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BBiioollooggiiccaa ll aa cctt iivviitt yy && PP hh yytt oocchh eemm iiccaa ll SStt uu dd yy oof f sseelleecctt eedd

MM eedd iicciinn aa ll PP llaa nn tt ss

In

ByMusa 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 SciencesQuaid-i-Azam University

Islamabad2010

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BBiioollooggiiccaa ll aa cctt iivviitt yy && PP hh yytt oocchh eemm iiccaa ll SStt uu dd yy oof f sseelleecctt eedd

MM eedd iicciinn aa ll PP llaa nn tt ss

By Musa Khan

Department of Plant SciencesQuaid-i-Azam University

Islamabad2010

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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

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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 Pakistanfor 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

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ii

In memory of my dear mother

(July 2008)

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ABBREVIATIONS

BuOH Butanol

C1I Chk1 InhibitorC2I Chk2 InhibitorCdc Cell division controlCdc25A/B/C Cell-devision-cycle 25A/B/CCdk Cyclin-dependent-kinasesChk1 Checkpoint-kinase 1Chk2 Checkpoint-kinase 2CKI Cyclin dependent kinase inhibitorDPPH 1, 1-diphenyl-2-picrylhydrazyl

EtOAc Ethyl acetateGA Gallic acidHUVEC Human umbilical vein endothelial cellsIC50 Concentrations which inhibits by 50 %IpC50 Concentrations which inhibits proliferation by 50 %IR Ionizing radiationp21 Protein 21p53 Protein 53PARP Poly (ADP-ribose) polymerasePIC Protease inhibitor cocktailPMSF PhenylmethylsulfonylfluoridRB Retinoblastoma proteinROS Reactive oxygen speciesSPE Solid phase extractionTHF TetrahydrofuraneTNF Tumour necrosis factorUV-light Ultraviolet-Light

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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 211.6.3.4 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

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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 261.8.5 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 p21 CIP 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

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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 461.9.6.1 (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

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



2.2.3 Biological testing 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


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2.10 Colebrookea oppositifolia Smith (Labiateae) 77


2.10.2 Chemical constituents 772.11 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


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2.20 Pinus roxburghii Sargent (Pinaceae) 87


2.21 Punica granatum Linn. (Punicaceae) 882.21.1 Ethnobotanical uses 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

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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

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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 Flavonoidsfinger 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

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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 106Figure 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

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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

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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 (H 2O) extracts. The BuOH extract

inhibited cell proliferation most efficiently (IC 50 < 2.77 g extract weight/ml medium,

which corresponded to 250 g dried root/ml). The IC 50s for the EtOAc and H 2O extracts

were 16.65 g/ml and 104.25 g/ml, respectively (corresponding for both extract types to

>7.5 mg dried root/ml). The chemical composition of the BuOH extract was analyzed by

preparative TLC and quantified by RP-HPLC and it was estimated that it contained 3.73

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

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

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

4.68 M. The BuOH extract and berberine induced the intra-S-phase checkpoint causing

the accumulation of HL-60 cells in S-phase. In contrast to a very recent report by Liu et

al, (2006), It is found that the anti-cancer effects of berberine and the extract are not due

to genotoxicity but correlate with -tubulin acetylation, strong activation of Chk2,

phosphorylation of Ser177-Cdc25A and its subsequent degradation as well as the

consequent inactivation of Cdc2 (CDK1) and furthermore, the down-regulation of the

proto-oncogene cyclin D1. The molecular effects were observed at low concentrations

(11.1 g BuOH extract/ml; 1.4 g berberine/ml) which inhibited ~ 50 % of the HL-60

cells proliferation after 24 h treatment, hence supporting the mechanistic conjunction.

Mallotus philippensis is a well known medicinal plant of Pakistan. It possesses different

classes of chemical compounds with unique pharmacological activities. Roots of Mallotus

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Summary

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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 (IC 50

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 R f = 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 beenobserved 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

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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 theleaves 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.

