ZIA-UR-REHMAN MASHWANIprr.hec.gov.pk/jspui/bitstream/123456789/7173/1/Zia-ur...ZIA-UR-REHMAN...

ETHNOBOTANICAL EVALUATION AND BIOLOGICAL

SCREENING OF SELECTED MEDICINAL PLANTS FROM

GALLIYAT, WESTERN HIMALAYA, PAKISTAN

ZIA-UR-REHMAN MASHWANI

DEPARTMENT OF PLANT SCIENCES

QUAID-I-AZAM UNIVERSITY

ISLAMABAD, PAKISTAN

2015

ETHNOBOTANICAL EVALUATION AND BIOLOGICAL

SCREENING OF SELECTED MEDICINAL PLANTS FROM

GALLIYAT, WESTERN HIMALAYA, PAKISTAN

ZIA-UR-REHMAN MASHWANI

Thesis submitted to

The Department of Plant Sciences

Quaid-i-Azam University Islamabad

In the partial fulfillment of the requirements for the degree of

Doctor of Philosophy

In

Plant Sciences/ Plant Systematics and Biodiversity

DEPARTMENT OF PLANT SCIENCES

QUAID-I-AZAM UNIVERSITY

ISLAMABAD, PAKISTAN

2015

CERTIFICATE

This thesis submitted by Zia-ur-Rehman Mashwani is accepted by the Department

of Plant Sciences, Quaid-i-Azam University Islamabad as satisfying the thesis

requirement for the degree of Doctor of Philosophy in Plant Sciences (Plant Taxonomy).

Supervisor ……………………………………

Prof. Dr. Mir Ajab Khan

Co-supervisor……………………………….

Dr. Mushtaq Ahmad

External Examiner…………………………….

External Examiner……………………………

Chairperson………………………………….

Prof. Dr. Asghari Bano

Dated: …………………..

In the name of Allah, the Beneficent, the Merciful

With Him are the keys of the unseen, the treasures that none knoweth but He. He knoweth whatever there is on the earth and in the sea. Not a leaf doth fall but with His knowledge. There is not a grain in the darkness (or depths) of the earth, nor anything fresh or dry (green or withered) but is (inscribed) in a record clear (to those who can read).”

(VI. 59)

i

ACKNOWLEDGEMENT

I bow my head before Almighty Allah, The omnipotent, The omnipresent, The

merciful, The most gracious, The compassionate, The beneficent, who is the entire and

only source of every knowledge and wisdom endowed to mankind and who blessed me

with the ability to do this work. It is the blessing of Almighty Allah and His Prophet

Hazrat Muhammad (Sallallaho Alaihe Wasallam) which enabled me to achieve this goal.

I would like to take this opportunity to convey my cordial gratitude and

appreciation to my worthy and reverently supervisor Prof. Dr. Mir Ajab Khan, Ex Dean,

Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad and co-supervisor

Dr. Mushtaq Ahmad, Assistant Professor, Department of Plant Sciences, Quaid-i-Azam

University Islamabad. Without whose constant help, deep interest and vigilant guidance,

the completion of this thesis was not possible. I am really indebted to them for their

accommodative attitude, thought provoking guidance, immense intellectual input,

patience and sympathetic behavior.

.

I offer my cordial and profound thanks to Prof. Dr. Asghari Bano, Dean, Faculty

of Biological Sciences and Chairperson, Department of Plant Sciences, Q.A.U,

Islamabad, for providing me all the possible research facilities during the present studies.

Words are lacking to express my thanks and appreciation to Prof. Dr. Muhammad

Arshad, Chairman, Department of Botany, PMAS Arid Agriculture University

Rawalpindi and all other faculty member and my colleagues in the department include

Dr. Rahmattullah Qureshi (Assoc. Prof.), Dr. Abida Akram (Assoc. Prof.), Dr. Noshin

Ilyas (Assist Prof.), Dr. Naveed Iqbal Raja (Assist Prof.), Dr. Yamin Bibi (Assist Prof.),

Ms. Mubashirah Munir (Lect.), and Ms. Saira Asif (Lect.) for their accommodative

behavior, co-operation and well wishes which played a significant role in completion of

my study.

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Sincere thanks are extended to Dr. Muhammad Zafar, Herbarium Botanist,

Department of Plant Sciences, Q.A.U., for his encouraging behavior, co-operation and

support, rendered for completing my thesis.

This thesis is very much the product of my personal efforts and professional

collaboration with Dr. Iqbal Choudry Director, HEJ research institute of Chemistry,

International center for Chemical and Biological Sciences (ICCBS), University of

Karachi, Mr. Akhtar Nazman, PhD scholar, Infectious diseases Laboratory, Department

of Biotechnology, Quaid-i-Azam University, Islamabad and Dr. Samiullah, help in

performing to perform variety of biological assays.

I am thankful to Higher Education Commission (HEC) Pakistan for the financial

assistance through indigenous scholarship scheme (Batch IV) throughout my research

project.

It was all about the research friendly environment maintained by all lab fellows

during research work and I pay my heartiest thanks to Dr. Khalid Ahmad, Dr. Kifayat

Ullah, Dr. Abdul Nazir, Khalid Khattak, Fazli Rehman, Afzal, all other research scholars,

Sufyan, and Farooq for their good company. Thanks to my friends and all others for their

help and kind attitude, includes Zahid Ullah (plant identification), Dr. Arshad Mehmood

Abbassi (accompany in field trips) Mubarak Ali Khan (performed statistical analysis),

Naseer Ali Shah (data interpretation), Muhammad Ali, Naveed Alam and Muhammad

Rahim.

Last but not least, I really acknowledge and offer my heartiest gratitude to my

family. Parents are one’s first teachers and mine taught me to appreciate the immense

power, beauty and mystery of life.

Zia-ur-Rehman Mashwani

iii

Dedicated to

My supervisor

Prof. Dr. Mir Ajab Khan,

Dean (Ex.) Faculty of Biological Sciences

for his life-long contribution to the field of Plant Taxonomy in

Pakistan by initiating research in the era when others were

unacquainted and in very short resources.

iv

TABLE OF CONTENT

Acknowledgement i

Dedication iii

List of Figures vii

List of Tables x

List of Abbreviation xi

Abstract xiii

1 INTRODUCTION………………………………………………………….. 1-27

1.1 ETHNOBOTANY…………………………………………………… 1

1.1.1 Background………………………………………………………. 1

1.1.2 Ethnobotany in Global Perspective………………………………. 1

1.1.3 Ethnobotany in Pakistani Perspective…………………………….. 3

1.2 BIOLOGICAL SCREENING……………………………………… 5

1.2.1 in-vitro ANTIOXIDANT ASSAYS…………………………………. 5

1.2.1.1 Background………………………………………………………. 5

1.2.1.2 Free Radical……………………………………………………… 5

1.2.1.3 Antioxidants……………………………………………………… 6

1.2.1.4 Sources of Natural Antioxidants…………………………………. 7

1.2.2 in-vitro ANTIBACTERIAL ASSAY……………………………… 7

1.2.2.1 Background……………………………………………………….. 7

1.2.2.2 Bacterial Pathogenicity in Human and Challenges……………..... 8

1.2.2.3 Plant based Antimicrobials………………………………………. 8

1.2.2.4 Status of Antibacterial Research in Pakistan…………………….. 14

1.2.3 in-vitro ANTILEISHMANIAL ACTIVITY………………………… 16

1.2.3.1 Leishmaniasis…………………………………………………… 16

1.2.3.2 Transmission, pathogenesis and life cycle of Leishmaniasis…… 16

1.2.3.3 Herbal antileishmanial agents…………………………………….. 17

1.2.4 in-vitro ANTIGLYCATION ASSAY……………………………… 18

1.2.4.1 Background……………………………………………………… 18

1.2.4.2 Insight of Glyaction process……………………………………… 18

1.2.4.3 Plant-Derived Antiglycation agents……………………………….. 20

1.2.5 IMMUNOMODULATORY STUDIES…………………………… 21

1.2.5.1 Background……………………………………………………… 21

1.2.5.2 Plant based Immunomodulatory studies………………………… 22

1.2.6 in-vitro ANTICANCER ASSAY………………………………… 24

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1.2.6.1 Background……………………………………………………… 24

1.2.6.2 Cancer Epidemiology in Pakistan………………………………… 24

1.2.6.3 Medicinal Plants as a source of anticancer agents……………… 25

1.3 OBJECTIVE……………………………………………………………… 18

1.3.1 General Objective………………………………………………… 18

1.3.2 Specific Objective………………………………………………… 18

2 MATERIAL AND METHOD………………………………………….….19-35 2.1 Geography of Galliyat…………………………………………… 19

2.2 Ethnobotanical Documentation ……………………………….. 23

2.2.1 Plant Collection………………………………………………. 24

2.2.2 Ethnobotanical Data Preservation....………………………… 24

2.3 Biological Screening of Medicinal Plants…………………………… 25

2.3.1 Extract Preperation……………..…………………………… 25

2.3.2 ANTIBACTERIAL ASSAY …………………………………… 26

2.3.2.1 Samples preparation……………………………………… 26

2.3.2.2 Nutrient Media for bacterial growth…………………… 26

2.3.2.3 Bacterial Species…………………………………………. 26

2.3.2.4 Procedure………………………………………………. 26

2.3.3 ANTIOXIDANT ASSAYS……………………………………… 27

2.3.3.1 DPPH Free Radical Scavenging Activity……………….. 27

2.3.3.2 ABTS Radical Cation Assay……………………………. 28

2.3.3.3 Hydroxyl Radical Scavenging Activity…………………… 29

2.3.3.4 Phosphomolybdenum Assay……………………………. 30

2.3.3.5 FRAP Assay……………………………………………… 30

2.3.4 ANTILEISHMANIAL ASSAY………………………………… 31

2.3.5 ANTIGLYCATION ASSAY…………………………………….. 32

2.3.6 IMMUNOMODULATORY STUDIES………………………. 32

2.3.6.1 Preparation of luminol and Lucigenin…………………….. 32

2.3.6.2 Isolation of Human Polymorphoneutrophils (PMNs)…… 33

2.3.6.3 Chemiluminescence assay………………………………. 33

2.3.7 ANTI-CANCER ASSAY ……………………………………… 33

2.3.7.1.1 SRB assay for Human Cancer Lung Cell Line (LU-1) and

human prostate adenocarcinoma cells (LNCap-1)… 33

2.3.7.1.2 Metabolic Impairment Assays (MTT assay) for Human

Prostate Cancer Cell Line PC-3)……………… 34

2.3.8 STATISTICAL ANALYSIS…………………………………… 35

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3 RESULT…………………………………………………………………….……36-98 3.1 ETHNOBOTANICAL INVESTIGATION…………………………… 36

3.2 BIOLOGICAL SCREENING………………………………………… 58

3.2.1 Antibacterial Activity…………………………………………… 58

3.2.2 DPPH Radical Scavenging Activity……………………………… 63

3.2.3 ABTS+ Radical Scavenging Activity…………………………… 67

3.2.4 Hydroxyl Radical Scavenging Activity………………………….. 74

3.2.5 Phosphomolybdinum Assay…………………………………….. 81

3.2.6 The Ferric Reducing Ability of Plasma (FRAP)………………… 88

3.2.7 Anti-Leishmanial activity………………………………………… 89

3.2.8 Antiglycation activity…………………………………………… 91

3.2.9 Immunomodulatory studies……………………………………… 93

3.2.10 Metabolic Impairment Assays (MTT assay) for Human Prostate Cancer

Cell Line (PC-3)…………………………………………… 95

3.2.11 Sulforhodamine B (SRB) assay for Human Cancer Lung Cell Line

(LU-1) and human prostate adenocarcinoma cells (LNCap-1)… 97

4 DISCUSSION………………………………………………………………...99-113

4.1 ETHNOBOTANICAL INVESTIGATION……………………… 99

4.2 ANTIBACTERIAL ASSAY……………………………………… 103

4.3 ANTIOXIDANT ACTIVITY……………………………………… 105

4.4 ANTILEISHMANIAL ACTIVITY……………………………….. 108

4.5 ANTIGLYCATION ACTIVITY…………………………………. 109

4.6 IMMUNOMODULATORY STUDIES…………………………… 111

4.7 ANTI-CANCER ACTIVITY……………………………………… 111

5 REFRENCES………………………………………………………114-138

6 ANNEXES 139-159 Annex 1……………………………………………………………… 139

Annex 2……………………………………………………………… 141

Annex 3……………………………………………………………… 153

Annex 4……………………………………………………………… 155

Annex 5…………………………………………………………..…. 157

Annex 6……………………………………………………………… 159

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LIST OF FIGURES

Fig 1.1 Trade chain of medicinal plants showing its possible origin from Greece.. 4

Fig 1.2 describe the pathways of ROS production, the process of lipid peroxidation, the

role of glutathione (GSH) and other antioxidants……………………… 6

Fig. 1.3 Life cycle of the Leishmania parasite………………………………… 10

Fig. 1.4 Protein glycation and the formation of advanced glycation end-products

(AGEPs)……………………………………………………………… 12

Fig 1.5 An overview of the role of glycation of protein and AGEPs in the development

of diabetic complications …………………………………………… 13

Fig 1.6 represent a timeline for development of anticancerous drugs from biological

sources………………………………………………………………… 17

Fig 2.1 Location Map of the Study Area (Galliyat, Pakistan)…………………… 22

Fig 3.1 showing the distribution of different plant families of the representative

medicinal plants used in folk medicine by the local community……… 38

Fig 3.2 showing the habit-wise distribution of medicinal plants in the study area 38

Fig 3.3 showing the large contribution of medicinal plants from angiosperm utilized by

the local community…………………………………..……………… 39

Fig 3.4 showing the contribution of various parts of the medicinal plants used by the

local inhabitant in the study area……………………………………… 39

Fig 3.5 Antibacterial activity in term of measuring zone of inhibition (mm) of all

medicinal plants used in the study against Bacillus subtilus. Values are the mean

from three replicates and there standard error…………………………… 60


medicinal plants used in the study against Klebsiella pneumonia. Values are the

mean from three replicates and there standard error……………………… 60


medicinal plants used in the study against Pseudomonas aeruginosa. Values are

the mean from three replicates and there standard error………………… 61


medicinal plants used in the study against Enterococcus aerogenes Values are

the mean from three replicates and there standard error………………… 61

Fig 3.9 p-values represent the high significance between the three combination group.

Group 1, 15 min/37°C and 15 min/RT, Group 2, 60min/37°C and 60min/RT,

Group 3, 120 min/37°C and 120 min/RT……………………………… 64

Fig 3.10 Effect of temperature on the antioxidant activity (IC50) of medicinal plants

extract used in the study at different incubation period………………. 66

Fig 3.11 % scavenging activity of Geranium collinum extract on ABTS radical

scavenging activity. Values are the mean from 03 replicates. Columns with

similar alphabets are not significantly different at P < 0.05…………… 69

Fig 3.12 % scavenging activity of Persicaria barbata extract on ABTS radical

scavenging activity. Values are the mean from three replicates. Columns with


Fig 3.13 % scavenging activity of Impatiens edgeworthii extract on ABTS radical



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Fig 3.14 % scavenging activity of Rubia cordifolia extract on ABTS radical scavenging

activity. Values are the mean from three replicates. Columns with similar

alphabets are not significantly different at P < 0.05…………………… 70

Fig 3.15 % scavenging activity of Clematis grata extract on ABTS radical scavenging



Fig 3.16 % scavenging activity of Geranium wallichianum extract on ABTS radical



Fig 3.17 % scavenging activity of Berberis lycium extract on ABTS radical scavenging



Fig 3.18 % scavenging activity of Artemisia vulgaris extract on ABTS radical scavenging


alphabets are not significantly different at P < 0.05…………………….. 72

Fig 3.19 % scavenging activity of Boerhavia procumbens extract on ABTS radical



Fig 3.20 % scavenging activity of Capsella bursa-pastoris extract on ABTS radical


similar alphabets are not significantly different at P < 0.05……………… 73

Fig 3.21 % scavenging activity of Geranium collinum extract on Hydroxyl radical



Fig 3.22 % scavenging activity of Persicaria barbata extract on Hydroxyl radical



Fig 3.23 % scavenging activity of Impatiens edgeworthii extract on Hydroxyl radical



Fig 3.24 % scavenging activity of Rubia cordifolia extract on Hydroxyl radical



Fig 3.25 % scavenging activity of Clematis grata extract on Hydroxyl radical scavenging


alphabets are not significantly different at P < 0.05……………………… 78

Fig 3.26 % scavenging activity of Geranium wallichianum extract on Hydroxyl radical



Fig 3.27 % scavenging activity of Berberis lycium extract on Hydroxyl radical



Fig 3.28 % scavenging activity of Artemisia vulgaris extract on Hydroxyl radical



ix

Fig 3.29 % scavenging activity of Boerhavia procumbens extract on Hydroxyl radical



Fig 3.30 % scavenging activity of Capsella bursa-pastoris extract on Hydroxyl radical



Fig 3.31 % scavenging activity of Geranium collinum extract on Phosphomolybdinum

assay. Values are the mean from three replicates. Columns with similar alphabets

are not significantly different at P < 0.05……………………………… 83

Fig 3.32 % scavenging activity of Persicaria barbata extract on Phosphomolybdinum



Fig 3.33 % scavenging activity of Impatiens edgeworthii extract on Phosphomolybdinum



Fig 3.34 % scavenging activity of Berberis lycium extract on Phosphomolybdinum assay.

Values are the mean from three replicates. Columns with similar alphabets are

not significantly different at P < 0.05………………………………… 84

Fig 3.35 % scavenging activity of Artemisia vulgaris extract on Phosphomolybdinum



Fig 3.36 % scavenging activity of Rubia cordifolia extract on Phosphomolybdinum



Fig 3.37 % scavenging activity of Geranium wallichianum extract on

Phosphomolybdinum assay. Values are the mean from three replicates. Columns

with similar alphabets are not significantly different at P < 0.05………… 86

Fig 3.38 % scavenging activity of Boerhavia procumbens extract on



Fig 3.39 % scavenging activity of Clematis grata extract on Phosphomolybdinum assay.

Values are the mean from three replicates. Columns with similar alphabets are

not significantly different at P < 0.05…………………………………..... 87

Fig 3.40 % scavenging activity of Capsella bursa-pastoris extract on



Fig 3.41 Presenting the FRAP values of three different medicinal plant………… 88

Fig 3.42 % inhibition of L.major promastigotes at different concentration shown by the

medicinal plants used in the study…………………………………… 90

Fig 3.43 Antiglycation abilities of the medicinal plants by inhibition of formation of

AGE’s in BSA/Glucose system………………………………………… 92

Fig 3.44 Modulation of Oxidative brust from immune cell (Human

polymorphoneutrophils) by the extract of medicinal plants used in the study 94

Fig 3.45 Cytotoxicy activity profile of medicinal plants on Human prostate cancer cell

line (PC-3)…………………………………………………………… 96

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Fig 3.46 The cytotoxicity activity profile of three medicinal plants extracts for LU-1

human cell line showing the % survival……………………………… 97

Fig 3.47 The cytotoxicity activity profile of three medicinal plants extracts for LNCap-1

human cell line showing the % survival……………………………… 98

LIST OF TABLES

Table 2.1 Comparison of demographic situation and informants in the different

villages………………………………………………………………………33

Table 2.2 Plants along with their codes used in the current study…………………. 36

Table: 3.1 showing the medicinal plants used in the area and their ethnomedicinal

uses………………………………………………………………… 52

Table 3.2 Minimal Inhibitory Concentration (MIC) values of each plants against all four

bacteria used in the study…………………………………… 62

Table 3.3 IC50 values calculated for the medicinal plants and Ascorbic acid (standard) at

varying temperature and incubation period (min)………………… 66

Table 3.4 IC50 values calculated for all plants and standard ascorbic acid in ABTS+

radical scavenging activity…………………………………………… 68

Table 3.5 IC50 values calculated for all plants and standard ascorbic acid in Hydroxyl

radical scavenging activity………………………………………… 75

Table 3.6 IC50 values calculated for all plants and standard ascorbic acid in

Phosphomolybdinum assay………………………………………… 82

Table 3.7 Antileishmanial Activity of medicinal plants in term of IC50 along with the

Glucantime (standard)………………………………………………… 90

Table 3.8 Inhibitory capacities of the extracts of the medicinal plants on the formation of

Advanced Glycation Endproduct (AGE’s)…………………………… 92

Table 3.9 Immunomodulatory activity of medicinal plants in term of their IC50… 94

Table 3.10 IC50 values of medicinal plants representing their inhibitory action on Human

prostate cancer cell line (PC-3)……………………………………… 96

Table 3.11 IC50 values for medicinal plants for Human cell line LU-1 and LNCap-1…98

xi

LIST OF ABBREVIATIONS

Abbreviations Full Name

μg Micro gram

μl Micro liter

٠CCl3 Trichloromethyl radical

b.w Body weight

β Beta

AGEs Advanced Glycation End-Product

AV Artemisia vulgaris

BaCl2 Barium chloride

BaSO4 Barium sulphate

BL Berberis lycium

BHT Butylated hydroxyl toluene

BSA Bovine serum albumin

°C. Centigrade

CBp Capsella bursa-pastoris

CFU Colony Forming Unit

CG Clematis grata

CuSO4 Copper sulphate

dH2O Distilled water

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DPA Diphenylamine

DPPH 1, 1-diphenyl-2-picryl-hydrazyl

Fe Iron

Fe2Cl3.6H2O Hydrated Ferric chloride

FeCl3 Ferrous chloride

FeSO4-EDTA Ferrous sulphate EDTA

FRAP Ferric Reducing Ability of Plasma

g Gram

GA Gallic acid

GC Geranium collinum

GW Geranium wallichianum

HBSS++ Hank’s balance salt solution

HCl Hydrochloric acid

H2O2 Hydrogen peroxide

H2SO4 Sulphuric acid

HCl Hydrochloric acid

BHA Hydroxyanisole

IC50 Fifty percent inhibition concentration

IE Impatiens edgeworthii

LD50 Fifty percent death

LDL Low density lipoprotein

LNCaP Human prostate cancer cell line

xii

LSD Least significant difference

LU 1 Human lung carcinoma

M Molar

mg milli gram

MgCl2 Magnesium chloride

MIC Minimum inhibition concentration

mL milliliter

mm millimeter

mM Milli molar

MTT 3-(4, 5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazoliumbromide

N Normal

Na2CO3 Sodium carbonate

NaCl Sodium chloride

NAD Nicotinamide adenine dinucleotide

NADP Nicotinamide adinine dinucleotide phosphate

reduced

NADPH Nicotinamide adinine dinucleotide phosphate

NaH2PO4 Sodium dihydrogen phosphate

NaOH Sodium hydroxide

NBT Nitroblue tetrazolium

NH4OH Ammonium hydroxide

NO Nitric oxide

O2 Superoxide

OD Optical density

OH Hydroxyl

PBS Phosphate buffer

PC3 Prostate cancer cells

PB Persicaria barbata

pH Log of hydrogen ion conc.

