Biological activity & Phytochemical Study of selected ...

BBiioollooggiiccaall aaccttiivviittyy && PPhhyyttoocchheemmiiccaall SSttuuddyy ooff sseelleecctteedd

MMeeddiicciinnaall PPllaannttss

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

BBiioollooggiiccaall aaccttiivviittyy && PPhhyyttoocchheemmiiccaall SSttuuddyy ooff sseelleecctteedd

MMeeddiicciinnaall PPllaannttss

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

i

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

ii

In memory of my dear mother

(July 2008)

iii

ABBREVIATIONS

BuOH Butanol C1I Chk1 Inhibitor C2I Chk2 Inhibitor Cdc Cell division control Cdc25A/B/C Cell-devision-cycle 25A/B/C Cdk Cyclin-dependent-kinases Chk1 Checkpoint-kinase 1 Chk2 Checkpoint-kinase 2 CKI Cyclin dependent kinase inhibitor DPPH 1, 1-diphenyl-2-picrylhydrazyl

EtOAc Ethyl acetate GA Gallic acid HUVEC Human umbilical vein endothelial cells IC50 Concentrations which inhibits by 50 % IpC50 Concentrations which inhibits proliferation by 50 % IR Ionizing radiation p21 Protein 21 p53 Protein 53 PARP Poly (ADP-ribose) polymerase PIC Protease inhibitor cocktail PMSF Phenylmethylsulfonylfluorid RB Retinoblastoma protein ROS Reactive oxygen species SPE Solid phase extraction THF Tetrahydrofurane TNF Tumour necrosis factor UV-light Ultraviolet-Light

iv

Table of Contents

Acknowledgments i

Dedication ii

Abbreviations iii

Summary 1

Introduction 4

1.1 General introduction 4

1.2 Pharmacognosy 5

1.3 Bioassay guided isolation of natural products 5

1.4 Medicinal plants as a source of important drug 6

1.5 Secondary metabolites 10

1.5.1 Small molecules 10

1.5.1.1 Alkaloids 10

1.5.1.1 Alkaloids 10

1.5.1.3 Glycosides 12

1.5.1.4 Phenols 14

1.5.1.5 Phenazines 15

1.5.2 Big “small molecules” 15

2.5.2.1 Polyketides 15

2.5.2.2 Nonribosomal peptides 15

1.6 Technique used in phytochemistry 16

1.6.1 Chromatography 16

1.6.2 Capillary electrophoresis 20

1.6.3 Spectroscopic Techniques 20

1.6.3.1 NMR spectroscopy 20

1.6.3.2 Two-Dimensional Nuclear Magnetic Resonance Spectroscopy

(2DNMR) 20

1.6.3.3 Infrared Spectroscopy 21

1.6.3.4 Fourier transform infrared spectroscopy 21

1.6.3.5 Ultraviolet-visible spectroscopy 22

1.6.4. Liquid chromatography-mass spectrometry 23

1.6.5. Gas chromatography-mass spectrometry (GC-MS) 23

1.7 Development of Anticancer agents from Medicinal plants 23

v

1.8 Development of cancer 24

1.8.1 Self-sufficiency in growth signals 25

1.8.2 Insensitivity to antigrowth signals 26

1.8.3 Evading apoptosis 26

1.8.4 Limitless replicative potential 26

1.8.5 Sustained angiogenesis 26

1.8.6 Tissue invasion and metastasis 27

1.8.7 The cell cycle 28

1.8.7.1 Cell cycle phases (short summary) 29

1.8.7.2 Presence of cyclins and Cdks during single phases 29

1.8.8 Function and activation of (proto)-oncogenes/oncogenes 30

1.8.8.1 Oncogenes 30

1.8.8.2 Cyclin D1 30

1.8.8 3 Cdc25A (Cell-division-cycle 25A) 30

1.8.8 4 Function and activation of tumor suppressor genes 31

1.8.8 5 p53 (protein 53) 31

1.8.8 6 Activation of p53 31

1.8.8 7 P21CIP (protein 21) 31

1.8.8 8 Activation of p21CIP 32

1.8.8.9 RB 32

1.8.8.10 Activation of RB 33

1.8.9 Cell death 33

1.8.9.1 Apoptosis 33

1.8.9.2 Autophagy 35

1.8.9.3 Necroses 35

1.9 Bioassays Techniques 37

1.9.1 Apoptosis assays (Hoechst 33258 propidium iodide (HOPI) double-staining)

37

1.9.2 Western blot assay 37

1.9.2.1 Steps in a western blot 37

1.9.3 Fluorescence Activated Cell Sorting (FACS) assay 40

1.9.3.1 Flow cytometers 41

1.9.3.2 Application 42

1.9.4 Comet assay 42

vi

1.9.4.1 Experimental procedure 42

1.9.4.2 Principals 44

1.9.5 Total Phenolics or Folin-Ciocalteau Micro Method 44

1.9.5.1 Calibration curve 45

1.9.6 Antioxidant activity 46

1.9.6.1 (1, 1-diphenyl-2-picrylhydrazyl) (DPPH) 47

1.10 Selection of Medicinal plants species 48

1.10.1 Berberis lycium Royle (Berberidaceae) 49

1.10.2 Mallotus philippensis (Lam.) Muell. Arg. (Euphorbiaceae) 49

1.10.3 Adhatoda vasica Nees (Acanthaceae) 50

1.10.4 Albizia lebbeck (L.) Benth. (Mimosaceae) 51

1.10.5 Bauhinia variegata Linn. (Caesalpinaceae) 51

1.10.6 Bombax ceiba Linn. (Bombacaceae) 51

1.10.7 Calotropis procera (Willd.) R. Br. 1. c (Asclepiadaceae) 52

1.10.8 Carrisa opaca Staff ex Haines (Apocynaceae) 53

1.10.9 Caryopteris grata Benth. (Verbenaceae) 53

1.10.10 Cassia fistula Linn (Caesalpinaceae) 53

1.10.11 Colebrookea oppositifolia Smith (Labiateae) 54

1.10.12 Debregeasia salicifolia (D.Don) Rendle in Prain (Urticaceae) 54

1.10.13 Dalbergia sissoo Roxb. (Papilionaceae) 55

1.10.14 Dodonaea viscosa (L.) Jacq., Enum. Pl. Carib. (Sapindaceae) 55

1.10.15 Ficus palmata Forssk. (Moraceae) 56

1.10.16 Ficus racemosa L. (Moraceae) 57

1.10.17 Jasminum humile Linn. (Oleaceae) 57

1.10.18 Lantana camara L. (Verbenaceae) 58

1.10.19 Melia azedarach L. (Meliaceae) 58

1.10.20 Olea ferruginea Royle (Oleaceae) 59

1.10.21 Phyllanthus emblica L. (Euphorbiaceae) 59

1.10.22 Pinus roxburghii Sargent (Pinaceae) 60

1.10.23 Pyrus pashia Buch. & Ham. (Rosaceae) 60

1.10.24 Punica granatum L. (Punicaceae) 61

1.10.25 Rubus ellipticus Smith (Rosacceae) 61

1.10.26 Viburnum cotinifolium D. Don (Caprifoliaceae) 62

1.11 Objectives 63

vii

Chapter: 2 Review of Literature 64

2.1 Berberis lycium Royle (Berberidaceae) 64

2.1.1 Ethnobotanical uses 64

2.1.2 Chemical constituents 64

2.1.3 Biological testing 65

2.2 Mallotus philippensis (Lam.) Muell. Arg. (Euphorbiaceae) 69




2.3 Adhatoda vasica Nees in Wall (Acanthaceae) 73




2.4 Albizia lebbeck (L.) Benth. (Mimosaceae) 73




2.5 Bauhinia variegata Linn. (Caesalpinaceae) 74




2.6. Bombax ceiba Linn. (Bombacaceae) 74




2.7 Calotropis procera Linn. (Asclepiadaceae) 75




2.8 Carissa opaca Stapf ex Haines (Apocynaceae) 76



2.9 Cassia fistula Linn. (Caesalpinaceae) 77


viii



2.10 Colebrookea oppositifolia Smith (Labiateae) 77



2.11 Debregeasia salicifolia (D.Don) (Urticaceae) 78




2.12 Dalbergia sissoo Roxb. (Papilionaceae) 78




2.13 Dodonaea viscosa Linn. (Sapindaceae) 79




2.14 Ficus palmata Forssk. (Moraceae) 81



2.15 Ficus racemosa L. (Moraceae) 81




2.17 Lantana camara Linn. (Verbenaceae) 83




2.18 Melia azedarach Linn. (Meliaceae) 84




2.19 Phyllanthus emblica L. (Euphorbiaceae) 86


ix



2.20 Pinus roxburghii Sargent (Pinaceae) 87


2.21 Punica granatum Linn. (Punicaceae) 88




2.22 Rubus ellipticus Smith (Rosaceae) 89




2.23 Viburnum cotinifolium D. Don (Caprifoliaceae) 90



Chapter: 3 Materials & Methods 91

3.1 Reference Compounds 91

3.2 Plant Material 91

3.3 Anti bodies for western blot analyses 93

3.4 Miscellaneous Chemicals and Reagents 94

3.5 Cell culture and bacterial strains 95

3.6 Extraction 95

3.6.1 Extraction for Antioxidant and Total Phenolics Determination 95

3.6.2 Extraction of roots powder 95

3.6.3 Extraction for Flavonoids analyses 96

3.7 Chromatographic Methods 96

3.7.1 Thin Layer Chromatography (TLC) 96

3.7.1.1 Thin Layer Chromatography of Berberis lycium fractions

96

3.7.1.2 Thin Layer Chromatography for Flavonoids analyses 97

3.7.2 High Performance Liquid Chromatography (HPLC) 97

3.7.2.1 General HPLC Parameters 97

3.7.2.2 HPLC Method 97

3.7.2.3 Sample Preparation 98

x

3.7.3 Gas Chromatography and Mass Spectrometer 98

3.8 Biological Testing 99

3.8.1 Antineoplastic Activities 99

3.8.1.1 Anti-proliferation or Growth inhibition assay 99

3.8.1.2 Hoechst dye 33258 and propidium iodide double staining

(Apoptosis Assay) 99

3.8.1.3 Western blotting 99

3.8.1.4 Cell cycle distribution analysis (FACS analyses) 100

3.8.1.5 Single cell gel electrophoresis (SCGE)/Comet assay 101

3.8.1.6 Statistical analyses 102

3.8.2 Total Phenolics determination 102

3.8.3 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) test 102

3.8.4 Antibacterial Determination 103

Chapter. 4 Results and Discussion 104

4.1 Results 104

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. 104

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

4.1.1.4 Effect of BuOH extract, Berberine and Palmatine on cell

cycle distribution 115

4.1.1.5 Induction of apoptosis by extracts of B. lycium and

Berberine 118

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

xi

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. 137

4.1.3.1 Total Phenolics Determination. 137

4.1.3.2 Determination of Free radical scavenging activity 137

4.1.3.3 Flavonoids finger printing of selected Plants 140

4.1.4 Antibacterial and Free radical scavenging activities,

Flavonoids finger printing of Mallotus philippensis. 150

4.1.4.1 Antibacterial activities 150

4.1.4.2 Free radical scavenging activities 150

4.1.4.3 Flavonoids finger printing of Mallotus philippensis. 150

4.2 Discussion 155

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. 157

4.2.3 Total Phenolics, Free radical scavenging activity and Flavonoids finger

printing of selected Medicinal Plants 159

4.2.4 Antibacterial and Free radical scavenging activities, Flavonoids finger

printing of Mallotus Philippensis. 163

Chapter. 5. Conclusion 166

List of Publications 169

Plates 170

Chapter. 6. References 182

List of Figures

Figure 1 Examples of new medicinal plant drugs 9

Figure 2 Acquired capabilities of cancer 25

Figure 3 Cyclin and Cdks distribution during the cell cycle 29

Figure 4 DNA damage induced by UV-light and further the activation of p53 32

xii

Figure 5 Mechanism of Apoptosis 36

Figure 6 Alkaloids of Berberis lycium 65

Figure 7 Compounds of Mallotus philippensis 72

Figure 8 TLC of Berberis lycium extracts 105

Figure 9 RP-HPLC Chromatogram of alkaloids standards 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 110

Figure 14 Alkaloids percentage in Berberis lycium 112

Figure 15 Anti-proliferative effect of B. lycium extracts and its alkaloids 114

Figure 16 Analysis of cell cycle proteins 115

Figure 17 Cell Cycle Distribution of HL-60 cells upon treatment with of BuOH extract

and berberine for 48 h 117

Figure 18 Induction of apoptosis by the B. lycium extracts and berberine 120

Figure 19 Western blot analysis of pro-apoptotic mediators and effectors 121

Figure 20 The genotoxicity of increasing concentrations of BuOH extract and berberine

122

Figure 21 Comet assay 123

Figure 22 Induction of stress response by the BuOH extract and Berberine 125

Figure 23 Anti-proliferative effect of Mallotus phillipensis extracts 127

Figure 24 Induction of apoptosis by the Mallotus phillipensis Hexane fraction 128

Figure 25 Analysis of cell cycle proteins 129

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

Mallotus phillipensis 130

Figure 27 Induction of stress response by Mallotus phillipensis 131

Figure 28 GC/MS chromatogram of hexane soluble fraction of Mallotus phillipensis 136

Figure 29 Gallic acid standard curve 138

Figure 30 Total Phenolics and Extract yield per gram 139

Figure 31 Antioxidant cure of Ascorbic acid 139

Figure 32 Flavonoids finger printing of standard and selected plants 145

Figure 30 Percentage of Flavonoids in Plant samples 146

Figure 34 Types of Flavonoids in each sample 147

xiii

Figure 35 Antibacterial activities of Mallotus philippensis 151

Figure 36 Free radical scavenging activity of Mallotus philippensis 153

Figure 37 Flavonoids finger printing of Mallotus philippensis 154

List of Tables

Table 1 Reference compounds 91

Table 2 Investigated Plants species 92

Table 3 Anti bodies for western blot analyses 93

Table 4 Miscellaneous Chemicals and Reagents 94

Table 5 Parameters for HPLC-PDA analyses of Alkaloids 97

Table 6 Gradient elution systems used for HPLC separations 98

Table 7 Gas Chromatograph and Mass Spectrometer conditions 98

Table 8 10% Polyacrylamide Gel Preparation 101

Table 9 Linearity study of standard curve for standard compounds 111

Table 10 Percent composition of active alkaloids in Berberis lycium 111

Table 11 Comparative total Phenolic, extract yield per gram and IC50 Values 140

Table 12 Appearance of standards under UV 265nm 148

Table 13 Qualitative analyses of plants samples for Flavonoids types 149

Table 14 Antibacterial activities of roots and flower powder extract 152

Summary

1

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 anti-

neoplastic 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 µg extract weight/ml medium,

which corresponded to 250 µg dried root/ml). The IC50s for the EtOAc and H2O extracts

were 16.65 µg/ml and 104.25 µg/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

µM Berberine and 1.51µM Palmatine per 1 mg dried root. Therefore, HL-60 cells were

exposed to the respective concentrations of berberine and palmatine. Berberine showed an

IC50 < 1.87µM after 72 h of incubation, while palmatine had no significant effect up to

4.68 µM. 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 α-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 µg BuOH extract/ml; 1.4 µg 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

Summary

2

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

3

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

4

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


5

technique that directed towards physiologically relevant molecular targets.

Pharmacognosy encapsulates all of the relevant fields into a distinct interdisciplinary

science.

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 structure–activity 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, Alzheimer’s 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


6

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 1981–2002.

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


7

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, I–IV). The drugs are, Arteether (I, or Artemotil®) is an effective anti-

malarial 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


8

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 Alzheimer’s disease and theirfore has

been approved for the treatment of Alzheimer’s 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, 4-

hydroxyphenylpyruvate 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).


9

O

O

OH

OCH2CH3

O

H

I Arteether

O

O

N

OH

II Galatamine

NO2OO

O CF3

III Nitisinone

S

S

O

O

O

OH

H

N+

IV Tiotropium

O

O OO

OH

VII Calanolide A

O

N

OO

HO

HO

OH

HO2C

HO

HH

V M6G or morphine-6-glucuronide

N

O

N

NH2

O

O

HOVII Exatecan

N

N

N

NH

H

OH

CO2CH3

H3CO

H3CO2C

FF

HH

OAc

VI Vinflunine

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.


10

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 animal’s 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


11

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.


12

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 beta-

carotenes.


13

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 α-glycosides or β-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 α-amylase can only hydrolyze α-linkages; others,

like emulsin, can only affect β-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


14

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.


15

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 gram-

positive 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


16

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) Techniques by difference in bed shape.

ii) 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


17

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


18

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


19

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


20

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


21

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 one-

dimensional NMR. It has also known that Jean Jeener first proposed the first two-

dimensional experiment, COSY, in 1971, who was a professor at Université 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 tea-

leaves. It has been understood that a well trained manpower can be used more sparingly,

at a significant cost saving (Luypaert et al., 2003).


22

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


23

(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.


24

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


25

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).


26

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,


27

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 that’s 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 ìm 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


28

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/G1-

phase, 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.


29

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


30

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. Hyper-

phosphorylation 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


31

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 G1-

phase, 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 p53-

dependent 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).


32

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 i s

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 hypo-

phosphorylated 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


33

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


34

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 ced-

4 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


35

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.


36

2. Cell shrinkage, and 2. Cell starts swelling and nucleus condensation theorganellesare damaged.

3. Nucleus fragmentation and 3. Cell lysis, the organelles and building apoptotic bodies. the chromatin are destroyed.

Phagocytosis and no Phagocytosis and inflammation inflammation

Figure 5. Mechanism of Apoptosis


37

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 3-

D 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


38

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


39

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. Two-

dimensional (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").


40

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


41

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.


42

Optical system: commonly used are lamps (mercury, xenon); high-power water-

cooled 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.


43

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 37°C. 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 4°C 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 4°C) 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 DNA-


44

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


45

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 µL into separate cuvettes, and

to each add 1.58 mL water, and then add 100 µL of the Folin-Ciocalteu reagent, and mix

well. Wait for between 30 sec and 8 min, and then add 300 µL of the sodium carbonate

solution, and shake to mix. Leave the solutions at 20°C 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 40°C 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


46

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

C6−C3−C6 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


47

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-2-

picrylhydrazyl (α,α-diphenyl-β-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]

N N

NO2

NO2

O2N..

1. DiPhenylicrylhydrazyle (free radical)

N N

NO2

NO2

O2N

2. Diphenylepicrylhydrazine (nonradical)

H

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


48

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 α-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).


49

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-)10-25-

flowered, 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 obovoid-

subglobose, 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 yellow-

brownish 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


51

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.


52

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


53

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


54

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 crenulate-

serrulate, apex long acuminate, adaxially rugulose and puberulent, abaxially densely-

tomentose to lanate-tomentose. Panicles 10-15 cm, branches 4-7 cm; verticillasters 10-18-

flowered, 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


55

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, 1-4-

seeded. 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


56

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


57

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 ovate-

lanceolate 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: April-

June. 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.


58

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


59

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: August-

November.

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 linear-

oblong, 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,


60

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, linear-

lanceolate, 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, 7–13-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


61

rounded. Stamens slightly shorter than petals. Pome brown, with pale dots, sepals

caducous. Fl. Mar–Apr, fr. Aug–Sep.

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 1–3 m tall. Branchlets purplish brown or brownish, pubescent, with sparse, curved

prickles and dense, purplish brown bristles or glandular hairs. Leaves imparipinnate, 3-

foliolate; petiole 2–6 cm, petiolule of terminal leaflet 2–3 cm, lateral leaflets subsessile,

petiolule and rachis purplish red bristly, pubescent, with minute prickles; stipules linear,


62

7–11 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. Mar–Apr, fr. Apr–May. 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.


63

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.

Chapter 2 Review of Literature

64

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 Küpeli, 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).

N

O

O

+

OCH3

CH3O

I Berberine

N

O

O

+

OCH3

CH3O

II Palmatine


65

MeN NMe

MeOOMe

III Sindamine

OMe

O

O CHO

O

OH

MeN

O

NMe

CHO

MeO

MeO

OMe

OH

IV Chenabine

ON

OOO

N

O

O

H

V Berbamine

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


66

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

berberine’s 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 berberine’s 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


67

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 berberine’s antiarrhythmic effect

is unclear, but an animal study indicated it may be due to suppression of delayed after-

depolarization 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).


68

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 p53-

independent (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 a-

tubulin 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


69

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).


Its many chemical constituent of Mallotus philippensis include β-sitosterol, stigmasterol,

bergenin, and alpha–amyrin. (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:A-

friedoolean-3-en-2-one (Kikuchi and Toyoda, 1967), 2 α-hydroxy-D:A-friedooleanan-3-

one and 3 α-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), anti-

tumor (Arisawa et al., 1990), and human immunodeficiency virus (HIV) reverse

transcriptase inhibitory activities have been described (Nakane et al., 1991) Anthelmintic,


70

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 (5,7-

dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1 ,2-

chromine), 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:A-

friedoolean-3-en-2-one ( IC50 = 292 mol ratio/ 32 pmol/TPA); 3α-hydroxy-D:A-

friedoolean -2-one (IC50 = 288); positive control, curcumin (IC50 = 343); Epstein-


71

Barrvirus early antigen (EBV-EA) activation induced by 12-O-tetradecanoyl phorbol 13-

acetate (TPA) used in the experiment.