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Chapter 1 Introduction

4

INTRODUCTION

1.1 General introductionThe 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 19 th 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 propercollection, 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

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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 sourcesincluding unicellular and multi cellular organism like bacteria, fungi, plants, and marine

organisms. In broad way, Pharmacognosy that study various parameter which includes

both botanical dietary supplements, including herbal remedies (Cardellina, 2002; Tyler,

1999), and searching for single chemically and pharmacologically active compound that

can be use as drug and may proceed through further development into Food and Drug

Administration (FDA)-approved medicines. According to Bruhn and Bohlin the

definition of pharmacognosy may proceed as a molecular science that explores

naturally occurring structureactivity relationships with a drug potential (Bruhn and

Bohlin, 1997).

1.3 Bioassay guided isolation of natural productsAs natural sources have many useful and important bioactive compounds and many have

been discovered using bioactivity directed fractionation and isolation (BDFl). The

research of pharmacognosy or isolation of natural products facilitated by newly

development of new bioassay methods. It has been found that the bioactive compounds

are mostly plant secondary metabolites, which become medicine after processing to pure

compounds; some are very useful dietary supplements, and many useful commercial

products. Further modification of the active compounds lead to enhance the biological

profiles and a large number of such compounds which are approved or undergoing

clinical trials for clinical uses against different diseases like pulmonary diseases, cancer,

HIV/AIDS, malaria, Alzheimers and other diseases (Butler., 2004; Newman et al .,

2003).Crude herbs are used as drugs in different country of the world and therefore it take

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a basic part of many traditional medicines worldwide. In Asia, traditional Chinese

medicine (TCM), Korean Chinese medicine, Japanese Chinese medicine (kampo),

ayurvedic medicine (India) and jamu (Indonesia), phytotherapy and hoemeopathy in

Europe, Alternative medicines are typically named when herbal therapies use with

various other traditional remedies in America. Integrative medicine came into being when

the alternative medicine, mainly the aforementioned traditional and folk medicines used

worldwide, with conventional medicine (Western medicine).

1.4 Medicinal plants as a source of important drug Different type of isolation methods have been used to obtain pharmacologically active

compounds that can use as drug for different diseases. The methods which includes

isolation from plants and other natural sources, combinatorial chemistry, synthetic

chemistry, and molecular modeling (Geysen et al . , 2003; Ley Baxendale, 2002 and

Lombardino and Lowe, 2004). Although there is much research in molecular modeling,

combinatorial chemistry, and other synthetic chemistry techniques which has been

funding by pharmaceutical companies and organizations, natural products which have

much complicated structural formulas and particularly medicinal plants, remain an

important source of new drugs, new chemical entities (NCEs) and new drug leads, (Butler,

2004; Newman et al ., 2000, 2003). According to survey in 2001 and 2002, approximately

one quarter of the best-selling drugs in the world were natural products or derived from

natural products (Butler, 2004). It has also been reported that approximately 28% of

NCEs between 1981 and 2002 were natural products or natural product-derived natural products (Newman et al ., 2003) and another survey during this period 20% of NCEs were

considered natural product mimics, meaning that the synthetic compound was derived

from the study of natural products (Newman et al ., 2003). On the bases of this report it

has been assumed that research on natural products accounts for approximately 48% of

the NCEs reported from 19812002.

Further more it has been known that natural products also provide a starting point for

laboratory syntheses with diverse structures and often with multiple stereo centers that

can be challenging synthetically (Koehn and Carter, 2005; Clardy and Walsh, 2004;Peterson and Overman, 2004; Nicolaou and Snyder, 2004). Natural products shows many

structural features in common (e.g., aromatic rings, chiral centers, degree of molecule

saturation, complex ring systems, and number ratio of heteroatoms) which have been

shown to be very important to drug discovery efforts ( Feher and Schmidt, 2003; Piggott

and Karuso, 2004; Clardy and Walsh, 2004; Koehn and Carter, 2005; Lee and Schneider,

2001). Many synthetic and medicinal chemists are working in the creation of natural

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product and natural-product like libraries that resembles the structural features of natural

products with the compound-generating potential of combinatorial chemistry ( Eldridge et

al ., 2002; Burke et al ., 2004; Hall et al ., 2001a; Ganesan, 2004; Tan, 2004). Some natural

products that are isolated from medicinal plants can serve not only as new drugs

themselves but can also be made useful by further necessary modification by medicinal

and synthetic chemists.