PMNs Polymorhoneutrophils

RBCs Red blood cells

RC Rubia cordifolia

ROO• Peroxy radicals

ROS Reactive oxygen species

RLU Relative Luminescence Unit

RT Rutin

SRB Sulforhodamine B

TBA Thiobarbituric acid

TBARS Thiobarbituric acid reactive substances

TCA Trichloroacetic acid

TLC Thin layer chromatography

TPC Total phenolic contents

TPTZ 2,4,6-tripyridyl-s-triazine

UV Ultra violet

WHO World health organization

xiii

ABSTRACT

The present project was conducted with aim to provide information on

ethnomedicinal uses, biological screening, antioxidant potential, antileishmanial,

antiglycation, immunomodulatory and anticancer activates of the medicinal plants from

Galliyat region of western Himalaya, Pakistan.

The present study provides information on the ethnobotanical uses of medicinal

plants in the Galliyat area of Western Himalaya, Pakistan. This study includes 45

medicinal plants including 38 angiosperms, 4 gymosperms and 3 pteridophytes. The

inhabitants of the area utilized different parts of the plants for the medication purposes

involve; leaves (19 sp.), root (11 sp.), fruit (7 sp.), flower (7 sp.), bark (6 sp.), seed (5

sp.), aerial parts (4 sp.), whole plants (3 sp.), rhizome (3 sp.) and wood (2 sp.). Major

source of the ethnobotanical data were old age peoples and traditional practitioners.

Women have more knowledge as compared to the males.

The biological screenings of medicinal plants were carried out for 10 selected

medicinal plants include: Geranium collinum Steph. ex Willd., Persicaria barbata (L.)

Hara, Impatiens edgeworthii Hook. f., Rubia cordifolia L., Clematis grata Wall.,

Geranium wallichianum D.Don ex Sweet., Berberis lycium Royle., Artemisia vulgaris L.,

Boerhavia procumbens Banks ex Roxb., and Capsella bursa-pastoris (L.) Medic.

Antibacterial activities of selected medicinal plants showed different response against

bacterial species. Geranium collinum exhibited the maximum antibacterial activity

against Klebsiella pneumoniae and Enterococcus aerogenes respectively. While

Persicaria barbata showed the maximum activity against Bacillus subtilus and

Pseudomonas aeruginosa respectively.

Antioxidant potential of these medicinal plants was investigated by using multiple

approaches include DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging assay at

modified experimental condition. Six conditions (15 min/25°C, 60 min/25°C, 120

min/25°C, 15 min/37°C, 60 min/37°C and 120 min/37°C) were selected. Higher activity

was observed at 120min > 60min > 15min represent the time dependent phenomenon.

Temperature also represents a significant impact on the antioxidant activity. However

more antioxidant activity was observed at Human body temperature (37°C) as compared

to room temperature (25°C). In ABTS (2, 2-azinobis (3-ethylbenzothiazoneline-6-

xiv

sulphonic acid) radical scavenging assay Geranium collinum represent the best activity.

Hydroxyl radical scavenging capacity produced very good result as majority of plants

produced significant results include Geranium collinum, Persicaria barbata, Clematis

grata, and Rubia cordifolia. During phosphomolybdinum assay, Boerhavia procumbens,

Artemisia vulgaris, Berberis lycium, Capsella bursa-pastoris, Persicaria barbata, and

Rubia cordifolia showed the significant results. Persicaria barbata exhibited the best

result for FRAP (Ferric ion reducing antioxidant power) assay.

The medicinal plants screened in antileishmanial assay produced interesting

results against Leishmania major. All plants produced highly significant results except

the Capsella bursa-pastoris which showed closed response to Glucantime (standard

drug). Antiglycation capabilities of the medicinal plants revealed that Persicaria barbata,

Geranium collinum and Berberis lycium have significant potential to inhibit the

formation of the advanced glycation end-products. In immunomodulatory studies

Geranium collinum, Artemisia vulgaris, Boerhavia procumbens, Capsella bursa-pastoris,

Clematis grata, Rubia cordifolia, and Persicaria barbata showed the significant

immunomodulatory effects.

Geranium collinum, Impatiens edgeworthi and Rubia cordifolia showed the

significant activity against human prostate cancer cell line (PC 3). Geranium collinum

was considered to present the significant result against the Human lung carcinoma (LU-

1). In-case of human prostate adenocarcinoma (LNCaP-1), Geranium collinum and

Geranium wallichianum produced the significant results.

It is concluded that Geranium collinum, Persicaria barbata and Geranium

wallichianum showed the best results of all performed activities in comparison to other

plants in the study. Geranium collinum enjoy the least literature background and all the

activities were performed for the first time for this plant. In some experiments it

displayed better response than reference compound used in the study. Many of the

biological activities were performed for the first time in case of various plants. The

potential of medicinal plants involving the aspects of ethnomedicinal investigation,

antioxidant activities, antiglycation assay, antileishmanial assay, immunomodulatory

studies and anticancer studies would prove to be useful for pharmaceutical industry,

students and scientific community for further research in this area.

Chapter: 1

INTRODCUTION

Chapter 1 Introduction

1

ETHNOBOTANY

1.1.1 BACKGROUND

Ethnobotany as the largest sub-discipline of ethnobiology, is the functional

relationship between humans and the plant environment in primitive settings

(Harshberger, 1896). With the passage of time, this field showed a comprehensive

evolution to cover functional association, ecological, cognitive, symbolic, and human–

plant relationship in a contemporary situation (Schultes and von Reis, 1995).

Development of Ethnobotany started from the 1st International Congress of Ethno-

biology (ICE) in 1988 held at Belem (Brazil), which was resulting in “Declaration of

Belem”. This declaration contains the Code of Ethics, which was later on adopted by the

International Society of Ethno-biology in 2005. In 1992, Legislation was developed

internationally to protect the intellectual property of various cultures and individuals with

respect to their knowledge about the plant usage. This legal framework also encompasses

about the various biological materials refer to any geographic or political territory. These

legislations were implemented by the United Nations in the form of UN convention on

Biodiversity (Secretariat of Convention on Biological Diversity, 2001).

Any ethnobotanical study covers these basic aspects:

i. Accumulation of ethnobotanical knowledge for conservation of

biodiversity and human civilization.

ii. Accumulation of ethnobotanical knowledge for agriculture, forestry and

for pharmaceutical industry.

iii. The synthesis and application of "I” and “II” aspects of biodiversity and

human endurance.

1.1.2 ETHNOBOTANY IN GLOBAL PERSPECTIVE

Plants used as medicine can be traced back over five millennia to prehistoric

documents of earlier cultures like in Egypt, China and India (Hill, 1952), but it is

definitely as old as humankind (Hamburger and Hostettmann, 1991). Initially these

medicinal plants were mainly consumed in the form of crude drug like, powders, teas,

tinctures and other formulations (Balick and Cox, 1997).


2

Use of medicinal plants in the primary health care system has an important

contribution and considered to be the major source of medicine for many populations.

Medicinal plants have not only the medicinal uses, but locally, they can too be

consumed for nutritional and ritual purposes (Abbink, 1995). The use of medicinal plants

in the traditional healing practices differs across various cultures. These indigenous

people acquired such knowledge from their elders. Hence such knowledge may have

limited authenticity. It is also said that this knowledge was acquired as part of the

doctrine of signatures (Bennett, 2007), which states that medicinal plants which show

some resemblances with the part of the body might be used for the treatment of that

organ/parts. The increased costs of conventional allopathic medication and

unapproachability of these facilities in a rural set-up compelled the local population to

shift toward the medicinal plants. There are estimated 35-70 thousand plant species

utilized in traditional medicinal system globally (Lewington, 1990; Fransworth &

Soejarto, 1991).

80% of the world population mainly rely on the traditional medicine as per World

Health Organization (WHO), particularly in rising countries, resources to build up a

primary health care arrangement are still lacking (WHO, 2001). Global annual market

value is close to USD 43 billion for medical specialty based on the medicinal plants

consumed by the local peoples (Elisabetsky, 1990). In the United States, data extracted

from the prescriptions assigned by community pharmacies during 1959-1980. 25% of the

prescriptions include plant extracts or their active constituents. These include 119

chemical constituents originated from 90 medicinal plants (Farnsworth et al., 1985).

Lange (2006) reported that during 1991-2003, annual trade of medicinal plants

was average 467,000 tons and a few countries dominate this deal. According to WHO

(2011), the world market for traditional medicine in 2008 was estimated to be worth USD

83 billion. Estimates indicate that global medicinal plant business will touch USD 5

trillion by 2050 (Shinwari, 2010).


3

Thither are 422,000 seed plants reported globally (Govaerts, 2001), out of which

approximately 50,000 (Schippmann, et al., 2002) or 53,000 (Hamilton, 2004) flowering

plants are utilized for medicinal uses. According to Schultes (1978) total isolated

secondary metabolites estimated to be 12,000. At least 119 chemical substances from 90

plant species are important drugs used all over the globe. Many of them contain

compounds derived from or modeled after naturally occurring lead molecules (Cox,

2000), approximately 74 % of the medicinal plant information comes from the traditional

use of medicinal plants (Farnsworth et al., 1985). 11% of the total 252 drugs declared

basic and essential by WHO is exclusively from plant origin (Rates, 2001). Among

anticancer and anti-infective drug which already in the market or under trial, 60% are of

natural origin (Shu, 1998).

1.1.3 ETHNOBOTANY IN PAKISTANI PERSPECTIVE

Pakistan has more than 6,000 plant species of phanerogams (Ali &Qaiser, 1986), out

of which about 400–600 were regarded as important medicinal plants (Hamayaun et al.,

2005). The Himalayan mountainous range consists of 18% land cover in the Indo-Pak

subcontinent, reported to have about eight thousands plant species of flowering plants

and about 22% (about 1748 plants) were known to be medicinal value (Kala, 2005). In

Himalaya about 70% of the medicinal flora consist of wild species, 70-80% of the human

population in the region rely on traditional usage of medicinal plants for primary health

care (Pie and Manandhara, 1987).

Traditional knowledge about the medicinal plants considered to be part of culture

in Pakistan, as it is practiced among the major component of its population (Qureshi et

al., 2009a). This knowledge is considered to be originated from Greece (Fig. 1.1), where

the oldest ancient Greek philosophers and Muslims during the era of Islamic civilization

were regarded as founders. Muslim scholar brought this knowledge to the subcontinent

and also exercised for hundreds. This knowledge mainly depends upon the use of

medicinal plants. Ethnic and cultural diversity, varied climate and rich medicinal plant

resources made Pakistan as an area of interest in term of ethno-medicinal knowledge of

plants (Shinwari and Khan, 2000).


4

Source: Shinwari & Qaisar (2011),Pak. J. Bot., 43: 5-10SI

Fig 1.1: Trade chain of medicinal plants showing its possible origin from Greece.

In early 1950’s, 84% of various populations in Pakistan were dependent on the

indigenous remedies from medicinal plants (Hocking, 1958). But, Ahmad (2003)

reported that approx. 10% of the persons involved in the collection of medicinal plants as

primary profession and about 30% peoples were involved in secondary occupation.

Medicinal plants collected by peoples also contribute about 600 species as the major Non

Timber forest Products (Shinwari, 2010). About 500 medicinal plants were reported to be

commonly used in primary health care practices and some 350 plants were traded to

International and national market to earn millions of US dollars (Ahmad, 2003).

The field of ethnobotany in Pakistan was considered to be virgin in the late 20th

century (Shinwari and Khan, 2000) but with the onset of the 21st century this field gains

much attraction from the researchers and this popularity can be traced by the huge

number of research reports published on the indigenous knowledge of medicinal plants

by the various rural communities in Pakistan.


5

1.2 BIOLOGICAL SCREENING

1.2.1 in-vitro ANTIOXIDANT ASSAYS

1.2.1.1 Background

Free radical Biochemistry was led to birth directly after the World War II (1935-

45), when two nuclear bombs (Hiroshima and Nagasaki) led to enormous disaster in

terms of deaths of complete population, and the people who took to survive were with a

shorter life span. Gerschman et al., (1954) speculated that this disastrous effect of those

ionizing radiation led to the formation of free radicals (Reactive oxygen species). This

idea serves as innovation for the following scientist imaginations.

1.2.1.2 Free Radical

Free radicals are the molecules or the fragments of the molecules have one or

more free electrons in atomic or molecular orbital have unpaired valence electrons or an

open electron shell (Halliwell & Gutteridge, 1999). These free electron(s) gave a

significant level of reactivity to these atoms or molecules. Free radicals of O2 form the

important group of the free radicals formed in the living organism (Miller et al., 1990).

Normal cellular metabolism resulted in the formation of reactive oxygen species (ROS)

and reactive nitrogen species (RNS) normally. Both of these are well known for their

dual function either as deleterious or beneficial (Valko et al., 2006). At low and

moderate concentrations the role of ROS considered to be beneficial, plays various

physiological roles in cellular responses (Fig 1.2). The damaging effect of these free

radicals can cause biological damage and is known as oxidative stress or nitrosative stress

(Ridnour et al., 2005; Valko et al., 2001; Kovacic & Jacintho, 2001).

Important free radicals in biological systems consist of nitric oxide (NO·), peroxyl

(RO2·), hydroxyl (OH-) and superoxide (O2·-). Hypochlorous acid (HOCl), peroxynitrite

(ONOO-), singlet oxygen 1O2, hydrogen peroxide (H2O2) and ozone (O3) are not free

radicals but lead to the formation of free-radical formation in biological system.


6

Source: M. Valko et al. The Int. J. of Biochem. & Cell Bio. 39 (2007) 44–84

Fig 1.2 Description of the pathways of ROS production, the process of lipid peroxidation,

the role of glutathione (GSH) and other antioxidants .

Free radicals caused many human diseases. The defense mechanism of human

beings are always vulnerable due to significant account of free radicals from outer world

(Hughes, 2000).

1.2.1.3 Antioxidants

Substances which can inhibit the oxidation of other molecules by free radicals are

regarded as antioxidants (Sies, 1996) and biological antioxidant is a substance which


7

prevents or delays the process of oxidation when available in low concentration in

comparison to oxidizable substance (Halliwell and Gutteridge, 1995).

1.2.1.4 Sources of Natural Antioxidants

Most natural antioxidants are from plant species which contain compounds that

possess antioxidant activity (Prat, 1992). Antioxidant compounds are synthesized in

plants as secondary metabolites which serve as defense mechanism and neutralized the

reactive oxygen species (ROS) to prevent the oxidative damage. Phenolics contribute a

number of antioxidant actions like hydrogen donation, free-radical scavenging, metal ion

chelation, singlet oxygen quenching, and also act as substrate for hydroxyl and

superoxide. Hence, the plants antioxidant activities were mainly correlated with their

corresponding phenolic compounds (Velioglu et al., 1998). These occur in various parts

in plants like leaves, fruit, stem, bark, root, pollen, seeds and flowers. Flavonoids e.g.,

catechins, flavones, isoflavones, flavonols and falvanones, coumarins and polyfuntional

organic acids, cinnamic acid derivatives were considered to be the most commonly

occurring natural antioxidants (Prat, 1992). In the food industries, medicinal plants can

serve as a useful alternative to synthetic antioxidants in food processing. Hence, the

demand of studies to evaluate plants essential oil and their extracts with potential

antioxidant activities is growing day-by-day (Rasooli, 2007).

1.2.2 in-vitro ANTIBACTERIAL ASSAY

1.2.2.1 Background

Human discovered the presence of micro-organisms long ago, and it was known

to the people of certain civilizations that there were certain plants which had therapeutic

potential against these microbes, which is now-a-days considered to be the antimicrobials

(Maciel et al., 2002). Out of the total plants species existed on the earth were estimated to

be 250,000-500,000, only a small fraction (1-10%) were known to be consumed as food

by human beings and animals (Cowan, 1999). Medicinal plants with antiseptic properties

were well recognized since ancient times. Medicinal plants were screened and


8

characterized for their potent activities traced back to the 20th century (Hoffman & Evans

1911; Martindle 1910).

1.2.2.2 Bacterial Pathogenicity in Human and Challenges

Primary cause of the early death is the infectious diseases around the globe with a

rate of more than 50 thousands of killings per day particularly in developing countries

(Davis, 1994). WHO (2002) reported that in the industrialized nations more than 30% of

the persons suffer from food-borne diseases in each year and at least two million people

were died of diarrhea in 2000 worldwide. Many pathogenic microorganisms, such as

Klebsiella pneumonia, Pseudomonas aerigenosa, and Enterococcus Sp. were known to

be reasons of these food borne complications and diseases (Deak & Beuchat, 1996). In

recent years, the resistance to the drugs used against these diseases causing bacterial

strains become increasing in world over (Piddock and Wise, 1989) and this happens due

to the indiscriminate uses of antibiotics.

Regardless of the fact that a huge investment was done for the development and

production of antibiotics but to combat the bacterial diseases are still major goal in the

medicine because of the indiscriminate use of conventional drugs result into the

emergence of resistant bacterial strains (Kenneth and George, 2004), this resistance to

drugs now becoming more important and leading toward a global issue (Westh et al.,

2004). Companies involving in pharmaceutical formulations were now motivated to

invest and develop new drugs against this current challenges (Nascimento et al., 2000). In

the United States hospitals, out of 2 million patients infected with bacterial diseases about

70% patients were resistant to those drug which were used in treatment earlier (Anon,

2004)

1.2.2.3 Plant based Antimicrobials

Medicinal plants used in the treatment of diseases triggered the production of

various medicines (Iwu, 2002; Tyler, 1997). These medicinal plants were considered to

be the large unexploited sources of antimicrobial agents have massive therapeutic

potential (Cowan, 1999; Chopra et al., 1992), because these are used not only to stop the


9

growth of disease causing agents but also to kill them without showing any side-effect

(Cohen, 1992). Antimicrobial studies on plants have evolved from multidisciplinary

investigations (Rabeet al., 2002; Eldeen et al., 2006).

For this purpose plants were screened for their potential antibacterial activity, and

certain plants showed the high level of inhibition were regarded to be the novel source of

antibiotic model (Afolayan, 2003). The diversity of plant species and compounds they

contained evoked researchers to screening wide variety of plants with the intention of

getting clinically significant drugs, with different biological activities and novel

structures (Belay et al., 2011).