O O

O

O

OH

OH

OH

HO OH

Me

Me

Me

Me

I 4'-Hydroxyisorottlerin

O

O

Me

Me

Me

MeMe

OH

OH

OH

HO

O

O

H

H

II Kamalachalcones C

O

O

O

O

O

HO

MeMe

MeMe

MeMe

MeMe

Me

OH

OH

OH

HO

HO

O

O

OO

HO

III Kamalachalcone D

OH OHO HO

OHOH

Me

Me

Me

MeMe

OCO Pr i

IV Mallotophilippen A

OH OHO HO

OHOH

Me

Me

Me

MeMe

OCO

V Mallotophilippen B

Me

O

OOH

HO

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


72

O

O

O

OH

VII 3 -prenylrubranine

O

OOH

HO

VIII Red compound

O O

HO OH

OH

OH

O

O

IX Isorottlerin

Figure 2 Compounds of Mallotus philippensis

2.3 Adhatoda vasica Nees (Acanthaceae)


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).


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.


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


73

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)


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).


The leaves of Albizia lebbeck are good source of saponins (Pal et al., 1995).


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).


74

2.5 Bauhinia variegata Linn. (Caesalpinaceae)


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).


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).


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)


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


75

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.


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.


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 blood-

pressure elevating action in anaesthetized dog (Misra, 1966).

2.7 Calotropis procera Linn. (Asclepiadaceae)


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).


76


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).


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 anti-

inflammatory 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 Ž100%.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)


77


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)


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).


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-5-

hydroxymethyl-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)


78


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)


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)


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-12-

ene,b-sitosterol, stigmasterol, oleanolic acid and lupeol in extracts of Debregeasia

salicifolia


Ahmed et al. (2006) has identified that leaves extracts has IC50 values more than 100

µg/ml of DPPH radical scavenging activity while comparing with Ascorbic acid IC50 =

1.75

2.12 Dalbergia sissoo Roxb. (Papilionaceae)


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


79

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.


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, ß-sitosterol and ß-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)


Hajare et al (2000) reported marked antipyretic and moderate analgesic activities by

Dalbergia sissoo leaves. Ethanolic extract of D. sissoo leaves possesses anti-

inflammatory 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)


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


80

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.


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 Abdel-

Mogib et al (2001) and Ahmad et al (1987).


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


81

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)


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.


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)


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.


82


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).


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).


83

2.17 Lantana camara Linn. (Verbenaceae)


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).


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), α-amyrin , β-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 β-O-senecioyl-oleanolic acid, 22β

hydroxyl-oleanonic acid , 19α-hydroxy ursolic acid and a new triterpenoid 3β-

isovaleroyl-l9α-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,


84

isonuomioside A, calceolarioside E and derhamnosylverbascoside (Taoubi et al., 1997),

martynoside and verbascoside (Syah et al., 1998), theveside (Ford and Bcndall, 1980),


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)


Persian Lilac” is a fast growing tree of the plains and foot-hills, cultivated along road-

sides 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


85

(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).


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, 3-

bis-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).


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 (El-

Lakwah et al., 1995).


86

2.19 Phyllanthus emblica L. (Euphorbiaceae)


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).


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-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-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 A—C

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-b–Dglucose,

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


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


87

(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)


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)


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


88

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).


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.


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.(Meléndez et al., 2006; Mathabe et al., 2007). Jiménez

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 Alzheimer’s diseases (Hartman et al., 2006).


89

2.22 Rubus ellipticus Smith (Rosaceae)


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 squash-

making. 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.


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


90

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)


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)


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)


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).

Chapter 3 Materials & Methods

91

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 Purity factor Company Berberine chloride dihydrate 98.92% Phyto Lab GmbH & Co, Germany Palmatine chloride 96.98% Phyto Lab GmbH & Co, Germany Berbamine dihydrochloride >95% Sigma-Aldrich (Schnelldorf,

Germany) Codeine hydrochloride >99% Heilmittelwerk Wien, Austria Kampferol >99% Roth, Karisruhe, Germany Myricetin >99% Roth, Karisruhe, Germany Catechin >98% Roth, Karisruhe, Germany Vitexin 96.30% Roth, Karisruhe, Germany Orientine 97.70% Roth, Karisruhe, Germany Isoquercetin >99% Roth, Karisruhe, Germany Hyperosid >99% Roth, Karisruhe, Germany isovitexin >99% Roth, Karisruhe, Germany Luteolin-7-glucoside 97.70% Extrasynthese, Geney, France Rutin 96.80% Merk, Darmstadt, Germany Kampferol-7-neohesperidoside

97.30% Roth, Karisruhe, Germany

Quercetin >99% Roth, Karisruhe, Germany Luteolin 98.10% Roth, Karisruhe, Germany Apigenin 93.80% 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 Quaid-

i-Azam University Islamabad. The voucher specimens of the collected plant materials are

deposited at the Herbarium of Pakistan, of the same department.


92

Table 2 Investigated Plants species

S/No Name Family name Locality Parts Acc. No. 1 Berberis lycium Berberidacea Margalla

Hills Roots 125174

2 Mallotus phillipensis Euphorbiaceae Margalla Hills

Roots 125263

3 Rubus ellipticus Rosaceae Margalla Hills

Aerial 125272

4 Bauhinia variegata Caesalpinaceae Margalla Hills

Aerial 125275

5 Caryopteris grata Verbenaceae Margalla Hills

Aerial 125262

6 Colebrookea oppositifolia

Labiateae Margalla Hills

Aerial

7 Phyllanthus emblica. Euphorbiaceae Campus Aerial 1252282

8 Melia azedarach Meliaceae Campus Aerial 125266

9 Ficus racemosa Moraceae Campus Aerial 125270 10 Dodonaea viscosa Sapindaceae Campus Aerial 125284

11 Jasminum humile Oleaceae Margalla Hills

Aerial 22265

12 Albizia lebbeck Mimosaceae Campus Aerial 125311

13 Pinus roxburghii Pinaceae Margalla Hills

Aerial 125274

14 Olea ferruginea Oleaceae Margalla Hills

Aerial 125280

15 Bombax ceiba Bombacaceae Margalla Hills

Aerial 125371

16 Cassia fistula Caesalpinaceae Campus Aerial 125281

17 Lantana camara Verbenaceae Campus Aerial 125264

18 Punica granatum Punicaceae Campus Aerial 125265

19 Pyrus pashia Rosaceae Margalla Hills

Aerial 125370

20 Dalbergia sissoo Papilionaceae Campus Aerial 125277

21 Debregeasia salicifolia Urticaceae Margalla Hills

Aerial 125271

22 Adhadoda vasica Acanthaceae Campus Aerial 125276

23 Carissa opaca Apocynaceae Margalla Hills

Aerial 125279

24 Viburnum cotinifolium Caprifoliaceae Margalla Hills

Aerial 125312

25 Ficus palmata Moraceae Margalla Hills

Aerial 125269

26 Calotropis procera Asclepiadaceae Campus Aerial 125283


93

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 Company Cdc2-p34 (17) Santa Cruz (Santa Cruz, CA, USA) Cdc25A (M-191) Santa Cruz (Santa Cruz, CA, USA) phospho-Cdc25A-(phSer17) Santa Cruz (Santa Cruz, CA, USA) α-tubulin Santa Cruz (Santa Cruz, CA, USA) PARP Santa Cruz (Santa Cruz, CA, USA) β-tubulin Santa Cruz (Santa Cruz, CA, USA) cleaved Caspase-3(Asp17) Signaling (Danvers, MA, USA) pospho-p38-MAPK (Thr180/Tyr182)

Signaling (Danvers, MA, USA)

p38-MAPK Signaling (Danvers, MA, USA) cyclin D1 Signaling (Danvers, MA, USA) p21 Signaling (Danvers, MA, USA) phospho-Cdc2(phTyr15) Signaling (Danvers, MA, USA) Chk2 Signaling (Danvers, MA, USA) phospho-Chk2 (Thr68) Signaling (Danvers, MA, USA) γH2AX (phSer139) Calbiochem, (San Diego, CA, USA) phoshpho-Cdc25A-(phSer177) Abgent (San Diego, CA, USA) Cdc25A phospho Ser17 Santa Cruz (Santa Cruz, CA, USA) Caspase-2 (H-19) Santa Cruz (Santa Cruz, CA, USA) Cdc25A (F-6) Santa Cruz (Santa Cruz, CA, USA) β-Actine Sigma (St. Louis, MO Acetylated α-tubulin Sigma (St. Louis, MO


94

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 Company Uses 1, 1-Diphenyl-2-picrylhydrazyl Sigma-Aldrich (Schnelldorf,

Germany) Free radical

Gallic acid BDH, Poole, England Total phenolics

Ascorbic acid Sigma-Aldrich (Schnelldorf, Germany)

Free radical

Hoechst 33258 HO, Sigma, St Louis, MO, USA Apoptoses

propidium iodide PI, both Sigma, St Louis, MO Apoptoses Trice buffer Sigma-Aldrich (Schnelldorf,

Germany) Western blotting

Triton X-100 Sigma-Aldrich (Schnelldorf, Germany)

Western blotting

Phenyl methyl sulfonyl fluoride (PMSF)

Sigma-Aldrich (Schnelldorf, Germany)

Western blotting

Protease inhibitor cocktail (PIC) Sigma-Aldrich (Schnelldorf, Germany)

Western blotting

Sodium dodecyl sulfate (SDS) Sigma-Aldrich (Schnelldorf, Germany)

Western blotting

Amonium per sulfate (APES) Sigma-Aldrich (Schnelldorf, Germany)

Western blotting

N, N, N', N'-tetra methyle ethylene diamine

Bio-Red laboratories, India Western blotting

Acrylamide/Bis solution Bio-Red laboratories, India Western blotting

RPMI 1640 medium Life Technologies (Paisley, Scotland, UK)

Cell Prolifiration

Heat inactivated fetal calf serum Life Technologies (Paisley, Scotland, UK)

Cell Prolifiration

Glutamine Life Technologies (Paisley, Scotland, UK)

Cell Prolifiration

Pencillin-streptomycin Life Technologies (Paisley, Scotland, UK)

Cell Prolifiration

Agarose Gibco, Paisley, Scotland Comet assay Ethidium bromide Sigma-Aldrich, Munich,

Germany Comet assay

Folin-Ciocalteu’s phenol reagent MERK, Darmastadt Germany Total phenolics

2-Aminoethyle diphenyl borinate Sigma-Aldrich (Germany) Flavonoids detection

Nutreint Broth medium MERK, Darmastadt Germany Antibacterial assay

Nutreint Agar medium MERK, Darmastadt Germany Antibacterial assay


95

3.5 Cell culture and bacterial strains

The ATCC (American Type Culture Collection, Manassas, VA, USA) brand culture HL-

60 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 ºC. 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,


96

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 re-

dissolved 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.


97

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 Shimadzu SCL-10AD VP system controller Degasser Shimadzu DGU-14A Degasser Autosampler Shimadzu SIL-10AD VP Auto Injector Pump Shimadzu SPD 10AD VP Liquid Chromatograph Flow 1.3 ml/min Stationar Phase

Guard Column: Hypersil ODS C18, 5μ, 4 x 4 mm. Column: Hypersil ODS C18, 5μ, 125 x4 mm.

Detector Shimadzu SPD 10AD VP Diode Array Detector

Software 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 1-

heptansulfonate 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)


98

Table 6 Gradient elution systems used for HPLC separations

S/No Time

(min) Flow rate (ml/min)

Eluent A%

Eluent B%

1 0 1.3 75 25 2 12 1.3 30 70 3 13 1.3 10 90 4 15 1.3 10 90 5 17 1.3 75 25 6 25 1.3 75 25

A= buffer, B=Acetonitril

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 μg/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 μm (Agilent J&W DB-

5ms Ultra Inert) Mode Electron ionization (EI) Scan Mode Mass range Scanned 25-800 amu Source tempratue 230 Cº Scan time 0 - 60 min Transfer line temperature 280 Cº Mass data processed soft ware

Agilent Chemstation

GC Agilent 5890N GC system Injection mode Split mode 10:1 Injection temperature 250CºInjection volume 1 μl Carrier gas Helium; Flow rate: 1.5 mL/min Oven temperature 120 Cº - 300 Cº


99

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 µg

BuOH extract /ml and 1.4 µg berberine/ml medium. In another set of experiment HL -60


100

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 ºC, 4 min) and the pellets lysed in 150 µl 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 ºC, 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 ºC 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 (0.2-

0.3 : 1000), at 4 Cº 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), α-tubulin, PARP and β-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 γH2AX

(phSer139) from Calbiochem, (San Diego, CA, USA), and phoshpho-Cdc25A-

(phSer177) from Abgent (San Diego, CA, USA), and against acetylated α-tubulin and β-

actin were from Sigma (St. Louis, MO).

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 µg/ml BuOH extract, 0.7 µg/ml (1.86 µM) berberine, or 0.28µg/ml

(0.75 µM) 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 re-

suspended in 5 ml cold PBS, after centrifugation (600 rpm, 5 min) the culture were again


101

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 Cº 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

Chemical quantity Stalking gel Distilled water 6 mL Tris buffer 0.6 (pH 6.8) 2.52

mL Sodium dodecyl sulfate (SDS) 10% 100 μL Acrylamide/Bis solution 30% 1.32

mL Ammonium per sulfate (APES) 50 μL N, N, N', N'-tetra methyl ethylene diamine 10 μL Seperating gel Tris buffer 1.6 M (pH 8.8) 2.5 mL Distilled water 4.04

mL Sodium dodecyl sulfate (SDS) 10% 100 μL Acrylamide/Bis solution 30% 3.3 mL Ammonium per sulfate (APES) 50 μL N, N, N', N'-tetra methyl ethylene diamine 10 μL

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 μl

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 μL 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 4°C 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 μL ethidium bromide (20

µg/ml, Sigma-Aldrich, Munich, Germany) and the percentage DNA in tail was analyzed


102

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 Dunnett´s 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 Folin–Ciocalteu (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 µL 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 µL into separate

cuvettes from each calibration solution, sample or blank and to each add 1.58 ml distill

water, and then pipet 100 µL of the Folin-Ciocalteu reagent. After mixing the reagent and

sample well, kept for 30 sec and 8 min. Add 300 µL of the sodium carbonate solution,

and shacked well to mix. Left the solutions at 40°C 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


103

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 (1000µl) was

mixed with different concentration of each test compound (2.5-200µg/ml, 500µl) 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 (2.5-15µg/ml,

500µl) 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).

Chapter 4 Results & Discussion

104

4.1 Results


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-EtOAc-

isopropanol-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.


105

Figure 8 TLC of Berberis lycium extracts

1. H2O Fraction (1 mg/ mL) 6 µl

2. Ethyl acetate.fraction (1 mg/ mL) 6µl

3. Ethyl acetate.Fraction (1 mg/ mL) 8 µl,

4. Standard palmatine chloride (0.125 mg/mL) 4µl

5. Standard berberine chloride dihydrate (0.25 mg/mL) 4 µl

6. Standard berbamine dihydrochloride (1 mg/mL) 2µl

7. BuOH Fraction (1mg/mL) 4µl

8. BuOH Fraction


106

0.0 5.0 10.0 15.0 20.0 min

-25

0

25

50

75

100

125

150

mAU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

barA.Press.(Status)280nm,4nm (1.00)

4.48

3/10

7131

8

5.52

4/29

216.

121/

1070

4

6.91

7/19

547.

465/

1398

58.

098/

1403

618.

585/

1053

69.

209/

1068

637

9.65

9/85

0449

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.

9 A 1

2

4

3


107

0.0 5.0 10.0 15.0 20.0 min

-25

0

25

50

75

100

125

mAU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95


4.52

4/97

1034

5.57

0/52

834

6.15

7/12

530 6.

568/

4705

6.97

9/38

797.

558/

3920

7.75

4/27

518.

066/

3488

50

9.33

7/13

6112

9.69

3/93

3662

Minutes

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

extract.

9B


108

0.0 5.0 10.0 15.0 20.0 min

-25

0

25

50

75

100

125

150

mAU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95


4.52

4/10

9024

6

5.55

4/19

3428

6.15

3/23

925 6.

447/

7921

6.56

4/95

206.

983/

1840

17.

209/

6015

7.54

6/49

385

8.06

2/41

5770

9.35

0/15

4142 9.73

4/47

4556

Minutes

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

9C


109

0.0 5.0 10.0 15.0 20.0 min

-25

0

25

50

75

100

125

150

mAU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

barA.Press.(Status)280nm4nm (1.00)

4.50

1/10

9499

0

8.07

7/42

709 8.

350/

5232

8.51

7/26

538.

681/

3630

28.

875/

4745

9.12

8/11

131

9.40

8/10

378

9.78

2/15

2207

Minutes

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

extract

9D


110

200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm

0.0

1.0

2.0

mAU(x100) 9.54

20

9

24

8

30

2

38

0

22

9

26

4

34

7

200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm

0.0

0.5

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1.5

2.0mAU(x100)

9.13

20

2

21

2

24

9

30

2

37

8

34

6

22

6

26

5

20

6

200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm

0.0

1.0

2.0

mAU(x100) 8.05

25

6

30

4

37

927

9

34

4

200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm

0.0

0.5

1.0

1.5mAU(x1,000)

4.54 20

1

26

2

39

7

20

6

28

5

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

3. Berbamine

1. Berberine

2. Palmatine

4. Codeine


111

Table 9 Linearity study of standard curve for standard compounds

Compounds Calibration curve Slope Intercept R2 Range of injection

amount (μg/mL)

Berberine y= 34.59x+16.59 34.59 16.59 0.9994 10-100

Palmatine y=34.24x+44.368 34.24 44.37 0.9974 1-50.00

Berbamine y=3.0872x+22.946 3.0872 22.95 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 Berberine % Palmatine %

BuOH Fraction 18.04 ± 0.01 2.80

Ethyl acetate Fraction 0.54 ± 0.0015 0.04 ± 0.00255

H2O Fraction 2.76 ± 0.026 0.93 ± 0.065


112

Percent composition of active alkaloids

Berberine Palmatine0

5

10

15

20

BuOH Fraction

H2O Fraction

EtOAc Fraction

% a

ge

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 µg 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 µg/ml, corresponding to 0.5–1.5 mg dried root weight/ml medium)

and palmatine (0.28-0.83 µg/ml, corresponding to 0.5–1.5 mg dried root weight/ml


113

medium). The IC50 for berberine was less than 3.7 µM (= 1.4 µg/ml, corresponding to an

equivalent of ~ 1 mg dried roots of B. lycium/ml medium) after 48 h and < 1.9 µM 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

Contro

l17

.535

.046

.6

Contro

l17

.535

.046

.6

Contro

l17

.535

.046

.60

25

50

75

100

*

* *

*

* *

*

* *

extract concentration (µg/ml)

% c

ell

pro

life

rati

on

Treatment with BuOH extract

Contro

l2.8 5.

611

.1

Contro

l2.8 5.

611

.1

Contro

l2.8 5.

611

.10

25

50

75

100

* *

** *

* * **


% c

ell

pro

life

rati

on


114

Treatment with H2O extract

Contro

l69

.510

4.3

139.020

8.5

Contro

l69

.510

4.3

139.020

8.5

Contro

l69

.510

4.3

139.020

8.5

0

25

50

75

100


* ** * *

*

* *

* *

* *% c

ell

pro

life

rati

on

Treatment with Palmatine

Contro

l 0.2

80.

550.8

3

Contro

l 0.2

80.

550.8

3

Contro

l 0.2

80.

550.8

30

25

50

75

100 *

drug concentration (µg/ml)

% c

ell

pro

life

rati

on

Treatment with Berberine

Contro

l0.7 1.

42.1

Contro

l0.7 1.

42.1

Contro

l0.7 1.

42.1

0

25

50

75

100

**

**

*

**

drug concentration (µg/ml)

% c

ell

pro

life

rati

on

Figure 15 Anti-proliferative effect of B. lycium extracts and its alkaloids: (a) EtOAc

extract (17.5, 35.0 and 46.6 µg/ml medium); (b) BuOH extract (2.8, 5.6 and 11.1 µg/ml);

(c) H2O extract, (69.5, 104.3, 139.0 and 208.5 µg/ml); (d) Berberine (0.7, 1.4, and 2.1

µg/ml); and (e) Palmatine (0.28, 0.55 and 0.83 µg/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).


115

Figure 16 Analysis of cell cycle proteins: HL-60 cells (1x106 cells) were seeded into T-

25 tissue culture flasks and allowed to grow for 48 h when cells were incubated with

11.1µg BuOH extract/ml medium (left side panels) and 1.4 µg 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 β-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 µg BuOH extract/ml (corresponding 0.5 mg dried root

/ml), and 0.7 µg berberine/ml (which is contained in 5.5 µg 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.


116

Control

BuOH (5.6 μg /mL)


117

Berberine (0.7 μg/ mL equ.)

Cell cycle distribution afterBuOH extract treatment (48 h)

G0-G1 S G2-M0

10

20

30

40

50 Control5.6 µg*

*

% c

ells

Cell cycle distribution afterBerberine treatment (48 h)

G0-G1 S G2-M0

10

20

30

40

50

60Control0.7 µg

*

*

*

% c

ells

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 µg/ml BuOH extract and 0.7 µg/ml berberine and then subjected to

FACS analysis. Experiments were done in triplicate. Error bars indicate SEM, and

asterisks significance (p<0.05).


118

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 µg/ml) were shown earlier to induce H2AX

phosphorylation (γH2AX) in osteosarcoma cells indicating genotoxicity (Liu et al.,

2009). In the present study we demonstrate that 0.7 and 1.4 µg/ml berberine and the

corresponding concentration of BuOH extract specifically induced apoptosis in HL-60

cells without concomitant induction of γH2AX (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 α-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.


119

1 2

3 4


120

EtOAc extract treatment (48 h)

Contro

l5.9

11

.7

17.5

0

10

20

30

*

*

*


% a

po

pto

tic

ce

lls

BuOH extract treatment (48 h)

Contro

l2.8 5.6 11

.10

10

20

30

*

**


% a

po

pto

tic

ce

lls

H2O extract treatment (48 h)

Control

69.5

139.