Sometime new chemical structures are very difficult to found during drug discovery from

medicinal plants, in such cases known compounds with new biological activity can provide

important drug directions. Molecular target play important rule in drug discovery, since the

sequencing of the human genome, a lot new molecular targets have been identified as

important and useful in various diseases (Kramer and Cohen, 2004). The developments

of high-throughput screening technique may show to the point and more selective activity

directed towards these targets, when use the reported compounds from medicinal plants.

It has also be known that the compounds isolated from traditionally used medicinal

plants shown to act on newly validated molecular targets, one example is indirubin,

which targeted and inhibit cyclin dependent kinases (Eisenbrand et al ., 2004; Hoessel et

al ., 1999) and another example is kamebakaurin, which has been shown to target and

inhibit NF-nB (Lee et al ., 2002; Hwang et al ., 2001). There are many known compounds

which shown to act on novel molecular targets, this development leads to produce

interest in members of these frequently isolated plant compound classes. There are many

examples but some are cucurbitacin I, from the National Cancer Institute (NCI)

Diversity Set of many known compounds and it is found to be highly selective in

inhibiting the JAK/STAT3 pathway in case of tumors with activated STAT3 (Blaskovich

et al ., 2003), another example is h-lapachone, which also selectively kills cancer cells

over normal cells by direct activation of checkpoint during the cell cycle (Li et al ., 2003),

and betulinic acid is also the same type of compound, with selective melanoma

cytotoxicity which control the cell cycle by the activation of p38 (Tan et al ., 2003;

Cichewicz and Kouzi, 2004; Pisha et al ., 1995).

According to a review article by (Balunas and Kinghorn, 2005), Four new drugs which

have been derived from medicinal plants, and have been introduced recently to the U.S.

market (Fig. 1, IIV). The drugs are, Arteether ( I , or Artemotil ) is an effective 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

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many derivatives of artemisinin which are used in Europe in different stages or clinical

trials as anti-malarial drugs (Van Agtmael et al ., 1999).

Galantamine or galanthamine ( II , Reminyl ) is a also an ethno botanical directed

isolated natural product in Russia in the early 1950s, which is first isolated from

Galanthus woronowii Losinsk. ( Amaryllidaceae ) (Pirttila et al ., 2004; Heinrich and Teoh,

2004). This compound (Galantamine) is effective in Alzheimers disease and theirfore has

been approved for the treatment of Alzheimers disease, it take part in slowing the process

of neurological degeneration through inhibiting acetylcholinesterase (AChE) and it also

well bind nicotinic acetylcholine receptor (nAChR) and modulating the same. (Pirttila et

al ., 2004; Heinrich and Teoh, 2004;).

An other compound, Nitisinone (III, or Orfadin ) is discovered very recently and has

been isolated from medicinal plant-derived, it shows a characteristic to control the rare

inherited disease, tyrosinaemia, which shows the usefulness of natural products as leadstructures (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 oftyrosine 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 lastingeffects while comparing with other available COPD medications (Barnes, 2002; Mundy

and Kirkpatrick, 2004).

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O

O

OH

OCH 2 CH 3

O

H

I Arteether

O

O

N

OH

II Galatamine

NO 2OO

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

NH 2

O

O

HOVII Exatecan

N

N

N

NH

H

OH

CO 2 CH 3

H 3 CO

H 3 CO 2 C

F

F

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.

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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 metabolitesAll those organic compounds present in plants and in animals that are not working in the

normal growth, development or reproduction of organisms but produced in different

metabolic processes. Secondary metabolites are not essential for life as compare to

primary metabolites, that the absence of secondary metabolites results not in failure of

life, but in long-term impairment of the organism's survivability/fecundity or aesthetics,

or perhaps in no significant change at all but it is useful for animals ailments andnormalizes 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 AlkaloidsAlkaloids 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

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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.

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1.5.1.2 TerpenoidsThe 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.