Plant derived secondary metabolites were considered to be the best candidate for

control of bacterial pathogens (Raghavendra, et al., 2006). These secondary metabolite

belongs to the various groups of the phytochemicals and showed inhibition against the

variety of microbe in-vitro (Cowan, 1999). Among these phytochemicals, flavonoids

recently gained the interest of researcher in the field of medicine because of their

beneficial properties including antimicrobial activities (Harborne and Baxter, 1999;

Havsteen, 1983). The component of flavonoids like flavones are reported to be the reason

of the plant antimicrobial activity (Tsao et al., 1982)

1.2.3 in-vitro ANTILEISHMANIAL ACTIVITY

1.2.3.1 Leishmaniasis

Leishmaniasis, a vector borne disease caused by the several parasitic species of

Leishmania, infects human being and mammals (Gilles, 1999). Human leishmaniasis is

caused by over 20 different Leishmania species and sub-species that belong to the genus

Leishmania. The disease affects approximately 12 million people from 88 countries, with

an increasing prevalence of 1.5-2.0 million newly diagnosed cases every year and 350

million people at risk. It is manifested mainly in three clinical forms: cutaneous, muco-

cutaneous and visceral leishmaniasis (WHO/TDR, 2012, Singh and Sivakumar, 2004).


10

1.2.3.2 Transmission, pathogenesis and life cycle of Leishmaniasis

Leishmaniasis is transmitted while Leishmania infected female sandflies take

blood from healthy people (Fig 1.3). Two transmission ways are possible; zoonotic-in

case when parasite is transferred from infected non-human reservoir host to healthy

individuals (accidental hosts) or anthroponotic-where humans are the sole reservoir hosts.

Congenital, parenteral and sexual transmissions are also suspected (Gilles, 1999, Davies

et al., 2003).

The drug used for the treatment like amphotericin B (Bristol Myers Squibb) and

Glutamine (Aventus) was reported to have toxicity and also there were reports of

resistance against these drugs. Other problems with these include their cost and lengthy

treatment periods which make them ineffective with the passage of time (Lima et al.,

2010; Clements and Peacock, 1990; Gallis et al., 1990 Katlama et al., 1985).

Figure 1.3 :Life cycle of the Leishmania parasite.


11

1.2.3.3 Herbal antileishmanial agents

Several researches have conducted on the antileishmanial activity of crude extracts,

essential oil, or chemically defined compounds derived from plants in-vitro against

promastigotes and amastigotes or in-vivo against Leishmania infected animals.

Ethnobotanical background of medicinal plants forms a firm base for their

screening against Leishmania parasite. Sawadogo et al., (2011) screened five ethno-

medicinal plants from Burkina Faso used against Leishmania donovani to determine their

potential antileishmanial activity. The same study was conducted in Benin for 10

different traditional medicinal plants to treat parasitic infections. (Bero et al., 2011), eight

traditional Peruvian medicinal plants species were screed against Leishmania. (Gonzalez-

Coloma et al., 2012). Tempone et al., (2008) used 16 plants selected on ethnomedicinal

grounds were analyzed Leishmania chagasi, L. amazonensis. 64 Malian traditional

medicinal plants extracts were used in assay against Leishmania major (Ahuaet al.,

2007). On the other hand, some authors have investigated antileishmanial potential of

single medicinal plant. Sadeghi-Nejad et al., (2011) used ethanolic and methanolic leaf

extracts of Satureja khuzestanica and different extracts from three Cuban Pluchea

species (García, M. et al., 2011) against Leishmania major promastigotes.

Essential oil extracted from certain plants also been reported to exhibit promising

antileishmanial activity. Essential oil of Artemisia absinthium and Echinops kebericho

against Leishmania aethiopica and L.donovani) (Tariku, Y. et al., 2011), Essential oil

from Chenopodium ambrosioides against Leishmania donovani (Monzote et al., 2007),

Ocimum gratissimum essential oil produced mainly eugenol progressively inhibited

Leishmania amazonensis growth. (Ueda-Nakamura, et al., 2006)

1.2.4 in-vitroANTIGLYCATION ASSAY

1.2.4.1 Background

Diabetes mellitus, an endocrine gland disorder characterized by chronic

hyperglycemia, resulted from an absolute or relative deficiency of or resistance to insulin.


12

It affects 1–2% of the population, which accounts about 100 million diabetic patients

globally and this may be doubled after the next 10-15 years (Nathan, 1993).

1.2.4.2 Insight of Glycation process

Hyperglycemia involves a direct reaction, Maillard or browning reaction, takes

place between body structural proteins & sugars. Protein glycation is the initial step of

the Maillard reaction, also known as nonenzymatic glycosylation (Ahmad & Ahmed,

2006).

Source: Ahmad & Ahmed (2006). The Journal of nutrition, 136(3), 796S-799S.

Figure 1.4 Protein glycation and the formation of advanced glycation end-products (AGEs)

As schematically illustrated in Fig 1.5, Proteins were initially non-enzymatically

combined with glucose to form glycated proteins. These glycated proteins by reacting

with the dicarbonyl derrivatives like 3-deoxyglucosone which resulted in complex cross-

linked, fluorescent and heterogeneous molecule called advanced glycation end-products

(AGEs) (Sell & Monnier, 1989). These amadori products and glucose also followed

various processes of auto-oxidation and produced the free radicals (Hunt et al., 1993).

Those free radicals destroy the proteins, nucleic acid and lipids and also take part in

tissue destruction as a symptom of diabetes (Bonnefont-Rouselot, 2002).


13

The increased process of glycation produces the AGEs which turn to accumulate

in the tissues follow the conformational changes by changing enzyme activity resulted in

altering the half-life of proteins, immunogenicity and cross linking protiens (Vlasara and

Palace, 2002). Hence, the AGEs accumulation resulted in oxidative stress (Fig 1.5) which

plays a role in pathology of diabetes (Ahmed, 2005).

Source: Ahmed, 2005. Diabetes Res. Clin. Pract. 67:3–21.

Fig 1.5 An overview of the role of glycation of protein and AGEPs in the development of

diabetic complications

1.2.4.3 Plant-Derived Antiglycation Agents

The plant-derived anti-glycative compounds appear attractive candidates for the

improvement of new generation therapeutics for the treatment of diabetic complications

and prophylaxis of aging, and points to the importance of an antioxidant-rich diet, as part

of the overall diabetes management strategy (Odjakova et al., 2012). Synthetic anti-

glycation compound, Aminoguanidine (AG), which prevents the formation of AGEs was


14

withdrawn from the crucial phase III of clinical trials because of safety concerns and

apparent lack of efficacy (Thornalley, 2003). The anti-glycation capacity of numerous

medicinal herbs and dietary plants was comparable with (Ardestani and Yazdanparast,

2007), or even stronger than that of aminoguanidine (Jang et al., 2010; Tsuji-Naito etl.,

2009; Farsi et al., 2008).

Polyphenols are the most abundant dietary antioxidants (Landis-Piwowar et al.,

2007). Several studies revealed that polyphenols significantly correlates with the phenolic

content of a plant (Gugliucci et al., 2009; Peng et al., 2008; Ardestani and Yazdanparast,

2007). In microalgae this ability to inhibit AGEs is different from other plants and is not

promoted by polyphenols (Sun et al., 2010). Polyunsaturated fatty acids and carotenoids

act as an anti-glycation agent in that case (Chen,, 1996). Recently, Polysaccharides got

much attention because of their unique biological activities (Schepetkin and Quinn, 2006)

and was found to be strong radical scavenger (Yang et al., 2008)

In traditional medicinal systems, variety of medicinal plants used as herbal drugs

for the treatment of diabetes. Hence, medicinal plants used in diabetes treatment were

reported to screened for their anti-glycation potential globally, like in China (Chen et al.,

2011; Wang, 2009; Xi et al., 2008; Tang et al., 2004) and in India (Gupta et al., 2008). In

recent years, this trend to screen the medicinal plants especially with traditional

knowledge background are utilize for diabetes.

1.2.5 IMMUNOMODULATORY STUDIES

1.2.5.1 Background

The vertebrates have been benefitted with a remarkable defense mechanism in

order to protect the body from the foreign invading agents and cancer, known as immune

system (Chandra, 1997). This system comprised of two components, non-specific or

innate and specific or adaptive system. Earlier one is considered to be the 1st line of

defense and later as 2nd line of defense to protect the body in case of re-exposure to the

same agent (Mayer, 2006). Immunomodulation refers to any process in which an immune

response is altered to a desired level (Calne, 1960). This process may be specific (one


15

gene or agent) or it may be non-specific (impact on entire immune system response)

(Sell, 1987). The concept of immunomodulation was developed by Jenner in 1796, when

he undertook the first vaccination (Baron, 1838). Hence, researchers started to screen the

therapeutic agents/therapies which could help the immune response but it took one

century to achieve this landmark.

Macrophage cells, peripheral blood mononuclear and polymorphonuclear

neutrophils form the first line of defense to eliminate the invading agent/microbes.

Phagocytosis also plays an important role in the defense mechanism accomplished in

multiple steps during infection (Beutler, 2004). Sometime, it also contributes in the

pathology of various immunological disorders after establishing the phagocytes and

microbes interaction. Therefore, various agents can be used to inhibit this process of

phagocytes coupled with ROS production as a step to treat a variety of immune disorders

(Afonsoet al., 2007).

1.2.5.2 Plant based Immunomodulatory studies

Medicinal plants can serve as an alternate choice in chemotherapy of different

disorders due to their potential immunomodulatory effect. This effect has been

considered to be of much pronounced in case of host-defense mechanism activation

during the phase of weaken immune responses or during autoimmune complication

where immunosuppression requires selectively (Ganjuet al., 2003). Many herbal

preparations were reportedly shown to have impact on changing immune response and

also known to have immunomodulatory activity.

Plant extracts with potiential immunomodulatory activities have been widely

investigated in this time in various parts of the world. Plant used for their

immunomodulatory activity include like Adhatoda vasica (Vinothapooshan and Sundar,

2011), Ficus carica (Patil et al., 2010), Cissampelos pareira (Bafna and Mishra, 2010),

Mangifera indica (Makare et al., 2001). There are a number of natural agents (herbs)

which are used for the enhancing of the body’s response to disease.


16

1.2.6 in-vitro ANTICANCER ASSAY

1.2.6.1 Background

Cancer is a key public health hazard in both developing and non-developing part

of the world (WHO 2004). In United States, it was considered as the second major cause

of mortality (Hoyer et al., 2005), where ¼ of deaths occured due to cancer. It may be

caused by incorrect diet, genetic predisposition and through the environment (Reddya et

al., 2003). Carcinoma of the breast, colon and prostate carcinoma were highly prevalent

malignancies in the Western nations. Approximately half of the total death in Western

countries accounted for cancer-related among men and women (Jemal et al., 2010). It

was documented that 12.7 million cancer cases and 7.6 million cancer deaths around the

world in 2008. Breast cancer among females and lung cancer in males were considered as

the most frequent diagnosed cancer (Jemal et al., 2011)

Mutagenic events coupled with random events of mutations and selection derived

cancer. Nowell (1976) first time described that cancer was an evolutionary process and

his views were supported broadly after three decades of research (Crespi and Sammer,

2005; Merlo et al., 2006).

1.2.6.2 Medicinal Plants as a source of anticancer agents

Cancer was treated with medicinal plants have a long history (Hartwell, 1982).

There are so many anticancer agents with good efficacy were derived from the plant

sources. This can be proved by the fact that currently available 60% of the anticancerous

drugs were previously derived from the biological sources included medicinal plants,

microorganisms and marine biota (Cragg et al., 2005; Newman et al., 2003).

The search of novel anticancer agents from the plants sources starts with the

discovery of Vinca alkaloids (vincristine and vinblastine) and podophyllotoxin in early

1950s. The major initiative was taken up by National Cancer Institute (NCI) in the United

States during early 1960 to collect the medicinal plants extensively, with major focus on

temperate region of the world. NCI reported large number of novel therapeutic agents

from medicinal plants after screening with wide range of cytotoxic effect (Cassady and


17

Douros, 1980) but, it took almost 30 years (1960s-1990s) to being passed from the

clinical trials. Due to some reasons this program by NCI was stopped in 1982 and re-

launched in 1986. This time the major focus was the sub-tropical and tropical area

globally.

About 1,00,000 chemotypes were screened in the 50 years but Food and Drug

Administration (FDA) approved only 7 anticancerous agents derived from the plant

sources after successful result in clinical trials (Figure 1.6).

Source: Ma and Wang. 2009. Drug Discovery Today . 14(23/24)

Fig 1.6 Represent a timeline for the development of anticancerous drugs from

biological sources

About 35,000 medicinal plants with 114,000 extracts from 20 different countries

were screened by the NCI for their therapeutic potential against cancer (Cragg and Boyd,

1996). In United States, 92 commercial drugs were available for the treatment of cancer

from natural sources and globally there were 62% anticancer drugs from natural origin

were available during 1983-94 (Cragg et al., 1997).


18

1.3 OBJECTIVE

1.3.1 General Objectives

To collect and preserve ethnobotanical knowledge of the medicinal plants in the

biodiversity rich selected study area of Western Himalaya, Pakistan.

To screen some selected Ethnobotanicals for their biological activities as an

approach to drug discovery.

1.3.2 Specific Objectives

To gather and validate the Ethnobotanical information on medicinal plants

To seek out the source of natural antioxidants

To place plants with antibacterial agents that deal with microbial resistance

To determine the efficacy of medicinal plants to cure Leishmaniasis

To screen plant species with antiglycation properties to treat Diabetes

To designate plant species as immunomodulator, an alternate choice for

chemotherapy

To search for anticancerous drugs used in cancer chemotherapy and cancer

chemoprevention

Chapter 2

MATERIALS AND METHODS

Chapter 2 Material and Methods

19

2.1 Geography of Galliyat.

The survey was conducted in Galliyat, a high mountainous region situated in

District Abbottabad, Khyber Pakhtunkhwa (KP) province, Pakistan. The area is divided

into various territories and each of which is known as “Galli” means “a mountainous

tract”, hence the area is collectively called as Galliyat (plural of Galli). These include

Nathia Galli, Bara Galli, Changla Galli, Donga Galli and Ayubia National Park (Plate 1).

It lies between 33°-56′ and 34°-21′ N, 72°-55′ and 73°-29′ E with an altitude ranges from

7000 to 9500 feet (Arshad, 1991).

The study area is part of country’s richest biodiversity center in the Western

Himalaya and a source of ethnobotanical knowledge. Most of the population of the

country is rural with low literacy rate and they also lack modern health facilities. Hence

they are more dependent upon natural resources, especially plants for their health

maintenance and to compensate their low income as well. Topographically the area

mainly comprises hills and gradients and thus very little accessed for research studies

(Ahmed et al. 2013). The region falls under Subtropical and Moist temperate forests

consisting of fairly dense woods of conifers (Plate 2-3), sometimes mixed with broad

leaved trees and a mixture of luxuriant shrubs and herbs (Shafique, 2003). Champion et

al., (1965) and Hussain and Ilahi (1991) mentioned this area as Himalayan moist

temperate forests. Dominant gymnosperms composition of a forest includes Pinus

wallichiana, Cedrus deodara, Abies pindrow and Taxus wallichiana. The forest canopy

may be varying from dense (Plate 4) to break with open grassy patches. Due to cool and

humid weather for most of the year, the vegetation in the area comprises a spacious kind

of trees, herbs, shrubs and climbing irons. Ground cover comprises a wide variety of

angiosperms along with ferns and mosses.

The average rainfall varies from 100–130 mm in northern parts. A large part of

the winter precipitation from the western disturbance is received in the form of snow. The

northern parts receive little rain, but heavy snowfall in the winter (Khan et al., 2010).

This area is populated by several ethnic groups (Syed, Abbasi, Gujar, and Awan), all

speaking the Hindko dialect of the Western Punjabi, and belonging in turn to the Indo-

Aryan (Indic) language family spoken in Northern Pakistan.


20

PLATE 1: The scatter human population enjoying the diverse moist temperate forests in

Galliyat

PLATE 2: A panoramic view of Ayubia National Park (Galliayat)


21

PLATE 3: Diversity of Coniferous forest along the road connecting Galliyat to another

region of the Country

PLATE 4: Thick strands of coniferous forest form an attractive landscape in Galliyat


22

Fig 2.1: Location Map of the Study Area (Galliyat, Pakistan)


23

2.2 ETHNOBOTANICAL DOCUMENTATION

An ethnomedicinal survey was posted out to record the traditional uses of

medicinal plants to heal various disorders by the local inhabitant in Galliyat. A total of 8

field visit were conducted during May to September 2010-2012 in the study area. Each

trip was 5-7 days long.

Ethnobotanical data was collected through well-versed semi-structured

interviews, questionnaires, focus group conversation, participant observation and walk-

in-the-woods methods (Cotton, 1996; Martin, 1995). These involved a total of 124 key

informants ranged from 25–60 years. Demographic data of the informants is presented in

the table 2.1. These include farmers, herders, shepherds, housewives, Hakims (local

herbalists), Pansaries (local medicinal plants sellers), plant collectors and other pure

cultured persons (Cotton, 1996; Martin, 1995). Informed consent was obtained before the

start of interviews from each general informant and traditional healer who participated in

this study. Interviews were conducted in local language and run independently for each

informant.

Table 2.1: Demographic information of local inhabitant in five different villages of Galliyat.

Demographic data Namli

Mera Donga Gali Kalabagh

Nathia

Gali Ayubia

No. of Families 36 55 21 42 25

Sample size

(No. of Informants) 25 42 11 32 14

Informants average age 43.54

±16.38

39.72

±15.94 45±15.11

39.65

±12.38

45.83

±9.73

Average family

members 14.92 ±5.64 12.44 ±6.65

15.67

±6.38

14.75

±6.25 10 ±2.48

Average/month/head

expenditure (in PKR) 931 ±212 1063 ±370 941 ±194 1166 ±537

1420

±457

All semi-structured interviews were followed by independent walk-in-the-woods

which gave an opportunity for more discussion with the informant and the practical

identification of traditionally used medicinal plants in the natural environment. This


24

method was combined with the participant observation, practice through which reliable

information was obtained on the how of collection and preparation of specific remedial

parts (Cotton, 1996; Alexiades, 1996). In addition, focus group discussions were also

designed so as to gain further information on medicinal plant knowledge of the

community and prove the reliability of the data collected through semi-structured

interviews (Martin, 1995).

2.2.1 PLANT COLLECTION

Interviews and discussions were all followed by a voucher specimen collection

that was held with the help of traditional healers and local field assistants. Specimens

were air-dried, numbered, labelled and pressed. Identification of specimens was

performed by Plant taxonomist at Plant Taxonomy and Biosystematics Laboratory,

Department of Plant Sciences, Quaid-I-Azam University (QAU), Islamabad, using

taxonomic keys and floras (Stewart, 1958: Nasir & Ali, 1971-91: Ali & Nasir, 1970-

2002: Qureshi & Choudhri, 1987: Ali & Qaiser, 1986: Parker, 1918). Comparison with

authenticated herbarium specimens was also done in the Herbarium of Pakistan, Quaid-I-

Azam University (ISL). Voucher specimens were deposited for future reference at the

Herbarium of Pakistan (ISL) Pakistan.

2.2.2 ETHNOBOTANICAL DATA PRESERVATION

The data recorded in the field was transferred to the window based computer

program (Microsoft office-Excel-Version 2010). Data was arranged in a tabulated form

for presentation. Graphical representation was based on numerical data extracted.


25

2.3 BIOLOGICAL SCREENING OF MEDICINAL PLANTS

Amid ethnobotanically documented medicinal plants were selected for various biological

activities (Table 2.2). These plants were selected on the basis of their rich ethnobotanical

uses, literature examination to best of our knowledge present in previous literature and

negligible biologically screened.

Table 2.2: Selected medicinal plants for biological activities.

S.No. Plant Name Codes used

1 Geranium collinum GC

2 Persicaria barbata PB

3 Impatiens edgeworthii IE

4 Rubia cordifolia RC

5 Clematis grata CG

6 Geranium wallichianum GW

7 Berberis lycium BL

8 Artemisia vulgaris AV

9 Boerhavia procumbens BP

10 Capsella bursa-pastoris CBp

2.3.1 Extract preparation

Fresh plant material of one (01) kg was collected for each specimen and under the

shade dried and then grind in 50 mesh sized Wiley Mill to obtain the powdered samples.

This powder is then used for the extract preparations. 500 g of powder was added with 95

% pure methanol at room temperature and placed for three (03) days and then whole the

process was repeated. The extracts obtained were filtered using Whatman filter paper 1

and then filtrates were pooled together. The pooled filtrate was concentrated with rotary

evaporator (Laborota 4000 Germany) at 40 ℃ under lowered pressure. Final dried and

concentrated extract was weighed. Which were stored in refrigerator for further

processing in various biological assays.


26

2.3.2 ANTIBACTERIAL ASSAY

Antibacterial activities of a sample can be determined by using many different

techniques available (Linton, 1983). These included, three major methods antibacterial

testing approaches could be used a) agar dilution assay b) agar diffusion assay and c)

bioautographic assay. Out of these three approaches, the agar diffusion assay was the

most widely used by the microbiologists and was also adopted in the current study.

2.3.2.1 Samples preparation

Samples were prepared by dissolving the 12.5mg, 10mg, 7.5mg, 5mg, 2.5mg of

plant extract in of 01 ml of DMSO and then vortex apparatus (VM300 Japan) was used to

mix the extract in the DMSO for 3-5 min, until the sample fully dissolved.