220

8.5

0

10

20

30

**

extract concentration (µg/ ml)

% a

po

pto

tic

ce

lls

Berberine treatment (48 h)

Contro

l0.7

1.4

2.1

0

10

20

30

**

*


% a

po

pto

tic

ce

lls

Figure 18 Induction of apoptosis by the Berberis lycium extracts and berberine:

(1) Control (2) BuOH extract 11.1 μg/ml (3) EtOAc extract 17.5 μg/ml (4) H2O extract

208.5 μg /ml. Error bars indicate SEM, asterisks significance (p<0.05).


121

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


122

1 2

3 4

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 µM 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 μL 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


123

NaCl, 10 mM Tris, 100 mM Na2EDTA, pH 10.0) at 4°C 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 μL ethidium bromide (20 µg/ml, Sigma-

Aldrich, 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 1.4µg/ mL; 3. B. lycium (BuOH fraction 11.14µg/ ml) 4.

Positive control (H2O2); Statistical analysis: Dunnett´s test.

C o m e t a s s a y

a fte r c e ll tre a tm e n t fo r 4 8 h

2O2

50 µM

HC

ontrol

2.8

5

.5

11.1

0.4

0.7

1.4

0

5

1 0

1 5

2 0

2 5

3 0 B u O H

B e rb e r in e

c o n c e n tra tio n (µ g /m l)

% t

ail

DN

A

Figure 21 Comet assay: The genotoxicity of increasing concentrations of BuOH extract

and berberine was investigated in logarithmically growing HL-60 cells. 50 µM 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 µg BuOH extract /ml and 1.4 µg berberine/ml medium (Fig. 22). Also Chk2


124

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 α-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.


125

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

Berberine:


126

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 n-

Hexane, 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).


127

Treatment with Hexan extract

Control 1

1.5 2

Control 1

1.5 2

Control 1

1.5 2

0

25

50

75

100

* * *

*

*

*

*

extract concentration (mg/ml)

% c

ell

pro

life

rati

on

Treatment with EtOAc extract

Control 1

1.5 2

Control 1

1.5 2

Control 1

1.5 2

0

25

50

75

100

* *

**


% c

ell

pro

life

rati

on

Treatment with BuOH extract

Control 1

1.5 2

Control 1

1.5 2

Control 1

1.5 2

0

25

50

75

100* * * * * * *


% c

ell

pro

life

rati

on

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).


128

Hexan extract treatment (48 h)

Control 1

1.5

0

10

20

30

*


% a

po

pto

tic

ce

lls

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).

2

C 1


129

Figure 22 Analysis of cell cycle proteins: HL-60 cells (1x106 cells) were seeded into T-

25 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


130

1.5 mg/ml dry roots equivalent /ml medium

Cell cycle distribution afterBuOH extract treatment (48 h)

G0-G1 S G2-M0

10

20

30

40

50 Control1.5 mg/ml

% c

ells

Figure 26 Cell Cycle Distribution of HL-60 cells upon treatment with hexane

Fraction of Mallotus phillipensis:


131

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)


132

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).

H

HH

H

HO

I Lupeol

O

O

Me

Me

Me

MeMe

OH

OH

OH

HO

O

O

H

H

II Kamalachalcones C

H

HH

H

HO

OH

III Betulin


133

(IV)

(V)


134

(VI)

(VII)


135

(VIII)

(IX)


136

(X)

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


137

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 µg/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-2-

picrylhydrazyl (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).


138

Galic acid Curve

y = 0.0004x + 0.046

R2 = 0.9991

0

0.2

0.4

0.6

0.8

0 500 1000 1500 2000 2500

Concentration (mg/l)

Ab

sorb

ance

Figure 29 Gallic acid standard curve


139

R. e

llip

ticu

s.

B. v

arie

gata

C. g

rata

C. o

ppos

itifo

lia

P.

embl

ica.

M. a

zade

reca

. F

. rec

emos

a

D. v

isco

sa

J. h

umil

e

A. l

abbe

ck

P. r

oxbu

rgii

O. f

erru

gine

a

Bom

bex

ceib

a

C. f

istu

la

L. c

amar

a

P. g

rana

tum

P. p

ashi

a D

. sis

so

D. S

alic

ifol

ium

A. V

asic

a

C. o

paca

V. c

otin

ifol

ium

. F

. Pal

mat

a

C. p

roce

ra

0

500

1000

1500

2000

2500(m

g/g)

( extract yeild (mg/g) Gallic acid equavalent (mg/g)

Figure 30 Total Phenolics and Extract yield per gram:

Antioxidant curve of Ascorbic acid

0 5 10 15 20 250

20

40

60

80

100

Concentration in ugIC50= 5.75 ug

% i

nh

ibit

ion

Figure 31 Antioxidant cure of Ascorbic acid


140

Table 11 Comparative total Phenolic, extract yield per gram and IC50 Values.

S/No Plants name Amount of extract/g

Total Phenolics a (mg of GAE/g dw)

IC50

1. Rubus ellipticus 0.137 ± 0.001 1558 2.54 2. Bauhinia variegata 0.13 ± 0.01 1775 3.26 3. Caryopteris grata 0.197 ± 0.001 1450 3.29 4. Colebrookea oppositifolia 0.07 ± 0.01 383 3.36 5. Phyllanthus emblica 0.255 ± 0.001 2160 3.58 6. Melia azedarach 0.21 ± 0.01 2120 7.32 7. Ficus racemosa 0.115 ± 0.001 1775 9.14 8. Dodonaea viscosa 0.19 ± 0.01 1600 10.26 9. Jasminum humile 0.137 ± 0.001 1050 11.32 10. Albizia lebbeck 0.25 ± 0.001 1850 12.1 11. Pinus roxburghii 0.085 ± 0.001 308 15.13 12. Olea ferruginea 0.11 ± 0.01 450 16.26 13. Bombax ceiba 0.09 ± 0.01 725 24.56 14. Cassia fistula 0.21 ± 0.01 1383 25.03 15. Lantana camara 0.185 ± 0.001 1358 30.65 16. Punica granatum. 0.165 ± 0.001 1817 32.38 17. Pyrus pashia. 0.225 ± 0.001 2027 32.50 18. Dalbergia sissoo 0.16 ± 0.01 1025 32.56 19. Debregeasia salicifolia 0.077 ± 0.001 167 33.37 20. Adhadoda vasica 0.117 ± 0.001 426 39.27 21. Carissa opaca 0.15 ± 0.01 800 40.26 22. Viburnum cotinifolium 0.137 ± 0.001 500 41.33 23. Ficus palmata 0.115±0.001 883 64.26 24. Calotropis procera 0.13±0.001 500 125

dw dry weight of the original sample.

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-7-

neohesperidoside, 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


141

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.

O

OO

O

H

H

OH

1F Kaempferol-7-neohesperidoside

O

O O

O

O

O

O

O

O

O

H

HH

H

H

H

O O

OO

O

H

H

OH

H

HO

HO

2F Myricetin

O O

O

O

H

H

H

O

OH

H

3F Catechin

O

OH

OH

O

OHO

O

O

O

OH

H

H

HH

OH

4F Vitexin

OOH

O

OHO

O

O

O

H

H

HH

OH

OH

OH

5F Orientin

O O

OO

O

H

H

O

O

O

O

O

O

H

H

H

H OH

HH

H

H

H

F6 Isoquercitrin


142

O O

OO

O

H

H

H

O

O

O

O

O

O

H

H

H

H OH

7F Hyperoside

O

O

O

OH

O

H

H

HOOO

O

OH

H

HOH

8F Isovitexin

O O

OO

O

H

OH

H

O

O

O

O

O

H

H

H

H

9F Luteolin 7-O-glucoside

O

OO

O

H

H

OH

11F Kaempferol-7-neohesperidoside

O

O O

O

O

O

O

O

O

O

H

HH

H

H

H

O O

OO

H

H

O

O

O

O

O

O

H

H

H

H

H

HO

OO

O

O

O H

H

H

10F Rutin

O O

OO

O

H

H

OH

H

HO

12F Quercetin


143

O O

OO

O

H

H

OH

H

13F Luteolin

O O

OO

O

H

H

H

14F Apigenin

A B

A B

A B


144

A B


145

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.


146

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-7-

neohesperidoside; F12 Quercetin; F13 Luteolin and F14 Apigenin)

PERCENTAGE OF FLAVONOIDS

100

29.16

4.16 0

258.33

54.16

0

29.1616.66

0 012.5

33.33

58.33

020406080

100120

Pheno

lic A

cids F1 F2 F3 F4 F5 F6 F7 F8 F9

F10 F11 F12 F13 F14

Flavonoids

%ag

e

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)


147

R. e

llip

ticu

s.

B. v

arie

gata

C. g

rata

C. o

ppos

itifo

lia

P. e

mbl

ica

M. a

zeda

rach

F. r

acem

osa

D. v

isco

saJ.

hum

ile

A. l

ebbe

ck

P. r

oxbu

rghi

i

O. f

erru

gine

aB

. cei

ba

C. f

istu

la

L. c

amar

a P

. gra

natu

m

P. p

ashi

a .

D.

siss

oo

. D. S

alic

ifoli

aA

. Vas

ica

C. o

paca

V. c

otin

ifol

ium

F. P

alm

ata

C. p

roce

ra

Phenolic Acid

F1

F2

F4

F5

F7

F8

F10

F11

F14

Figure 34. Types of Flavonoids in each sample


148

Table 12 Appearance of standards under UV 265nm

S/No Standard R /time Colour (Reagent A)

Colour (Reagent B)

1 Kaempferol 0.81 light green light green 2 Myricetin 0.73 orange dark green 3 Catechin 0.74 dark green dark brown 4 Vitexin 0.65 light green light green 5 Orientin 0.48 light green light green 6 Isoquercitrin 0.52 orange orange 7 Hyperoside 0.54 orange dark brown 8 Isovitexin 0.57 light green light green 9 Luteolin 7-glucoside 0.57 Fluorescent yellow orange 10 Rutin 0.42 Fluorescent yellow orange 11 Kamferol-7-

neohesperidoside 0.55 light olive green light olive green

12 Quercetin 0.87 yellow orange 13 Luteolin 0.9 Fluorescent yellow fluorescent yellow 14 Apigenin 0.86 light green light green


149

Table 13 Qualitative analyses of plants samples for Flavonoids types

Plant Flavonoids detected

Phenolic acid

F1 F2 F4 F5 F6 F7 F8 F9 F10 F11 F14

R. ellipticus. Five + + - + - - - - - + - +

B. variegata Five + - - - + - - - - - - -

C. grata Two + - - - + - - - - - - -

C. oppositifolia Four + - - - - - - - - - + -

P. emblica Three + + - + - - - - - - - -

M. azedarach Four + - - - - - - + - + - -

F. racemosa Eight + - - + - - - + - + - -

D. viscosa Six + - - - - - - - - + - +

J. humile Five + + - + - - + - - - - -

A. lebbeck Six + - - + - - + - - + - -

P. roxburghii Four + + - - - - - + - - - -

O. ferruginea Three + - - + - - - + - - - +

B. ceiba Three + + - + - - - - - - + -

C. fistula Four + - - + - - - + - - - -

L. camara Five + + - + - - - - - - - -

P. granatum Five + + + + - - - - - - - -

P. pashia Five + - - - + - - + - - - -

D. sissoo Three + - - - + - - + - - - -

D. salicifolia Five + - - - + - - + - - + -

A. vasica Seven + - - - + - - + - - - -

C. opaca Ten + - - + + - - + - + - -

V. cotinifolium Five + - - + - - - + - - - -

F. palmata Seven + + + - - + - + - -

C. procera Five + - - + - - - + - - - -

(+) = 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


150

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-2-

picrylhydrazyl) 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 µg/ml respectively) see Figure 36, Ascorbic acid was used as standard

antioxidant (IC50 = 5.75 µg /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


151

diphenyl borinate solution) and reagent B (5% ethanolic solution of polyethylene glycol-

400) 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.

Bacillus subtilis Staph. aureus Salmonella setubal

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

mg

/ml

kamala extract

roots extract

Figure 35 Antibacterial activities of Mallotus philippensis.


152

Table 14. Antibacterial activities of roots and flower powder extract

Concentration

(mg/ml)

Inhibition zone (mm)

(roots extract)

Inhibition zone (mm)

(flower powder or kamala) B1 B2 B3 B4 B5 B1 B2 B3 B4 B5

15.00

- 24.0 - - 6.0 - 24 20 - -

12.50 - 22.0 - - 5.5 - 22 20 -

10.00 - 21.5 - - 5.0 - 21.5 20 - - 7.50 - 21.5 - - 4.0 - 21.5 18 - - 5.00 - 20.0 - - 4.0 - 20.0 17 - - 3.00 - 19.5 - - 4.0 - 19.5 17 - - 2.00 - 19.0 - - 4.0 - 19.0 15 - - 1.00 - 17.0 - - 0.0 - 17 13 - -

Ciprofloxacine (2 mg/ml)

35.0 39.0 22.0 - 36.0 35.0 39.0 22.0 29.0 36.

DMSO - - - - - - - - - -

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

A

20 40 60 80 100 120 140 160 180 200 2200.0

0.1

0.2

0.3

0.4

0.5

0.6 (Flower powder) (Leaves)

Abs

orba

nce

at (

517

nm)

Concentration (ug/ml)

B


153

20 40 60 80 100 120 140 160 180 200 220

20

30

40

50

60

70

80

90

100Flower powder (Leaves)

% in

hibi

tion

Concentration(ug/ml)

C

Ascorbic acid Leaves Kamala

0

5

10

15

20

25

30

35

40

45

50

µg

/m

l

Figure 36 Free radical scavenging activity of Mallotus philippensis


154

A B

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)


155

4.2 Discussion


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 inhibiting-

and 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 HL-

60 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 µg/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 single-

layers 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 µM

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


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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 µM), 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 α-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


158

(big and small) have been found in the different parts of the plant. Rottlerin (5, 7-

dihydroxy-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 TRAIL-

mediated 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:A-

friedoolean-3-en-2-one ( IC50 = 292 mol ratio/ 32 pmol/TPA); 3α-hydroxy-D:A-

friedoolean -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-O-

tetradecanoyl 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


159

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,




160


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-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). 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 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-L-

rhamno-Dglucoside (Sharma et al., 2001). Dipentadecyl ketone, glycerol 1, 3-bis-undec-

9- 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 1-

cinnamoyl-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.


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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;


162

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.


163

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 3.12-

6.25 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


164

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


165

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.

Chapter 5 Conclusions

166

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 µg/ml) and palmatine (0.3-0.7 µg/ml). The IC50 for berberine was 1.2

µg/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 proto-

oncogene 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 pro-

apoptotic effect by acetylation of α-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 proto-

oncogene 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 anti-

neoplastic effects have been triggered by induction apoptosis through caspase-2

activation. Hexane fraction of M. phillipensis analyzed with GC/MS and it has been


167

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 (HL-

60) 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,




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,


168

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.

List of publications

169

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 21-

23, 2008 at Lahore College for woman University Lahore, Pakistan. p. 174-

181.

2. Musa Khan Dawar, Rizwana Aleem Qureshi. In Vitro Antibacterial and

Antioxidant Activity of Mallotus philippinensis (Lam.) Müll.-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 Hölzl, Sabine Bauer, Katharina Viola,

Wolfgang Mikulits, Rizwana Aleem Quereshi, Siegfried Knasmüller, Michael

Grusch, Brigitte Kopp, Georg Krupitza. Berberine and a Berberis lycium

extract inactivate Cdc25A and induce α-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)

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 References

182

Abbasi, A. M., Khan, M. A., Ahmad, M., Zafar, M., Khan, H., Muhammad, N.,

Sultana, S. Medicinal plants used for the treatment of jaundice and hepatitis

based on socio-economic documentation. AFR J BIOTECHNOL, 8:1643-

1650.

Abdel-Mogib, M., Basaif, S. A., Asiri, A. M., Sobahi, T. R., Batterjee, S. M. 2001.

New clerodane diterpenoid and flavonol-3-methyl ethers from Dodonaea

viscosa. PHARMAZIE, 56:830–1.

Abesundara, K. J. M., Matsui, T., Matsumoto, K. 2004. A-glucosidase inhibitory

activity of some Sri Lanka plant extracts, one of which, Cassia auriculata,

exerts a strong anti hyperglycemic effect in rats comparable to the therapeutic

drug acarbose. J. Agr. Food Chem., 52:2541-2545.

Abraham, Z., Bhakuni, D. S.; Garg, H. S.; Goel, A. K.; Mehrotra, B. N.; Patnaik, G.

K. 1986. Screening of Indian plants for biological activity: Part XII. Indian J.

Exp. Biology, 24:48-68..

Afaq, F. A., Sarfaraz, S. 2005. Pomegranate fruit juice for chemoprevention and

chemotherapy of prostate cancer. Proc. Natl. Acad. Sci. .U S A, 102:14813-

14818.

Ahluwalia, V. K., Seshadri, T. R. 1963. Dalbergenone from the heartwood of

Dalbergia sissoo. Curr. Sci., 32:455.

Ahluwalia, V. K., Sachdev, G. P., Seshadri, T. R. 1965. Chemical components of

immature green pods of Dalbergia sissoo. Indian J. Chem., 3:474.

Ahmad, I., Ahmad, M., Ahmad, A., 1994. Antimicrobial activity of Dodonaea

viscosa oil. Fitoterapia, 65:167-168.

Ahmad, N. S., Farman, M., Najmi, M. H., Mianand, K. B., Hasan, A. 2006. Activity

of polyphenolic plant extracts as scavengers of free radicals and inhibitors of

xanthine oxidase. J. Basic & App. Sci., 2:1.

Ahmad, S. S. 2007. Medicinal wild plants from Lahore-Islamabad motorway (m-2).

PAKISTAN J BOT., 39:355-375.


183

Ahmad, V. U., Fatima, I., Fatima, A. 1987. The sapogenins from Dodonaea viscosa.

Fitoterapia, 58:361-362.

Ahmed, S. A., Siddiqui, S. A., Zaman, A., 1974. Flavones from Colebrookia

oppositifolia. Indian J. Chem., 12:1327-8

Ahmed, Z. F., El-Moghazy Shoaib, A. M., Wassel, G. M., E1-Sayyad, S. M. 1972.

Phytochemical study of Lantana camara, Terpenes and lactones II. PLANTA

MED., 22:34.

Akbar, E., Riaz, M., Malik, A. 2001. Ursene type nortriterpene from Debregeasia

salicifolia. Fitoterapia, 72:382-385.

Akhter, M. H., Sabir, M., Bhide, N. K. 1979. Possible mechanism of antidiarrhoel

effect of berberine. Indian J. Med. Res., 70:233-241.

Akihisa, T., Tokuda, H., Ichiishi, E., Mukainaka, T., Toriumi, M., Ukiya, M. 2001.

Anti-tumor promoting effects of multiflorane-type triterpenoids and cytotoxic

activity of karounidiol against human cancer cell lines. Cancer Lett., 173:9-14.

Albrecht, M., Jiang, W., Kumi-Diaka, J. 2004. Pomegranate extracts potently

suppress proliferation, xenograft growth, and invasion of human prostate

cancer cells. J Med Food, 7:274-283.

Alche, L. E., Barquero, A. A., Sanjuan, N. A., Coto, C. E. 2002. An antiviral principle

present in a purified fraction from Melia azedarach leaf aqueous extract

restrains herpes simplex virus type-1 propagation. Phytotherapy Res., 16:348-

352.

Alche, L. E., Berra, A., Veloso, M. J., Coto, C. E. 2000. Treatment with meliacine, a

plant derived antiviral, prevents the development of herpetic stromal keratitis

in mice. J. Med. virol., 61:474-480.

Alche, L. E., Assad, F. K., Meo, M., Coto, C. E., Maier, M. S. 2003. An antiviral

meliacarpin from leaves of Melia azedarach. zeitschrift-fur-naturforschung-

Section-C,- Biosciences, 58: 215-219.

Al-Dubai, A. S., Al-khulaidi, A. A. 1996.Medicinal and Aromatic Plants of Yemen


184

(In Arabic), Sana’a, Yemen: Obadi Center for studies and Publishing.

Ali, M. N., Khan, A. A. 1978. Pharmacognostic studies of Berberis lycium Royle and

its importance as a source of raw material for the manufacture of berberine in

Pakistan, PAK J. FORE., 26.

Ali, M., Sharma, S. K., 1996. Heterocyclic constituents from Berberis lycium roots.

Ind. J. Hetero. Chemistry, 6:127-130.

Ambastha, S. P., The wealth of India, vol. 2B. Publication and Information

Directorate. New Delhi: CSIR, pp.118.

Amin, A. H., Subbaiah, T. V., Abbasi, K. M., 1969. Berberine sulfate: antimicrobial

activity, bioassay, and mode of action. Can J Microbiol, 15:1067-1076.

Amin, A. H., Mehta, D. R. 1959. A bronchodilator alkaloid (vasicinone) from

Adhatoda vasica Nees. Nature, 184:1317.

Anila, L., Vijayalakshmi, N. R. 2000. Beneficial effects of flavonoids from Sesamum

indicum, Emblica officinalis and Momordica charantia. PHYTOTHER. RES,

14:1-4.

Anonymous. 1950.In: Wealth of India. Raw materials, vol 3. CSIR, New Delhi.

Anonymous, 1992. The wealth of India, Vol-III. Publication and information

Directorate (CSIR), New Delhi, p. 337-343.

Ansari., M. M. 2004. Pharmacognostical and phytochemical studies on some

traditional plant drugs’(PhD Theses), Jamia Hamdardar (Hamdard Nagar)

New Delhi, 110062.

Ansari, M. A., Razdan, R. K., Tandon, M., Vasudevan, P. 2000. Larvicidal and

repellent actions of Dalbergia sissoo Roxb. (F. Leguminosae) oil against

mosquitoes. Bioresource Tech. 73:207-211.