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

1.5.1.3 GlycosidesIt 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, toxicsubstances are often bonded to glucuronic acid to increase their water

solubility; the resulting glucuronides are then excreted.

ii) Type of glycosidic bond : It classified as -glycosides or -glycosides which

depending on bong geometry that whether the glycosidic bond lies "below" or

"above" the plane of the cyclic sugar molecule. On the bases of this particular

geometry some enzymes like -amylase can only hydrolyze -linkages; others,

like emulsin, can only affect -linkages

iii) Type of aglycone . Glycosides are also classified according to the chemicalnature 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

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some enzyme. They are stored in the vacuole but if the plant is attacked

they are released and become activated by enzymes in the cytoplasm.

These remove the sugar part of the molecule and release toxic hydrogen

cyanide. Storing them in inactive forms in the cytoplasm prevents them

from damaging the plant under normal conditions. An example of these is

amygdalin from almonds. They can also be found in the fruits (and wilting

leaves) of the rose family (including cherries, apples, plums, almonds,

peaches, apricots, raspberries, and crabapples).

Flavonoid glycosides: In this type of glycosides the aglycone units are

flavonoids e.g. Hesperidin (aglycone: Hesperetin, glycone : Rutinose),

Rutin (aglycone: Quercetin, glycone: Rutinose), Querctrin (aglycone:

Quercetin, glycone: Rhamnose).

Phenolic glycosides: The aglycone is a simple phenolic structure e.g.

Arbutin found in Arctostaphylos uva-ursi.

Saponin glycosides: The characteristic of saponin glycoside that they

normally produce soap-like foaming when shaken in aqueous medium, and

structurally saponin gycosides composed of one or more hydrophilic

glycoside moieties combined with a lipophilic triterpene derivative.

Saponin glycosides are found in liquorice ( Glycyrrhiza glabra).

Steroidal glycosides: The aglycone part is a steroidal nucleus. e.g. the

glycosides of Digitalis, Scilla and Strophanthus . These glycosides are

more effective in heart diseases.

Steviol glycosides: The glycosides found in Stevia rebaudiana bertoni and

about 300 times sweetest than sucrose. e.g. stevioside and rebaudioside A,

are used as natural sweeteners in many countries.

Thioglycosides: These glycosides contain sulfur e.g. sinigrin and sinalbin

found in black and white mustard respectively.

1.5.1.4 PhenolsPhenols or Phenolic are hydroxyl group (-OH) containing class of chemical compounds

where the (-OH) bonded directly to an aromatic hydrocarbon group. Phenol (C 6H5OH) is

considered the simplest class of this group of natural compounds. Other examples are

Resveratrol, Polyphenols (flavonoids and tannins), Gallic acid, Eugenols etc.

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1.5.1.5 PhenazinesIt 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 C 13H10 N2O 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 aquinone imine related to phenazine, and has antibiotic activity especially toward gram-

positive bacteria

1.5.2 Big small molecules

1.5.2.1 PolyketidesSecondary 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 polyketideantibiotic 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 peptidesIt 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

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from the pouring of newly added eluent or solvent. The eluent is slowly passed through

the column by opening the tap to move the component of the slurry of organic

compounds. It always useful to use a spherical eluent reservoir or an eluent-filled and

stoppered separating funnel is put on top of the column.

The stationary phase differently retained the individual components from each other and

separates them while they are running at different velocities through the column with the

eluent and therefore one compound can be elute at the end of the column at a time. A

series of fractions is collected during the entire chromatography process. The composition

of the eluent flow can be monitored thoroughly and therefore each fraction is analyzed for

dissolved compounds. For this purpose analytical chromatography, UV absorption, or

fluorescence technique can be used. Colored compounds (or fluorescent compounds with

the help of an UV lamp) can be seen through the column glass wall as moving bands.

Column chromatography divided into two phases i.e. Stationary phase or adsorbent and

mobile phase or eluent.

Stationary phase: The stationary phase or adsorbent is a solid material in column

chromatography. Mostly silica gel is used as stationary phase for column

chromatography and another is alumina which is second used stationary phase. In

the past cellulose powder has often been used. Also possible are affinity

chromatography or expanded bed adsorption (EBA) and ion exchange

chromatography, reversed-phase chromatography (RP). The finely ground

powders or gels are used as the stationary phases and/or are microporous for an

increased surface, while in EBA a fluidized bed is used.

