and orientin were detected in Bauhinia variegata; Phenolic acid and Luteolin were found in Colebrookea oppositifolia. Among the non medicinal plants, Caryopteris grata has showed strong free radical scavenging activity. Its ethno botanical and phytochemical data are not reported in literature. I have found Phenolic acid and orientin in the aerial part of Caryopteris grata by thin layer chromatography. The aerial parts of the plants species under investigation were analyzed to determine flavonoids by thin layer chromatography and standard flavonoids qualitatively and found Vitexin, Rutin and Apigenin for the first time in Rubus ellipticus; Orientin in Bauhinia variegata; Orientin in Caryopteris grata; Kamferol-7-neohesperidoside in Colebrookea oppositifolia; Vitexin in Phyllanthus emblica; Isovitexin in Melia azedarach; Vitexin,
Isovitexin and Rutin in Ficus racemosa; Rutin and Apigenin in Dodonaea viscosa;

Kaempferol, Vitexin and Hyperoside in Jasminum humile; Vitexin, Hyperoside and Rutin in Albizia lebbeck; Kaempferol and Isovitexin in Pinus roxburghii; Vitexin, Isovitexin and Apigenin in Olea ferruginea; Kaempferol, Vitexin and Kamferol-7-neohesperidoside in Bombax ceiba; Vitexin and Isovitexin in Cassia fistula; Kaempferol and Vitexin in Lantana camara; Vitexin and Myricetin in Punica granatum; Orientin and Isovitexin in Pyrus pashia.; Orientin and Isovitexin in Dalbergia sissoo; Luteolin, Orientin and Isovitexin in Debregeasia Salicifolia; Orientin and Isovitexin in Adhatoda vasica; Vitexin, Orientin, Rutin and Isovitexin in Carissa opaca; Vitexin and Isovitexin in Viburnum cotinifolium; Vitexin, Orientin, Rutin and Isovitexin in Ficus Palmata; Vitexin and Isovitexin in Calotropis procera.

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All plants species have shown Phenolic acids bands. Vitexin and Isovitexin were present in maximum numbers of plants samples (58.33 and 54.8 % percent respectively), Catechin, Luteolin-7-glucoside, Quercetin and Luteolin were not detected in any sample.

4.2.4 Antibacterial and Free radical scavenging activities, Flavonoids finger printing of Mallotus Philippensis. Kamala, a red powder consisting of glandular hairs from the capsules of Mallotus philippensis. It has been used as a drug and dye and has long been used as an anthelminticum and cathartic in traditional medicine (Satyavati et al., 1987; Srivastava et al., 1967; Gupta et al., 1984 and an orange dye for silk (Lounasmaa et al., 1975). Kamala, coating the fruit is commonly administered in curd for the elimination of intestinal worms and also for skin irritation, ringworm, and freckles (Usmanghani et al., 1997). In literature different scientist isolated small and high molecular weight compounds from kamala. Flavonoids such as Kamalachalcones A and B have been isolated by Toshiyuk et al (1998) from Kamala. Furusawa et al (2005) has reported a new flavanone, 4-hydroxy isorottlerin; 5, 7-dihydroxy-8-methyl-6-prenylflavanone and two new chalcone derivatives, Kamalachalcones C and D from Kamala. Daikonya et al (2002 and 2004) has reported Phloroglucinol derivatives, Mallotophilippens A and B; Mallotophilippens C, D and E that suppressed the NO production and iNOS gene expression. Rottlerin (McGookin et al., 1937) and several other useful compounds have been isolated so far from Kamala. Zaidi et al, 2009 has reported five compounds from kamala powder (M. Philippensis) and studied their activity against Helicobacter pylori. Among the isolated compounds from the genus Mallotus, rottlerin is considered the most potent bactericidal compound with minimum bactericidal concentration (MBC) value of 3.126.25 mg/l against different clinical H. pylori isolates including different Pakistani and Japanese strains, seven metronidazole resistant (MR) strains and nine clarithromycin resistant (CR). Strains were analyzed by E test and the minimum inhibitory concentration MR (~256 mg/l) and (MIC) values of CR (8-256 mg/l). Comparative study of roots extract and Kamala (M. philippensis) were made against five strains of bacteria; Bacillus subtilis, Staphylococcus aureus, Salmonella setubal, Staphylococcus epidermidis and Escherichia coli. Flower powder (Kamala or Kamara) extract has shown activities against Gram positive bacteria, Bacillus subtilis and