Antarkar, D. S., Ashok, B. V., Doshi, J. C., Athavale, A. V., Vinchoo, K. S., Natekar,

M. R., Thathed, P. S., Ramesh, V., Kale, N. 1980. Doublebind clinical trial of

Arogyawardhani-an ayurvedic drug in acute viral hepatitis. Ind. J. Med.


185

Research, 72:588-593.

Arisawa, M., Fujita, A., Suzuki, R., Hayashi, T., Morita, N., Kawano, N., Koshimura,

S. 1986. Studies on cytotoxic constituents in pericarps of Mallotus japonicus,

Part II. J. Nat. Prod., 49:298-302.

Arisawa, M., Fujita, A., Hayashi, T., Morita, N., Kikuchi, T., Tezuka, Y. Studies on

cytotoxic constituents in pericarps of Mallotus japonicus. IV. 1990 a. Chem.

Pharm. Bull., 38:698-00.

Arisawa, M., Fujita, A., Morita, N., Koshimura, S. 1990 b. Cytotoxic and antitumor

constituents in pericarps of Mallotus japonicus. PLANTA MED., 56:377-3 79.

Arun, K. Yadav, V. T. 2008. Anticestodal activity of Adhatoda vasica extract against

Hymenolepis diminuta infections in rats. J. Ethnopharmaco., 119:322-324.

Asif, A., Kakub. G., Mehmood, S., Khunum, R., Gulfraz, M. 2007. Wound healing

activity of root extracts of Berberis lyceum Royle in rats. Phytother. Res., 21:

589-591

Asmawi, M. Z., Kankaanranta, H., Moilanen, E., Vapaatalo, H. 1993. Anti-

inflammatory activities of Emblica officinalis Gaertn. Leaf extracts. J.

Pharmacy and Pharmaco., 45:581-584.

Attaway, J. A. 1994. Citrus juice flavonoids with anticarcinogenic and antitumor

properties. In Food phytochemicals for cancer prevention I, Fruits and

vegetables; Huang, M. T., Osawa, T., Ho, C. T., Rosen, R. T., Eds.; American

Chemical Society: Washington, DC, pp 240-248.

Babbar, O. P., Chhatwal, V.K., Ray, I. B., Mehra, M. K. 1982. Effect of berberine

chloride eye drops on clinically positive trachoma patients. Indian J. Med.

Res., 76:S83-S82.

Baker, J. T., Borris, R. P., Carte, B., Cordell, G. A., Soejarto, D. D., Cragg, G. M.,

Gupta, M. P., Iwu, M. M., Madulid, D. R., Tyler, V. E. 1995. Natural products

drug discovery and development: new perspectives on international

collaboration. J. Nat. Prod., 58:1325-1357.


186

Bandopandhyay, M., Dhingra, V. K. Mukerjee, S. K., Pardeshi, N. P. 1972.

Triterpenoids and other components of Mallotus philippinencis.

Euphorbiaceae. PHYTOCHEMISTRY., 11:1511.

Banerji, A., Murti, V. V. S., Seshadri, T. R., Thakur, R. S. 1963. Chemical

components of the flowers of Dalbergia sissoo: Isolation of 7-

methyltectorigenin, a new isoflavone. Indian J. Chem., 1:25-27.

Banerji, A., Murti, V. V. S., Seshadri, T. R. 1965. Occurrence of 7, 40-

dimethyltectorigenin in the flowers of Dalbergia sissoo. Curr. Sci., 34:431.

Banerji, A., Murti, V. V. S., Seshadri, T. R. 1966. Isolation of sissotrin, a new

isoflavone glycoside from the leaves of Dalbergia sissoo. Indian J. Chem.,

4:70-72.

Barnes, P. J. 2002. New treatments for COPD. Nat. Rev. Drug Dis., 1:437-446.

Barnes, P. J., Belvisi, M. G., Mak, J. C., Haddad, E. B., O’Connor, B. 1995.

Tiotropium bromide (Ba 679 BR), a novel long-acting muscarinic antagonist

for the treatment of obstructive airways disease. Life Sci., 56:853-859

Barry, V. C., Conalty, M. L., Rylance, H. J., Smith, F. R.1955. Antitubercular effect

of an extract of Adhatoda vasica. Nature, 176:119-20.

Barton, D. H. R., De Mayo, P., Warnhoff, E. W., Jeger, O., Perold, G. W. 1954.

Triterpenoids. Part XIX. The constitution of lantadene B. J. Chem. Soci.,

3689-3692.

Barua, A. K., Chakrabarti, P., Chowdhury, M. K., Basak, A., Basu K. 1976. The

structure and stereochemistry of lantanilic acid, the β,β-dimethylacryloyl ester

of lantaninilic acid, isolated from Lantana camara. PHYTOCHEMISTRY,

15:987-989.

Barua, A.K., Chakrabarti, P., Chowdhury, M. K., Barak, A., Basu, K., Ray, S., Saha,

S. K. 1985. The structure and stereochemistry of lantanilic acid. J. Ind.

Chem. Soc., 62:298-305.

Barua, A. K., Chakrabarti, P., Sanyal, P. K., Das, B. 1969. Triterpenoids. XXXII.


187

Structure of lantic acid: a new triterpene from Lantana camara. J. Indian

Chem. Soc., 46: 100-102.

Baslas, R. K., Agha, R. 1985. Isolation of a hypoglycemic principle from the bark of

Ficus glomerata Roxb. Him. Chem. Pharma. Bull., 2:13.

Basu, A., Chaudhuri, A.K. 1991. Preliminary studies on the anti-inflammatory and

analgesic activities of Calotropis procera root extract. J. Ethnopharmacol.,

31:319.

Basu, A., T. Sen., Ray, R. N., Nag Chaudhuri, A. K. 1992. Hepatoprotective effects of

Calotropis procera root extract on experimental liver damage in animals.


Baud, L., Ardaillou, R. 1986. Reactive oxygen species: Production and role in the

kidney. AM. J. PHYSIOL., 251:1765.

Beckett, A., Belthle, F., Fell, K., Lock, M. 1954. The active constituents of raspberry

leaves. J. Pharm. Pharmacol., 6:785-796.

Begum, S., Raza, S.M., Siddiqui, B.S., Siddiqui, S. 1995. Triterpenoids from the

aerial parts of Lantana camara. J. Nat. Prod., 58:1570-1574.

Begum, S., Wahab, A., Siddiqui, B. S., Qamar, F. 2000. Nematicidal Constituents of

the Aerial Parts of Lantana camara. J. Nat. Prod., 63:765-767.

Begum, S., Wahab, A., Siddiqui, B. S. 2003. Pentacyclic Triterpenoids from the

Aerial Parts of Lantana camara. Chem. Pharm. Bull., 51:134-137.

Begum, S., Zehra, S. Q., Siddiqui, B. S., Wahab, A. 2006. Triterpenoidal Secondary

Metabolites from Lantana Camara L.. Helv. Chim. Acta, 89:1932-1941.

Begum, S., Qamar, Z. S., Siddiqui, B. S., 2008. Two New Pentacyclic Triterpenoids

from Lantana camara LINN. Chem. Pharm. Bull., 56:1317-1320.

Benencia. F., Courreges. M..C., Coto, C., and Coulombie, F.C. 1997.

Immunomodulatory activities of Melia azedarach L. leaf extracts on human

monocytes. J. Herb Spices and Med. Plants. 5(3):7-13.


188

Benencia, F., Courreges, M. C., Coulombie, F. C., Massouh, E. J. 1992. Effect of

Melia azedarach fresh leaf aqueous extract on mice hematological parameters.


Bhakta, T., Mukherjee, P. K., Mukherjee, K., Banerjee, S., Mandal, S. C., Maity, T.

K., Pal, M., Saha, B. P. 1999. Evaluation of hepatoprotective activity of

Cassia fistula leaf extract. J. Ethnopharmacol., 66:277-82.

Bhakta, T., Banerjee, S., Mandal, S. C., Maity, T. K., Saha, B. P., Pal, M. 2001.

Hepatoprotective activity of Cassia fistula leaf extract. Phytomedicine. 8:220-

224.

Bhakumi, D. S., Dhar, M. L., Dhar, M. M., Dhawan, B. N., Gupta, B., Srimal, R. C.,

1971. Indian J. Exp. Biology., 2:91.

Bhakuni, R. S., Shukla, Y. N. Thakur, R. S.1987. Chemical examination of the roots

of Rubus ellipticus. Indian Drugs, 24:272.

Bhaskara, R. R., Murugesan, T., Pal, M., Saha, B. P., Mandal, S.C. 2003. Antitussive

potential of methanol extractof stem bark of Ficus racemosa L. PHYTOTHER.

RES, 17:1117-1118.

Bhattarai, N. K. 1992. Medical ethnobotany in the Karnali Zone, Nepal. Eco.Botany,

46:257-261.

Birdsall, T. C., Kelly, G. S. 1997. Berberine: Therapeutic potential of an alkaloid

found in several medicinal plants. Altern. Med. Rev., 2:94-103.

Biswas, S., Talukder, G., Sharma, A. 1999. Protection against cytotoxic effects of

arsenic by dietary supplementation with crude extract of Emblica officinalis

fruit. PHYTOTHER. RES, 13:513-516.

Blois, M.S. 1958. Antioxidant determinations by the use of a stable free radical,

Nature, 81:1199-1200.

Bonassi, S., Fenech, M., Lando, C. 2001. Human MicroNucleus Project: International

database comparison for results with the cytokinesis- block micronucleus

assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria


189

and host factors on the frequency of micronuclei. Env Mol Mutagenesis,

37:31.

Bonfil, R. D., Russo, D. M., Binda, M. M., Delgado, F. M., Vincenti, M. 2002. Higher

antitumor activity of vinflunine than vinorelbine against an orthotopic murine

model of transitional cell carcinoma of the bladder. Uro. Onco., 7:159-166.

Borkowski, B., Lutomski, J., Skrzydlewska, E., Zygmunt, B.1994. Rosliny lecznicze

w fitoterapii, IRiPZ, oznan pp. 470-471.

Bracke, M. E., Bruyneel, E. A., Vermeulen, S. J., Vennekens, K. I., Marck, V. V.,

Mareel, M. M. 1994. Citrus favonoid effect on tumor invasion and metastasis.

Food Technol., 48:121-124.

Brodie, C., Blumberg, P. M. 2003. Regulation of cell apoptosis by protein kinase c

delta. Apoptosis, 8:19-27.

Bruhn, J. G., Bohlin, L. 1997. Molecular pharmacognosy: an explanatory model.

Drug Disc. Today, 2:243-246.

Buckheit Jr, R. W., White, E. L., Fliakas-Boltz, V., Russell, J., Stup, T. L., Kinjerski,

T. L., Osterling, M. C., Weigand, A., Bader, J. P. 1999. Unique anti-human

immunodeficiency virus activities of the nonnucleoside reverse transcriptase

inhibitors calanolide A, costatolide, and dihydrocostatolide. ANTIMICROB

AGENTS CH., 43:1827-1834.

Burke, M. D., Berger, E. M., Schreiber, S. L. 2004. A synthesis strategy yielding

skeletally diverse small molecules combinatorially. J. Amer. Chem. Soc.,

126:14095-14104.

Burn, J., Camb, M., Withell, E. 1941. A principle in raspberry leaves which relaxes

uterine muscle. Lancet., 6149-6151.

Burn, J. H. and Withell, E. R. 1941. A principle in raspberry leaves which relaxes

uterine muscle. Lancet., 5:1-3.

Burnette, W. N. 1981. “Western blotting’: electrophoretic transfer of proteins from

sodium dodecyl sulfate - polyacrylamide gels to unmodified nitrocellulose and


190

radiographic detection with antibody and radioiodinated protein A”.

Analytical Biochemistry (United States: Academic Press) 112:195-203.

Butler, M. S. 2004. The role of natural product chemistry in drug discovery. J. Nat.

Prod., 67:2141-2153.

Caceres, A., Giron, L.M., Alvarado, S.R., Torres, M. F. 1987. Screening of

antimicrobial activity of plants popularly used in Guatemala for treatment of

dermatomucosal diseases. J Ethnopharmacol., 20:223-237.

Cardellina II, J. H. 2002. Challenges and opportunities confronting the botanical

dietary supplement industry. J. Nat. Prod., 65:1073-1084.

Carpinella, M. C., Herrero, G. G., Alonso, R. A. and S. M. Palacios. 1999. Antifungal

activity of Melia azedarach fruit extract. Fitoterapia., 70:296-298.

Cerda, B., Espin, J. C., Parra, S. 2004. The potent in vitro antioxidant ellagitannins

from pomegranate juice are metabolised into bioavailable but poor antioxidant

hydroxy-6H-dibenzopyran-6-one derivatives by the colonic microflora of

healthy humans. Eur. J. Nutr., 43:205-220.

Chandrashekher, C. H., Latha, K. P., Vagdevi, H. M., Vaidya, V. P. 2008.

Anthelmintic activity of the crude extracts of Ficus racemosa. Int. J. Green

Pharmacy, 2:100-103.

Chawla, H. M., Chibber, S. S. 1981. Chemistry of Dalbergia Species. J. Sci. Ind. Res,

40:313.

Chhabra, S. C., Mahunnah, R. L. A., Mshiu, E. N. 1991. Plants used in traditional

medicine in Eastern Tanzania. V. Angiosperms (Passifloraceae to

Sapindaceae). J. Ethnopharmacol., 33:143-157.

Chidambara Murthy, K. N., Jayaprakasha, G. K., Singh, R. P. 2002. Studies on

antioxidant activity of pomegranate (Punica granatum) peel extract using in

vivo models. J Agric Food Chem., 50:4791-4795.

Chintawar, S. D., Somani, R. S., Veena, S., Kasture, S.B. 2002. KastureNootropic

activity of Albizzia lebbeck in mice. J. Ethnopharmacol., 81:299-305


191

Choedon, T., Mathan, G., Arya, S., Kumar, V.L., Kumar, V. 2006. Anticancer and

cytotoxic properties of the latex of Calotropis procera in a transgenic mouse

model of hepatocellular carcinoma. World J. Gastroenterol., 12:2517-2522.

Chopra, R. N., Chopra, I. C., Handa, K. L., Kapoor, L. D. 1988. Indignenous drugs of

India. Kolkata: UN Dhur and Sons. (1958), pp. 503.

Chopra, R. N., Nayar, S. L., Chopra, I. C.1956 a. Glossary of Indian Medicinal Plants

(CSIRPublication, New Delhi, p. 74.

Chopra, R. N., Nayer, S. L., Chopra, I. C. 1956 b. Glossary of Indian Medicinal

Plants, New Delhi, CSIR, 90-91.

Choudhary, D. N., Singh, J. N. Verma, S. K., Singh, B. P. 1990. Antifertility effects

of leaf extracts of some plants in male rats. Indian J. Exp. Biol., 28:714-716.

Choudhry, V. P., Sabir, M., Bhide, V. N. 1972. Berberine in giardiasis. Indian

Pediatrics, 9:143-146.

Christina, A. J. M., Haja Najumadeen, N. A., Vimal kumar, S., Manikandan, N.,

Tobin, G. C., Venkataraman, S., Murugesh, N. 2006. Antilithiatic effect of

Melia azedarach on ethylene glycol induced nephrolithiasis in rats. PHARM.

BIOL., 44:480-485.

Chun, Y. T., Yip, T. T., Lau, K. L., Kong, Y. C. 1978. A biochemical study on the

hypotensive effect of berberine in rats. Gen. Pharmac., 10:177-182.

Cichewicz, R. H., Kouzi, S. A. 2004. Chemistry, biological activity, and

chemotherapeutic potential of betulinic acid for the prevention and treatment

of cancer and HIV infection. Med. Res. Rev., 24:90-114.

Ckless, K., Schlottfeldt, J. L., Pasqual, M. 1995. Inhibition of in-vitro lymphocyte

transformation by the isoquinoline alkaloid berberine. J. Pharm. Pharmacol.,

47:1029-1031.

Clardy, J., Walsh, C. 2004. Lessons from natural molecules. Nature, 432:829-837.

Coppen, J. J. W., Hone, G. A. 1995. Gum naval stores: turpentine and rosin from pine


192

resin. FAO, Non-wood Forest Products No. 2, 62 pp.

Cragg, G. M., Newman, D. J. 2004. A tale of two tumor targets: topoisomerase I and

tubulin. The Wall and Wani contribution to cancer chemotherapy. J.

Nat.Prod., 67:232-244.

Creagh, T., Ruckle, J. L., Tolbert, D. T., Giltner, J., Eiznhamer, D. A., Dutta, B.,

Flavin, M. T., Xu, Z. Q. 2001. Safety and pharmacokinetics of single doses of

(+)-calanolide A, a novel, naturally occurring nonnucleoside reverse

transcriptase inhibitor, in healthy, human immunodeficiency virus-negative

human subjects. Anti. Agen. Chemo., 45:1379-1386.

Creasey, W.A. 1979. Biochemical effects of berberine. Biochem. Pharmacol.

28:1081-1084.

Currens, M. J., Gulakowski, R. J., Mariner, J. M., Moran, R. A., Buckheit Jr., R. W.,

Gustafson, K. R., McMahon, J. B., Boyd, M. R., 1996. Antiviral activity and

mechanism of action of calanolide A against the human immunodeficiency

virus type-1. J. Pharm. Exp.Therap., 279:645-651.

Cutler, R. G. 1984. Antioxidants, aging, and longevity. In Free Radicals in Biology;

Pryor, W. A., Ed.; Academic Press: New York, pp. 371-428.

Czygan, F. Ch., 1995. Die Himbeere-Rubus idaeus L. Portrait einer Arzneipflanze. Z.

PHYTOTHER. RES, 16:366-374.

Daikonya, A., Katsuki, S., Wu, J.-B., Kitanaka, S. 2002. Anti-allergic Activity of

New Phloroglucinol Derivatives from Mallotus philippensis

(Euphorbiaceae).Chem. Pharm. Bull., 50:1566-1659.

Das, C., Poi, R., Chowdhury, A. 2005. HPTLC determination of vasicine and

vasicinone in Adhatoda vasica. PHYTOCHEM. ANALYSIS, 16, 90-92.

De, S., Ravishankar, B., Bhavsar, G. C. 1993. Plants with hepatoprotective activity-a

review. Ind. Drugs, 30, 355-363.

Deranjyagala, S. A., Wijesundera, R. L. C., Weerasna, O. V. D. S. J. 1988. Antifungal

activity of Ficus racemosa leaf extract and isolation of the active compound.


193

J. Nation. Sci. Council, 26, 19-26.

Dewick, P. M. 2002. Medicinal Natural Products: a Biosynthetic Approach, 2nd ed.

John Wiley and Sons, Chichester, England.

Dhiman, A. K. 2003. Sacred Plants and their Medicinal Uses, (Daya publishing

House, Delhi, pp. 125-127.

Dhingra, V. K., Seshadri, T. R., Mukerjee, S. K. 1974. Isotectorigenin from the bark

of Dalbergia sissoo. Indian J. Chem., 12:1118.

Dhir, H., Roy, R. K., Sharma, A., Talukdar, G. 1990. Modification of clastogenicity

of lead and aluminium in mouse bone marrow cells by dietary ingestion of

Phyllanthus emblica fruit extract. Mut. Research, 241:305-312.

Dorsch, W., Wagner, H. 1991. New antiasthmatic drugs from traditional medicine.

Int. Arch. Allergy App. Immunol., 94:262-5.

Dubey, V. K., Jagannadham, M. V. 2003. Procerain, a stable cysteine protease from

the latex of Calotropis procera. PHYTOCHEMISTRY., 62:1057-1071.

Dugas, A. J., Castaeda-Acosta, J., Bonin, G. C. 2000. Evaluation of the total peroxyl

radical-scavenging capacity of flavonoids: Structure–activity relationships. J.

Nat. Prod., 63:327.

Dutta, B. K., Rahman, I., Das, T. K. 1998. Antifungal activity of Indian plant extracts.

Mycoses, 41:535-536.

Dutta, S., Ghatak, K. L., Ganguly, S. N. 1997. Isolation and Structure Elucidation of

New Pentacyclic Triterpene Acid from the Leaves of Rubus ellipticus. Nat.

Pro. Sciences, 32:108-110.

Eisenbrand, G., Hippe, F., Jakobs, S., Muehlbeyer, S. 2004. Molecular mechanisms of

indirubin and its derivatives: novel anticancer molecules with their origin in

traditional Chinese phytomedicine. J. Can. Res. Clin.Onco., 130:627-635.

El-Dagwy, A. A. 1996. Encyclopedia for Production of Aromatic and Medicinal

Plants, Vol. 1. Imprimerie Atlas, Cairo, pp. 192.


194

Eldridge, G. R., Vervoort, H. C., Lee, C. M., Cremin, P. A., Williams, C. T., Hart, S.

M., Goering, M. G., O’Neil-Johnson, M., Zeng, L. 2002. High-throughput

method for the production and analysis of large natural product libraries for

drug discovery. Anay.Chem. 74:3963-3971.

El-Lakwah, F. A., Mohamed, R., Darwish, A. A. 1995. Evaluation of toxic effect of

chinaberry. Annals of Agri. Sci., 33:389-398.

Esposito Avella, M., Diaz, A., de Gracia, I., de Tello, R., Gupta, M. P. 1991.

Evaluation of traditional medicine: effects of Cajanus cajan L. and of Cassia

fistula L. on carbohydrate metabolism in mice. Rev. Med. Panama., 16:39-45.

Fair, J. D.; Kormos, C. M. 2008. Flash column chromatograms estimated from thin-

layer chromatography data. J. Chromatogr., 1211:49-54.

Farag, S. F., Ahmed, A. S., Terashima, K., Takaya, Y., Niwa, M. 2001. Isoflavonoid

glycosides from Dalbergia sissoo. PHYTOCHEMISRY., 57:1263-1268.