2.3.2.2 Nutrient Media for bacterial growth

Inoculums were prepared to grow bacteria by using nutrient broth (70122 Fluka,

Sigma-Aldrich) which was prepared by adding 0.8g of nutrient broth in 100ml of distilled

water. Nutrient agar medium (N9405 Fluka, Sigma-Aldrich) was added 2g nutrient agar

in 100ml distilled water and then media was sterilized by autoclave.

2.3.2.3 Bacterial Species

In the present study four clinical isolates of bacterial species were used. These

were obtained from Microbiology Research Lab. (MRL), Quaid-i-Azam University

Islamabad as a gift. These included two Gram positive bacteria species Bacillus subtilus,

Enterococcus aerogenes and two Gram negative bacterial species Pseudomonas

aeruginosa, Klebsiella pneumoniae. These species were kept at 4°C in nutrient broth and

24 hours old cultures were used. Before that turbidity of the broth was adjusted following

McFarland 0.5 BaSO4 turbidity test by addition of sterile saline solution until a standard

density of 106 CFU per ml was attained. Then inoculum used for nutrient agar.

2.3.2.4 Procedure

Sterilized nutrient media was used for the seeding of bacterial inoculum after

cooling till 45 °C. The 75ml of nutrient agar media was poured in petri-plates (14 cm)

and wait till solidify. The bacterial cultures were seeded on the plate with sterile ear-


27

plugs. Eight (08) wells were prepared in each petri-plate with the help of a sterilized cork

borer (8mm). Micropipette was used to take 100μl of different extracts were transferred

into the wells. Five wells were occupied with the five different extract concentrations,

two with the standards (Penicillin (≥98.0(N)Sigma-Aldrich) and chloramphenicol

(≥98%(TLC)Sigma-Aldrich) were used as positive control in concentration of 1mg/ml)

and in last well DMSO (negative control) was added. Petri-plates were placed in

incubator with temperature adjusted at 37°C and after 24 hours the clear zones around

each well were observed. Experiment was done in triplicate. This experiment was repeted

each time for different strains. The zone of inhibition formed around each well was

calculated by its diameter and subtracting the zone formed by the DMSO (negative

control) to get the actual zone of inhibition. Antibacterial activities of the various extracts

were recorded in term of measuring the zone of inhibition which represents the mean of

three replicates.

2.3.3 ANTIOXIDANT ASSAYS

There are many analytical methods used to evaluate antioxidant potential of the

biological system. The methods based on objectives fall into two major categories, a) to

evaluate the intensity of oxidative stress in various bio-molecules like nucleic acid,

protein or lipids, b) to investigate the capacities of a substrate to scavenge various free

radicals and its measurements (Sanchez-Moreno, 2002).

2.3.3.1 DPPH Free Radical Scavenging Activity

DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay was used to determine the free

radicals scavenging abilities of the selected medicinal plants. This assay was the

modification of previously described assay by Gyamfi et al. (1999). Three different time

periods (120mins, 60mins and 15mins) were used instead of a single (15min as

mentioned in original protocol) and two different temperature conditions were used

including room temperature (25°C) and human body temperature (37°C) which was not

previously mentioned. The aim of these modifications was to evaluate the impact of

temperature and incubation time gradient on the assay result and productivity.


28

Samples were prepared by following the 2-fold serial dilution in the methanol.

The various concentrations of the extract used in the study were 250μg/ml, 125μg/ml,

62.5μg/ml, 31.25μg/ml and 15.625μg/ml. DPPH solution was prepared by adding 3.2mg

of DPPH in 100ml of methanol. This will be kept as a stock solution for further used. A

small quantity (2.8ml) of DPPH solution was added to the tubes with 0.2ml of extract

concentration. This mixture was shaked well and then placed in the dark at both the

temperatures (25°C and 37°C) separately allowing differential reaction time. This form

six different combinations include 120mins/25°C, 60mins/25°C, 15mins/25°C,

120mins/37°C, 60mins/37°C and 15mins/37°C. After incubation time, color change was

observed and measurements were taken at 517nm by using the UV-spectrophotometer

(CE 2021 USA). In this experiment, methanol was used as a blank and solution of DPPH

was control. The results were recorded for each fraction in triplicate. Percent scavenging

activity was calculated by the formula as given below.

% Scavenging activity

= [(control absorbance-sample absorbance) ×100 / (control absorbance)]

The results reported were expressed as their IC50 calculated with the help of

computerized software GraphPad Prism (Version 5).

2.3.3.2 ABTS Radical Cation Assay

ABTS assay was performed using the method of Re et al. (1999). In this assay

ABTS (2, 2-azinobis(3-ethylbenzothiazoneline-6-sulphonic acid) at 7.4mM was treated

with Potasium persulphate of 2.45mM to generate the free radicals. The mixture was than

diluted till the final absorbance of the solution was achieved about 1 (about 0.98%) at

414nm of UV spectrophotometer after 3ml of the solution was taken in glass cuvettes.

Ethanol was used as a blank in the experiment. Plants extract was than divided in various

concentration ranged from 16.625 to 250µg/ml and 100µl of each concentration was

transferred to the cuvettes which already contains 3ml of solution. This mixture was then

thoroughly shacked. The absorbance with the help of UV-spectrophotometer (CE 2021

USA) was recorded after the interval of 1 min and 6 min. Ascorbic acid was used as


29

standerd in this assay. The antioxidant capacities were determined by following the

equation given below:

%ABTS scavenging activity =

[(control absorbance-sample absorbance) / (control absorbance)] ×100.



2.3.3.3 Hydroxyl Radical Scavenging Activity

Hydroxyl radical was regarded as an important free radical was investigated in the

test samples by following the Deoxy-ribose method as previously described by the Nagai

et al. (2005). Two fold serial dilutions were used to form a dilution series for this

experiment. Five dilutions in methanol were formed for both plant extract and gallic acid

(standard). These dilutions were ranged from 250µg/ml (highest) to 15.625 µg/ml

(lowest). Reaction was started by adding 450µl of Sodium Phosphate buffer (0.2mM at

pH= 7.0), 150µl of 2-deoxyribose (10mM), 150µl of FeSO4-EDTA (10mM), 150µl of

H2O2 (10mM) and immediately added with 500ml of distilled H2O. Then the mixture was

added with 57µl of the various extract concentrations in the mixture. This reaction was

withstood for incubation period of four hours at 37°C. After that 750µl of TCA (2.8%),

750µl of TBA (1%) and NaOH (50mM) was added which stopped the reaction. Finally

the complete mixture was boiled for 10 sminutes followed by cooling. Their absorbance

was measured at 520nm using UV-spectrophotometer (CE 2021 USA) with triplicate

reading. Means were calculated from the triplicate measurement and antioxidant activity

was evaluated by following equation.

% Hydroxyl radical scavenging activity

= (1- absorbance of sample / absorbance of control) × 100.




30

2.3.3.4 Phosphomolybdenum Assay

Phosphomolybdenum assay was used to investigate the antioxidant activities of

the selected medicinal plants by following the procedure previously mentioned by Prieto

et al. (1999). In this experiment, various concentrations of sample plants and standards

(Ascorbic Acid) were initially developed by using the 2-fold serial dilution method to get

the final concentrations ranged from 15.625µg/ml to 250µg/ml in methanol. First of all, a

reagent solution was prepared by adding sodium phosphate (28mM), sulphuric acid

(0.6M) and ammonium molybdate (4mM). 0.1ml of sample concentration was added to

1ml of reagent solution and then allowed to mix in a test tube. These test tubes were

capped with the aluminum foil and placed in a water bath for 90min at 95°C, followed by

cooling up to 25°C. At 765nm absorbance was noted by using UV-spectrophotometer

(CE 2021 USA). Equation given below was used to calculate the % activity:

% activity = (1 - absorbance of sample / absorbance of control) x 100



2.3.3.5 FRAP ASSAY

The antioxidant power of the medicinal plants were investigated by FRAP method

as previously described by the Benzie and Strain (1996). Initially the stock solution of the

reagent was prepared by adding acetate buffer (300mmol/L at pH 3.6), TPTZ (2,4,6-

tripyridyl-s-triazine) solution (10mmol/L) in HCl (40mmol/L) and Fe2Cl3.6H2O

(20mmol/L). The working solution was prepared fresh by mixing acetate buffer (25ml),

TPTZ (2.5ml) and Fe2Cl3.6H2O and was heated to 37°C before using and aqueous Fe2+

was used for calibration. In this study FRAP reagent alone will serve as a blank. Now,

each plant extract (50µl) and deionized water (150µl) was 1.5µl of FRAP reagent to get a

sample, incubated at 37°C. After 4 min the absorbance was noted at 593nm. FRAP values

were measured by using the calibration curve. All measurements were carried out in four

repetitions for each concentration of the extract.


31

2.3.4 ANTILEISHMANIAL ASSAY

Leishmanicidal activity was performed as following the previously described

procedure of Zhai protocol (1999). In this assay, an already established Leishmania

culture was taken from National Institute of Health (NIH), Islamabad as a gift and

performed these experiments in Infectious Disease Laboratory, Quaid-i-Azam University,

Islamabad. Media 199 (Invitrogen) with heat inactivated fetal calf serum (Sigma-Aldrich)

was used to culture of parasite. At 26°C the growth and incubation of parasite was done.

Leishmanial parasites were collected from the log phase and then were centrifuged for

three minutes at three thousands rpm. After centrifugation supernatant was removed

parasites were transferred in fresh medium to dilute. Parasite count was performed by

addition of trypan blue and formaldehyde to solution of parasite with 20µl and 200µl

respectively. Only 20µl of this mixture was taken and parasites were counted under the

neubar counting chamber (HBG Germany). Ultimately, the parasites final concentration

of 2X106 parasites per ml was attained.

Samples were prepared by dissolving plant extract into DMSO to achieved the

final concentration of 10mg/ml was treated as stock solution. Two-fold serial dilution

was used for this stock solution later-on. In various wells of 96-well microtiter plate was

added with 180µl of media 199. 20µl of stock solution for each tested plant was added in

the first well, following serial dilution and 20µl from the last well was discarded. Except

two rows, all other were supplied with 100µl of Leishmania parasites. The one out of the

two rows left, was added with DMSO (negative control) and other with a standard drug

Glucantime (Aventus-France) (positive control). These were also serially diluted in

media 199. This plate was incubated for 72 hours at 24°C in a shaker-incubator (GLSC

OS1). After that, 20µl formed each well was taken on neubauer counting chamber and

microscopic live parasite count was carried out. Window based Gaphpad Prism (Version

5) was used to calculate the IC50 of the plant extract exhibited the potential antilieshmanil

activity.


32

2.3.5 ANTIGLYCATION ASSAY

The assay was performed by following the previously described procedure by

Mastuda et al. (2003). This procedure was based on the inhibition of fluorescent

development of BSA (Bovine serum albumin - purchased from Merck) mediated by

methylglyoxal. 96 well microtiter plate was used in the assay. Reaction mixture was

prepared and 60µl of that was put in every well. This 60µl consist of three components

included 20µl of 50mg/ml of glucose anhydrous, and 14mM of magnesium oxide, 20µl

of 10mg/ml Bovine serum albumin and 20µl of the plant extract. 60µl of blank contained

40µlsodium phosphate buffer and 20µl of BSA while negative control was contained

20µl of 0.1M sodium phosphate buffer, 20µl of BSA and 20µl of 30mM NaN3. Rutin

(Flex Pharma) was used as positive control in the study. The plate was incubated for 09

days at 37°C. After completion, each well was added with 60µl of 100% TCA, followed

by centrifugation at the rate of 15000rpm at 4°C for four minutes. Then 5%TCA was

used to wash each pellet, which contained advanced glyacation end-product bounded

with BSA were dissolved in Phosphate buffer solution (60µl). Supernatant was discarded

included interfering substances, glucose and inhibitors. Advanced glyacation end-product

(AGEs) formation was noticed at 370-440nm with the help of spectrophotometer. The

percent inhibitions of AGEs were determined as follow:

% Inhibition = [1 - (Absorbance of extract/Absorbance of control)] x 100.




2.3.6.1 Preparation of Luminol and Lucigenin

Luminol (1.8 mg) and 1ml of sodium borate buffer were mixed and vortexed for

5-10 min. 09ml of Hank’s balance salt solution [Ca2+ and Mg2+] (HBSS++) was added to

luminol (180µg) per ml. Then 25.5 mg of lucigenin was dissolved in 10 mL of distill

water. Both were stored at -20 ℃ for later use.


33

2.3.6.2 Isolation of Human Polymorphoneutrophils (PMNs)

Heparinized venous blood (20 mL) was collected aseptically from healthy adult

male volunteers (25-38 years age) and by centrifuge (2-16 sigma Germany) based on

density gradient was used to isolate neutrophils (Mesaik et al., 2006). Whole blood

(20ml) was layered on Lymphocytes Separation Medium (LSM) (15ml) follow

centrifugation sediment the erythrocytes and Polymorphoneutrophils and band Mono

Nuclear Cells (MNCs) were above the LSM. The lymphocytes were aspirated by using a

clean Pasteur pipette and centrifuge again with equal volume of LSM for 10 min at 300 g

at 24 ℃, washed the cell and again suspended in Hank’s balance salt solution [Ca2+ and

Mg2+-free] (HBSS-) and stored in ice for later use. Cells were counted by using trypan

blue and finally got the concentration of 1X106 cells per ml.

2.3.6.3 Chemiluminescence assay

Chemiluminescence assay was performed as previously describe by

Mahomoodally et al. (2007) and Mesaik et al. (2006). Whole blood (25µl) was diluted in

sterile phosphate buffer solution (1:50), maintained the pH at 7.4 was taken along with

Polymorphoneutrophils (1X106) was suspended in Hank’s balance salt solution

(HBSS++). Plant extract (6.25-100µg/ml) was added in the 25µl of reaction mixtures for

30 min at 37℃ in luminometer have manual thermostat chamber. To each well 25 µL

Phorbol Myristate Acetate (PMA), then 25 µl of luminol or lucegenin (7 x 10-5 M)

luminol or lucegenin was added. 96 well microtiter plates was added with HBSS++ to

200µl and HBSS++ was used as control alone. Results monitored in Luminometer were

treated as chemiluminescence relative luminescence unit (RLU) and values were taken

with the intervals of 30s and 1sec. Ibuprofen (Abbott) was used as a positive control in

the experiment. Graphpad Prism software was used to calculate IC50 values

2.3.7 ANTI-CANCER ASSAY

2.3.7.1 Sulforhodamine B (SRB) assay for Human Cancer Lung Cell Line (LU-1)

and human prostate adenocarcinoma cells (LNCap-1)

Sulforhodamine B (SRB) is a protein staining assay used for in-vitro cytotoxicity

evaluation. This assay was based on bright pink colored SRB (aminoxanthene dye) binds


34

to various amino acid components of TCA bounded proteins. This assay was

experimentally proved for a large scale screenings (Vichai & Kirtikara, 2006).

The assay was conducted by following the standard protocol provided by Skehan

et al. (1990) for the determination of cellular toxicity and effect of extracts or compounds

on the capabilities of the cultured cell, ultimately the total protein was quantified which

was considered as % of the surviving cells. The medicinal plants extracts were screened

for their cytotoxic effects against two cancer cell lines including Human prostate cancer

cell line (LNCaP) and Human lug carcinoma (LU 1) purchased from American Type

Culture Collection (ATCC). Extract was dissolved in DMSO to get the final

concentration of 20µg/ml. 96 well plate microtiter plate was for seeding of the cancer

lines (3X10-4 cell/ml). Six different concentrations after diluted serially in 10µl of DMSO

(10%) were put in each well. Microtiter plates were incubated in air atmosphere

humidified 5% CO2, at 37 °C in incubator (09411 Newzealand) for 72 hours and SRB

staining was used to determined cell viability. The samples concentrations inhibited the

50% of the cell growth were determined as IC50 values and negative control was

represented by DMSO (0.5%), while colchicine was used as positive control.

2.3.7.2 Metabolic Impairment Assays (MTT assay) for Human Prostate Cancer Cell

Line PC-3)

MTT assay based on ability of action of mitochondrial enzyme dehydrogenase

action (reduction of tetrazolium salt into formazan) was used to measure metabolic

capabilities of cells. MTT assay was used to assess cytotoxicity on PC-3 cell line as

described previously (Kumi‐Diaka, 2002).

96 well plates were used to seed the PC-3 cancer cells and normal cells.

Concentrations (3.3X105 cell/ml) of the cells were taken as per final volume of

200µl/well. This was followed by the incubation of these plates were carried out at 37°C

for 24 hours in incubator humified with CO2 (10%). Fresh media was used to replace old

media. Medicinal plants extract (10µl) was added and standard drug doxorubricin for

positive control and for negative control no drug was added. Than-after plates were again


35

incubated at 37°C for next 48 to 72 hours in incubator humifies with CO2 (10%). After

completion of the incubation period the media was removed and fresh media was added

with MTT salt (50µl). Again incubation was followed at 37°C 4 hours in incubator

humified with CO2 (10%). This MTT salt and media was removed and formazan product

(insoluble) was dissolved in DMSO (50µl). At 540nm absorbance was measured. Finally

IC50 was calculated as cytotoxic activity of medicinal plants.

2.3.8 STATISTICAL ANALYSIS

The data was recoded in Microsoft Office-Excel (version 2010) and analysis of

various in vitro assays were carried out, obtained data and then analyze the data with the

help of computerized software GraphPad prism window 5 (GraphPad, 2007.), Origin 7.5

(OriginLab®), and BioStat (2009 AnalystSoft Inc.) to calculate the IC50 values. One-way

analysis of variance was used to determine different concentration effect in various

experiments with the help of computerized statistical software SPSS 13 (SPSS® Inc.).

Significance of these results was noted at the 0.05% level of probability. Paired t-test and

R2 statistics were applied by using Microsoft Office-Excel (version2010).

3. RESULTS

Chapter 3 Results

36

Research work was conducted during 2009-2012 in the Galliyat area of Western

Himalaya and 45 medicinal plants were investigated for ethnobotanical evaluation.

3.1 ETHNOBOTANICAL INVESTIGATION

Plants provide a major source of medicine in the Galliyat, Pakistan. Medicinal

plants from Galliyat are sold to other part of Pakistan for commercial purposes. A bulk of

population still relies on medicinal plants for curing different ailments. Ethnobotanical

survey revealed that the various areas of Galliyat are enriched with the useful medicinal

plants. The results show that the inhabitants of the area utilized 45 plants species

medicinally. The ethnobotanically valued plant species include Angiosperms (38 sp.),

gymnosperms (4 sp.), and Pteridophytes (3 sp.) (Fig. 3.1). Majority of the plants in the

study are perennial herbs (32 sp.), than by annual herbs (5 sp.), tree (5 sp.) and shrubs (4

sp.) (Fig 3.2).

In the present study, angiosperms are comprised of 21 families (Fig. 3.3),

Asteraceae (8 sp.), Araceae (2 sp.), Balsaminaceae (2 sp.), Berberidaceae (2 sp.),

Brassicaceae (1 sp.), Fumariaceae (1 sp.), Gentianaceae (2 sp.), Geraniaceae (2 sp.),

Nyctaginaceae (1 sp.), Paeoniaceae (1 sp.), Plantaginaceae (2 sp.), Polygonaceae (3 sp.),

Ranunculaceae (1 sp.), Rosaceae (2 sp.), Rubiaceae (1 sp.), Rutaceae (1 sp.),

Saxifragaceae (1 sp.), Solanaceae (1 sp.), Urticaceae (2 sp.), Valerianaceae (1

sp.),Violaceae (1 sp.). Pteridophytes consist of two families, Pteridaceae (2 sp.) and

Dryopteridaceae (1 sp.). Gymnosperms are also represented by two families, Pinaceae (3

sp.), and Taxaceae (1 sp.). Asteraceae is the largest represented family with 8 species.

The inhabitant of the area prefer different parts of the plants for the medication purposes

(Fig. 3.4) involve; leaves (19 sp.), root (11 sp.), fruit (7 sp.), flower (7 sp.), bark (6 sp.),

seed (5 sp.), aerial parts (4 sp.), whole plants (3 sp.), rhizome (3 sp.) and wood (2 sp.).

Results of the field work also suggest that the source of indigenous knowledge

pertinent to the use of plant species for medicinal purposes in the area. Majority (70%) of

the folk medicinal knowledge come from people above the age of 50 years and local

hakeems. While 24% from the age group of 15-50 years, while the rest of 6 % were from

Chapter 3 Results

37

age group below 15 years. Gender-wise, women (esp., Old aged) have more traditional

knowledge than men.

The dependency of people in the area on medicinal plants referred as decreased

significantly in last 10 years due to allopathic medication and tourism upsurge in the area.