Mobile Phase: It is either a pure solvent or of different solvents mixture. It is very

precisely studied so that the retention factor value of the compound of interest is

roughly around 0.2 - 0.3, it can be minimizing the time and the amount of eluent

to run the chromatography. The chosen of good eluent system is very important so

that the different compounds can be separated easily and effectively. The eluent

system is optimized in small scale pretests, in each case often using thin layer

chromatography (TLC) providing the same stationary phase.

The time required to run a column can be minimizes by maximizes the flow rate

of the eluent and thereby minimizes diffusion, which results a better separation,

see Van Deemter's equation for assistance. Although there are many technique to

maximize the column run rate, for example a simple laboratory column can be

runs by gravity flow which can be increased by extending the fresh eluent filled

column above the top of the stationary phase or negatively controlled with the tap

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controls. A pump can also be used for better achievement of flow rates or

compressed gas (e.g. air, nitrogen, or argon) can also be used to push the solvent

through the column (flash column chromatography) ( Still et al , 1978).

A spreadsheet that assists in the successful development of flash columns has been

developed. The spreadsheet calculate the retention volume as well as the band

volume of analytes, the fraction numbers expected to contain each analyte, and the

resolution between adjacent peaks. This information allows users to select optimal

parameters for preparative-scale separations before the flash column itself is

attempted (Fair and Kormos, 2008).

1.6.1.1.2 Planar Chromatography

Planar chromatography is also a separation technique in which the stationary phase is a

plane or present as a plane. A paper can be used as a plane, which may serves as such or

impregnated with stationary bed (paper chromatography), Glass plate can also be used on

which a layer of solid particles spread (thin layer chromatography). The traveling of

different compounds in the sample mixture travel with different velocities according to

how strongly they interact with the stationary phase as compared to the mobile phase. The

Retardation factor (R f ), 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

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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

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chromatography (RPLC). It has been known that the "normal phase" has very few

applications as compared to RPLC which has been used considerably more.

Such technique in which no pressure is used to accelerate the mobile phase through the

stationary phase are named as fast protein liquid chromatography which come under the

broad heading of chromatography.

The above mentioned chromatographic techniques are always used in phytochemistry

research. There are different other chromatographic techniques are also used e.g.,

Supercritical fluid chromatography, Affinity chromatography, Size exclusion

chromatography, Chiral chromatography, Ion exchange chromatography, Countercurrent

chromatography etc.

1.6.2 Capillary electrophoresisCapillary 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

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temperature and reaction mechanism and can also provide useful information regarding

protein and nucleic acid structure and function.

1.6.3.2 Two-Dimensional Nuclear Magnetic Resonance Spectroscopy (2DNMR)Two-dimensional NMR is useful as compared to one-dimensional NMR because the two

dimensional spectra provide more information than one dimensional spectra about a

molecule and are gives a detail information regarding the structure of a molecule,

particularly in case of molecules that are too complicated to work with using 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 Universit Libre de

Bruxelle. This work of Jean Jeener was further studied by Walter P. Aue, Enrico

Bartholdi and Richard R. Ernst, who published their work in 1976 (Martin and Zekter,

1988). There are other types of two-dimensional NMR such as exchange spectroscopy

(EXSY), J-spectroscopy, Nuclear Overhauser effect spectroscopy (NOESY), total

correlation spectroscopy (TOCSY) and heteronuclear correlation experiments, such as

HMBC, HMQC, and HSQC.

1.6.3.3 Infrared SpectroscopyInfrared 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).

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1.6.3.4 Fourier transform infrared spectroscopyFourier 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 directlyaffects the absorption in the visible ranges. Molecules undergo electronic transitions in

these ranges of the electromagnetic spectrum. This technique apposite the fluorescence

spectroscopy, in that fluorescence involved with transitions of molecule from the excited

state to the ground state, while in UV-visible spectroscopy the absorption measures

transitions from the ground state to the excited state.( Skoog, et al ., 2007).