Feher, M., Schmidt, J. M. 2003. Property distributions: differences between drugs,

natural products, and molecules from combinatorial chemistry. J. Chem. Infor.

Comp. Scie., 43:218-227.

Fenech, M. 2005. The Genome Health Clinic and Genome Health Nutrigenomics

concepts: Diagnosis and nutritional treatment of genome and epigenome

damage on an individual basis. Mutagenesis, 20:255.

Ferdous, M., Rouf, R., Shilpi, J. A., Uddin, S. J. 2008. Antinociceptive activity of the

ethanolic extract of Ficus racemosa Lin. (Moraceae). Ori. Pharm. Exp. Med.,

8:93-96.

Ford, C. W., Bcndall, M. R. 1980. Identification of the iridoid glucoside theveside in

Lantana camara (Verbenaceae) and determination of its structure and

stereochemistry by means of NMR. Aust. J. Chem., 33:509-518.

Forestieri, A. M., Monforrte, M. T., Ragusa, S., Trovata, A., Lauk, L. 1996. Anti-

inflammatory, analgesic and antipyretic activity in rodents of plant extracts

used in African medicine. PHYTOTHER. RES, 10:100-106.


195

Frankel, E. N.; Kanner, J.; German, J. B.; Parks, E.; Kinsella, J. E. 1993. Inhibition of

oxidation of human low-density lipoprotein by phenolic substances in red

wine. Lancet, 341:454-457.

Frantz, S. 2005. 2004. Approvals: the demise of the blockbuster? Nat. Rev. Drug Dis.,

4:93-94.

Frantz, S., Smith, A. 2003. New drug approvals for 2002. Nat. Rev. Drug Dis., 2:95-

96.

Fujita, A., Hayashi, T., Arisawa, M., Shimizu, M., Morita, N., Kikuchi, T., Tezuka, Y.

1988. Studies on cytotoxic constituents in pericarps of Mallotus japonicus,

part III. J. Nat. Prod., 51:708-712.

Fukuda, K., Hibiya, Y., Mutoh, M. 1999. Inhibition of activator protein 1 activity by

berberine in human hepatoma cells. PLANTA MED., 65:381-383.

Fukuda, K., Hibiya, Y., Mutoh, M. 1999. Inhibition by berberine of cyclooxygenase-2

transcriptional activity in human colon cancer cells. J. Ethnopharmacol.,

66:227-233.

Fukuda, Y., Sakai, K., Matsunaga, S., Tokuda, H., Tanaka, R. 2005. Cancer

chemopreventive activity of lupane- and oleanane-type triterpenoids from the

cones of Liquidamber styraciflua. Chem Biodiv., 2:421-8.

Furusawa, M., Ido, Y., Tanaka, T., Ito, T., Nakaya, K., Ibrahim, I. 2005. Novel,

complex flavonoids from Mallotus philippensis (Kamala tree). Helv Chim

Acta; 88:1048-58

Gajmer, T., Singh, R., Saini, R. K., Kalidhar, S. B. 2002. Effect of methanolic extracts

of neem (Azadirachta indica A. Juss) and bakain (Melia azedarach L.) seeds

on oviposition and egg hatching of Earias vittella (Fab.) (Lepidoptera:

Noctuidae). J. Appl. Entomol., 126:238-243.

Ganesan, A., 2004. Natural products as a hunting ground for combinatorial chemistry.

Current Opin. in Biotech., 15:584-590.


196

Gatto, B., Sanders, M. M., Yu, C., Wu, H. Y., Makhey, D., LaVoie, E. J., Liu, L. F.

1996. Identification of topoisomerase I as the cytotoxic target of the

protoberberine alkaloid coralyne. Cancer Res, 56:2795-2800.

Getie, M., Gebre-Mariam, T., Rietz, R., Hohne, C., Huschka, C., Schmidtke, M. 2003.

Evaluation of the anti-microbial and anti-inflammatory activities of the

medicinal plants Dodonaea viscosa, Rumex nervosus and Rumex abyssinicus.


Getie, M., Gebre-Mariam, T., Rietz, R., Neubert, R. H. 2002. Evaluation of the

release profiles of flavonoids from topical formulations of the crude extract of

the leaves of Dodonea viscosa (Sapindaceae). PHARMAZIE, 57:320-2.

Geysen, H. M., Schoenen, F., Wagner, D., Wagner, R. 2003. Combinatorial

compound libraries for drug discovery: an ongoing challenge. Nat. Rev. Drug.

Dis., 2:222-230.

Ghani, A. 1998. Medicinal Plants of Bangladesh.Asiatic Society of Bangladesh,

Dhaka.

Ghisalberti, E. L., 2000. Lantana camara L. (Verbenaceae). Fitoterapia, 71(5):467-486.

Ghosh, A. K., Bhattacharyya, F. K., Ghosh, D. K. 1985. Leismania donovani:

amastigote inhibition and mode of action of berberine. Exp. Parasitol, 60:404-

413.

Gil, M. I., Tomas-Barberan, F. A., Hess-Pierce, B. 2000. Antioxidant activity of

pomegranate juice and its relationship with phenolic composition and

processing. J. Agric. Food Chem., 48:4581-4589.

Giuseppe, V., Alessandra, P., Marzia, M., Paola, C., Vittoria, F., Michela, M.,

Emanuela, M. 2009. Rottlerin: a multifaced regulator of keratinocyte cell

cycle. Exp. Dermatology, 18:516-521.

Godbole, S. H., Pendse, G. S. 1960. Antibacterial property of some plants. Indian J.

Pharm., 22:39-42.

Gopal, H. 1972. Chemical constituents of Salmalia malabarica Schott and endl.


197

Flowers. J. Pharm. Sci., 61:807-808.

Grange, J. M., Snell, N. J. 1996. Activity of bromohexine and ambroxiol,

semisynthetic derivatives of vaccine from the Indian shrub Adhatoda vasica,

against Mycobacterium tuberculosis in vitro. J. Ethnopharmacol., 50:49-53.

Graul, A. I. 2001. The year’s new drugs. Drug News and Perspectives, 14:12-31.

Greenwald, P. 2002. Cancer chemoprevention. B. Med. Journal, 324:714-718.

Grusch, M., Polgar, D., Gfatter, S., Leuhuber, K., Huettenbrenner, S., Leisser, C.

2002. Maintenance of ATP favours apoptosis over necrosis triggered by

benzamide riboside. Cell Death Differ., 9:169-178.

Gudej, J., 2003. Kaempferol and quercetin glycosides from Rubus idaeus L. leaves.

Acta Polon. Pharm., 60:313-316.

Gudej, J., Rychlinska, I. 1996. Flavonoid compounds from the leaves of Rubus idaeus

L. Herba Pol., 42:257-261.

Gudej, J., Tomczyk, M., Urban, E., Tomczykowa, M., 1998.Analysis of chemical

composition of Rubus saxatilis L. leaves. Herba Pol., 44:340-344.

Gulfraz, M., Mehmood, S., Ahmad, A., Fatima, N., Parveen, Z., Williamson. 2008.

Comparison of the Antidiabetic Activity of Berberis lycium Root Extract and

Berberine in Alloxan-induced Diabetic Rats. PHYTOTHER. RES., 22:1208-

1212.

Guo, C., Wei, J., Yang, J. 2008. Pomegranate juice is potentially better than apple

juice in improving antioxidant function in elderly subjects. Nutr. Res., 28:72-

77.

Guo, R., Wei, P., Liu, W. 2007. Combined antioxidant effect of rutin and vitamin C in

Triton X-100 micelles. J. Pharm. Biochem. Anal., 43:1580.

Gupta, A. K., Vidyapati, T. J., Chauhan, J. S. 1979. 5,7-Dihydroxyflavanone-4′-O-α-

L-rhamnopyranosyl-β-D-glucopyranoside from Bauhinia variegata. Ind. J.

Chem., 18:85-86.


198

Gupta, A. K., Chauhan, J. S. 1984. Constituents from the stem of Bauhinia variegata.

Nat. Acad. Sci. Lett., 7:15-16.

Gupta, A. K., Vidyapati, T. J., Chauhan, J. S. 1980. Chemical examination of the stem

of Bauhinia variegata. PLANTA MED., 38:174-176.

Gupta, C. M., Bhaduri, A. P., Khanna, N. M., 1971. Biologically active quinazolones

and related compounds. J. Sci. Ind. Res., 30:101-106.

Gupta, K. C., Chopra, I. C. 1954. Anti-tubercular action of Adhatoda vasica (N.O.

acanthacea). Indian J. Med. Res., 42:355-8.

Hajare, S. W., Chandra, S., Tandan, S. K., Sarma, J., Lal, J., Telang, A. G. 2000.

Analgesic and antipyretic activities of Dalbergia sissoo leaves. Ind. J.

Pharmacology, 32:357-360.

Hajare, S. W., Chandra, S., Sharma, J., Tandan, S.K., Lal, J., Telang, A.G. 2001.

Anti-inflammatory activity of Dalbergia sissoo leaves. Fitoterapia, 72:131-

139.

Hall, D. G., Manku, S., Wang, F. 2001a. Solution and solid-phase strategies for the

design, synthesis, and screening of libraries based on natural product

templates: a comprehensive survey. J. Comb. Chem., 3:125-150.

Hall, M. G., Wilks, M. F., Provan, W. M., Eksborg, S., Lumholtz, B. 2001b.

Pharmacokinetics and pharmacodynamics of NTBC (2-(2-nitro-4-fluoro-

methylbenzoyl)-1, 3-cyclohexanedione) and mesotrione, inhibitors of 4-

hydroxyphenyl pyruvate dioxygenase (HPPD) following a single dose to

healthy male volunteers. B. J. Clin. Pharm., 52:169-177.

Halliwell, B., Aeschbach, R., Loliger, L. 1995. The characterization of antioxidants.

Food Chem. Toxicol., 33:601.

Hamayun, M., Khan, S. A., Iqbal, I., Rehman, G., Hayat, T., Khan, M. A. 2005.

Ethnobotanical Profile of Utror and Gabral Valleys, District Swat, Pakistan.

Eth. Leaflets.

Harsh, A. L., Nag, T. N. 1988 Flavonoids with antimicrobial activities of arid zone


199

plants. Geobios. India, 15:32-35.

Hartman, R. E., Shah, A., Fagan, A. M. 2006. Pomegranate juice decreases amyloid

load and improves behavior in a mouse model of Alzheimer’s disease.

Neurobiol. Dis., 24:506-515.

Hedberg, I., Hedberg, O., Madati, P. J., Mshigeni, K., Mshiu, E. N., Samuelsson, G. J.

1983. Inventory of plants used in traditional medicine in Tanzania. Part III.

Plants of the families Papilionaceae–Vitaceae. Ethnopharmacol., 9:237-260.

Hedrick, U. P. 1972. Sturtevant’s Edible Plants of the World. Dover Publications

ISBN 0-486-20459-6

Heinrich, M., Teoh, H. L. 2004. Galanthamine from snowdrop the development of a

modern drug against Alzheimer’s disease from local Caucasian knowledge. J.

Ethnopharmaco., 92:147-162.

Hertog, M. G.; Hollman, P. C. H.; Katan, M. B.; Kromhout, D. 1993. Dietary

antioxidant flavonoids and risk of coronary heart disease. Lancet., 342:1007-

1011.

Ho, L. H., Taylor, R., Dorstyn, L., Cakouros, D., Bouillet, P., Kumar, S. 2009. A

tumor suppressor functions for caspase-2. Proc Natl Acad Sci USA 106:5336-

5341.

Hoessel, R., Leclerc, S., Endicott, J. A., Nobel, M. E., Lawrie, A., Tunnah, P., Leost,

M., Damiens, E., Marie, D., Marko, D., Niederberger, E., Tang, W.,

Eisenbrand, G., Meijer, L. 1999. Indirubin, the active constituent of a Chinese

antileukaemia medicine, inhibits cyclin-dependent kinases. Nat.Cell. Bio., 1:

60-67.

Hooker, J.D., 1882. Flora of British India. Reeve and Co. London, 3:640.

Huang, C. G., Chu. Z. L., Yang, Z. M. 1991. Effects of berberine on synthesis of

platelet TXA2 and plasma PGI2 in rabbits. Chung Kuo Yao Li Hsueh Pao,

12:526-528.

Hussain, Z., Waheed, A., Qureshi, R. A., Burdi, D. K., Verspohl, E. J., Khan, N.,


200

Hasan, M. 2004. The effect of medicinal plants of Islamabad and Murree

region of Pakistan on insulin secretion from INS-1 cells. PHYTOTHER RES.,

18:73-77.

Hwang, B. Y., Lee, J. H., Koo, T. H., Kim, H. S., Hong, Y. S., Ro, J. S., Lee, K. S.,

Lee, J. J. 2001. Kaurane diterpenes from Isodon japonicus inhibit nitric oxide

and prostaglandin E2 production and NF-nB activation in LPS-stimulated

macrophage RAW264.7 cells. PLANTA MED., 67:406-410.

Ihmels, H., K. Faulhaber, D., Vedaldi, F., Dall’Acqua, G. V. 2005. Intercalation of

organic dye molecules into double-stranded DNA. Part 2: The annelated

quinolizinium ion as a structural motif in DNA intercalators. Photochem.

Photobiol., 81:1107-1115.

Iizuka, N., Miyamoto, K. K., Okita, A., Tangoku, H., Hayashi, S., Yosino, T., Abe,

T., Morioka, S., Oka, H. M. 2000. Inhibitory effect of Coptidis Rhizoma and

berberine on the proliferation of human esophageal cancer cell lines. Cancer

Lett., 148:19-25.

Ikawa, M., Schaper, T. D., Dollard, C. A., Sasner, J. J. 2003. Utilization of Folin-

Ciocalteu phenol reagent for the detection of certain nitrogen compounds. J.

Agric. Food Chem. 51:1811-5.

Ikram, M., Haq, M. E., Warsi, S. A. 1966. Alkaloids of Berberis lycium. PAKISTA. J.

SCI. IND. R., 9:343-6.

Ishikura, N., Watanabe, Y., Teramoto, S., 1989. The formation of flavonoids in cell

suspension cultures of Prunus yedoensis Matsum. Botanical Magazine, Tokyo

102:547-560.

Itokawa, H., Susan, L., Natschke, M., Akiyama, T., Lee, K. H. 2008. Plant-derived

natural product research aimed at new drug discovery. J. Nat. Med., 62:263-

280.

Itokawa, H., Qiao, Z. S. 1995. Cytotoic limonoids and tetranortriterpinoids from

Melia azedarach. Chem. Pharm. Bull., 43:1171-1175.


201

Jabbar, A., Raza, M. A., Iqbal, Z., Khan, M. N. 2006. An Inventory of the

Ethnobotanicals used as Anthelmintics in the Southern Punjab (Pakistan). J.

Ethnopharm., 108:152-154.

Jacob, A., Pandey, M., Kapoor, S., Saroja, R. 1988. Effect of the Indian gooseberry

(amla) on serum cholesterol levels in men aged 35–55 years. Eur. J. Clin.

Nutrition, 42:939-944.

Jagadish, V. K., Rana, A. C. 2002. Preliminary study on antifertility activity of

Calotropis procera roots in female rats. Fitoterapia., 73:111-115.

Jahan, I. A., Nahar, N.,. Mosihuzzaman, M., Rokeya, B., Alic, L., Khan, A.K.,

Makhmur, T. Choudhary, M. I. 2009. Hypoglycaemic and antioxidant

activities of Ficus racemosa Linn. fruits. Nat. Prod. Research., 23:399-408.

Jantova, S., Cipak, L. M. 2003. Effect of berberine on proliferation, cell cycle and

apoptosis in HeLa and L1210 cells. J. Pharm. Pharmacol., 55:1143-1149.

Jantova, S., Letasiova, S., Brezova, V., Cipak, L., Labaj, J. 2006. Photochemical and

phototoxic activity of berberine on murine fibroblast NIH-3T3 and Ehrlich

ascites carcinoma cells. J. Photochem. Photobiol., 85:163-176.

Jeena, K. J., Kuttan, G., Kuttan, R. 2001. Antitumour activity of Emblica officinalis.

J. Ethnopharmacolo., 75:65-69.

Jiang, P., Burczynski, F., Campbell, C. 2007. Rutin and flavonois contents in three

buckwheat species Fagoprym esculeentum, F. Tataricum, F. homotroicum,

and their protective effects against lipid peroxidation. Food Res Inter., 40:356.

Jiménez Misas, C. A., Rojas Hernández, N. M., López Abraham, A. M. 1979.

Biological evaluation of Cuban plants. IV Rev Cubana Med Trop., 31:29-35.

Johns, S. R., Lamberton, J. A., Morton, T. C., Suares, H., Willing, R. I. 1983.

Triterpenes of Lantana-tiliaefolia - 24-hydroxy-3-oxours-12-en-28-oic acid, a

new triterpene. Aust. J. Chem., 36(12):2537-2547.

Kaneda, Y., Torii, M., Tanaka, T., Aikawa, M. 1991. In vitro effects of berberine

sulfate on the growth and structure of Entamoeba histolytica, Giardia lamblia,


202

and Trichomonas vaginalis. Ann Trop Med Parasitol., 85:417-425.

Kaneda, Y., Tanaka, T., Saw, T. 1990. Effects of berberine, a plant alkaloid, on the

growth of anaerobic protozoa in axenic culture. Tokai. J. Exp. Clin. Med.,

15:417-423.

Karnick, C. R. 1994. Pharmacopoeial standards of herbal plants. Vol. 1, 1st Ed.,

Satguru Publication, Delhi, India. pp: 51.

Karthikeyan, A., Shanthi, V., Nagasathaya, A. 2009. Preliminary phytochemical and

antibacterial screening of crude extract of the leaf of Adhatoda vasica L. Int J

Green Pharm., 3:78-80.

Kashman, Y., Gustafson, K. R., Fuller, R. W., Cardellina II, J. H., McMahon, J. B.,

Currens, M. J., Buckheit Jr., R. W., Hughes, S. H., Cragg, G. M., Boyd, M. R.

1992. The calanolides, a novel HIV-inhibitory class of coumarin derivatives

from the tropical rainforest tree, Calophyllum lanigerum. J. Med. Chem.,

35:2735-2743.

Kasture, V. S. Chopde, C. T. Deshmukh, V. K. 2000. Anticonvulsive activity of

Albizzia lebbeck, Hibiscus rosa sinesis and Butea monosperma in

experimental animals. J. Ethnopharmaco., 71:65-75.

Katiyar, S. K., Meeran, S. M., Katiyar, N., Akhtar, S. 2008. p53 cooperates berberine

induced growth inhibition and apoptosis of non-small cell human lung cancer

cells in vitro and tumor xenograft growth in vivo. Mol. Carcinog., 48:24-37.

Kelloff, G. J., Crowell, J. A., Steele, V. E., Lubet, R. A., Malone, W. A., Boone, C.

W., Kopelovich, L., Hawk, E. T., Lieberman, R., Lawrence, J. A., Ali, I.,

Viner, J. L., Sigman, C. C. 2000. Progress in cancer chemoprevention:

development of diet-derived chemopreventive agents. J. of Nutrition,

130:467S-471S.

Keshri, G., Lakshmi, V., Singh, M. M. 2003. Pregnancy interceptive activity of Melia

azedarach Linn. In adult female Sprague-Dawley rats. Contaception, 68:303-

306.


203

Keshri, G., Bajpai, M., Lakshmi, V., Setty, B. S., Gupta, G. 2004. Role of energy

metabolism in the pregnancy interceptive action of Ferula assafoetida and

Melia azedarach extracts in rat. Contraception, 70:429-432.

Khalil, N. M., Sperotto, J. S., Manfron, M P. 2006. Antiinflammatory activity and

acute toxicity of Dodonaea viscosa. Fitoterapia, 77:478-480.

Khan, M. S. Y., Shamshad, A., Jain, P. C. 1988. Chemical investigation of root bark

of Dodonaea viscosa Linn. J. Nat. Prod., 4:2-13.

Khan, M., Sultana, S. 2005. Chemomodulatory effect of Ficus racemosa extract

against hemically induced renal carcinogenesis and oxidative damage response

in wistar rats. Life Sci.; 77:1194-210.

Khan, M., Kihara, R. M., Omoloso, A. D. 2001. Antimicrobial activity of Horsfieldia

helwigii and Melia azedarach. Fitoterapia, 72:423-427.

Khare, C. P. Encyclopedia of Indian Medicinal Plants, (Springer, Germany) pp.305-

306.

Khosla, P. K., Neeraj, V. I., Gupta, S. K., Satpathy, G. 1992. Berberine, a potential

drug for trachoma. Rev Int Trach Pathol Ocul Trop Subtrop Sante Publique,

69:147-165.

Kikuchi, T., Toyoda, T. 1967. Isolation and structure determination of pachysandiol-

A and a note on the stereochemistry of cerin. Tetrahedron Lett, 33:3181-5.

Kim, E.H., Kim, S. U., Choi, K. S. 2005. Rottlerin sensitizes glioma cells to TRAIL-

induced apoptosis by inhibition of Cdc2 and the subsequent downregulation of

survivin and XIAP. Oncogene, 24:838-849.

Kinghorn, A. D. 2001. Pharmacognosy in the 21st century. J. Pharm. Pharmaco,

53:135-148.

Kinghorn, A. D., Su, B. N., Jang, D. S., Chang, L. C., Lee, D., Gu, J. Q., Carcache-

Blanco, E. J., Pawlus, A. D., Lee, S. K., Park, E. J., Cuendet, M., Gills, J. J.,

Bhat, K., Park, H. S., Mata-Greenwood, E., Song, L. L., Jang, M., Pezzuto, J.