Majority of the home-made traditional medication was used by the persons with age

group more than 50 years, than by the children with age group below 15 years (esp.,

infants) and the least consumption was observed in the age group 15-50 years. The

availability of the medicinal plants also decreasing near the human settlements,

environmental changes, due to increased market pressure and non-sustainable harvesting

methods. Tourism is the major industry in the area, and medicinal plant collection is

mainly performed as a side business in the area.

Chapter 3 Results

38

Fig 3.1 showing the large contribution of medicinal plants from angiosperm utilized

by the local community

Fig 3.2 showing the habit-wise distribution of medicinal plants in the study area

4 3

38

0

5

10

15

20

25

30

35

40

Gymnosperm Pteridophytes Angiosperm

54

5

32

0

5

10

15

20

25

30

35

Annual Herb Shrub Tree Perrenial Herb

Chapter 3 Results

39

Fig 3.3 showing the distribution of different plant families of the representative

medicinal plants used in folk medicine by the local community

Fig 3.4 showing the contribution of various parts of the medicinal plants used by the

local inhabitant in the study area

0

1

2

3

4

5

6

7

8

Ara

ceae

Ast

erac

eae

Bal

sam

inac

eae

Ber

ber

idac

eae

Bra

ssic

acea

e

Dry

op

teri

dac

eae

Fum

aria

ceae

Ge

nti

anac

eae

Ge

ran

iace

ae

Nyc

tagi

nac

eae

Pae

on

iace

ae

Pin

acea

e

Pla

nta

gin

acea

e

Po

lygo

nac

eae

Pte

rid

acea

e

Ran

un

cula

ceae

Ro

sace

ae

Ru

bia

ceae

Ru

tace

ae

Saxi

frag

acea

e

Sola

nac

eae

Taxa

ceae

Urt

icac

eae

Val

eria

nac

eae

Vio

lace

ae

2

8

2 2

1 1 1

2

1 1 1

3

2

3

2

1

2

1 1 1 1 1

2

1 1

3, 5% 3, 5%4, 6%

19, 28%

7, 10%

11, 16%

7, 10%

5, 8%

6, 9% 2, 3%

Rhizome Whole plant Aerial parts Leaves Flowers Root Fruit Seed Bark Wood

40

Table: 3.1 showing the medicinal plants used in the area and their ethnomedicinal uses

Botanical Name Vernacular

Name

Family Habit Part used Flowering

Period

Ethnomedicinal Uses

Pteridophytes

1 Dryopteris ramosa

L. PLATE 5

Pakha Dryopteridaceae Herb Young

leaves

Dec-Mar Used as a vegetable against gastric

ulcer, constipation.

2 Adiantum incisum

L.

Phunka Pteridaceae Herb Leaves Mar-Apr Juice is extracted from the leaves

used for treatment of dysentery

and chronic diarrhea, also

recommended for Jaundice.

3 Adiantum venustum

L. PLATE 6

Kakwa Pteridaceae Herb Leaves,

rhizome

Mar-Apr Plants are boiled and its decoction

is used for body temperature,

headache and scorpion stings

treatment. The rhizome is used in

forming paste and used in to treat

cuts and wounds.

41

Gymnosperm

4 Abies pindrow

Royle.

Paludar,

partal

Pinaceae Tree Stem and

bark

Apr The leaves are used as substitute

for tea. Tea is made up of stem

bark and recommended for

stomach disorder and vomiting

5 Cedrus deodara

Roxb. ex D.Don PLATE 7

Deodar Pinaceae Tree Wood,

bark

Oct-Nov Bark is used in different remedies

to treat fever, diarrhea and skin

disorders while wood is used in

pulmonary diseases

6 Pinus wallichiana

Jackson PLATE 8

Kail, biar Pinaceae Tree Twigs,

Leaves,

Bark and

Wood

Apr- Jun Oil is extracted from the plant

applied on wound and ulcer.

Wood is also used for ulcer and

cough.

7 Taxus wallichiana Barmi,

Thuna

Taxaceae Tree Fruit and

leaves

Apr- May Leaves are used in asthma,

epilepsy, bronchitis and also as an

aphrodisiac medicine

42

PLATE 5: Dryopteris ramosa

PLATE 6: Adiantum venustum

PLATE 7: Cedrus deodara

PLATE 8: Pinus wallichiana

43


Name


Period

Ethnomedicinal Uses

Angiosperms

8 Arisaema

flavum (Forsk.)

Schott

Ad bis Araceae Herb Rhizome Jun- Jul. Rhizome of this poisonous plant is

used in treatment of snake bite

9 Arisaema

jacquemontii Blume PLATE 9

Hathphees Araceae Herb Fruit and

rhizome

Jun-Jul. Fruits and rhizomes are considered

to be poisonous. A small amount

of rhizome powder is used during

meal can relieve pains. There are

so many other preparation used

this rhizome powder to treat

psychic and nervous problems.

10 Achillea millefolium

L.

Sultani

booti

Asteraceae Herb Whole

plant

Jun-Sept Decoction are prepared to treat

piles and headache. The aerial

parts are considered to be

digestive tonic and diuretic.

Menstrual problems were also

treated with tincture

44

11 Artemisia dubia L. Kakamush Asteraceae Herb Aerial

parts.

Feb-Mar Commonly used for stomachache

and digestive complaints.

12 Artemisia

vulgaris L. PLATE 10

Chaagu Asteraceae Herb Leaves Aug-Nov Leaves are used to treat menses

complications, insomnia and to

kill internal parasitic worms.

13 Chrysanthemum

leucanthemum L. PLATE 11

Chitti

phulari

Asteraceae Herb Flowers

and leaves

Aug-Sept The Plants is used as pesticide and

insecticide. Flowers are used to

treat the digestive problems

14 Cichorium intybus

L.

Handh Asteraceae Herb Flowers,

leaves,

root

Jun-Oct The roasted root is used as a

coffee adulterant. Fresh flowers

are used to prepare decoction to

treat gravels. Latex is applied on

warts to destroy them

15 Saussuria

hetromalla L.

Kali Ziri Asteraceae Herb Roots Mar-Jun Roots are used as tonic and also to

treat various complications

included kidney, liver and chest

disorders.

16 Senecio

chrysanthemoides

DC. PLATE 12

Chitta

howla

Asteraceae Herb Flowers Jul-Sept Rhizome is used to treat various

respiratory disorders and asthma

45

PLATE 9: Arisaema jacquemontii

PLATE 10: Artemisia vulgaris

PLATE 11: Chrysanthemum leucanthemum

PLATE 12: Senecio chrysanthemoides

46


Name


Period

Ethnomedicinal Uses

17 Taraxacum

officinale Weber. PLATE 13

Dudal

Bumbola,

Haand

Asteraceae Perennial

Herb

Flowers,

Leaves

and roots.

Febr-Apr Flowers are used to made tea used

in the treatment of urinary and gall

bladder disorders and jaundice.

Leaves are consumed by diabetic

patients while root paste is applied

on swelling and joints.

18 Impatiens bicolor

Royle

Buntil Balsaminaceae Annual

Herb

Fruit,

Seed

Jul-Aug Fruits and seed are considered to

be tonic and diuretic and have

cooling impacts on the body. The

juice of the plant is used to treat

burns.

19 Impatiens

edgeworthii Hook. f. PLATE 14

Peela

Buntil

Balsaminaceae Annual

Herb

Leaf Jul-Sept The plant is used internally for

gonorrhea and externally for

burns.

20 Berberis

lycium Royle PLATE 15

Simlu Berberidaceae Shrub Bark and

branches

Apr-Jun Stem bark is used in the treatment

of diabetes and fever.

21 Podophyllum

hexandrum Wall.

Ex Royle PLATE 16

Ban Kakri Berberidaceae Perennial

Herb

Fruit Apr-May Fruit are used to treat liver

disorders and also as tonic

47

22 Capsella bursa-

pastoris (L.)

Medic.,

Chambraka Brassicaceae Herb Seeds Mar Jun Plant seeds are used as diuretic,

stimulant and astringent

23 Corydalis govaniana

Wall.

Bhutyata Fumariaceae Perennial

Herb

Root Jun-Aug The plant roots are utilized in the

treatment of cutaneous infection,

syphilis and various intestinal

disorder due to worms

24 Gentianodes kurroo

(Royle) Omer.

Nil Kanth Gentianaceae Perennial

Herb

Root Sept-Nov Roots are used in the treatments

of urinary and stomach infections

25 Swertia chirata L. Chiraita Gentianaceae Herb Aerial

parts

Jul-Aug Dried aerial parts are crushed for

powder, recommended for various

digestive disorders, also used in

various skin disorders

26 Geranium

collinum Steph. ex

Willd., PLATE 17

Rattanjot

Geraniaceae Perennial

Herb

Whole

plant.

Jul-Aug The plants are used to treat

wounds, swellings, tumors, nerves

problem both internally and

externally. Plant decoction is used

as antipyretic tonic and to cure

cold and cough

48

PLATE 13: Taraxacum officinale

PLATE 14: Impatiens edgeworthii

PLATE 15: Berberis lycium

PLATE 16: Podophyllum hexandrum

49


Name


Period

Ethnomedicinal Uses

27 Geranium

wallichianum

D.Don ex Sweet. PLATE 18

Chatty Char Geraniaceae Perennial

Herb

Flowers

and leaves

Jul-Sept Leaves and flowers are used to

cure eye vision problems and also

used for blood purification.

Crushed roots are used in

backache, strengthening muscles

and bones along with milk and

sugar.

28 Boerhavia

procumbens Banks

ex Roxb., PLATE 19

Punarnava Nyctaginaceae Perennial

Herb

Root Aug-Sept Dried root powder is snuffed to

treat flu, and if it is taken along

with honey is considered useful

against asthma and cough

29 Paeonia emodi

Wall.

Mamaikh Paeoniaceae Perennial

Herb

Root May-Jun Crushed roots are mixed with

roots of Geranium wallichianum,

milk and sugar to treat internal

body pain and backache.

30 Plantago lanceolata

Linn.,

Isabgool Plantaginaceae Perennial

Herb

Leaves Aug-Sept Leaves extract is applied on

wounds and also used in

dysentery, mouth diseases and

sore throat.

50

31 Plantago major L. PLATE 20

Achar Plantaginaceae Perennial

Herb

Leaves

and seeds

Aug-Sept Seeds are used in dysentery.

Leaves paste is considered to be

effective in bleeding esp., uterine

bleeding after pregnancy. Leaves

can also be used to settle the skin

discoloration by some injury.

32 Persicaria

barbata (L.) Hara PLATE 21

Pulpulak Polygonaceae Perennial

Herb

Leaves May-Jun Leaves are used to kill the fishes

when crushed with sand.

33 Rumex hastatus D.

Don

Khatta

Hulla,

Khatimal

Polygonaceae Shrub Leaves

and young

tender

branches

Jun-Oct Leaves are used for relief in-case

of Urtica dioica irritation, also

used in dysentery, scorpion sting.

Leaves in the area are famous as

wild vegetable

34 Rumex nepalensis

D.Don PLATE 22

Kho Polygonaceae Perennial

Herb

Leaves

and roots

Mar-Aug Root decoction is applied to treat

bones dislocation and paste is used

for swollen gums. Colic is also

treated with leaves.

51

PLATE 17: Geranium collinum

PLATE 18: Geranium wallichianum

PLATE 19: Boerhavia procumbens

PLATE 20: Plantago major

52


Name


Period

Ethnomedicinal Uses

35 Clematis

grata Wall., PLATE 23

Dhund Ranunculaceae Woody

Climber

Vegetative

portion.

Aug-Sept Paste is prepared for external

application on joints and also on boils.

Leaves extract is also used as

insecticide

36 Fragaria nubicola

Lindil.ex. Lacaita

Knachii Rosaceae Perennial

Herb

Fruits and

leaves

Apr-Jun Fruit has a very pleasant strawberry

flavor. Fruit and leaves are mixed with

that of Berberis lycium and used for the

treatment of stomach ulcer and also as

antiseptic

37 Rubus ellipticus Smith

Garachey Rosaceae Shrub. Fruits,

leaves and

roots

Feb-May Fruit is delicious and edible by local

inhabitants. Fresh extract of the leaves

is used for the treatment of Urticaria.

Decoction of root is useful in dysentery

and whooping coughs

38 Urtica dioca L. Bichu booti Urticaceae Perennial

Herb

Leaves. Jul-Aug The plant is considered to be irritable

and poisonous. Leaves and roots are

used in the treatment of Chambal

(condition in which white spots were

formed on the body)

53

39 Zanthoxylum

alatum L. PLATE 24

Timbar Rutaceae Shrub Bark,

branches

and fruit

Mar-Apr Stem is used as toothbrushes. And

young leaves are considered to be

useful in gum diseases.

Fruits are used to cure Ulcer and also

used as stomachic and carminative

40 Bergenia ciliata

(Haw.) Sternb PLATE 25

Butpia Saxifragaceae Perennial

Herb

Leaves

and

rhizome.

Mar-May Rhizome is crushed to powder and

utilized in various digestive disorders

and stomach ulcer. It is also

recommended in muscular

complication and rheumatism when

used with butter because it is reported

to cause dryness. Rhizome paste is

applied to swollen joints. It is also

reported to be anticancerous.

41 Atropa acuminata

Royle ex miers

Chella

lubar

Solanaceae Perennial

Herb

Root,

Leaves

Jun-Jul The plant action is regarded as

antispasmodic, stimulant and sedative,

used in the treatment of cough

54

PLATE 21: Persicaria barbata

PLATE 22: Rumex nepalansis

PLATE 23: Clematis grata

PLATE 24: Zanthoxylum alatum

55

42 Urtica pilulifera

Linn.,

Bichu booti Urticaceae Annual

Herb

Aerial parts Mar-Jun Leaves are used as herbal tea.

Leaves are used in the

treatment of

stomach/intestinal pains, liver

and circulatory system

diseases. It is also reported to

be used against cancer

43 Rubia cordifolia L., PLATE 26

Chero Rubiaceae Perennial

climbing

herb

Root Jun-Nov Roots are used for blood

purification, swellings,

wounds, bone fracture,

uterine tumor and bleeding

control

44 Valeriana jatamansi

Jones (V. wallichii

DC.) PLATE 27

Mushkbala Valerianaceae Perennial

Herb

Rhizome,

Leaves

Mar-May Rhizome juice is used in the

treatment of headache and

used in eye complication.

Paste is prepared from the

plant used for boils externally

and internally for cramps and

irritable bowel syndrome.

Dried leaves are placed in

56

clothes as an insecticide.

45 Viola canescens

Wall ex Roxb. PLATE 28

Banafsha,

Phulnaqsha

Violaceae Perennial

herb

Whole

plant,

flowers

Mar-Jun The flowers are used in the

form decoction recommended

in nervous disorders, epilepsy

and common cold. The plant

is also known for anticancer

properties

57

PLATE 25: Berginia ciliata

PLATE 26: Rubia cordifolia

PLATE 27: Valeriana jatamansii

PLATE 28: Viola canescens

Chapter 3 Results

58

3.2 BIOLOGICAL SCREENING

3.2.1 ANTIBACTERIAL ACTIVITY

In this experiment four bacterial strains have been used including two Gram positive

(Bacillus subtilus, Enterococcus aerogenes) and two Gram negative (Pseudomonas

aeruginosa, Klebsiella pneumoniae) to determine the antibacterial potential of the

medicinal plants. The various plants show variable results against all four bacteria by

forming zones of inhibition of variable diameters. The maximum and minimum inhibition

shown by each bacterium is calculated by measuring highest and lowest mean value zone

of inhibition respectively. These inhibitions are presented against each plant by

maximum inhibition followed by minimum along with their standard error value. The

zone of inhibition for Bacillus subtilus are as follows, Geranium collinum (7.3±0.233

mm-16.67±0.88 mm) Persicaria barbata (12.33±0.33 mm- 28.75±0.33 mm), Impatiens

edgeworthii (4.33±0.577 mm-9.67±0.33 mm), Rubia cordifolia (6.0±0.33 mm-11±0.3

mm), Clematis grata (13.0±0.154 mm-16.33±0.577 mm), Geranium

wallichianum (13.2±0.23 mm-16.33±0.577 mm), Berberis lycium (8.33±0.667 mm-

13.33±0.667 mm), Artemisia vulgaris (7.0±0.5 mm-23.0±0.577 mm), Boerhavia

procumbens (8.0±1 mm-23.0±0.577 mm), Capsella bursa-pastoris (7.0±0.33 mm-

13.2±0.3 mm) (Fig 3.5). In case of Klebsiella pneumonia, the inhibition of bacterial

growth in term of zone of inhibition are Geranium collinum (9.67±0.577 mm-16.67±1.52

mm) Persicaria barbata (10.0±1 mm- 14.76±3.179 mm), Impatiens

edgeworthii (6.67±0.577 mm-11.67±0.33 mm), Rubia cordifolia (6.0±0.17 mm-8.2±0.577

mm), Clematis grata (7.6±0.67 mm-10.667±0.73 mm), Geranium



procumbens (6.13±0.33 mm-14.54±0.811 mm), Capsella bursa-pastoris (7.0±0.17 mm-

9.2±0.577 mm) (Fig 3.6).

The inhibition shown by different plants against Pseudomonas aeruginosa includes,

Geranium collinum (12.33±0.88 mm-20.33±2.645 mm) Persicaria barbata (11.0±0.577

mm- 21.33±0.33 mm), Impatiens edgeworthii (6.13±0.33 mm-12.0±0.577 mm), Rubia

cordifolia (4.13±0.33 mm-6.342±0.23 mm), Clematis grata (8.6±0.577 mm-11.67±0.677

Chapter 3 Results

59

mm), Geranium wallichianum (8.33±0.33 mm-11.67±0.577 mm), Berberis

lycium (5.67±0.881 mm-9.3±0.33 mm), Artemisia vulgaris (5.21±0.577 mm-9.3±0.577

mm), Boerhavia procumbens (5.07±0.577 mm-10.667±0.577 mm), Capsella bursa-

pastoris (3.8±0.33 mm-5.342±0.23 mm) (Fig 3.7). Growth of bacterium Enterococcus

aerogenes is inhibited by medicinal plants include, Geranium collinum (11.33±3.17 mm-

19.0±0.577 mm) Persicaria barbata (11.0±1.52 mm- 14.0±0.881 mm), Impatiens

edgeworthii (7.67±0.333 mm-12.0±0.577 mm), Rubia cordifolia (3.53±0.23 mm-

6.24±0.44 mm), Clematis grata (5.7±0.577 mm-8.0±0.577 mm), Geranium



procumbens (2.0±0.13 mm-8.55±0.16 mm), Capsella bursa-pastoris (3.53±0.23 mm-

6.24±0.04 mm) (Fig 3.8).

Minimal inhibitory concentrations have also been measured for each plant against all

four bacteria used in the current study (table 3.2). The highest MIC value against Bacillus

subtilus was shown by plants Persicaria barbata, Clematis grata, Geranium

wallichianum, Berberis lycium, Boerhavia procumbens with least concentration

(2.5mg/ml) used. And lowest Mic was displayed by Capsella bursa-pastoris with higher

concentration (7.5mg/ml). In-case of Klebsiella pneumonia MIC values were of

Geranium collinum, Geranium wallichianum, Berberis lycium was calculated as 2.5

mg/ml and for Rubia cordifolia and Artemisia vulgaris (7.5 mg/ml). Against

Pseudomonas aeruginosa, the highest MIC value was calculated as 2.5 mg/ ml for Rubia

cordifolia, Clematis grata and Capsella bursa-pastoris and lowest 7.5 mg/ml for

Artemisia vulgaris, Impatiens edgeworthi and Artemisia vulgaris. MIC against

Enterococcus aerogenes were displayed by Geranium collinum, Persicaria barbata

Artemisia vulgaris, Clematis grata and Persicaria barbata with 2.5mg/ml and lowest

7.5mg/ml by Geranium wallichianum, Berberis lycium and Boerhavia procumbens.

Chapter 3 Results

60

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

Zone

of i

nhib

ition

(mm

)

Concentration (mg/ml)

GC

PB

IE

RC

CG

GW

BL

AV

BP

CBp

2.5 57.5 10

12.5

Bacillus subtilus

Fig 3.5 Antibacterial activity in term of measuring zone of inhibition (mm) of all medicinal

plants used in the study against Bacillus subtilus. Values are the mean from three replicates

and there standard error.

0

2

4

6

8

10

12

14

16

18

20

Zon

e of

inhi

bitio

n (m

m)


GC

PB

IE

RC

CG

GW

BL

AV

BP

CBp

2.5 57.5 10

12.5

Klebsiella pneumoniae


plants used in the study against Klebsiella pneumonia. Values are the mean from three

replicates and there standard error.