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Staphylococcus aureus (MICs 0.7 and 0.6 mg/ml), while it has not shown any response against the remaining bacterial strains up to maximum concentration of 15 mg/ml. Roots extract was effective against one Gram positive bacteria Bacillus subtilis and one Gram negative bacteria Salmonella setubal (MICs 1.00 and 2.00 mg/ml) respectively but it has not shown any activity against the remaining bacterial strains up to maximum concentration of 15 mg/ml. It has been concluded that there are difference in chemical composition between the roots and Kamala powder that inhibit bacterial strains in two different ways. Similarly a comparative study was made to determine the free radical scavenging capacity of Kamala powder and the leaves of Mallotus philippensis. Since both extracts have Phenolic compounds but the leaves extract was more active than Kamala powder in scavenging free radicals. It was important to know about the flavonoids in the leaves of Mallotus philippensis. A simple test of thin layer chromatography was performed to determine the flavonoids qualitatively and found vitexin, Isovitexin and Rutin in it. Rutin (quercetin-3-rhamnosyl glucoside) is a kind of flavonoid glycoside found in buckwheat, many vegetables, fruits, and plant-derived beverages such as tea and wine (Manach et al., 1997). Rutin or in other words Vitamin P has antiviral, antihypertensive and antiplatelet properties, due to its high radical scavenging activity and antioxidant capacity it strengthen the capillaries (Guo et al., 2007). Several other properties of rutin such as cytoprotective, antispasmodic, hypolipidaemic and anticarcinogenic have also been reported. All these mentioned properties are much useful for protecting the stability of the genetic material and preventing diseases (Yang et al., 2008). The micronucleus (MN) assay is an efficient biomarker for diagnosing genome instability in the cell (Bonassi et al., 2001). Fenech, (2008) has suggested that MN frequency can be normalized or reduced on supplementation with specific micronutrients such as rutin, a-tocopherol, ascorbic acid, and that there is an optimal level of micronutrient intake for minimizing genome damage rate. Furthermore, La Casa et al (2000) clearly indicate that rutin significantly reduced the gastric mucosal damage produced by intragastric instillation of the necrotizing agent, and increased GPx activity. Robak and Gryglewski (1988) have shown that rutin is a scavenger of the SOD-sensitive free radicals, which are generated during the activity of xanthine-oxidase. Dugas et al (2000) measured the antioxidant activity of a series of flavonoids against peroxyl radicals generated. In their study, the most active compound was the quercetin, followed by rutin. They suggest that potential role for dietary intake of rutin and quercetin containing foods in lowering the risk of 164

Chapter 4

Results & Discussion

certain pathophysiologies that have been associated with free radical-mediated disease. There are also studies that show a dose-response effect in inhibiting low density lipoprotein (LDL) per oxidation of rutin (Jiang et al., 2007; Ne`gre-Salvayre et al 1995. Moreover, Milde et al.(2000 suggested that rutin is a promising flavonoid for reducing the risk of atherosclerosis due to its inhibiting on LDL oxidation. It has been concluded from the study that the flavonoids of the leaves are more effective than the flavonoids of Kamala powder in scavenging free radical.

165

Chapter 5

Conclusions

Twenty seven plants species have been studied. Roots of three plants (Berberis lycium, Mallotus philippensis and Ziziphus nummularia) were studied for antineoplastic activity against p53 deficient human leukemia cell lines (HL-60). Roots extract of Ziziphus nummularia did not showed activity. Roots of Berberis lycium (BuOH fraction) and Mallotus philippensis (Hexane fraction) have shown good anti proliferation activity against HL-60 cell lines.

Antineoplastic activity of Berberis lycium: BuOH, EtOAc and H2O fractions of Berberis lycium roots were analyzed for its chemical constituents through thin layer chromatography and reverse phase high performance liquid chromatography. Berberine and palmatine have been detected in the samples. The calculated berberine content was 18.04 %, 0.54 % and 2.76 % and palmatine content was 2.80 %, 0.04 % and 0.93 % in the BuOH, EtOAc and H2O extracts, respectively. To evaluate which of the major constituents of the BuOH extract may have caused growth inhibition, HL-60 cells were treated with the measured equivalent concentrations of berberine (0.6-1.8 g/ml) and palmatine (0.3-0.7 g/ml). The IC50 for berberine was 1.2 g/ml after 48 h. Palmatine did not inhibit cell growth after 48 h. The inhibition of HL-60 proliferation that was observed upon treatment with BuOH extract or berberine was preceded by the induction of p21wafand by a dramatic down regulation of the protooncogene cyclin D1 after 48 h. BuOH extract and berberine caused a reduction of G1 cells and accumulation of cells in the S phase during cell cycle and caused a similar proapoptotic effect by acetylation of -tubulin, which is indicative for tubulin polymerization. Tilting the fine-tuned equilibrium of polymerized/de-polymerized microtubule is incompatible with normal cell division and this causes not only cell cycle arrest but also apoptosis.