M. 2004. Natural inhibitors of carcinogenesis. PLANTA MED., 70:691-705.


204

Kinjo, J., Furusawa, J., Baba, J., Takeshita, T., Yamasaki, M., Nohara, T.1987.

Studies on the constituents of Pueraria lobata. III. Isoflavonoids and related

compounds in the roots and the voluble stems. Chem. Pharma. Bulletin,

35:4846-4850.

Kirtikar, K. R., Basu, B. D. 1933 a. Indian medicinal plants. Allahabad: LM Basu

Publication, pp. 2422.

Kirtikar, K. R., Basu, B. D. 1935. Indian Medicinal Plants, Lolit Mohan Basu,

Allahabad, India, pp:1606.

Kirtikar, K. R., Basu B. A. 1991. Indian Medicinal plants. Vol-II, 2nd edi,periodical

experts book agency, NewDelhi, pp.856-860.

Kirtikar, K. R, Basu, B. D. 1933 b. Indian Medicinal Plants 2nd ed. Vol. 1 Lalit Mohan

Basu, 49-Leader Road, Allahabad, pp. 818-9.

Kirtikar, K. R., Basu, B. D. 1995. Indian Medicinal Plants,Vol. I, International Book

Distributors, Dehradun, India. pp. 641-643.

Kirtikar, K. R., Basu, B. D. 1918. “Indian Medicinal Plants,” ed. by Basu S. N.,

Panini Office, Bhuwaneswari Asrama, Bahadurganj, Allahabad, India, pp.

984.

Kirtikar, K. R., Basu, B. D. 1935. Terminalia chebula. In: Kirtikar KR, Basu BD (eds)

Indian medicinal plants, vol 1, 2nd edn. Lalit Mohan Basu Publications,

Allahabad, pp 1020-102.

Kishore, N., Chopra, A. K., Khan ,O. 1997. Antimicrobial properties of Calotropis

procera Ait. in different seasons. A study in vitro. Biol Mem., 23:53.

Kobayashi, Y., Yamashita, Y., Fujii, N., Takaboshi, K., Kawakami, T., Kawamura,

M., Mizukami, T., Nakano, H. 1995. Inhibitors of DNA topoisomerase I and II

isolated from the Coptis rhizomes. PLANTA MED., 61:414-418.

Koehn, F. E., Carter, G. T. 2005. The evolving role of natural products in drug

discovery. Nat. Rev. Drug Dis., 4:206-220.


205

Kramer, R., Cohen, D. 2004. Functional genomics to new drug targets. Nat.Rev.Drug

Dis., 3:965-972.

Krey, A. K., Hahn, F. E. 1977. Berberine: complex with DNA, Science

166(1969)755–757.M.W. Davidson, I. Lopp, S. Alexander, W.D. Wilson, The

interaction of plant alkaloids with DNA. II. Berberinium chloride. Nucleic

Acids Res., 4:2697-2712.

Krzaczek, T., 1984. Phenolic acids in some tannin drugs of the Rosaceae family.

Farm. Pol., 40:475-477.

Kumar, A., Ram, J., Samarth, R. M., Kumar, M. 2005. Modulatory influence of

Adhatoda vasica Nees leaf extract against gamma irradiation in Swiss albino

mice. PHYTOMEDICINE., 12:285-93.

Kumar, S., Dewan, S., Sangraula, H., Kumar, V.L. 2001. Anti-diarrhoeal activity of

the latex of Calotropis procera. J. Ethnopharmaco., 76:115-118.

Kumar, V. P., Chauhan, N. S., Padh, H., Rajani, M. 2006. Non-timber Forest Products

of Nepal. J. Ethnopharmaco., 107:182-188.

Kumar, V. L., Arya, S. 2006. Medicinal uses and pharmacological properties of

Calotropis procera. In: J.N. Govil, Editor, Recent Progress in Medicinal

Plants 11, Studium Press, Houston, Texas, USA, pp: 373-388.

Kumar, V. L., Basu, N. 1994. Anti-inflammatory activity of the latex of Calotropis

procera. J. Ethnopharmaco., 44:123-125.

Kuo, C. L., Chou, C. C., Yung, B. Y. 1995. Berberine complexes with DNA in the

berberine induced apoptosis in human leukemic HL-60 cells. Cancer Lett.,

93:193-200.

Kuo, Y. H., Lee, P. H., Wein, Y. S. 2002. Four new compounds from the seeds of

Cassia fistula. J. Nat. Prod., 65:1165-1167.

La Casa, C., Villegas, C., de la Lastra, A. 2000. Evidence for protective and

antioxidant properties of rutin, a natural flavone, against ethanol induced

gastric lesions. J ethnoparmacol., 71:45.


206

Labreque, G.C., 1983. Botanicals and their derivatives in vector control. In:

Whitehead, D.L., Bowers, U.S. (Eds.), Natural Products for Innovative Pest

Management. Pergamon, New York, pp. 451.

Lad, V., Frawley, D. 1986. The Yoga of Herbs. Santa Fe, NM: Lotus Press, 135-136.

Lahiri, P. K., Pradhan, S. N. 1964. Pharmacological investigation of vasicinol an

alkaloid from Adhatoda vasica Nees. Ind. J. Exp. Biol., 2:219-223.

Lampronti, I., Khan, M. T. H., Bianchi, N., Borgatti, M., Gambar, R. 2004. Inhibitory

effects of medicinal plant extracts on interactions between DNA and

transcription factors involved in inflammation. Minerva Biotecno., 16:93-99.

Lanner, R. M. 1981. The piñon pine, a natural and cultural history. University of

Nevada Press, pp 208.

Lansky, E. P., Newman, R. A. 2007. Punica granatum (pomegranate) and its potential

for prevention and treatment of inflammation and cancer. J. Ethnopharmaco.,

109:177-206.

Lansky, E. P., Jiang, W., Mo, H. 2005a. Possible synergistic prostate cancer

suppression by anatomically discrete pomegranate fractions. Invest. New

Drugs, 23:11-20.

Lansky, E. P, Harrison, G., Froom, P., Jiang, W. G. 2005b. Pomegranate (Punica

granatum) pure chemicals show possible synergistic inhibition of human PC-3

prostate cancer cell invasion across Matrigel. Invest. New Drugs, 23:121-122.

Lateef, M., Iqbal, Z., Khan, M.N., Akhtar, M.S., Jabbar, A. 2003. Anthelmintic

activity of Adhatoda vasica roots. Int. J. Agric. Biol., 5:86-90.

Lau, W.S. 1999. Infrared characterization for microelectronics. World Scientific.

Lee, D., Bhat, K. P. L., Fong, H. H. S., Farnsworth, N. R., Pezzuto, J. M., Kinghorn,

A. D. 2001. Aromatase inhibitors from Broussonetiapapyrifera. J. Nat. Prod.,

64:1286-1293.

Lee, J. H., Koo, T. H., Hwang, B. Y., Lee, J. J. 2002. Kaurane diterpene,


207

kamebakaurin, inhibits NF-kappa B by directly targeting the DNA-binding

activity of p50 and blocks the expression of antiapoptotic NF-kappa B target

genes. J. Bio. Chem., 277:18411-18420.

Lee, J. -H., Kim, S. -D., Lee, J. -Y., Kim, K. -N., Kim, H. -S. 2005. Analysis of

flavonoids in concentrated pomegranate extracts by HPLC with diode array

detection. F. Sci. Biotech.,. 14:171-174.

Leet, J. E., Hussain, S. F., Minard, R. D., Sharma, M. 1982. Sindamine. Punjabine and

gilgitine: three new secobisbenzylisoquinoline alkaloids. Heterocycles,

19:2355-60.

Leet, J. E, Elango, V., Hussain, S. F., Maurice, S. 1983. Chenabine and Jhelumine:

secobisbenzyleisoquinolines or simple isoquinoline benzylisoquinoline

dimmers. Heterocycles, 20:426-9.

Lei, F., Zhang, X. N., Wang, W., Xing, D. M., Xie, W. D., Su, H., Du, L. J. 2007.

Evidence of anti-obesity effects of the pomegranate leaf extract in high-fat diet

induced obese mice. Int. J. Obes., 31:1023-9.

Letasiova, S., Jantova, S., Cipak, L., Muckova, M. 2006. Berberine-antiproliferative

activity in vitro and induction of apoptosis/necrosis ofthe U937 and B16 cells.

Cancer Lett., 239:254-262.

Letasiova, S., Jantova, S., Miko, M., Ovadekova, R., Horvathova, M. 2006. Effect of

berberine on proliferation, biosynthesis of macromolecules, cell cycle and

induction of intercalation with DNA, dsDNA damage and apoptosis in Ehrlich

ascites carcinoma cells. J. Pharm. Pharmacol., 58:263-270.

Ley, S. V., Baxendale, I. R. 2002. New tools and concepts for modern organic

synthesis. Nat. Rev. Drug Dis., 1 (8):573-586.

Li, R. W., Leach, D. N., Myers, S. P., Lin, G. D., Leach, G. J., Waterman, P, G. 2004.

A new anti-inflammatory glucoside from Ficus racemosa L. Planta Med.,

70:421-6

Li, R. W., Leach, D. N., Myers, S. P., Lin, G. J., Waterman, P. G. 2004. A new anti-


208

inflamatory glucoside from Ficus racemosa L. Planta Med., 421-6.

Li, T.,K., Bathory, E., LaVoie, E. J., Srinivasan, A. R., Olson, W. K., Sauers, R. R.,

Liu, L. F., Pilch, D. S. 2000. Human topoisomerase I poisoning by

protoberberines: potential roles for both drug–DNA and drug-enzyme

interactions. Biochem., 39:7107-7116.

Li, X. K., Motwani, M., Tong, W., Bornmann, W., Schwartz, G. K., Huanglian, A.

2000. Chinese herbal extract, inhibits cell growth by suppressing the

expression of cyclin B1 and inhibiting CDC2 kinase activity in human cancer

cells. Mol. Pharmacol., 58:1287-1293.

Li, Y., Sun, X., LaMont, J. T., Pardee, A. B., Li, C. J. 2003. Selective killing of

cancer cells by beta-lapachone: direct checkpoint activation as a strategy

against cancer. Proceedings of the National Academy of Sciences of the

United States of America, 100:2674-2678.

Lim, J. H., Park, J. W., Choi, K. S., Park, Y. B., Kwon, T. K. 2009. Rottlerin induces

apoptosis via death receptor 5 (DR5) upregulation through CHOP-dependent

and PKC d-independent mechanism in human malignant tumor cells.

Carcinogenesis, 5:729-736.

Lin, J. G., Chung, J. G., Wu, L. T. 1999. Effects of berberine on arylamine N-

acetyltransferase activity in human colon tumor cells. Am. J. Chin. Med.,

27:265-275.

Lin, J. S., Yang, J. T., Chen, S., Fan, F. S., Yu, J. L., Yang, C. C., Lu, M. C., Kao, A.

C., Huang, H. F., Lu, J. G. 2007. Berberine induces apoptosis in human HSC-

3 oral cancer cells via simultaneous activation of the death receptor-mediated

and mitochondrial pathway. Anticancer Res., 27:3371-3378.

Lin, J. P., Yang, J.S., Lee, J. H., Hsieh, W. T., Chung, J. G. 2006. Berberine induces

cell cycle arrest and apoptosis in human gastric carcinoma SNU-5 cell line.

World J. Gastroenterol., 12:21-28.

Lin, S.Y., Lin, Chung, J. G., Lin, J. P., Chen, G. W., Kao, S. T., 2006. Down-

regulation of cyclin B1 and up-regulation of Wee1 by berberine promotes


209

entry of leukemia cells into the G2/M-phase ofthe cell cycle. Anticancer Res.,

26:1097-1104.

Liu, Z., Liu, Q., Xu, B., Wu, J., Guo, C., Zhu, F., Yang, Q., Gao, G., Gong, Y., Shao,

C. 2009. Berberine induces p53-dependent cell cycle arrest and apoptosis of

human osteosarcoma cells by inflicting DNA damage. Mutat Res., 9:75-83.

Loken, M. R. 1990. Immunofluorescence Techniques in Flow Cytometry and Sorting

(2nd ed.). Wiley. pp. 341-53.

Lombardino, J. G., Lowe III, J. A. 2004. The role of the medicinal chemist in drug

discovery then and now. Nat. Rev. Drug Dis., 3:853-862.

Lotsch, J., Geisslinger, G. 2001. Morphine-6-glucuronide: an analgesic of the future?

Clin. Pharmacokinetics, 40:485-499.

Lounasmaa, M., Widen, C. J., Tuff, C. M., Huhtikangas, A. 1975. On the

phloroglucinol derivatives of Mallotus philippinensis. PLANTA MED., 28:16-

31.

Louw, P. G. J. 1943. Lantanin, the active principal of Lantana camara L. Part I.

Isolation and preliminary results on the determination of its

constitution.Onderstepoort. J. Vet. Sci. Animal Ind., 18:197-202.

Luximon-Ramma, A., Bahorun, T., Soobrattee, M. A., Aruoma, O. I. 2002.

Antioxidant activities of phenolic, proanthocyanidin, and flavonoid

components in extracts of Cassia fistula. J Agric Food Chem., 50:5042-5047.

Luypaert, J.; Zhang, M. H.; Massart, D. L. 2003. Feasibility study for the use of near

infrared spectroscopy in the qualitative and quantitative analysis of green tea,

Camellia sinensis (L.). Analytica Chimica Acta, 478(2), Elsevier, pp. 303-312.

Mahadevan, N., Venkatesh, S., Suresh, B, 1998. Anti-inflammatory activity of

Dodonaea viscosa. Ancient. Sci. Life, 18:152-156.

Mahathir M., 2002. “Compendium of Medicinal Plants used in Malaysia,” Vol. 2,

Herbal Medicine Research Centre, Institute for Medical Research, Jalan

Pahang, Kuala Lumpur, pp. 85.


210

Mahla M., Singh, R., Suhag, P., Bharti, Kalidhar, S.B. 2002. Biological efficacy of

Melia azedarach roots against Earias vittella larve. J. Med. Aromatic Plant

Sci., 24:726-728.

Malik, A., Mukhtar, H. 2006. Prostate cancer prevention through pomegranate fruit.

Cell Cycle, 5:371-373.

Manach, C., Morand, C., Demigne, C. 1997. Bioavailability of rutin and quercetin in

rats. FEBS Lett., 409:12.

Mandal, S. C., Maity, T., Das, J., Shaha, B. P., & Pal, M. 1998. Antihepatotoxic

activity of Ficus racemosa against paracetamol-induced acute liver damage in

rats. Nat. Prod. Sci., 4:174-179.

Mandal, S. C., Maity, T. K., Das, J., Saba, B. P., Pal, M. 2000. Anti-inflammatory

evaluation of Ficus racemosa Linn. leaf extract. J. Ethnopharmacol., 72:87-

92.

Mandal, S. C., Maity, T. K., Das, J., Pal, M., Saha, B. P. 1999. Hepatoprotective

activity of Ficus racemosa leaf extract on liver damage caused by carbon

tetrachloride in rats. PHYTOTHER RES., 13:430-2

Mandal, S. C., Maity, T. K., Das, J., Pal, M., Saba, B. P. 1999. Hepatoprotective

activity of Ficus racemosa L. leaf extract. PHYTOTHER. RES., 13:430-432.

Mantena, S. K., Sharma, S.D., Katiyar, S. K. 2006. Berberine, a natural product,

induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in

human prostate carcinoma cells. Mol. Cancer Ther., 5:296-308.

Mantena, S. K., Sharma, S. D., Katiyar, S. K. 2006. Berberine inhibits growth,

induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells

by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane

potential and cleavage of caspase 3 and PARP. Carcinogenesis, 27:2018-2027.

Marczal, G. 1963. Qualitative studies of the tannin content of Rubus idaeus leaf.

Herb. Hung., 2:347-357.

Marin-Neto, J. A., Maciel, B. C., Secches, A. L., Gallo, L. 1988. Cardiovascular


211

effects of berberine in patients with severe congestive heart failure. Clin.

cardiology, 11:253-260.

Martin, G. E; Zekter, A. S. 1988. Two-Dimensional NMR Methods for Establishing

Molecular Connectivity; VCH Publishers, Inc: New York, pp.59.

Mascolo, N., Sharma, R., Jain, S. C., Capasso, F. 1988. Ethnopharmacology of

Calotropis procera flowers. J. Ethnopharmacol., 22:211.

Mathabe, M. C., Nikolova, R. V., Lall, N., Nyazema, N. Z. 2006. Antibacterial

activities of medicinal plants used for the treatment of diarrhoea in Limpopo

Province, South Africa. J. Ethnopharmacol., 105:286-293.

Mathias, L., Vieira, I. J. C., Braz-Filho, R., Rodrigues-Filho, E. 1998. A new

isoflavone glycoside from Dalbergia nigra. J. Nat. Prod. 61:1158-1161.

Mathur, R., Sharma, A., Dixit, V. P., Varma, M. 1996. Hypolipidaemic effect of fruit

juice of Emblica officinalis in cholesterol fed rabbits. J. Ethnopharmaco.,

50:61-68.

McGookin, A., Reed, F. P., Robertson, A. 1937. Rottlerin. Part I. J. Chem. Soc., 748-

755.

Mehra, P. N., Karnik, C. R. 1968. Pharmacognostic studies on Bombax ceiba Linn.

Indian J. Pharm., 30:284.

Meister, A., Anderson, M. E. 1983. Glutathione. Annu. Rev. Biochem., 52: 711.

Meléndez, P. A., Capriles, V. A. 2006. Antibacterial properties of tropical plants from

Puerto Rico. PHYTOMEDICINE., 13:272-276.

Mertens-Talcott, S. U., Jilma-Stohlawetz, P., Rios, J. 2006. Absorption, metabolism,

and antioxidant effects of pomegranate (Punica granatum L.) polyphenols

after ingestion of a standardized extract in healthy human volunteers. J. Agric.

Food Chem., 54:8956-8961.

Miana, G. A. 1973. Tertiary dihydroprotoberberine alkaloids of Berberis lycium,

Phytochemistry., 12:1822-1823.


212

Milde, J., Elstner, E. F., Graßmann, J. 2000. Synergistic inhibition of lowdensity

lipoprotein oxidation by rutin, g-terpinene, and ascorbic acid. Phytomedicin.,

11:105.

Mishra, V., Khan, N. U., Singhal, K.C. 2005. Potential antifilarial activity of fruit

extracts of Ficus racemosa Linn. against Setaria cervi in vitro. Indian J. Exp.

Biol., 43:346-50.

Misra, R. B., Mishra, S. S., Misra, R. K. 1968. Pharmacology of Bombax

malabaricum D. C. Indian J. Pharm., 30:165.

Mishra, M. B., Tewari, J. P, Mishra, S. S. 1966. Studies in indigenous uterotonic

drugs (a preliminary note). Ind. J. Physiol. & Pharmacol., 10:59-60

Mitchell, G., Bartlett, D. W., Fraser, T. E., Hawkes, T. R., Holt, D. C., Townson, J.

K., Wichert, R. A. 2001. Mesotrione: a new selective herbicide for use in

maize. Pest Manag. Sci., 57:120-128.

Mondal, S. C., Mukherjee, P. K., Saha, K., Das, J., Pal, M., Saha, B. P. 1997.

Hypoglycemic activity of Ficus racemosa leaves in streptozotocin-induced

diabetic rats. Nat. Prod. Sci., 3:38-41.

Morsy, T. A., Rahem, M. A., Allam, K. A. 2001. Control of Musa domestica third

instar larvae by the latex of Calotropis procera (Family: Asclepiadaceae). J.

Egyp. Soc. Parasitology, 31:107-110.

Mossa, J, S., Tariq, M., Mohsin, A., Ageel, A. M., al-Yahya, M. A., al-Said, M. S.,

Rafatullah, S. 1991. Pharmacological studies on aerial parts of Calotropis

procera. Am. J. Chin. Med., 19:223-31.

mossambicensis, Melia azedarach and azadirachta indica against Plasmodium

falciparum. Afr. J. of Health Sci., 2:309-311.

Mothana, R. A. A., , Abdo, S. A. A., Hasson, S., Althawab. F. M. N., Alaghbari, S. A.

Z. Lindequist, U. 2008, Antimicrobial, Antioxidant and Cytotoxic Activities

and Phytochemical Screening of Some Yemeni Medicinal Plants. eCAM

Advance Access published online.


213

Muhaisen Hasan, M. H., Ilyas, M., Mushfiq, M., Parvee, M., Basudan, O. A. 2002.

Flavonoid from Viburnum cotinifolium. J. Chem. Res., 10:480-481.

Mujtaba, G., Khan, M. A., Ahmad, M., Zafar, M., Khan, A. A. 2009. Observations on

antifertility and abortifacient herbaldrugs. Afr. J. Biotech., 8:1959-1964.

Mukerjee, S. K., Saroja, T., Seshadri, T. R. 1971. Dalbergichromene, a new

neoflavonoid from stem-bark and heartwood of Dalbergia sissoo.

Tetrahedron, 27:799-803.

Mukherjee, P. K., Saha, K., Murugesan, T., Mandal, S. C., Das, J., Pal, M., Saba, B.

P. 1998. Screening of antidirrhoeal profile of some plant extracts of a specific

region of West Bengal. J. Ethnopharmacol., 13:430-432.

Mundy, C., Kirkpatrick, P. 2004. Tiotropium bromide. Nat. Rev. Drug Dis., 3:643.

Nadkarni, A. K., Nadkarni, K. M. 1982. Indian materia medica vol 1. Bombay:

Popular Prakashan, pp. 432.

Nadkarni, K. M. 1980. Indian Material Mediea. 3rd Ed. (Ed. A.K. Nadkarni), Popular

Parakashan Depot, Bombay, India, pp. 180-190.