Chapter 3 Results

61

0

2

4

6

8

10

12

14

16

18

20

22

24

Zone

of i

nhib

ition

(mm

)


GC

PB

IE

RC

CG

GW

BL

AV

BP

CBp

2.5 57.5 10

12.5

Pseudomonas aeruginosa


plants used in the study against Pseudomonas aeruginosa. Values are the mean from three


0

2

4

6

8

10

12

14

16

18

20

Zone

of i

nhib

ition

(mm

)


GC

PB

IE

RC

CG

GW

BL

AV

BP

CBp

2.5 57.5 10

12.5

Enterococcus aerogenes


plants used in the study against Enterococcus aerogenes Values are the mean from three


Chapter 3 Results

62

Table 3.2: Minimal Inhibitory Concentration (MIC) values of each plants against all

four bacteria used in the study

Plants Name

MIC (mg/ml)

Bacillus

subtilus

Klebsiella

pneumonia

Pseudomonas

aeruginosa

Enterococcus

aerogenes

Geranium collinum 5 2.5 5 2.5

Persicaria barbata 2.5 5 5 2.5

Impatiens edgeworthii 5.5 5 7.5 5

Rubia cordifolia 5 7.5 2.5 5

Clematis grata 2.5 5 2.5 2.5

Geranium wallichianum 2.5 2.5 5 7.5

Berberis lycium 2.5 2.5 7.5 7.5

Artemisia vulgaris 5 7.5 7.5 2.5

Boerhavia procumbens 2.5 5 5 7.5

Capsella bursa-pastoris 7.5 5 2.5 5

Chapter 3 Results

63

3.2.2 DPPH Radical Scavenging Activity

2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) radical scavenging activity has

been performed to evaluate the radical scavenging ability of the medicinal plants extract

at varying temperatures and incubation period, and to know about the effect of this

variation in temperature and incubation time on the activity is shown by the medicinal

plants extracts. Two ranges {human body temperature (37°C) and other room

temperature (RT)} was selected for temperature, and three durations (15, 60, 120 mins)

were for incubation time.

The whole experiment was divided into three groups with same incubation

period and different temperature. First group include the IC50 values of 15min/37°C and

15min/RT, second was 60 min/37°C and 60 min/RT, and third was 120 min/37°C and

120 min/RT. Three (03) Paired T-tests were used for the statistical comparison of the

significances of IC50 results shown by plants and standard in all three groups. p-values

were calculated to know about the significances of the results in each group, which were

0.0047, 0.0036 and 0.0040 respectively for each group at p-value ≥0.01 (Fig 3.). These

values were considered as highly significant and proved the hypothesis that IC50 values

was significant at 37°C<RT and also at 120min<60min<15min.

Ascorbic acid was used as standard compound in this assay and all other values

were compared with this value at specific condition about their significances. IC50 values

for the samples tested at different conditions were calculated by using the standard

regression line method and were tabulated (Table. 3.3).

Results showed a concentration dependent phenomenon in the assay. Highest

concentration used was 250µg/ml and followed the 2-fold serial dilution to achieve the

final concentration of 15.625µg/ml. And the different combinations were used for the

evaluation of this assay confirmed that most significant results were displayed by all

plants and standard at 120min of incubation time as compare to other two time variable.

Assay also showed a temperature dependent behavior and results were more significant at

37°C than RT. The best combination in all used was the 120 min incubation time at 37°C.

Chapter 3 Results

64

1 2 3

0.000

0.001

0.002

0.003

0.004

0.005

p-va

lue

Combination Groups

Piared t-Test

Fig 3.9 p-values represent the high significance between the three combination group. Group 1, 15

min/37°C and 15 min/RT, Group 2, 60min/37°C and 60min/RT, Group 3, 120 min/37°C and 120 min/RT

0

20

40

60

80

100

120

140

160

An

tio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015

Incubation time

Ascorbic Acid

0

20

40

60

80

100

120

140

160A

ntio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015

Incubation time (min)

Geranium collinum (GC)

0

500

1000

1500

2000

2500

3000

Antio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015


Persicaria barbata (PB)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Antio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015


Impatiens edgeworthi (IE)

Chapter 3 Results

65

0

250

500

750

1000

1250

1500

1750

2000

Antio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015


Rubia cordifolia (RC)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Antio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015


Clematis grata (CG)

0

300

600

900

1200

1500

1800

2100

2400

2700

3000

An

tio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015


Geranium wallichianum (GW)

0

200

400

600

800

1000

1200

1400

1600

An

tioxi

da

nt a

ctiv

ity (

IC5

0)

37°C

RT

120

6015


Berberis lycium (BL)

0

250

500

750

1000

1250

1500

1750

2000

2250

2500

2750

3000

3250

An

tioxi

da

nt a

ctiv

ity (

IC5

0)

37°C

RT

120

6015


Artemisia vulgaris(AV)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

An

tioxi

da

nt a

ctiv

ity (

IC5

0)

37°C

RT

120

6015


Boerhavia procumbens (BP)

Chapter 3 Results

66

0

500

1000

1500

2000

2500

3000

3500

An

tio

xid

an

t a

ctivity (

IC5

0)

37°C

RT

120

6015


Capsella bursa-pastoris (CBp)

Fig 3.10 Effect of temperature on the antioxidant activity (IC50) of medicinal plants extract

used in the study at different incubation period

Table 3.3 IC50 values calculated for the medicinal plants and Ascorbic acid (standard) at

varying temperature and incubation period (min)

Human Body Temp. (37°C) Room Temperature (25°C)

15 60 120 15 60 120

Ascorbic Acid 134.62 50 5.8 147.28 66.95 28.52

Geranium collinum

(GC)

63.23 52.73 44.09 139.44 87.09 64.63

Persicaria barbata

(PB)

2363.53 403.7 256.92 2921.06 430.39 260.4

Impatiens edgeworthi

(IE)

825.03 365.21 290.83 1758.39 959.31 718.77

Rubia cordifolia

(RC)

1729.99 513.32 341.71 1876.24 1131.36 981.23

Clematis grata

(CG)

842.1 542.43 245.93 4234.71 3289.57 2227.32

Geranium

wallichianum (GW)

851.42 599.79 404.37 2772.02 1373.92 652.3

Berberis lycium

(BL)

1503.32 316.19 81.71 1322.83 1079.54 823.38

Artemisia vulgaris

(AV)

386.11 98.9 35.58 3012.25 2952.73 1244.84

Boerhavia

procumbens (BP)

556.06 262.66 163.81 4704.92 2818.11 715.77

Capsella bursa-

pastoris (CBp)

1072.65 551.44 348.93 3644.01 1868.92 942.62

Chapter 3 Results

67

3.2.3 ABTS+ RADICAL SCAVENGING ACTIVITY

ABTS (2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid) was considered to be

peroxidase substrate, when any peroxyl radical or any other oxidant with available H2O2

produced to ABTS-(meta stable radical cation) and was intensely colored. So, capacity of

any substance to decrease the color after direct reaction with ABTS- was regarded as

antioxidant capacity.

Ascorbic acid was used as standard to determine the ABTS+ radical scavenging

activity. Among the plants used in the study, only Geranium collinum showed the highly

significant activity (with IC50 value 17.06 µg/ml) better than the standard ascorbic acid

(126.10 µg/ml) used and Artemisia vulgaris showed the least scavenging activity (with

IC50 value 1461.93 µg/ml). All other plants were with the moderate activity. The IC50

values for each plant are presented in table 3.4. The regression lines for all plants used in

the study were also calculated with their respective R2 value (Annex 3). The percent

scavenging activity for each plant was also plotted against the standard to know about the

significance of results achieved (Fig 3.12 to fig 3.21). The results suggested that the

activity is concentration dependent and with decrease in concentration, scavenging

activity also fell down.

On the basis of this activity, all the medicinal plants used in the screening were fall in

following descending order, showed by a plant extract. Each plant is given with the

corresponding IC50 value (table 3.4 ) coupled with the respective R2 value. Geranium

collinum (IC50 = 17.06 µg/ml, R2= 0.682) > Berberis lycium (IC50 = 214.70 µg/ml,

R2=0.982) > Capsella bursa-pastoris(IC50 = 254.83 µg/ml, R2=0.8789) > Persicaria

barbata (IC50 = 301.49 µg/ml, R2= 0.9728) > Clematis grata (IC50 = 315.24 µg/ml, R2=

0.9517) > Boerhavia procumbens (IC50 = 407.67 µg/ml, R2= 0.9291) > Impatiens

edgeworthii (IC50 = 642.14 µg/ml, R2= 0.972) > Rubia cordifolia (IC50 = 890.55 µg/ml,

R2= 0.8944) > Geranium wallichianum (IC50 = 976.85 µg/ml, R2=0.9825) > Artemisia

vulgaris(IC50 = 1461.93 µg/ml, R2= 0.9714). .

Chapter 3 Results

68

Table 3.4: IC50 values calculated for all plants and standard ascorbic acid in ABTS+

radical scavenging activity

Species IC50 (µg/ml-) R2 value

Ascorbic Acid 126.10 0.9622

Geranium collinum 17.06* 0.9562

Persicaria barbata 301.49 0.9728

Impatiens edgeworthii 642.14 0.972

Rubia cordifolia 890.55 0.8944

Clematis grata 315.24 0.9517

Geranium wallichianum 976.85 0.9825

Berberis lycium 214.70 0.982

Artemisia vulgaris 1461.93 0.9714

Boerhavia procumbens 407.67 0.9291

Capsella bursa-pastoris 254.83 0.9569

*showing the highly significant activity

Chapter 3 Results

69

0

10

20

30

40

50

60

70

80

90

100

c

bc

% s

cava

ngin

g ac

tivity

e

de

ab

cd

ab

cd

a

ab

Concentration (µg/ml)

GA

GC

15.62531.5

62.5125250

Fig 3.11 % scavenging activity of Geranium collinum extract on ABTS radical scavenging

activity. Values are the mean from 03 replicates. Columns with similar alphabets are not

significantly different at P < 0.05.

0

10

20

30

40

50

60

70

80

90

100

de

ee% s

cava

ngin

g ac

tivity

e

de

cd

d

cd

cd

ab


GA

PB

15.62531.5

62.5125250

Fig 3.12 % scavenging activity of Persicaria barbata extract on ABTS radical scavenging

activity. Values are the mean from three replicates. Columns with similar alphabets are not


Chapter 3 Results

70

0

10

20

30

40

50

60

70

80

90

100

de de

d

dede

% sc

avan

ging

act

ivity

e

cd

de

cd

ab


GA

IF

15.62531.5

62.5125250

Fig 3.13 % scavenging activity of Impatiens edgeworthii extract on ABTS radical

scavenging activity. Values are the mean from three replicates. Columns with similar

alphabets are not significantly different at P < 0.05.

0

10

20

30

40

50

60

70

80

90

100

edede

de de

% s

cava

ngin

g ac

tivity

e

cd

de

cd

ab


GA

RC

15.62531.5

62.5125250

Fig 3.14 % scavenging activity of Rubia cordifolia extract on ABTS radical scavenging

activity. Values are the mean from three replicates. Columns with similar alphabets are

not significantly different at P < 0.05.

Chapter 3 Results

71

0

10

20

30

40

50

60

70

80

90

100

de

dd

cd

d

% s

cava

ngin

g ac

tivity

e

cd

de

cd

ab


GA

CG

15.62531.5

62.5125250

Fig 3.15 % scavenging activity of Clematis grata extract on ABTS radical scavenging



0

10

20

30

40

50

60

70

80

90

100

ee e

de

e% s

cava

ngin

g ac

tivity

e

cd

de

cd

ab


GA

GW

15.62531.5

62.5125250

Fig 3.16 % scavenging activity of Geranium wallichianum extract on ABTS radical



Chapter 3 Results

72

0

10

20

30

40

50

60

70

80

90

100

dede de

c

d

% s

cava

ngin

g ac

tivity

e

cd

de

cd

ab


GA

BL

15.62531.562.5

125250

Fig 3.17 % scavenging activity of Berberis lycium extract on ABTS radical scavenging



0

10

20

30

40

50

60

70

80

90

100

de dedede de

% s

cava

ngin

g ac

tivity

e

cd

de

cd

ab


GA

AV

15.62531.5

62.5125250

Fig 3.18 % scavenging activity of Artemisia vulgaris extract on ABTS radical scavenging



Chapter 3 Results

73

0

10

20

30

40

50

60

70

80

90

100

de dede

d

d

% s

cava

ngin

g ac

tivity

e

cd

de

cd

ab


GA

BP

15.62531.5

62.5125250

Fig 3.19 % scavenging activity of Boerhavia procumbens extract on ABTS radical



0

10

20

30

40

50

60

70

80

90

100

ef ef

e

c

e

% s

cava

ngin

g ac

tivity

bc

cd

de

cd

ab


GA

CBP

15.62531.562.5

125250

Fig 3.20 % scavenging activity of Capsella bursa-pastoris extract on ABTS radical



Chapter 3 Results

74

3.2.4 HYDROXYL RADICAL SCAVENGING ACTIVITY

In living organism when hydrogen peroxide react with Fe2+ generate the hydroxyl

radicals. This is widely acceptable phenomenon known as Fenton reaction. The resultant

hydroxul radical swere considered to be highly reactive (Fe2+ + H2O2 → Fe3+ + HO− +

HO*).

Gallic acid (IC50= 5.32 µg/ml, R2= 0.9478) was used as standard to determine the

radical scavenging activity. Among the plants used in the study, Geranium collinum,

Persicaria barbata, Clematis grata, and Rubia cordifolia showed the highly significant

activity. Geranium collinum (with IC50 value 5.19 µg/ml) better than the standard Gallic

acid (5.32 µg/ml) used and Impatiens edgeworthii showed the least scavenging activity

(with IC50 value 126.70 µg/ml). All other plants were with the significant to moderate

activity. The IC50 values for each plant are presented in table 3.5. The regression lines for

all plants used in study were also calculated with their respective R2 value (Annexure

IV). The percent scavenging activity for each plant was also plotted against the standard

to know about the significance of results achieved (Fig 3.22 to fig 3.31). The result

suggested that the activity is concentration dependent and with decrease in concentration,

scavenging activity also fell down.

On the basis of this activity, all the medicinal plants used in the screening fall in

following decending order showed by a plant extract. Each plant is given with the

corresponding IC50 value (table 3.5) coupled with the respective R2 value. Geranium

collinum (IC50 = 5.19 µg/ml, R2= 0.9368) > Persicaria barbata (IC50 = 5.43 µg/ml, R2=

0.9714 ) > Clematis grata (IC50 = 7.08 µg/ml, R2= 0.878 ) > Rubia cordifolia (IC50 =

8.28 µg/ml, R2= 0.9026) > Berberis lycium (IC50 = 32.18 µg/ml, R2= 0.8538) > Artemisia

vulgaris(IC50 = 49.93 µg/ml, R2= 0.8038) > Geranium wallichianum (IC50 = 51.78

µg/ml, R2= 0.8914) > Boerhavia procumbens (IC50 = 61.65 µg/ml, R2= 0.9554) >

Capsella bursa-pastoris(IC50 = 71.87 µg/ml, R2= 0.8826) > Impatiens edgeworthii (IC50 =

126.70 µg/ml, R2= 0.8794).

Chapter 3 Results

75

Table 3.5: IC50 values calculated for all plants and standard ascorbic acid in

Hydroxyl radical scavenging activity

Plant Species IC50 (µg/ml-) R2 value

Gallic Acid 5.32 0.9478

Geranium collinum 5.19* 0.9368

Persicaria barbata 5.43* 0.9714


Rubia cordifolia 8.28* 0.9026

Clematis grata 7.08* 0.878


Berberis lycium 32.18 0.8538

Artemisia vulgaris 49.93 0.8038

Boerhavia procumbens 61.65 0.9554

Capsella bursa-pastoris 71.87 0.8826


Chapter 3 Results

76

0

10

20

30

40

50

60

70

80

90

100

c

bc

% s

cava

ngin

g ac

tivity

c

bc

bc

bb

abab

ab


GA

GC

15.62531.5

62.5125250

Fig 3.21 % scavenging activity of Geranium collinum extract on Hydroxyl radical



0

10

20

30

40

50

60

70

80

90

100

c

cd

c

% s

cava

ngin

g ac

tivity

c

bc

bb

abab

ab


GA

PB

15.62531.5

62.5125

250

Fig 3.22 % scavenging activity of Persicaria barbata extract on Hydroxyl radical



Chapter 3 Results

77

0

10

20

30

40

50

60

70

80

90

100

d

de

bc

cd

c

% sc

avan

ging

act

ivity

c

b

bc

ab

ab


GA

IP

15.62531.5

62.5125250

Fig 3.23 % scavenging activity of Impatiens edgeworthii extract on Hydroxyl radical



0

10

20

30

40

50

60

70

80

90

100

c

bc

b

ab

b

% s

cava

ngin

g ac

tivity

c

b

bc

ba

ab


GA

RC

15.62531.562.5

125250

Fig 3.24 % scavenging activity of Rubia cordifolia extract on Hydroxyl radical



Chapter 3 Results

78

0

10

20

30

40

50

60

70

80

90

100

c

bc

bc

b

b

% s

cava

ngin

g ac

tivity

c

b

bc

ab

ab


GA

CG

15.62531.5

62.5125250

Fig 3.25 % scavenging activity of Clematis grata extract on Hydroxyl radical scavenging



0

10

20

30

40

50

60

70

80

90

100cd

c

cd

bc

c

% s

cava

ngin

g ac

tivity

bc

b

bc

ab

ab


GA

GW

15.62531.5

62.5125

250

Fig 3.26 % scavenging activity of Geranium wallichianum extract on Hydroxyl radical



Chapter 3 Results

79

0

10

20

30

40

50

60

70

80

90

100

cd

c

c

b

bc

% s

cava

ngin

g ac

tivity

c

b

bc

ab

ab


GA

BL

15.62531.562.5

125250

Fig 3.27 % scavenging activity of Berberis lycium extract on Hydroxyl radical scavenging



0

10

20

30

40

50

60

70

80

90

100

bc

cd

c

b

bc

% s

cava

ngin

g ac

tivity

c

b

bc

ab

ab


GA

AV

15.62531.5

62.5125250

Fig 3.28 % scavenging activity of Artemisia vulgaris extract on Hydroxyl radical



Chapter 3 Results

80

0

10

20

30

40

50

60

70

80

90

100

cd

cd

c

b

c

% sc

avan

ging

act

ivity

c

b

bc

ab

ab


GA

BP

15.62531.5

62.5125

250

Fig 3.29 % scavenging activity of Boerhavia procumbens extract on Hydroxyl radical



0

10

20

30

40

50

60

70

80

90

100

cdd

c

bcbc

% sc

avan

ging

act

ivity

c

b

bc

ab

ab


GA

CBP

15.62531.5

62.5125

250

Fig 3.30 % scavenging activity of Capsella bursa-pastoris extract on Hydroxyl radical



Chapter 3 Results

81

3.2.5 PHOSPHOMOLYBDINUM ASSAY

The phosphomolybdinum antioxidant activity was based on the Mo (VI) reduction

to Mo (V) by sample and finally resulted at acidic pH with a product of phosphate/Mo

(V).

Ascorbic acid with (IC50 = 4.78, R2 = 0.92) was used as standard to determine the

Phosphomolybdinum activity. Among the plants used in the study, Boerhavia

procumbens (IC50 = 6.11 µg/ml), Artemisia vulgaris (IC50 = 7.12 µg/ml), Berberis lycium

(IC50 = 7.27 µg/ml), Capsella bursa-pastoris(IC50 = 7.73 µg/ml), Persicaria barbata

(IC50 = 8.31 µg/ml), Rubia cordifolia (IC50 = 8.35 µg/ml) showed the highly significant

activity. While Geranium wallichianum showed the least scavenging activity with IC50

value 139.23 µg/ml. All other plants were with the moderate activity. The regression

lines for all plants used in study were also calculated with their respective IC50 and R2

value (Annexure V). The result suggested that the activity is concentration dependent and

with decrease in concentration, scavenging activity also fell down (Fig 3.32 to fig 3.41).

On the basis of this activity, all the medicinal plants used in the screening fall in

following descending order showed by a plant extract. Each plant is given with the

corresponding IC50 value (Table 3.6) coupled with the respective R2 value. Boerhavia

procumbens (IC50 = 6.11 µg/ml, R2= 0.9509) > Artemisia vulgaris (IC50 = 7.12 µg/ml,

R2= 0.9198) > Berberis lycium (IC50 = 7.27 µg/ml, R2= 0.8401) > Capsella bursa-

pastoris(IC50 = 7.73 µg/ml, R2= 0.9213) > Persicaria barbata (IC50 = 8.31 µg/ml, R2=

0.9849) > Rubia cordifolia (IC50 = 8.35 µg/ml, R2= 0.8397) > Impatiens edgeworthii (IC50

= 45.22 µg/ml, R2= 0.8005) > Clematis grata (IC50 = 63.85 µg/ml, R2= 0.8645) >

Geranium collinum (IC50 = 89.89 µg/ml, R2= 0.9212) > Geranium wallichianum (IC50 =

139.23 µg/ml, R2=0.7987).