Antineoplastic activity of Mallotus philippensis: The inhibition of HL-60 cells proliferation that was observed upon treatment with hexane extract of Mallotus philippensis was preceded by the down regulation of the protooncogene Cdc25A and cyclin D1 after 48 h. The hexane fraction induced apoptosis 18% after 48h of treatment with 1.5 mg dry roots equivalent /ml medium. I monitored the ability of M. phillipensis hexane fraction and the observation indicates that the antineoplastic effects have been triggered by induction apoptosis through caspase-2 activation. Hexane fraction of M. phillipensis analyzed with GC/MS and it has been 166

Chapter 5

Conclusions

detected different compounds in the fraction. Mass spectrometric data of some compounds have been co-related with already reported compounds from different parts of the same species. Unknown compound (GC Rf = 39.9, 45.66, 43.905 and 47.735 minutes respectively) have been detected. It has been confirmed from the present antineoplastic assay that hexane fraction is active against p53 deficient human leukemia cell lines (HL60) and the activity was due to compound/compounds other than rottlerin. Total Phenolics, Free radical scavenging activities and Flavonoids finger printing: Twenty four plant species were studied for total Phenolics, free radical scavenging activities and flavonoids finger printings. Out of twenty four, eighteen plants species have medicinal importance, which includes Bauhinia variegata, Cassia fistula, Bombax ceiba, Calotropis procera, Carissa opaca, Adhatoda vasica, Albizia lebbeck, Colebrookea oppositifolia, Dalbergia sissoo, Dodonaea viscosa, Ficus palmata, Ficus racemosa, Lantana camara, Melia azedarach, Phyllanthus emblica, Punica granatum, Rubus ellipticus and Viburnum cotinifolium and the remaining six species, Jasminum humile, Olea ferruginea, Pinus roxburghii, Pyrus pashia, Caryopteris grata and Debregeasia salicifolia were randomly selected. Phyllanthus emblica has shown highest amount of total Phenolics. Gallic acid was used as standard Phenolic compounds. Pyrus pashia has shown highest amount of total Phenolics among the randomly selected plant species. Rubus ellipticus has shown comparatively highest capacity in scavenging free radicals. Its activity was strong than standards ascorbic acid. Flavonoids finger printing of the plant samples have shown the presence of Vitexin, Rutin and Apigenin for the first time in Rubus elepticus; Orientin in Bauhinia variegata; Orientin in Caryopteris grata; Kamferol-7-neohesperidoside in Colebrookea oppositifolia; Vitexin in Phyllanthus emblica; Isovitexin in Melia azedarach; Vitexin, Isovitexin and Rutin in Ficus racemosa; Rutin and Apigenin in Dodonaea viscosa; Kaempferol, Vitexin and Hyperoside in Jasminum humile; Vitexin, Hyperoside and Rutin in Albizia lebbeck; Kaempferol and Isovitexin in Pinus roxburghii; Vitexin, Isovitexin and Apigenin in Olea ferruginea; Kaempferol, Vitexin and Kamferol-7-neohesperidoside in Bombax ceiba; Vitexin and Isovitexin in Cassia fistula; Kaempferol and Vitexin in Lantana camara; Vitexin and Myricetin in Punica granatum; Orientin and Isovitexin in Pyrus pashia.; Orientin and Isovitexin in Dalbergia sissoo; Luteolin, Orientin and Isovitexin in Debregeasia Salicifolia; Orientin and Isovitexin in Adhatoda vasica; Vitexin, Orientin, Rutin and Isovitexin in Carissa opaca; Vitexin and Isovitexin in Viburnum cotinifolium; Vitexin, 167

Chapter 5

Conclusions

Orientin, Rutin and Isovitexin in Ficus palmata; Vitexin and Isovitexin in Calotropis procera. All plants species have shown Phenolic acids bands. Vitexin and Isovitexin were present in maximum numbers of plants samples (58.33 and 54.8 % percent), Catechin, Luteolin-7-glucoside, Quercetin and Luteolin were not detected in any sample.