Nadkarni, K. M. 1954. Indian Materia Medica, 3rd ed. Vol. 1. Bombay, Popular Book

Depot, pp. 432.

Nadkarni, K. M., Nadkarni, A. K., 1982. Indian Materia Medica, Vol. I, Bombay

Popular Prakashan, Bombay, India, pp. 457.

Nadkarni, K. M. 1954. Indian Materia Medica, (Popular Prakashan, Bombay, pp. 784-

785.

Nair, K. P. 1970. Giardiasis in children. Ped. Clin. India, 5:45.

Nakane, H., Arisawa, M., Fujita, A., Koshimura, S., Ono, K. 1991. Inhibition of HIV-

reverse transcriptase activity by some phloroglucinol derivatives. FEBS Lett.,

286:83-85.

Nam, K A., Lee, S. K. 2000. Evaluation of cytotoxic potential of natural products in

cultured human cancer cells. Nat. Prod. Sci., 6:183-188.


214

Nandkarni, A. K. 1976. Indian Materia Medica, 1. Popular Book Depot, Bombay, pp.

246.

Naovi, S. A. H., Khan, M. S.Y., Vohora, S. B. 1991. Antibacterial, antifungal and

anthelmintic investigations on Indian medicinal plants. Fitoterapia. 62:221-

228.

Naqvi, S. A, Khan, M. S., Vohora, S. B. 1991. Antibacterial, antifungal, and

antihelminthic investigations on Indian medicinal plants. Fitoterapia, 62:221-

228.

Nasir, E., Ali, S. I. 1971-91. Flora of West Pakistan No1-190. Pakistan Agri.Research

Council, Islamabad.

Nawwar, M. A., Hussein, S. A., Merfort, I. 1994. NMR spectral analysis of

polyphenols from Punica granatum. Phytochemistry., 36:793-798.

Ne‘gre-Salvayre, A., Mabile, L., 1995. Delchambre, J. Alpha-Tocopherol, ascorbic

acid, and rutin inhibit synergistically the copper-promoted LDL oxidation and

the cytotoxicity of oxidized LDL to cultured endothelial cell. Biol. Trace

Elem. Res., 47:81.

Newall, C. 1996. Herbal Medicines, A Guid for Healthcare professional.

Pharmaceutical press: London, 226.

Newman, D. J., Cragg, G. M., Snader, K. M. 2000. The influence of natural products

upon drug discovery. Nat. Prod. Rep., 17:215-234.

Newman, D. J., Cragg, G. M., Snader, K. M. 2003. Natural products as sources of

new drugs over the period 1981-2002. J. Nat. Prod., 66:1022-1037.

Nicolaou, K. C., Snyder, S. A. 2004. The essence of total synthesis. Proceedings of

the National Academy of Sciences of the United States of America,

101:11929-11936.

Niero, R., Cechinel, V., Souza, M., Montanari, J., Yunes, R., Monache, F. 1999.

Antinociceptive activity of nigaichigo side F1 from Rubus imperialis. J. Nat.

Prod., 62:1145-1146.


215

Nogueira, E., Rosa, G., Vassilief, V. 1998. Involvement of GABA α-benzodiazepine

receptor in the anxiolytic effect induced by hexanic fraction of Rubus

brasiliensis. J. Ethnopharmacol., 61:119-126.

O’Neill, M J., Lewis, J.A., Noble, H. M., Holland, S., Mansat, C., Farthing, J. E.,

Foster, G., Noble, D., Lane, S. J., Sidebottom, P. J., Lynn, S. M., Hayes, M.

V., Dix, G. J. 1998. Isolation of translactone-containing triterpenes with

thrombin inhibitory activities from the leaves of Lantana camara. J. Nat.

Prod., 61:1328-1331.

Ofulla, A. V. O., Chege, G. M. M., Rukunga, G. M., Kiarie, F. K., Githure, J., Kofi,

T. M. W. 1995. In vitro antimalarial activity of neem leaf extracts against

Plasmodium falciparum. African J. Health Sci., 2:309-311.

Ogunlana, E. O., Ramstad, E. 1975. Investigations into the antibacterial activities of

local plants. Planta Med., 27:354-358.

Ohigashi, H., Takamura, H., Koshimizu, K., Tokuda, H., Ito, Y. 1986. Search for

possible anti-tumor promoters by inhibition of 12-O-tetradecanoylphorbol-13-

acetate-induced Epstein-Barr virus activation; ursolic acid and oleanolic acid

from an anti-inflammatory Chinese medical plant. Cancer Let., 30:143-51.

Okouneva, T., Hill, B. T., Wilson, L., Jordan, M. A. 2003. The effects of vinflunine,

vinorelbine, and vinblastine on centromere dynamics. Mol. Can. Therap.,

2:427-436.

Okuda, T., Yoshida, T., Hatano, T., Iwasaki, M., Kubo, M., Orime, T., Yoshizaki, M.,

Naruhashi, N. 1992. Hydrolysable tannins as chemotaxonomic markers in the

Rosaceae. Phytochemistry., 31:3091-3096.

Olsson, M., Vakifahmetoglu, H., Abruzzo, P. M., Hogstrand, K., Grandien. A.,

Zhivotovsky, B. 2009. DISC-mediated activation of caspase-2 in DNA

damage-induced apoptosis. Oncogene, 28:1949-1959.

Ozarowski, A., Jaroniewski, W. 1989. Rooeliny lecznicze i ich praktyczne

zastosowanie, IWZZ, Warszawa pp. 184, 243.


216

Pal, B. C., Achari, B., Yoshikawa, K., Arihara, S. 1995. Saponins from Albizzia

lebbeck. Phytochemistry., 38:1287-1291.

Paliwa, J. K., Dwivedi, A. K., Singh, S., Gutpa, R. C2000. Pharmacokinetics and in-

situ absorption studies of a new anti-allergic compound 73/602 in rats. Int. J.

Pharm., 197:213-20.

Pan, W. D., Li, Y.J., Mai, L.T., Ohtani, K., Kasai, R., Tanaka, O., Yu, D.W. 1993.

Studies on triterpenoid constituents of the roots of Lantana camara. Yaoxue

Xuebao, 28:40-44.

Panda, S., Kar, A. 2003. Fruit extract of Emblica officinalis ameliorates

hyperthyroidism and hepatic lipid peroxidation in mice. Pharmazie, 58:753-

761.

Pandey, H. P., Verma, B. K. 2002. Biotoxicity screening of Melia azedarach L.

against mosquito (Anopheles stephensis Liston) larve. J. phytolog. Res.,

15:221-223.

Park, E. J., Pezzuto, J. M. 2002. Botanicals in cancer chemoprevention. Can. Meta.

Rev., 21:231-255.

Patel, A., Obiyan, J., Patel, N., Dacke, C. 1995. Raspberry leaf extract relaxes

intestinal smooth muscles in vitro. J. Pharm. Pharmacol., 47:1129.

Patel, A. V., Rojas-Vera, J., Dacke, C. G., 2004. Therapeutic constituents and actions

of Rubus species. Curr. Med. Chem., 11:1501-1512.

Patwardhan, S. A. and Gupta, A. S.1981. An octamethoxyflavone from Pogostemon

purpurascens. Phytochemistry., 20:1458-1459.

Perianayagam, J. B., Sharma, S. K., Joseph, A., Christina, A. J. M. 2004. Evaluation

of anti-pyretic and analgesic activity of Emblica officinalis Gaertn. J.

Ethnopharmacol., 95:83-85.

Pervez, K., Ashraf, M., Hanjra, A. H. 1994. Anthelmintic efficacy of Melia azedarach

against gastrointestinal nematodes in sheep. Applied parasitol., 35:135-137.


217

Peterson, E. A., Overman, L. E. 2004. Contiguous stereogenic quaternary carbons: a

daunting challenge in natural products synthesis. Proceedings of the National

Academy of Sciences of the United States of America, 101:11943-11948.

Petrera, E., Coto, C. E. 2003. Effect of meliacine, a plant derived antiviral, on tumor

necrosis factor alpha. Fitoterapia, 74:77-83.

Pezzuto, J. M., Song, L. L., Lee, S. K., Shamon, L. A., Mata-Greenwood, E., Jang,

M., Jeong, H. J., Pisha, E., Mehta, R. G., Kinghorn, A. D. 1999. Bioassay

methods useful for activity-guided isolation of natural product cancer

chemopreventive agents. In: Hostettmann, K., Gupta, M.P., Marston, A.

(Eds.), Chemistry, Biological and Pharmacological Properties of Medicinal

Plants from the Americas. Harwood Academic Press, Amster-dam, pp. 81-

110.

Piggott, A. M., Karuso, P. 2004. Quality, not quantity: the role of natural products and

chemical proteomics in modern drug discovery. Comb.Chem. High

Throughput Screening, 7:607-630.

Pirttila, T., Wilcock, G., Truyen, L., Damaraju, C. V. 2004. Long-term efficacy and

safety of galantamine in patients with mild-to-moderate Alzheimer’s disease:

multicenter trial. Eur. J. Neurology, 11:734-741.

Pisha, E., Chai, H., Lee, I. S., Chagwedera, T. E., Farnsworth, N. R., Cordell, G. A.,

Beecher, C. W., Fong, H. H., Kinghorn, A. D., Brown, D. M., Wani, M. C.,

Wall, M. E., Hieken, T. J., Das Gupta, T. K., Pezzuto, J. M. 1995. Discovery

of betulinic acid as a selective inhibitor of human melanoma that functions by

induction of apoptosis. Nat. Medicine, 1:1046-1051.

Piyanuch, R., Sukhthankar, M., Wandee, G., Baek, S. J. 2007. Berberine, a natural

iso-quinoline alkaloid, induces NAG-1 and ATF3 expression in human

colorectal cancer cells.Cancer Lett., 258:230-240.

Pullaiah, T. 2006. “Encyclopaedia of World Medicinal Plants,” Vol. 3, Regency

Publications, New Delhi, pp. 1211.

Quresh, R. A., Ghufran, M. A., Gilani, S. A., Sultana, K., Ashraf, M. 2007.


218

Ethnobotanical Studies of Selected Medicinal Plants of Sudhan Gali and

Ganga Chotti Hills, District Bagh, Azad Kashmir. Pakistan. J. Bot., 39:2275-

2283.

Qureshi, M. A., Qureshi, N. M., Arshad, R., Begum, R. 1991. A Study on the

Antisperm Activity in Extracts from Different Parts of Calotropis-

Procera.Pak. J. Zool., 23:161.

Qureshi, S. J., Khan, M. A., Ahmad, M. 2008. A survey of useful medicinal plants of

abbottabad in northern Pakistan. Trakia J. Sci., 6:39-51.

Rahman, N. N. K. M., Hassan, R. 1994. Bioactive components from Ficus glomerata.

Pure App. Chem., 66:2287-2290.

Rahuman, A. A., Venkatesan, P., Geetha, K., Gopalakrishnan, G., Bagavan, A.,

Kamaraj, C. 2008. Mosquito larvicidal activity of gluanol acetate, a tetracyclic

triterpenes derived from Ficus racemosa Linn. Parasitol Res., 103:333-9.

Rai, S. K., Mallavarapu, G. R., Rai, S. P., Srivastara, S., Sing, D., Mishra, R., Kuma,

S. 2005. Constituents of the flower oil of Carissa opaca growing in the

Aravalli mountain range at New Delhi. Flavour Fragr. J., 21:304-305.

Raj, R. K. 1975. Screening of indigenous plants for anthelmintic action against human

Ascaris lumbricoides: Part-II. Indian J. Physiol. Pharmacol., 19(1).

Rajkapoor, B., Jayakar, B., Murugesh, N., Sakthisekaran, D. 2006. Chemoprevention

and cytotoxic effect of Bauhinia variegata against N-nitrosodiethylamine

induced liver tumors and human cancer cell lines. J. Ethnopharmaco.,

104:407-409.

Ramachandran, N., Subramanian, A. G., Sankara, S. 1975. Isorhamnetin and

quercetin glycosides from Dodonaea viscosa and Sapindus emarginatus.

Indian J. Chem., 13:639-640.

Ramakrishna, N. V. S., Kumar, E. K. S. V., Kulkarni, A. S., Jain, A. K., Bhat, R. G.,

Parikh, S., Quadros, A., Deuskar, N., Kalakoti, B. S. 2001. Screening of

natural products for new leads as inhibitors of b-amyloid production: Latifolin


219

from Dalbergia sissoo. Indian J. Chem., 40:539-540.

Ramesh, P., Yuvarajan, C. R. 1995. Coromandelin, a new isoflavones apioglucoside

from the leaves of Dalbergia coromandeliana. J. Nat. Prod., 58:1240-1241.

Rana, A. C., Santana, D. D., Saluja, A. K. 1990. Pharmacological screening of the

Alcohlic Extract of the leaves of Rubus Ellepticus. Indian J. Pharmac. Sci.,

52:174-177.

Rani, M., Suhag, P., Kumar, R., Singh, R., Kalidhar, S.B. 1999. Chemical component

and biological efficacy of Melia azedarach stems. J. Med. Aromatic Plant Sci.,

21:1043-1047.

Rani, P., Khullar, N. 2004. Antimicrobial evaluation of some medicinal plants for

their anti-enteric potential against multi-drug resistant Salmonella typhi.

Phytother. Res, 18, 670-673.

Rao, P. S., Asheervadam, Y., Khalilullah, M., Murti, V.V.S. 1989. A revised structure

for the isoflavone lanceolarin. Phytochemistry., 28:957-958.

Rao, R. B., Murugesan, T., Pal, M., Saha, B. P., Mandal, S. C. 2003. Antitussive

potential of methanol extract of stem bark of Ficus racemosa Linn. Phytother

Res. 17:1117-8.

Rao, R. B., Anupama, K., Swaroop, K. R., Murugesan, T., Pal, M., Saha, B. P.,

Mandal, S. C. 2002. Evaluation of antipyretic potential of Ficus racemosa

bark. Phytomedicine., 9:731-733.

Rao, Y. K., Fang, S. –Hua, Tzeng, Y. –Min. 2008. Antiinflammatory Activities of

Flavonoids and a Triterpene Caffeate Isolated from Bauhinia Variegate.

Phytother. Res., 22:957-962.

Ratnasooriya, W. D., Jayakody, J. R., Nadarajah, T. 2003. Antidiuretic activitity of

aqueous bark extract of Sri Lankan Ficus racemosa in rats. Acta Biol. Hung.,

54:357-363.

Reddy, L., Odhav, B., Bhoola, K. D. 2003. Natural products for cancer prevention: a

global perspective. Pharmacol. and Therapeut., 99:1-13.


220

Renart, J., Reiser, J., Stark, G. R.1979. Transfer of proteins from gels to

diazobenzyloxymethyl-paper and detection with antisera: a method for

studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A.,

75:3116-3120.

Resmi, C. R., Venukumar, M. R., Latha, M. S. 2006. Antioxidant activity of Albizzia

lebbeck (Linn.) Benth. in alloxan diabetic rats. Ind. J. Physiol. Pharmacol.,

50:297-302.

Rhaman, W., Begum, S. J. 1966. Flavonoids from the white flowers of Bauhinia

variegata. Naturwissenschaften, 53:384.

Richards, R., Durham, D., Liu, X. 1994. Antibacterial activity of compounds from

Rubus pinfaensis. Planta Med., 60: 471-473.

Robak, J., Gryglewski, R. J. 1988. Flavonoids are scavengers of superoxide anions.

Biochem Pharmacol., 37:837.

Robbers, J. E., Tyler, V. E. 1999. In: Tyler’s Herbs of choice: the therapeutic use of

phytomedicinals, The Haworth Herbal Press, New York-London pp. 63-64,

194-195.

Rojas, A., Cruz, S., Ponce-Monter, H., Mata, R. 1996. Smooth muscle relaxing

compounds from. Dodenaea Viscosa. Planta Med., 62:154.

Rojas, A., Cruz, S., Rauch, V., Linares, M. R. 1995. Spasmolytic potential of some

plants used in mexicane tradional medican for the treatment of gastrointestinal

disorder. Phytomedicine., 2:51.

Rojas, A., Hernandez, L., Pereda-Miranda, R., Mata, R. 1992. Screening for

antimicrobial activity of crude drug extracts and pure natural products from

Mexican medicinal plants. J. Ethnopharmacol., 35:275-83.

Rojas-Vera, J., Patel, A. V., Dacke, C. G. 2002. Relaxant activity of raspberry (Rubus

idaeus) leaf extract in guinea-pig ileum in vitro. Phytother. Res., 16:665-668.

Roop, J. K. 2005. Extracts of Azadirachta indica and Melia azedarach seeds inhibit

follculogenesis in albino rats. Braz. J. Med. Biol. Res. 38:943-947.


221

Rosenblat, M., Volkova, N., Coleman, R., Aviram, M. 2006. Pomegranate by product

administration to apolipoprotein e-deficient mice attenuates atherosclerosis

development as a result of decreased macrophage oxidative stress and reduced

cellular uptake of oxidized low-density lipoprotein. J Agric Food Chem.,

54:1928-1935.

Roy, S., Barua, A. K. 1985. The structure and stereochemistry of a triterpene acid

from Lantana camara. Phytochemistry., 24:1607-1608.

Sachdev, K., Kulshreshtha, D. K. 1984. Dodonic acid, a new diterpenoid from

Dodonaea viscosa. Planta Med., 50:448-9.

Sachdev, K., Kulshrestha, D. K. 1983. A biologically active lipophilic flavonoids

from Dodnia viscosa. Phytochemistry., 22:1253.

Sack, R. B., Froelich, J. L. 1982. Berberine inhibits intestinal secretory response of

Vibrio cholera and Escherichia coli enteroxins. Infect. Immin., 35:471-475.

Saghir, M. I., Awan, A. A., Majid, S., Khan, M. A., Quereshi, S. J., Bano, S. 2001.

Ethnobotanical studies of Chikar and its Allied area of District Muzafarabad.

Online J. of Bio. Sciences, 1:1165-1170.

Said, H. M., 1996. Medicinal herbal-a textbook for medical students and doctors.

Volume 1. Hamdard Foundation, Nazimabad, Karachi-74600, Sindh, Pakistan.

Sallem, R., Ahmed, S. I., Shamim, S. M., Faizi, S., Siddiqui, B. S. 2002. Antibacterial

effect of Melia azedarach flowers on rabbits. Phytother Res., 16:762-764.

Salunke, B. K., Klotkar, H. M., Mendki P.S., Upasani S. M., Maheshwari V.L. 2005.

Efficacy of Flavonoids in controlling Callosobruchus chinensis (l.)

(Coleoptera: bruchidae), a post-harvest pest of grain legumes. Crop. Prot.,

24:888-893.

Salwa, F. F., Amany, S. A., Kenji, T., Yoshiaki, T., Masatake, N. 2001. Isoflavonoid

glycosides from Dalbergia sissoo. Phytochemistry., 57:1263-1268.

Samvatsar, S., Diwanji, V. B. 2000. Plant sources for the treatment of jaundice in the

tribals of Western Madhya Pradesh of India. J. Ethnopharmacol., 73:313-316.


222

Sanders, M., M., Liu, A.,A., Li, T.,K., Wu, H.,Y., Desai, S.,D., Mao, Y., Rubin, E.,H.,

LaVoie, E.,J., Makhey, D., Liu, L. F. 1998. Selective cytotoxicity of

topoisomerase-directed protoberberines against glioblastoma cells. Biochem.

Pharmacol., 56:1157-1166.

Sarg, T., Abdel-Monem, A., Abdel-Ghani, A., Badr W., Shams, G. 1999.

Phytochemical and Pharmacological Studies of Dalbergia sissoo Growing in

Egypt. Pharmac. Biology, 37:54-62.

Sastry, M. S., Mahadevan, V. 1963. Chemical Investigation of Lantana camara.

Current Sci. (India), 32:71-76.

Satyavati, G. V., Gupta, K. A., Tandon, N. 1987. Medicinal Plants of India, 2:201.

Saxena, A., Vikram, N. K. 2004. Role of selected Indian plants in management of

type 2 diabetes: a review. J. Altern. Complement Med., 10:369-378.

Schmidt, A., Mordhorst, T., Nieger, M. 2005. Investigation of a betainic alkaloid from

Punica granatum. Nat. Prod. Res., 19:541-6.

Seeram, N. P., Lee, R., Heber, D. 2004. Bioavailability of ellagic acid in human

plasma after consumption of ellagitannins from pomegranate (Punica

granatum L.) juice. Clin. Chim. Acta., 348:63-68.

Seeram, N. P., Henning, S. M., Zhang, Y. 2006. Pomegranate juice ellagitannin

metabolites are present in human plasma and some persist in urine for up to 48

hours. J. Nutr., 136:2481-2485.

Serafim, T. L., Oliveira, P. J., Sardao, V. A., Perkins, E., Parke, D., Holy, J. 2007.

Different concentrations of berberine result in distinct cellular localization

patterns and cell cycle effects in a melanoma cell line. Cancer Chemother.

Pharmacol., 61:1007-1018.

Seshadri, V., Bhatta, A. K., Rangaswami. S. 1971. Phenolic compounds of Bombax

malabaricum (root-bark). Curr. Science, 40:630-631.

Seshadri. V., Bhatta, A. K., Rangaswami. S. 1973. Phenolic components of Bombax

malabaricum. Indian J. Chem., 11, 825.


223

Setty, R., Quereshi, S., Viswanath, A. A., Swamy, A.H., Patil, T., Prakash, T., Prabhu,

K., Veeran, G. A. 2007. Hepatoprotective activity of Calotropis procera

flowers against paracetamol-induced hepatic injury in rats. Fitoterapia.,

78:451-4.

Sharanabasappa, A., Saraswati, B. P. 2004. Spermicidal activity of Melia azedarach:

In vitro and in vivo studies. Chem. Biol. Interface: Synergistic New Frontiers,

25:21-26.