Chapter 3 Results

82

Table 3.6: IC50 values calculated for all plants and standard ascorbic acid in

Phosphomolybdinum assay

Plant Species IC50 (µg/ml-) R2 value

Ascorbic Acid 4.78 0.92

Geranium collinum 89.89 0.9213

Persicaria barbata 8.31* 0.9840


Rubia cordifolia 8.35* 0.8397

Clematis grata 63.85 0.8645


Berberis lycium 7.27* 0.8401

Artemisia vulgaris 7.12* 0.9198

Boerhavia procumbens 6.11* 0.9509

Capsella bursa-pastoris 7.73* 0.9213


Chapter 3 Results

83

0

10

20

30

40

50

60

70

80

90

100

% sc

aven

ging

act

ivity

e

d

cd

bb

bb

ababab

AA

GC

15.62531.5

62.5125250


Fig 3.31 % scavenging activity of Geranium collinum extract on Phosphomolybdinum

assay. Values are the mean from three replicates. Columns with similar alphabets are not


0

10

20

30

40

50

60

70

80

90

100


% sc

aven

ging

act

ivity

bcbcbbbbb

abab

ab

AA

PB

15.62531.5

62.5125250

Fig 3.32 % scavenging activity of Persicaria barbata extract on Phosphomolybdinum



Chapter 3 Results

84

0

10

20

30

40

50

60

70

80

90

100

% sc

aven

ging

act

ivity

d

bc

bb

bbabab

abab

AA

IP

15.62531.562.5

125250


Fig 3.33 % scavenging activity of Impatiens edgeworthii extract on Phosphomolybdinum



0

10

20

30

40

50

60

70

80

90

100

% s

cave

ngin

g ac

tivity

c

bc

bc bcb

bb

ab

ab ab

AA

BL

15.62531.5

62.5125250


Fig 3.34 % scavenging activity of Berberis lycium extract on Phosphomolybdinum assay.

Values are the mean from three replicates. Columns with similar alphabets are not


Chapter 3 Results

85

0

10

20

30

40

50

60

70

80

90

100

% s

cave

ngin

g ac

tivity

bcbc

bb

b ababababab


AA

AV

15.62531.5

62.5125250

Fig 3.35 % scavenging activity of Artemisia vulgaris extract on Phosphomolybdinum



0

10

20

30

40

50

60

70

80

90

100

% sc

aven

ging

act

ivity

c

bc bc

bcb

bbabab

ab


AA

RC

15.62531.5

62.5125250

Fig 3.36 % scavenging activity of Rubia cordifolia extract on Phosphomolybdinum



Chapter 3 Results

86

0

10

20

30

40

50

60

70

80

90

100

% s

cave

ngin

g ac

tivity

ede

cd

c

bc

bcb

b

ab

ab

AA

GW

15.62531.5

62.5125

250


Fig 3.37 % scavenging activity of Geranium wallichianum extract on

Phosphomolybdinum assay. Values are the mean from three replicates. Columns with

similar alphabets are not significantly different at P < 0.05.

0

10

20

30

40

50

60

70

80

90

100

bc

% s

cave

ngin

g ac

tivity

c

bcbc

b

bbabab

ab


AA

BP

15.62531.5

62.5125250

Fig 3.38 % scavenging activity of Boerhavia procumbens extract on Phosphomolybdinum



Chapter 3 Results

87

0

10

20

30

40

50

60

70

80

90

100

cd

% sc

aven

ging

act

ivity

cd

c

bc

bbc

bb ab

ab


AA

CG

15.62531.562.5

125250

Fig 3.39 % scavenging activity of Clematis grata extract on Phosphomolybdinum assay.

Values are the mean from three replicates. Columns with similar alphabets are not


0

10

20

30

40

50

60

70

80

90

100c

% s

cave

ngin

g ac

tivity

bc

bcb b

bb

abab

ab


AA

CBP

15.62531.562.5

125250

Fig 3.40 % scavenging activity of Capsella bursa-pastoris extract on

Phosphomolybdinum assay. Values are the mean from three replicates. Columns with

similar alphabets are not significantly different at P < 0.05.

Chapter 3 Results

88

3.2.6 THE FERRIC REDUCING ABILITY OF PLASMA (FRAP)

The FRAP assay (ferric reducing ability of plasma), a test of the total antioxidant

power was carried out for the three medicinal plants viz Geranium collinum (GC),

Persicaria barbata (PB) and Geranium wallichianum (GW). The regression line was

used to determine their IC50 values. Persicaria barbata (PB) displayed the highest FRAP

value with IC50 (103.8 µg/ml) at R2 value 0.8434 among the three plants tested. Followed

by, Geranium wallichianum (GW) with IC50 (116.76 µg/ml) at R2 value and least by

Geranium collinum (GC) with 0.8732 IC50 (142.74 µg/ml) at R2 value 0.7986.

The FRAP values were plotted against the concentration (µg/ml) used of the

extract from three different medicinal plants (Fig 3.41)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

FRA

P V

alue

s


PB

GW

GC

1000500

200150

100

Fig 3.41 Presenting the FRAP values of three different medicinal plant Note: FRAP values represent the ODs (Optical Density Measurements)

Chapter 3 Results

89

3.2.7 ANTI-LEISHMANIAL ACTIVITY

The activity of all selected medicinal plants extracts were tested against

Leishmania major promastigotes. Glucantime has been used as the positive control in this

study and showed IC50=251 µg/mg on promastigotes (1x106 /100µ/well) of a log phase

culture. The results shown in table 3.7 represent that Geranium collinum (IC50=6.98

µg/mg) showed the best result of all plants tested against L. major promastigotes along

with the Berberis lycium (IC50=8.46 µg/mg) also with good inhibition. Capsella bursa-

pastoris (IC50=204.23 µg/mg) was with the least inhibition of growth of the promastigote

but comparatively significant to that of Glucantime (IC50=251 µg/mg).

On the basis of this experiment for the determination of anti-leishmanial activity

of medicinal plants used in the screening fall in following descending order, showed by a

plant extract alongwith the respective IC50 value. Geranium collinum (IC50=6.98 µg/mg),

Berberis lycium (IC50=8.46 µg/mg), Impatiens edgeworthi (IC50=12.5 µg/mg), Rubia

cordifolia (IC50=12.8 µg/mg), Boerhavia procumbens (IC50=15.8 µg/mg), Artemisia

vulgaris (IC50=16.2 µg/mg), Persicaria barbata (IC50=18.09 µg/mg), Geranium

wallichianum (IC50=23 µg/mg), Clematis grata (IC50=25 µg/mg), and Capsella bursa-

pastoris (IC50=204.23 µg/mg).

Overall results suggested that plants used in the current study exhibited anti-

leishmanial activity against L.major promastigotes. In comparison with Glucantime IC50

value (standard drug), majority of the plants fall in a significant activity (Fig 3.43). This

activity exhibited a concentration dependent phenomenon in case of majority of

medicinal plants used in the study.

Chapter 3 Results

90

0

10

20

30

40

50

60

70

80

90

100

110

% in

hibi

tion

of L

.Maj

or p

rom

astig

otes

GC

PB

IP

CG

GW

BL

AV

BP

CBp

100 50 25 12.5200 6.25

Concentration (µg)

Fig 3.42 % inhibition of Leishmania major promastigotes at different concentration

shown by the medicinal plants used in the study. Key for the legend used for the

medicinal plants is given in table 3.7

Table 3.7 Antileishmanial Activity of medicinal plants in term of IC50 along with the

Glucantime (standard)

Samples IC50 (µg/ml-)

Glucantime (+ control) 251

Geranium collinum (GC) 6.98*

Persicaria barbata (PB) 18.09

Impatiens edgeworthi (IE) 12.5

Clematis grata (CG) 25

Geranium wallichianum (GW) 23

Berberis lycium (BL) 8.46*

Artemisia vulgaris(AV) 16.2

Rubia cordifolia (RC) 12.8

Boerhavia procumbens (BP) 15.8

Capsella bursa-pastoris (CBp) 204.23

Chapter 3 Results

91

3.2.8 ANTIGLYCATION ACTIVITY

Inhibition of production of AGEs in the Glucose/BSA system was used to

evaluate the antiglycation capacities of medicinal plants extracts. Results suggested that

various extracts used in the study inhibited the formation of AGEs mediated by glucose

was a dose-dependent phenomenon.

Rutin was used as standard drug in the study which showed the 86% inhibition

and all other results were compared with this result for their significances. From the

results obtained, it was observed that the degree of antiglycation activities varied

considerably from plant to plant. Persicaria barbata (PB), Geranium collinum (GC) and

Berberis lycium (BL) showed the significant inhibition against AGE’s formation. The %

inhibitory capacity for these plants was 68.89, 62.06 and 54.23 observed respectively. All

other plants also inhibited the AGE’s formation non-significantly. These included

Impatiens edgeworthi (17.15%), Clematis grata (9.43 %), Geranium wallichianum

(3.17%), Artemisia vulgaris (13.9%), Rubia cordifolia (17.74%), Boerhavia procumbens

(9.52%) and Capsella bursa-pastoris (5.87%) (Table 3.8).

Overall results displayed a clear picture of the potential of the various medicinal

plants (Fig 3.43) extracts used in the study about their antiglycation activity. Out of all

medicinal plants used three plants i.e., Persicaria barbata, Geranium collinum, and

Berberis lycium with promising activities was identified after comparison with standard

drug in purified form.

Chapter 3 Results

92

10

20

30

40

50

60

70

80

90

100

Ant

igly

catio

n A

bilit

y (%

)

GC PB IE RC

CG

GW BL

BP

AV

CB

p

Rut

in

Fig 3.43 Antiglycation abilities of the medicinal plants by inhibition of formation of AGE’s in

BSA/Glucose system

Table 3.8: Inhibitory capacities of the extracts of the medicinal plants on the formation

of Advanced Glycation Endproduct (AGE’s)

Sample (0.5 mg/ml) Inhibition (%)

Geranium collinum (GC) 62.06*

Persicaria barbata (PB) 68.89*

Impatiens edgeworthi (IE) 17.15

Clematis grata (CG) 9.43

Geranium wallichianum (GW) 3.17

Berberis lycium (BL) 54.23*

Artemisia vulgaris(AV) 13.9

Rubia cordifolia (RC) 17.74

Boerhavia procumbens (BP) 9.52

Capsella bursa-pastoris (CBp) 5.87

Rutin 86

Chapter 3 Results

93


The preliminary screening results on human whole blood oxidative burst activity

were performed to evaluate the ability of the medicinal plants extracts to modulate the

immune system response of phagocytes and monocytes.

Ibuprofen, the standard drug was used in the study as a positive control. All the

plants extracts used in the study were compared with the drug to check their

significances. Ibuprofen modulate the immune response displayed an IC50 value11.8±1.9

µg/ml. With reference to this, majority of the plants in the study were found to modulate

the immune response significantly. displayed the moderate inhibitory activity. Four

plants Geranium collinum (GC), Artemisia vulgaris (AV), Boerhavia procumbens (BP)

and Capsella bursa-pastoris (CBp) showing the highly significant results with IC50 value

less than 2µg/ml. Clematis grata (CG) Persicaria barbata (PB) and Rubia

cordifolia (RC) also exhibited the very significant results with IC50 values 3.4 ± 0.2

µg/ml, 9.6±0.1 µg/ml, and 9.6 ± 0.8 µg/ml respectively. Only three medicinal plants

extracts were found to possess non-significant activity. Out of the three Berberis

lycium (BL) and Geranium wallichianum (GW) have IC50 value more than 200 µg/ml and

Impatiens edgeworthi (IE) with IC50 value 110.4±7.6 µg/ml (Table 3.9).

The results suggested the dose dependent behavior of the medicinal plants

(Fig 3.45) extract and the highly significant results emphasizing their potential as a

source of novel alternative immunomodulatory agents.

Chapter 3 Results

94

1.5

3.0

4.5

6.0

7.5

9.0

10.5

12.0

13.5

Viab

ility

of h

uman

pol

ymor

phon

eutr

ophi

ls

GC PB IE RC

CG

GW BL

BP

AV

CB

p

IBp

Fig 3.44 Modulation of Oxidative burst from immune cell (Human polymorphoneutrophils) by

the extract of medicinal plants used in the study. (The key for the code name of the

medicinal plants is presented in table 3.)

Table 3.9 Immunomodulatory activity of medicinal plants in term of their IC50

Samples IC50

Ibuprofen (+ control) (IBp) 11.8±1.9

Geranium collinum (GC) <2

Persicaria barbata (PB) 9.6±0.1

Impatiens edgeworthi (IE) 110.4±7.6

Rubia cordifolia (RC) 9.6 ± 0.8

Clematis grata (CG) 3.4 ± 0.2

Geranium wallichianum (GW) >200

Berberis lycium (BL) >200

Artemisia vulgaris(AV) <2

Boerhavia procumbens (BP) <2

Capsella bursa-pastoris (CBp) <2

Chapter 3 Results

95

3.2.10 Metabolic Impairment Assays (MTT assay) for Human

Prostate Cancer Cell Line (PC-3)

The MTT test is based on the mitochondrial enzymatic reduction of the tetrazolium

salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] to detect and

determine cell viability.

Medicinal plants extracts were tested against the Human prostate cancer cell line (PC-

3) to know about their cytotoxic effect by following the MTT assay. Optical absorbance

was used to measure the relative number of live cells, which give rise to % viable cell.

This value was used to calculate the IC50 of the medicinal plants samples were used in the

study. Doxorubicin, a reference drug was used as a positive control with the IC50 value

2.8±0.12 µg/ml. In comparison with this drug six plants exhibited the non-significant

activity have the IC50 more than 30 µg/ml. These plants extracts included Persicaria

barbata (PB), Clematis grata (CG), Geranium wallichianum (GW), Artemisia vulgaris

(AV), Boerhavia procumbens (BP) and Capsella bursa-pastoris (CBp) (Fig 3.46).

Among the plants used Geranium collinum (GC) showed the best activity with IC50 value

12.796±0.22 µg/ml. other plants included in the study are Impatiens edgeworthi (IE) with

IC50 value 14.1883±0.275 µg/ml Rubia cordifolia (RC) with IC50 value 20.09±0.395

µg/ml, Berberis lycium (BL) with IC50 value 24.149±0.981 µg/ml also displayed the

significant results (Table 3.10).

Chapter 3 Results

96

5

10

15

20

25

Cyt

otox

icity

act

ivity

pro

file

GC PB IE RC

CG

GW BL

BP

AV

CB

p Dx

Fig 3.45 Cytotoxicy activity profile of medicinal plants on Human prostate cancer cell

line (PC-3)

Table 3.10 IC50 values of medicinal plants representing their inhibitory action on

Human prostate cancer cell line (PC-3)

Samples IC50 (µg/ml)

Doxorubicin 2.8±0.12

Geranium collinum (GC) 12.796±0.22

Persicaria barbata (PB) > 30

Impatiens edgeworthi (IE) 14.1883±0.275

Rubia cordifolia (RC) 20.09±0.395

Clematis grata (CG) > 30

Geranium wallichianum (GW) > 30

Berberis lycium (BL) 24.149±0.981

Artemisia vulgaris (AV) > 30

Boerhavia procumbens (BP) > 30

Capsella bursa-pastoris (CBp) > 30

Chapter 3 Results

97

3.2.11 Sulforhodamine B (SRB) assay for Human Cancer Lung Cell Line

(LU-1) and human prostate adenocarcinoma cells (LNCap-1)

The Sulforhodamrne (SRB) assay was performed to evaluate the cytotoxicity for

the three medicinal plants viz Geranium collinum (GC), Persicaria barbata (PB) and

Geranium wallichianum (GW) against the human cell line, human lung carcinoma (LU-

1) and human prostrate adenocarcinoma (LNCaP). The cytotoxicity activity profile of

vinblastine (0.04 µg/ml) was used as a positive control in this experiment. Extracts with

IC50>25 µg/ml were considered as inactive (Table 3.11).

In case of LU-1, only Geranium collinum (GC) showed the significant activity

with IC50=15 µg/ml while the other two plants Persicaria barbata (PB) and Geranium

wallichianum (GW) showed the non-significant results with respect to their IC50

calculated (Fig 3.47).

0

15

30

45

60

75

90

105

% S

urvi

val


GC

PB

GW

50 2512.5

6.25

Fig 3.46 The cytotoxicity activity profile of three medicinal plants extracts for LU-1

human cell line showing the % survival

Chapter 3 Results

98

In case of LNCap-1, Geranium collinum (GC) showed the cytotoxicity activity

IC50=24 µg/ml, Geranium wallichianum (GW) 25 µg/ml were considered to be

significant at the border line. But, Persicaria barbata (PB) showed the non-significant

results with respect to IC50= 34 (Fig 3.48).

0

15

30

45

60

75

% S

urvi

val


GC

PB

GW

50 25

Fig 3.47 The cytotoxicity activity profile of three medicinal plants extracts for LNCap-1

human cell line showing the % survival

Table 3.11 IC50 values for medicinal plants for Human cell line LU-1 and LNCap-1

Cell Line Geranium

collinum

Persicaria

barbata

Geranium

wallichianum

LU-1 15 >50 >50

LNCap-1 24 34 25.1

4. DISCUSSION

Chapter 4 Discussion

99

4.1 ETHNOBOTANICAL INVESTIGATION

The Himalayas were famous for the most unusual and richest ecosystems on earth

with great variation in-terms of climate and altitudes ranged from alpine peaks to

foothills, form a variety of forest, and presented an example of direct impact of altitude

on vegetation (Mani, 1978). Himalayas are fed with monsoon rainfall mainly, coming

from Bay of Bengal are intercepted by the eastern side of these Himalayan mountains,

left the west with lesser precipitation. This moisture gradient also influences the

distribution and diversity of vegetation in the region (Kenderick, 1989). In Pakistan,

Himalaya range lies in the North West region of the country, gives shelter to a diverse

communities. The traditional knowledge of plants use by these communities have been

documented includes; medicinal plants in old re-growth forest (Adnan and Holscher,

2011), degraded and reforested sites (Adnan and Holscher, 2010), tribal communities in

Northern Himalaya ranges (Abbasi et al., 2010) medicinal plant biodiversity of lesser

Himalayas-Pakistan (Abbasi et al., 2011), ethnobotanical studies from Bagh, Kashmir,

Lesser Himalaya (Shaheen et al., 2011).

Galliyat region located in in Western Himalaya contains highly diverse

Himalayan moist temperate forest (Hussain & Ilahi, 1991). Ayubia National Park (ANP)

forms a small portion in the Galliyat gained the main focus in the past. Ethnobotanical

studies of Ayubia National Park was documented by Gilani et al., (2001), traditional

knowledge of herbs in ANP (Gilani et al., 2006), economic values of underutilized plant

in ANP (Ahmad and Javed, 2007), ethnobotany of fodder species in ANP (Jabeen, 1999),

ethnobotanical and management of fodder and fuel in ANP (Aumeeruddy et al., 2004),

People and plant workshop on applied ethnobotany in ANP (Aumeeruddy et al., 1998).

Hence, the present study was carried out with the aim to cover the complete Galliyat

region to collect and conserve the precious traditional knowledge.

The present study provides the information on the Ethnobotanical use of 45

medicinal plants comprises 38 angiosperms, 4 gymosperms and 3 pteridophytes. Familial

distribution of medicinal plants used in the study includes 21 angiosperms families and


100

gymnosperms and pteridophytes by two families each. Major source of the

ethnobotanical data was the people with older age and traditional practitioners and

women have more knowledge as compared to the males.

Ethnobotanical usages of the medicinal plants reported in this study were also

described earlier by various authors for some different usage in different communities

and tribes. Current uses of Dryopteris ramosa was in accordance with as reported earlier

by Arshad and Ahmad (2005) against gastric ulcer and constipation but some authors also

reported its multiple uses such as for healing purposes (Rauf et al., 2007). Adiantum

incisum in the area is used in chronic diarrhea and dysentery but also reported to be used

in the treatment for jaundice (Jan et al., 2009) and curing skin diseases, fever and

diabetes (Murad et al., 2011). Adiantum venustum was used in headache and scorpion

setting but it is considered poisonous if taken in large quantities (Ibrar and Hussain,

2009) and also mentioned by Sher et al., (2010) and Saqib and Sultan (2005), and ethno-

veterinary by Khan and Hanif (2006). Abies pindrow was reported previously to sell as

timber and fuel wood (Gilani et al., 2001). Cedrus deodara was reported to be used as

stomachic, antheliminthic, febrifuge (Shah and Khan, 2006).