Application

UV/Visible spectroscopy is widely used in the quantitative analysis of transition metal

ions and highly conjugated organic compounds solutions. It has also been used for the

detector for HPLC. The presence and absence of an analyte gives an indication which can

be considered to be proportional to the concentration. For perfect results, the instrument's

indication about an analyte in the unknown should be compared with the indication of a

standard; this is identical to the use of calibration curves. The response or indications

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(e.g., peak height) for a particular amount of concentration is known as the response

factor.

1.6.4. Liquid chromatography-mass spectrometryBoth 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.

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1.7 Development of Anticancer agents from Medicinal plantsIn 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 celllines 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

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been occurred. The percentage is more in developing countries i.e. 60 percent and it has

been calculated that more than half of all new cases and fatalities occurred in developing

countries. Due to poverty in development countries the poor medical infrastructure often

means that cancer is a sure-fire death sentence. The rates of survival from cancer in

developing countries are exceptionally poor. Most people do not seek medical help until

their disease is advanced and incurable; it is due to lack of awareness, stigma and reliance

on traditional healers mean. Cancer diseases are after cardiovascular diseases the second

common cause of death. Because of the dramatic development, cancer research has give

rise to a rich and complex body of knowledge. The primarily step was set in the discovery

of mutations in proto-oncogenes that produce oncogenes with dominant gain of function

(Cyclin D1 and Cdc25A described below and tumour suppressor genes with recessive loss

of function (p53 and RB describe below) (Bishop and Weinberg, 1996). This first mutation

of these degenerated cells helps them to get an advantage in proliferation and progression

compared to normal cells. Hanahan and Weinberg published few years ago The

Hallmarks of Cancer (figure 2). In this review they described six different capabilities

which each cell needs to degenerate in a malignant cancer cell.

1.8.1 Self-sufficiency in growth signalsSelf-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 thesesignals a normal cell and their receptors cannot start the proliferation machine, but many

oncogenes mimic these growth signals and initiate cell cycle on their own. For example

the epidermal growth factor receptor (EGFR) is upregulated in stomach, brain and breast

tumours. This liberation from dependence on exogenously derived signals disrupts a

critically important homeostatic mechanism that normally operates to ensure a proper

behaviour of various cell types within a tissue (Hanahan and Weinberg 2000).

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Figure 2. Acquired capabilities of cancer

Legend figure 2: Acquired capabilities of cancer. Most of cancer types have acquired the

same or near the same set of functional capabilities during their development (Hanahan

and Weinberg, 2000).

1.8.2 Insensitivity to antigrowth signalsTo 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) canstop 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,

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survival factor insufficiency, or hypoxia (Evan et al ., 1998). The effectors are regulated

by these sensors and could start, if necessary, the apoptotic machine. This hallmark has a

profound consequence, because until this step, degenerated cells could be disposed and

eliminated via the programmed cell death and the homeostasis is assured, but loss of this

function is another step for cancer development.

1.8.4 Limitless replicative potentialAll hitherto described capabilities together lead to an uncoupling of a cells growth

program from signals in its environment. After completing of a certain number of

doublings, they stop growing. Cancer cells have the ability to overcome this. They get

immortalized and thats a big advantage compared to normal cells (Hanahan and

Weinberg, 2000)

1.8.5 Sustained angiogenesis

Nutrients and other important substances are supplied by vasculature and they are crucialfor the function and survival of cells and tissue. Cells which are within 100 m of a

capillary blood vessel get nourish by this vessel. Because of this dependence on nearby

capillaries, cells within a tissue would have an intrinsic ability to encourage blood vessel

growth. To growth exuberantly and progress a lager size of tumor, cancer cells need this

ability to sustain angiogenesis. The two different types of angiogenesis initiating signals

are vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Each

of these factors binds to transmembrane tyrosine kinase receptors displayed by

endothelial cells (Fedi et al ., 1997). The ability to induce and sustain angiogenesis seems

to be acquired in a discrete step during tumour development, via an angiogenic switch

from vascular quiescence (Hanahan and Weinber

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