Sharma, A., Chibber, S. S., Chawla, H. M. 1979a. Caviunin 7-O-gentiobioside from

Dalbergia sissoo pods. Phytochemistry., 18:1253.

Sharma, A., Chibber, S. S., Chawla, H. M. 1979b. Isocaviunin from mature pods of

Dalbergia sissoo. Indian J. Chem., 18:472-473.

Sharma, A., Chibber, S. S., Chawla, H. M. 1980a. Isocaviunin 7-gentiobioside, a new

isoflavone glycoside from Dalbergia sissoo. Phytochemistry., 19:715.

Sharma, A., Chibber, S. S., Chawla, H. M. (1980b). Isocaviudin, a new isoflavone

glucoside isolated from Dalbergia sissoo. Indian J. Chem., 19:237-238.

Sharma, B. B., Gupta, D. N., Varshney, M. D., Anand, O. P. 1981., Indian J. Veter.

Medicine, 5:25.

Sharma, M. L., Atal, C. K. 1999. Oxytocic, thrombopoietic and broncho-dilatory

activities of Vasicine-A novel molecule isolated form Adhatoda vasica Nees.

In: Sairam TV (ed). Home Remedies. Vol. II. Penguin, New Delhi.

Sharma, O. P., Singh, A., Sharma, S. 2000. Levels of lantadenes, bioactive

pentacyclic triterpenoids, in young and mature leaves of Lantana camara var.

aculeata. Fitoterapia 71: 487-491.

Sharma, Om. P., Dawra, R. K., Ramesh, D. I990. A triterpenoid acid, lantadene D

from Lantana camara var. Aculeata. Phytochemistry., 29:3961.

Sharma, P. C., Yelne, M. B., Dennis, T. J. 2001. Data base on Medicinal plants used

in Ayurveda, (Documentation and Publication Division, Central Council for

Research in Ayurveda and Siddha, New Delhi, pp. 389-406.


224

Sheat, W. G. 1948. Propagation of Trees, Shrubs and Conifers. MacMillan and Co

Shi, M., Vivian, C. J., Lee, K. J., Ge, C., Morotomi-Yano, K., Manzl, C. 2009. DNA-

PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M

checkpoint maintenance. Cell, 136:508-520.

Shoji, S. 2001. Chemistry and cancer preventing activities of ginseng saponins and

some related triterpenoid compounds. J Korean Med Sci., 16:S28-S37.

Siddiqui, B.S., Raza, S.M., Begum, S., Siddiqui, S., Firdous, S. 1995. Pentacyclic

triterpenoids from Lantana camara. J. Nat. Prod., 38:681-685.

Siddiqui, B. S., Wahab, A., Begum, S. 2000. Two new pentacyclic triterpenoids from

the aerial parts of Lantana camara Linn. Heterocycles, 53:681-687.

Sidi, S., Sanda, T., Kennedy, R. D., Hagen, A. T., Jette, C. A., Hoffmans, R. 2008.

Chk1 suppresses a caspase-2 apoptotic response to DNA damage that by

passes p53, Bcl-2, and caspase-3. Cell, 133:864-877.

Simpson, M., Parsons, M., Greenwood, J., Wade, K. 2001. Raspberry leaf in

pregnancy: its safety and efficacy in labor. J. Midwifery Womens Health,

46:51-59.

Sing, M., Srivastava, S., Rawat, A. K. S. 2007. Antibacterial activity of Indian

Berberis species. Fitoterapia, 78:574-576.

Singh, A., Bhardwaaj, P., Varshneya, C., Telang, R. S. 2005. Anthelmintic activity of

leaves of Bauhinia variegate. Ind. Vet. Journal., 82:855-857.

Singh, B., Rastogi, R. B. 1970. The nature and distribution of cardenolides.

Phytochemistry, 9:315.

Singh, H., Saklani, A., Lal, B. 1990. Ethnobotanical observations on some

Gymnosperms of Garhwal Himalaya, Uttar Pradesh, India. Eco. Botany,

44:349-354.

Singh, N.P.; McCoy, M. T.; Tice, R. R. Schneider, E. L. 1988. A simple technique for

quantitation of low levels of DNA damage in individual cells. Exp. Cell Res.,


225

175:184-191.

Singh, R. P., Padmavathi, B., Rao, A. R. 2000. Modulatory influence of Adhatoda

vesica (Justicia adhatoda) leaf extract on the enzymes of xenobiotic

metabolism, antioxidant status and lipid peroxidation in mice. Mol. Cell

Biochem., 213:99-109.

Singh, R. H, Chaturvedi, G. N. 1966. Inhibition of formaldehyde induced arthritis by

certain indigenous drugs. Ind J Med Res, 54:188-195.

Singh, S.K., Singh, A., Tripathi, V. J., Finzi, P. V., J. 1996. Minor constituents of

Lantana camara. Indian Chem. Soc., 73:547.

Singleton, V. L., Orthofer, R., Lamuela-Raventos, R. M. 1999. Analysis of total

phenols and other oxidation substrates and antioxidants by means of Folin-

Ciocalteu reagent. Meth. Enzymol.; 299:152-178.

Singleton, V. L. Rossi, J. A. 1965. Colorimetry of total phenolics with

phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16:144-

158.

Skoog, 2007. Principles of Instrumental Analysis. 6th ed. Thomson Brooks/Cole., 169-

173.

Slinkard, K., Singleton, V. L. 1977. Total phenol analysis: Automation and

comparison with manual methods. Am. J. Enol. Vitic. 28:49-55.

Soares de Oliveira, J., Pereira Bezerra, D., Teixeira de Freitas, C. D., Delano Barreto

Marinho Filho, J., Odorico de Moraes, M., Pessoa, C., Costa-Lotufo, L. V.,

Ramos, M. V., 2007. In vitro cytotoxicity against different human cancer cell

lines of laticifer proteins of Calotropis procera (Ait.) R. Br. Toxicol In Vitro.,

21:1563-73.

Soltoff, S. P. 2007. Rottlerin: an inappropriate and ineffective inhibitor of PKCdelta.

Trends Pharmacol. Sci., 28:453-458.

Soltoff, S. P. 2001. Rottlerin is a mitochondrial uncoupler that decreases cellular ATP

levels and indirectly blocks protein kinase Cdelta tyrosine phosphorylation. J.


226

Biol. Chem., 276:37986-37992.

Sophia, D., Manoharan, S. 2007. Hypoglycemic activities of Fiscus racemosa Linn.

bark in alloxan-induced diabetic rats. Afr.J. Trad. Comp. Alter. Med., 4:279-

288.

Sporn, M. B., Dunlop, N. M., Newton, D. L., Smith, J. M. 1976. Prevention of

chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids).

Federation Proceedings, 35:1332-1338.

Sporn, M. B. 1999. Prevention of cancer in the next millennium: report of the

chemoprevention working group to the American Association for Cancer

Research. Cancer Res., 59:4743-58.

Srivastava, M. C., Singh, S. W., Tewari, J. P. 1967. Ind. J. Med. Res., 55:46-748.

Still, W. C., Kahn, M., Mitra, A. 1978. J. Org. Chem., 43:2923-2925.

Subbaiah, T. V., Amin, A. H. 1967. Effect of berberine sulphate on Entamoeba

histolytica. Nature, 215:527-528.

Suhag, P., Merra, Kalidhar, S. B. 2003. Phytochemical investigation of Melia

azedarach leaves. J. Med. Aromatic Plant Sci., 25:397-399.

Sun, D., Abraham, S. N., Beachey, E. H.1988. Influence of berberine sulfate on

synthesis and expression of Pap fimbrial adhesin in uropathogenic Escherichia

coli. Antimicrob Agents. Chemother, 32:1274-1277.

Surh, Y. J. 2003. Cancer chemoprevention with dietary phytochemicals. Nat. Rev.

Cancer., 3:768-780.

Swabb, E. A., Tai, Y. H., Jordan, L. 1981. Reversal of cholera toxin-induced secretion

in rat ileum by luminal berberine. Am. J. Physiol., 241:248-252.

Syah, Y. M., Pennacchio, M., Ghisalberti, E. L. 1998. Cardioactive phenylethanoid

glycosides from Lantana camara. Fitoterapia, 69:285-286.

Talapatra, S. K., Pradhan, D. K., Talapatra, B., 1978. Terpenoids and related

compounds: part XV. 3á-Hydroxyfriedel-2-one and 2â-acetoxyfriedel-3-one


227

(epicerin acetate), two new pentacyclic triterpenoids from cork waste, their

partial syntheses and one-step conversions to friedelin. Ind. J. Chem., 16:361-

5.

Talapatra, S. K., Shrestha, K. M., Talapatra, B. 1989. Chemical investigation of some

medicinal plants of Nepal: part II. Indian J. Chem., 28:880-881.

Tan, D. S. 2004. Current progress in natural product-like libraries for discovery

screening. Comb. Chem. High Throughput Screening.7:631-643.

Tanaka, R., Matsunaga, S. 1988. Triterpene dienols and other constituents from the

bark of Phyllanthus flexuosus. Phytochemistry., 27:2273-7.

Tanaka, R., Tabuse, M., Matsunaga, S. 1988. Triterpenes from the stem bark of

Phyllanthus flexuosus. Phytochemistry., 27:3563-7.

Tanaka, R., Minami, T., Tsujimoto, K., Matsunaga, S., Tokuda, H., Nishino, H. 2001.

Cancer chemopreventive agents, serratane-type triterpenoids from

Piceajezoensis. Cancer Lett., 172:119-26.

Tanaka, R., Nakata, T., Yamaguchi, C., Wada, S. –ichi., Yamada,T., Tokuda,.H.

2008. Potential Anti-Tumor-Promoting Activity of 3α-Hydroxy-D:A-

friedooleanan-2-one from the Stem Bark of Mallotus philippensis. Planta

Med., 74:413-416

Tanaka, T., Ito, T., Iinuma, M., Takahashit, Y., Naganaw, H. 1998. Dimeric chalcone

derivatives from Mallotus Philippensis. Phytochemistry., 48:1423-1427.

Taoubi, K., Fauvel, M. T., Gleye, J., Moulis, C., Fouraste, I. 1997. Phenylpropanoid

glycosides from Lantana camara and Lippia multiflora. Mania Med., 63:192.

Tapia, J. A., Jensen, R. T., García-Marín, L. J. 2006. Rottlerin inhibits stimulated

enzymatic secretion and several intracellular signaling transduction pathways

in pancreatic acinar cells by a non-PKC-delta-dependent mechanism. Biochim.

Biophys. Acta, 1763:25-38.

Thakur, C. P., Thakur, B., Singh, B., Singh, S., Sinha, P. K., Sinha, S. K. 1988. The

Ayurvedic medicines, Haritaki, Amla and Bahira reduce cholesterol induced


228

atherosclerosis in rabbits. Ind. J. Cardiology, 21:167-175.

Thammanna, X., Narayana Rao, K., Madhava Chetty, K., 1990. Angiospermic Wealth

of Tirumala. TTD Press, Tirupati.

The Wealth of India, 1988. Raw Materials, vol. VI, Publication and Information

Directorate (CSIR), New Delhi, 1962. 1988, pp. 337.

Tillman, D. M., Izeradjene, K., Szucs, K. S., Douglas, L., Houghton, J. A. 2003.

Rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related

apoptosis-inducing ligand-induced apoptosis via uncoupling of the

mitochondria independent of protein kinase C. Cancer Res., 63:5118-5125.

Towbin, H., Staehelin, T., Gordon, J. 1979. “Electrophoretic transfer of proteins from

polyacrylamide gels to nitrocellulose sheets: procedure and some

applications.”. Proc Natl Acad Sci U S A., 76: 4350-4354.

Tripathi, R. M., Das, P. K. 1977. Studies on anti-asthamatic and anti-anaphylactic

activity of Albizzia lebbeck. Ind. J. Pharmaco., 9:189-194.

Tripathi, R. M., Sen, P. C., Das, P. K. 1979. Further studies on the mechanism of

action of Albizzia lebbeck, an Indian indigenous drug used in the treatment of

atopic allergy. J. Ethno. Pharmaco., 1:397-406.

Tripathi, S. N., Shukla, P., Mishra, A. K., Udupa, K. N. 1978. Experimental and

clinical studies on adrenal function in bronchial asthma: with special reference

to treatment with Albizzia lebbeck. Qua. J. Sur. Sci., 14:169-176.

Tsao, A. S., Kim, E. S., Hong, W. K. 2004. Chemoprevention of cancer. CAA Cancer.

J. for Clinicians, 54:150-180.

Tyler, V. E. 1999. Phytomedicines: back to the future. J. Nat. Prod., 62:1589-1592.

Uddin, S. N., Uddin, K. M. A., Ahmed, F. 2008. Analgesic and antidiarrhoeal

activities of Treama orientalis Linn. in mice. Oriental Pharm. Exp.Med.,

8:187-191.

Uddin, S. N., Ali, M. E., Yesmin, M. N. 2008a. Antioxidant and Antibacterial


229

Activities of Senna tora Roxb. American J. Plant Physiol., 3:096-100.

Ukiya, M., Akihisa, T., Tokuda, H., Hirano, M., Oshikubo, M., Nobukuni, Y. 2002.

Inhibition of tumor-promoting effects by poricoic acids G and H and other

lanostane-type triterpenes and cytotoxic activity of poricoic acids A and G

from Poria cocos. J Nat Prod., 65:62-5.

Usmanghani, K., Saeed, A., Alam, M. T. 1997. “Indusynic Medicine, Research

Institute of Indusyunic Medicine,” A-952, Block H., North Nazimabad,

Karachi, pp. 285-287.

Van Agtmael, M. A., Eggelte, T. A., Van Boxtel, C. J. 1999. Artemisinin drugs in the

treatment of malaria: from medicinal herb to registered medication. Tre.

Pharmac. Sci., 20:199-205

Veerapur, V. P, Badiger, A. M., Joshi, S. D., Nayak, V. P., Shastry. C. S. 2004.

Antiulcerogenic activity of various extracts of Dodonaea viscosa (L) Jacq.

Leaves. Indian J. Pharm. Sci., 66:407-411.

Verma, R. C., Gupta, A. 2004. Effect of pre-treatments on quality of solar-dried amla.

J. Food Engin., 65:397-402.

Vinson, J., Zubik, L., Bose, P., Samman, N., Proch, J. 2005. Dried fruits: excellent in

vitro and in vivo antioxidants. J. Am. Coll. Nutr., 24:44-50.

Vohra, S. B., Dandia, P. C. 1992. Herbal analgesic drugs. Fitoterapia. 63:195-207.

Wachsman, M. B., Castilla, V., Coto, C. 1998. Inhibition of foot and mouth disease

virus (FMDV) uncoating by a plant-derived peptide isolated from Melia

azedarach L leaves. Arch. virol., 143:581-590.

Wagner, H., Ludwig, C., Grotjahn, L., Khan, M. S. Y.1987. Biologically active

saponins from Dodonaea viscosa. Phytochemistry., 26:697-701.

Wahab, S. M. A. E., Wassel, G. M., Ammar, N. M., Hanna, T. 1987. Flavonoid

constituents in the different organs of selected Bauhinia species and their

effect on blood glucose. Herba Hung., 26:27-39.


230

Wang, Y. X., Yao, X. J., Tan, Y. H. 1994. Effects of berberine on delayed after

depolarizations in ventricular muscles in vitro and in vivo. J. Cardiovasc.

Pharmacol., 23:716-722.

Warrier, P. K., Nambiar, V. P. K. 1995. Ramankutty, C.Indian medicinal plants, a

compendium of 500 species, (Orient Longman Limited, Hyderabad, pp. 10-14.

Watt, G. 1889. A dictionary of the economic products of India. Published under the

authority of His Majesty,s Secretary of State for India in Council, Kolkatta.

London: Yohn Murry, pp. 652.

Wititsuwannakul, D., Chareonthiphakorn, N., Pace, M., Wititsuwannakul, R. 2002.

Polyphenol oxidases from latex of Hevea brasiliensis: purification and

characterization. Phytochemistry., 61:115-121.

Wójcik, E., 1989. Phytochemical investigation of Rubus plicatus blackberry. Acta.

Polon. Pharm., 46:386-390.

Wu, J. F., Liu, T. P. 1995. Effects of berberine on platelet aggregation and plasma

levels of TXB2 and 6-keto-PGF1 alpha in rats with reversible middle cerebral

artery occlusion. Yao Hsueh Hsueh Pao, 30:98-102.

Yadava, R. N. Verma, V. 2003. A new biologically active flavone glycoside from the

seeds of Cassia fistula (Linn.). J. Asian Na.t Prod. Res., 5:57-61.

Yadava, R, N., Reddy, V. M. 2003. Anti-inflammatory activity of a novel flavonol

glycoside from the Bauhinia variegata. Nat. Prod. Res., 17:165-169.

Yang, F., Li, X. C., Wang, H. Q., Yang, C. R. 1996. Flavonoid glycosides from

Colebrookia oppositifolia. Phytochemistry., 42:867-869.

Yang, J., Guo, J., Yuan, J. 2008. In vitro antioxidant properties of rutin. LTW,

41:1060.

Yang, S. S., Cragg, G. M., Newman, D. J., Bader, J. P. 2001. Natural product-based

anti-HIV drug discovery and development facilitated by the NCI

developmental therapeutics program. J. Nat. Prod., 64:265-277.


231

Yesilada, E., Küpeli, E. 2002. Berberis crataegina DC, Roots exhibits potent anti-

inflammatory, analgesic and febrifuge effects in mice and rats. J.

Ethnopharmacol., 79:237-49.

Yousaf, Z., Shinwari, Z. K., Ali, S. M. 2004. Medicinally Important Flora of Dhibbia

Karsal Village (Mianwali District Punjab. Asian J. Plant Sci., 3:757-762.

Yu, D., Suzuki, M., Xie, L., Morris-Natschke, S. L., Lee, K. H. 2003. Recent progress

in the development of coumarin derivatives as potent anti-HIV agents. Med.

Res.Rev., 23:322-345.

Yuan, J., Shen, X. Z., Zhu, X. S. 1994. Effect of berberine on transit time of human

small intestine. Chung Kuo Chung His I Chieh Ho Tsa Chih, 14:718-720.

Zabihullah, Q., Rasheed, A., Akhter, N. 2006. Ethnobotanical survey in kot manzaray

baba valley. Pakistan. J. Pl. Sci., 12:115-121.

Zaidi, S. F. H., Yoshida, I., Butt F., Yusuf, M.A., Usmanghani, K., Kadowaki, M.,

Sugiyama, T. 2009. Potent Bactericidal Constituents from Mallotus

philippinensis against Clarithromycin and Metronidazole Resistant Strains of

Japanese and Pakistani Helicobacter pylori. Biol. Pharm. Bull., 32:631-636.

Zhang, R. X., Dougherty, D. V., Rosenblum, M. L. 1990. Laboratory studies of

berberine used alone and in combination with 1,3-bis(2-chloroethyl)-1-

nitrosourea to treat malignant brain tumors. Chin Med.J. (Engl.), 103:658-665.

Zhang, Y. -J., Tanaka, T., Iwamoto, Y., Yang, C. -R., Kouno, I. 2000. Novel

Norsesquiterpenoids from the Roots of Phyllanthus emblica. J. Nat. Prod.,

63:1507-1510.

Zhang, Y. -J., Tanaka, T., Iwamoto, Y., Yang, C. -R., Kouno, I. 2001. Novel

sesquiterpenoids from the roots of Phyllanthus emblica. J. Nat. Prod., 64:870-

873.

Zhang, Y. -J., Tanaka, T., Yang, C. -R., Kouno, I. 2001b. New Phenolic Constituents

from the Fruit Juice of Phyllanthus emblica. Chem. Pharm. Bull., 49:537-540.

Zhang, Y. -J., Abe, T., Tanaka, T., Yang, C. -R., Kouno, I. 2001c. Phyllanemblinins


232

A−F, New Ellagitannins from Phyllanthus emblica J. Nat. Prod., 64:1527-

1532.

Zhang, Y. -J., Abe, T., Tanaka, T., Yang, C. -R., Kouno, I. 2002. Two New Acyalted

Flavanone Glycosides from the Leaves and Branches of Phyllanthus

emblica.Chem. Pharm. Bull., 50:841-843.

Zhang, Y. J., Tanaka, T., Iwamoto, Y., Yang, C. R., Kouno, I. 2000. Phyllaemblic

acid, a novel highly oxygenated norbisabolane from the roots of Phyllanthus

emblica. Tetrahed. Letters, 41:1781-1784.

Zhang, Y. J., Nagao, T., Tanaka, T., Yang, C. R., Okabe, H., Kouno, I. 2004.

Antiproliferative activity of the main constituents from Phyllanthus emblica.

Bio. Pharmac. Bulletin, 27:251-255.

Zhao, Y-Y., Cui, C-B., Cai, B., Han, B., Sun, Q-S. 2005. A new phenanthraquinone

from the stems of Bauhinia variegata L. J. Asian Nat. Prod. Res., 7:835-838.

Zhou, H., Hamazaki , A., Fontana, J. D., Takahashi, H., Esumi, T., Andcheer, C.B.,

Tsujimoto, H., Fukuyama,Y. 2004. New ring C-Seco limonoids from Brazilian

Melia azedarach and their cytotoxicity. J. Nat. Product., 67:1544-1547.

Zhou, H., Hamazaki, A., Fontana, J. D., Takahashi, H., Wandscheer, C. B.,

Fukuyama, Y. 2005. Cytotoxic limonoids from Brazilian Melia azedarach.

Bio. Pharm. Bull., 28:1362-1365.

Zwemer, C., Shoemaker Jr, J., Hazard III, S., Davis, R., Bartoletti, A., Phillips, C.

2000. Hyperoxic reperfusion exacerbates postischemic renal

dysfunction.Surgery, 128:815-821.

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