Antibacterial and Free radical scavenging activities of Mallotus philippensis: Kamala, a red powder found on the surface of Mallotus philippensis has been comparatively studied with roots extract of Mallotus philippensis for antibacterial activity and aerial parts of Mallotus philippensis for free radical scavenging activity. Kamala extract has shown activities against Gram positive bacteria, Bacillus subtilis and Staphylococcus aureus (MICs 0.7 and 0.6 mg/ml), while it did not showed any response against the remaining bacterial strains up to maximum concentration of 15 mg/ml. Roots extract was effective against one Gram positive bacteria Bacillus subtilis and one Gram negative bacteria Salmonella setubal (MICs 1.00 and 2.00 mg/ml) respectively but it did not showed any activity against the remaining bacterial strains up to maximum concentration of 15 mg/ml. It has been concluded that there are difference in chemical composition between the roots and Kamala powder that inhibit bacterial strains in two different ways. The leaves extract was more active than Kamala powder in scavenging free radicals. Flavonoids finger printing of the leaves have shown the presence of vitexin, isovitexin and rutin. It has been confirmed from the present investigation that flavonoids of the leaves of Mallotus philippensis are more active than the flavonoids of kamala in scavenging the free radical. Future Prospect In summary, the work done was much significant. Berberis lycium was the most active medicinal plants and can be used for the treatment of various infectious deceases. However the amount use in crude form must be carefully studied. The alkaloids of Berberis lycium are much active and therefore need a comprehensive study regarding its side effect. Hexane soluble fraction of Mallotus philippensis (roots) contain very active compounds which still need to be explore. Rubus ellipticus contain strong anti oxidant compounds and therefore the plant is strongly recommended for further biological activities. 168

List of publications

List of Publication
1. Musa Khan Dawar, Fareeha Maheen, Rizwana Aleem Qureshi. Comparative study of total Phenolic and Free radical scavenging activities of reported and non reported medicinal plants of Margalla Hills, Islamabad. Proceeding of International Seminar Medicinal Plants: Isolation and Application May 2123, 2008 at Lahore College for woman University Lahore, Pakistan. p. 174181. 2. Musa Khan Dawar, Rizwana Aleem Qureshi. In Vitro Antibacterial and Antioxidant Activity of Mallotus philippinensis (Lam.) Mll.-Arg.

(Euphorbiaceae). Proceeding of International Seminar Medicinal Plants: Isolation and Application May 21-23, 2008 at Lahore College for woman University Lahore, Pakistan. p. 24-32. 3. Musa Khan, Benedikt Giessrigl, Caroline Vonach, Sibylle Madlener, Sonja Prinz, Irene Herbaceck, Christine Hlzl, Sabine Bauer, Katharina Viola, Wolfgang Mikulits, Rizwana Aleem Quereshi, Siegfried Knasmller, Michael Grusch, Brigitte Kopp, Georg Krupitza. Berberine and a Berberis lycium extract inactivate Cdc25A and induce -tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis. Mutation Research. (2010) 683: 123-130. 4. Musa Khan Dawar, Rizwana Aleem Qureshi, Fareeha Maheen, Amir Muhammad Khan. Comparative study of total Phenolic and Free radical scavenging activities of reported and non reported medicinal plants of Margalla Hills, Islamabad. Pakistan journal of botany (accepted)

169

Plate1. Berberis lycium Royle

Plate 2. Mallotus philippensis (Lam.) Muell. Arg.

Plate 3. Caryopteris grata Benth.

Plate 4. Debregeasia salicifolia (D.Don) Rendle in Prain

Plate 5. Ficus racemosa L.

Plate 6. Dodonaea viscosa (L.) Jacq.

Plate 7. Pinus roxburghii Sargent

Plate 8. Bauhinia variegata L.

Plate 9. Carissa opaca Stapf ex Haines

Plate 10. Dalbergia sissoo Roxb.

Plate 11. Rubus ellipticus Smith

Plate 12. Ficus palmata Forssk.

Plate 13. Olea ferruginea Royle

Plate 14. Adhatoda vasica Nees

Plate 15. Calotropis procera Lin.

Plate 16. Cassia fistula Linn.

Plate 17. Phyllanthus emblica L.

Plate 18. Jasminum humile Linn.

Plate 19. Punica granatum L.

Plate 20. Melia azedarach L.

Plate 21. Lantana camara L.

Plate 22. Pyrus pashia Buch. & Ham.

Plate 23. Albizia lebbeck (L) Benth

Plate 24. Bombax ceiba Linn.

Chapter 6

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