The ethnobotanical uses of Arisaema flavum are identical in comparison with

previous literature, fresh rhizome is reported to be applied to snake bite and scorpion

sting (Shah and Khan, 2006) which showed accordance with current results. Similarly,

Arisaema jacquemontii was also reported to be poisonous (Ali and Qaiser, 2009; Saghir

et al., 2001) and against snake bites (Zabihullah et al., 2006). Achillea millefolium

previously described as tonic and stimulant (Gilani et al., 2001) and diuretic, stimulant,

tonic (Qureshi et al., 2007a). Known usage of Artemisia dubia is as stomachic, purgative

(Ashraf et al., 2010) and for Artemisia vulgaris leaf extract was used for malaria and

fever (Ashraf et al., 2010). Chrysanthemum leucanthemum, which is one of the most

common plants in the area, was also reported to have insecticide and pesticide uses

(Abbasi et al., 2010; Hussain, 2003). The roots of Cichorium intybus are used in

indigestion, abdominal disorders (Khan and Khatoon, 2008). The folk lore use of

Saussuria hetromalla was previously known for healing wounds and burning (Matin et


101

al., 2001) and rhizomes can be crushed and given to increase milk flow in cattles (Shah et

al., 2012). Antipyretic properties of the Senecio chrysanthemoides are also mentioned by

Saghir et al., (2001). Taraxacum officinalewas earlier reported to be used as diuretic,

used for kidney and liver disorder (Hussain et al., 2008). Impatiens bicolor was

mentioned to used as diuretic, tonic and has cooling effect and also used to cure wounds

from burning (Gilani et al., 2006). Current results about the use of Impatiens edgeworthii

in traditional knowledge was in accordance with Qureshi et al., (2007a) because of the

near-by located study area in Himalayan ranges. But, was also reported to be used as

antipyretic (Saghir et al., 2001).

The bark of stem and root of Berberis lycium were earlier reported by Qureshi et

al., (2009) to be used against rheumatism joint and other pain. Gilani et al., (2006)

mentioned the use of Podophyllumemodi against liver disorders and also used as tonic,

while its healing potential in ulcer cuts and wounds were also known (Dar, 2003). Leaves

of Capsella bursa-pastoris were considered to be astringent in diarrhea (Hussain et al.,

2008). The root decoction of Corydalis govanianawas reported to treat the ophthalmic

diseases (Saqib and Sultan, 2005). Aerial parts of Swertia chirata were considered to

treat fever and malaria disease (Ahmad et al., 2004). Geranium wallichianum was earlier

known for use in backache, gout and strengthening of body muscles (Qureshi et al., 2009;

Gilani et al., 2006). Dried powder of root of Boerhavia procumbens were reported to

over-come the complication of dysmenorrhea (Qureshi and Bhatti, 2008). Rumex

nepalensis which was used as wild vegetable in the area with sound medicinal properties

in folk lore medicine was also reported to be antiseptic and its uses against allergy

(Ahmed et al., 2004).

Previously, it was known that seeds of Plantago lanceolata were used against

constipation (Dar, 2003), and abdominal problems (Khan and Khatoon, 2008). Persicaria

barbata was used against flu problem (Matin et al., 2001). Clematis grata was reported

to be germicidal (Abbasi et al., 2010). Roots of the Rubia cordifolia can be effective in

animal bites (Rauf et al., 2007). Rhizome of Valeriana jatamansi was reported earlier to

treat stomachache (Ibrar et al., 2007) and backache and rheumatic pain (Humayun et al.,


102

2006) However, the same plant used to treat epileptic disorders. Chambal was cured by

the application of Urticadioca (Gilani et al., 2006). The rhizome powder of Bergenia

ciliate was reported to be effective in ulcer (Qureshi et al., 2009) and treating wounds

(Humayun et al., 2006). However, Berginia powder was used effectively for stomach

cancer by the local inhabitant of Murree. The stem of the Zanthoxylum alatum were

reported to be used in the formation of “miswaks” to treat toothache, ripen fruit in cardiac

disorders (Qureshi et al., 2009). Decoction of the flower of Viola canescens was used for

cough, cold, fever (Gilani et al., 2006).

The present ethnobotanical investigation after comparing with the previous

literature revealed that most of the medicinal plants usages in the study area were not

previously described in the allied or other regions. Although some plants showed

overlapping uses with earlier reports in the nearby localities but the major portion

describes the novel uses of the plants in the study area. In present research work it has

been assessed that some of the medicinal plants which have high quality potentials have

been suggested to be promoted in the area through in-situ conservation. Keeping the view

of my perception through research work following plants may be further cultivated in the

area. The plants suggested for cultivation are: Arisaema jacquemontii, Artemisia vulgaris,

Berberis lycium, Geranium wallichianum, G. collinum, Paeonia emodi, Plantago

lanceolata, Persicaria barbata, Zanthoxylum alatum, Podophyllum hexandrum, Bergenia

ciliata, Atropa acuminata, Valeriana jatamansi and Viola canescens. Podophyllum

hexandrum is being endangered in the area and else-where in Pakistan.


103

4.2 ANTIBACTERIAL ASSAY

Recently, popular medicinal plants have attracted much attention about their

extracts and isolated bioactive ingredients. In developing countries these medicinal plants

are known to play a pivotal role in encompassing their primary healthcare needs. These

have been regarded as a source of novel antibacterial drugs with promising inhibitory

action against the variety of infectious microbes (Coelho de Souza et al., 2004).

Antibacterial activities were performed for all the selected medicinal plant against four

different bacterial species include two Gram positive and Gram negative bacterial

isolates. Nikaido (1999) reported that Gram negative bacteria present more resistance

than gram positive and this was possible because of presence of extra outer layer in the

Gram negative bacterial strains. This phenomenon was also reported by several workers

earlier about the more resistance of Gram negative than Gram positive against the plant

extract (Lin et al., 1999; Parekh and Chanda, 2006).

Antibacterial potential of any drug is measured in term of its Minimal Inhibitory

Concentration (MIC) value. It represents the lowest concentration upon which any drug

inhibits the growth of a bacteria or any micro-organism. MIC methods are widely used in

the comparative testing of new agents (European Committee on Antimicrobial

Susceptibility Testing, 2000). MIC values of the all the medicinal plant extracts on all

tested microorganisms are shown in table 3.2. The MIC values obtained in this study

from all plant extracts tested ranged from 2.5 to 7.5 mg/ml. The highest MIC value of

7.25 has been observed for Impatiens edgeworthi, Rubia cordifolia, Geranium

wallichianum, Berberis lycium, Artemisia vulgaris, Boerhavia procumbens and Capsella

bursa-pastoris depending on the bacterial strains. Except Impatiens edgeworthi, all other

plant extract displayed the minimum 2.5mg/ml MIC value and was bacterial strain

dependent. Clematis grata was with least MIC against three bacterial strains among four

tested.

Antibacterial activity was expressed in terms of the diameter of the zone of

inhibition calculated. All plants showed variable response against different strains tested.

Geranium collinum presented the maximum antibacterial activity at 12.5mg/ml


104

(maximum dose used in study) against Klebsiella pneumoniae with zone of inhibition

16.67±1.52 mm measured while Rubia cordifolia with least measurement of 8.2±0.577

mm. Like-wise, Persicaria barbata was with 28.75±0.33 mm at maximum dose of

12.5mg/ml and least was measured for Impatiens edgeworthii with 9.67±0.33 mm against

the Bacillus subtilus. In case of Pseudomonas aeruginosa, Persicaria barbata displayed

the highest 21.33±0.33 mm, and Capsella bursa-pastoris (3.8±0.33 mm-5.342±0.23 mm

was with lowest activity at maximum dose applied. For Enterococcus aerogenes, the

maximum dose applied for Geranium collinum produced the maximum result with

19.0±0.577 mm while least activity at the same dose was shared by Capsella bursa-

pastoris, and Rubia cordifolia with 6.24±0.44 mm diameter of zone.

No earlier reference was available for antibacterial screening of some medicinal

plants used in the current study. These include Geranium collinum, Persicaria barbata,

Impatiens edgeworthii, Clematis grata, and Boerhavia procumbens. In current

experiment, extract of Rubia cordifolia displayed different results from the previously

mentioned activity performed on natural dye produced by the R. cordifolia. Where

natural dye did not showed any antibacterial results (Ray and Majumdar, 1976).

Antibacterial activity evaluated for Geranium wallichianum was in agreement with

Ahmad et al., (2003) against the Bacillus subtilus, Klebsiella pneumoniae, and

Pseudomonas aeruginosa, and Ismail et al., (2012) against Bacillus subtilus and

Pseudomonas aeruginosa. Result shown by Berberis lycium was as reported by Singh et

al., (2007), who perform on Indian Berberis species. But, Results of Berberis lycium

were not in accordance with Res (2011), where author want to check antibacterial

efficacy of Berberis lycium in comparison with penicillin. Prior to current experiments of

antibacterial activity of Artemisia vulgaris, a wide literature was only available for

Artemisia absinthium. And same was in case of Boerhavia procumbens where literature

was previously available on the related species Boerhavia diffusa. Bazzaz and

Haririzadeh (2003) described Capsella bursa-pastoris as most active plant in comparison

with all other Iranian plants studied.

Antibacterial assay results revealed that the current study was performed for

selected medicinal plants from the region for the very first time in case of majority of


105

plants. There were some plants with known antibacterial activity but was mostly based on

other part of the world have different geo-climatic condition and that also contribute for

the variation in the potential of the activity. Antibacterial activity of medicinal plants can

contribute much for pharmaceutical industry. It can further be analyzed that if the

antibacterial activity of the present medicinal plants used against certain bacteria causing

infectious diseases reveals better inhibitory effect than the drugs already in use and

available in the market. This study will be helpful for the pharmaceutical companies to

introduce new plant based antibacterial drug to combat with the recent phenomenon of

multiple drug resistance.

4.3 ANTIOXIDANT ACTIVITY

Blois (1958) first time described the 2,2-diphenyl-2-picrylhydrazine (DPPH) free

radical scavenging activity. This assay was modified by numerous researchers later-on.

This assay was widely used assay for the determination of antioxidant potential of plant

samples. DPPH molecule was regarded as stable free radicals which donate a hydrogen

atom upon reaction with other compounds (Priyadarsini et al., 2003).

DPPH radical scavenging assay was performed to evaluate the antioxidant activity

of selected medicinal plants at varying experimental condition. This included series of

experiment were aimed to optimize condition for the assay to produce maximum

antioxidant results for a plants species. Two temperature conditions were selected after

Baumann et al., (1997) included room temperature (25 °C) and human body temperature

(37°C) and 120, 60 and 15min were considered as duration for incubation period. Six

conditions (15 min/RT, 60 min/RT, 120 min/RT, 15 min/37°C, 60 min/37°C and 120

min/37°C) were selected on the basis of different temperature and incubation period

combinations. Results obtained from the assay were subjected to analyze for their

respective IC50 values (Table 3.3). And subjected to paired t-test to check their

significances (Fig. 3.10). Ascorbic acid was used as a positive control in this assay and all

other results were taken into the comparison of this with lowest activity (IC50=147.28

µg/ml, R2=0.9251) at 15min/RT and highest at (IC50=5.8 µg/ml, R2=0.9611) at 120


106

min/37°C. Artemisia vulgaris represent the best results as compared to all other plants

(IC50= 35.58 µg/ml, R2=0.7654) at 120 min/37°C, while Boerhavia procumbens

(IC50=4704.92 µg/ml, R2= 0.9288) at 15 min/RT. Geranium collinum showed overall

significant results as compared to all other plants used in the study. The temperature

represents significant impact in the study produced more results at 37°C as compared to

the RT. Kim et al., (2000) reported the use high temperature for streaming ginseng

enhance the radical scavenging activity. But the temperature ranges they used are 100,

110, and 120 °C for 2 h using an autoclave. Likewise, assay also showed time dependent

phenomenon, more activity with increase in time 120min > 60min > 15min. Lee et al

(2002) also reported the time dependent phenomenon of antioxidant assays.

Rice-Evans and Miller (1994) developed first time ABTS scavenging assay and

latter-on modified by Re et al. (1999). Original assay was modified based on oxidation of

metmyoglobin activation with H2O2 in ABTS+ presence, to produce radical cation, and

reduced in presence of H+ donating antioxidants. The reaction of ABTS and potassium

persulphate produces ABTS+ (Blue/green) chomophores and now this method is widely

acceptable. Ascorbic Acid (IC50= 126.10 µg/ml, R2= 0.9622) was used as positive control

in this experiment. Geranium collinum (IC50=17.06 µg/ml, R2=0.9562) represented the

best activity in the experiment as compared to the standard Ascorbic acid. While

Artemisia vulgaris (IC50= 1461.93 µg/ml, R2= 0.9714) showed the least activity.

Scavenging capabilities of Hydroxyl radical of any medicinal plant extract was

directly relevant to antioxidant activity of that plant. This activity includes production of

OH- using Fention reaction (Fe3+ / ascorbate /EDTA /H2O2 system). This system is based

on oxidation process resulted in the formation of OH- which react with DMSO to yield

formaldehyde (Hussain et al., 1987). In this experiment, Gallic acid (IC50=5.32µg/ml,

R2=0.9478) was used as positive control. This assay produced very good result as

majority of plants produced significant results included Geranium collinum (IC50=5.19

µg/ml, R2=0.9368), Persicaria barbata (IC50=5.43 µg/ml, R2=0.9714), Clematis grata

(IC50=7.08 µg/ml, R2=0.878), and Rubia cordifolia (IC50=8.28 µg/ml, R2=0.9026). The

least activity was shown by the Impatiens edgeworthii with (IC50=126.70 µg/ml,

R2=0.8794).


107

Phosphomolybdinum assay included the formation of phosphomolybdenum

complex, in which reduction of Mo (VI) to Mo (V) took place by the sample extract.

Ultimately at acidic pH, this reduction resulted in the production of green phosphate Mo

(V) complex (Kanner 1994).Ascorbic acid (IC50= 4.78µg/ml, R2= 0.92) was used as

standard in this assay. Like previous assay many plants like Boerhavia procumbens

(IC50= 6.11µg/ml, R2= 0.9509) Artemisia vulgaris (IC50= 7.12 µg/ml, R2= 0.9198)

Berberis lycium (IC50= 7.27µg/ml, R2= 0.8401) Capsella bursa-pastoris (IC50=

7.73µg/ml, R2= 0.9213) Persicaria barbata(IC50= 8.31 µg/ml, R2= 0.9840) and Rubia

cordifolia(IC50= 8.35µg/ml, R2= 0.8397) showed the significant results. Geranium

wallichianum (IC50= 139.23 µg/ml, R2= 0.7987) represented the least activity.

FRAP was considered to be among the most rapid test and very useful technique

for determination of antioxidant analysis. This activity was estimated by measuring the

increase in absorbance caused by the formation of ferrous ions from FRAP reagent

containing TPTZ (2,4,6 – tri (2 – pyridyl) – s – triazine) and FeCl3.6H2O (Benzie and

Strain, 1996). FRAP assay was used for only three plants, Persicariabarbata was with

best result.

Certain plants used in the study were also screened for antioxidant potiential by

various workers using a variety of approaches. DPPH radical scavenging assay,

phosphomolybdate assay and ferric thio-cyanate assay was previously determined for

Impatien edgworthiiby Shahwar et al., (2012). DPPH radical scavenging, nitric oxide

radical scavenging, reducing power assays was determined for Artemisia

vulgaris(Temraz and El-Tantawy, 2008). ABTS+ , DPPH and ferric reducing/antioxidant

power (FRAP) was measured by Wojdyłoet al., (2007).

Rubia cordifolia was previously known for its antioxidant properties as mentioned

by several workers. Initially antioxidant activities were performed by Tripathi et al.,

(1995). Later on, they isolated an antioxidant compound Rubiadin (Tripathiet al., 1997).

ABTS radical scavenging activity was performed by Cai et al., (2004a) on medicinal

plants associated with cancer including Rubia cordifolia. While structural radical


108

activities were also performed on R. cordifolia by Cai et al., (2004b). in-vivo antioxidant

evaluation for R. cordifolia was also reported by Joharapurkar et al., (2003). For

Boerhavia procumbens, Ferric reducing antioxidant power (FRAP) assay,total

antioxidant activity, ferric thiocyanate assay and DPPH free radical scavenging activity,

were evaluated by Abbasi et al., (2012). Only superoxide anion radical assay was

reported for Berberis lyceum (Ahmed and Shakeel, 2012). Polyphenolics and ABTS

radical scavenging activity was determined for Capsella bursa-pastoris by Ivanovaet al.,

(2005).

The current study includes variety of antioxidant activities to get the clear picture

of the antioxidant profile of selected medicinal plants. As these antioxidant assays used in

the current study were based on the different free radical. This can be used in future as a

baseline to choose a medicinal plant with antioxidant capabilities. Which ultimately be

very helpful in complementary and alternative medicine to improve health care and in

prevention of diseases. To enhance the immunity of human body and being safe from free

radicals will helps and facilitate all the efforts being carried out for the prevention of

diseases and provision of good health by organization like World Health Organization

(WHO) and other to justify the slogan, “Prevention is better than cure” or “heath for all”.

4.4 ANTILEISHMANIAL ACTIVITY

Leishmaniasis is regarded as a major protozoal health problem worldwide (WHO,

2002). Control of Leishmaniasis remains a problem because no vaccines exist and

emergence of resistance to chemotherapy poses a growing challenge to medicine (Croft

and Yardley, 2002). The existing first line drug, second and third line drugs were

considered to be expensive and have toxicity and other serious side effects.

Developments of resistance to these drugs also pose potential threat. Because of the fact

that many conventional drugs were obtained from the plant sources were initially used in

crude form in traditional of folk healing practices (Mourice et al., 1999). Hence, recently

there has been a growing interest in search of new drug source from traditional system of

medicine globally (Rates, 2001).


109

The medicinal plants screened in this assay produced interesting results with

significant IC50 values in comparison with Glucantime (a standard drug) used as a

positive control against Leishmania major. Glucantime produced IC50 value as IC50=251

µg/mg and none of the plant screened crossed this value except the Capsella bursa-

pastoris (IC50=204.23 µg/mg) which showed about near response. As the IC50 value of

Capsella bursa-pastoris is not even cross the Glucantime. Hence, it cannot be regarded as

insignificant rather a moderate activity. Rest of the plant showed well significant activity

ranging from the highest Geranium collinum (IC50=6.98 µg/mg) to Clematis

grata (IC50=25 µg/mg). Previous literature survey revealed that only Rubia cordifolia and

Berberis lycium were earlier tested for their antileishmanial activity against Leishmania

donovani. Rubia cordifolia (Singhaet al., 1992) and Berberis lycium (Sharma et al.,

2009) was reported to be insignificant against L.donovani. Both the plants were in

significant activity in the current study, due to plants selection in different geo-climatic

condition and against different Leishmania species.

It can be concluded that this assay provides a useful addition in already available

information about the potent antileishmanial plants, which can further leads to identify

and isolate the antileishmial compound of significant therapeutic value.

4.5 ANTIGLYCATION ACTIVITY

Diabetes is a serious metabolic disorder with multiple complications. Role of free

radicals in diabetes has been widely discussed in early eighties (Halliwell and Gutteridge,

1989) and involvement of free radicals in the commencement of diabetes and diabetes

complication were experimentally apparent (Lipinsky, 2001). Hyperglycemia is the

distinguishing feature of diabetes, and persistent conditions in a diabetic patient lead to

the formation of oxidative stress due to multiple reasons included auto-oxidation of

glucose. This auto-oxidation generates free radicals, hydrogen peroxide, which is

considered to be highly reactive after interaction with biomolecules, which accelerates

the formation of Advanced Glyaction End-product (AGE’s). These AGE’s have a

tendency to accumulate in the tissue, and crosslinking with other macromolecules in


110

tissues result in the abnormalities in the cell and tissue function (Gluglianoet al, 1995). At

present, treatment of diabetes involves various therapeutic agents in addition to insulin.

However, due to the unwanted side effects and efficacies of these agents, there is a

demand of new alternatives against diabetes (Moller, 2001). Therefore, in term of

potential antidiabetic agents, plants were considered to be unexplored and rich source.

Due to lack of mechanism based in-vitro assay, only few plants were subjected to

thorough investigation (Habeck, 2003; Fabricant and Farnsworth, 2001; Oubreet al.,

1997).

Antiglycation capabilities of the medicinal plant were checked by their potential

to inhibit of the formation of the advanced glycation end-products (Choudharyet al.,

2010). Rutin was used as the positive control and all other plants were compared with the

result produced from that. Rutin produced 86% of the inhibition of AGE’s. Out of all

plants tested only three were found to have significant result includes Persicaria barbata,

Geranium collinum and Berberis lycium. The assay followed the concentration dependent

phenomenon as previously described by Choi et al., (2008)

No previous record was found about antiglycation activities of all these plants in

already published literature sources worldwide. Hence, It can be concluded that this assay

was performed for the first time to know about the antiglycation abilities of the plants.

This study will be helpful in identifying the new herbal sources to combat the diabetes.

The three mentioned plants with significant activity needs to be processed further in-vivo

to know about their efficacy. However, antiglycation activity of plants only converts the

already taken sugar into simpler form of glucose etc. for metabolic processes of the body.

So far no plant could have been able to rejuvenate the pancreas cell to produce insulin

again. Hence efforts are being made to find out a medicinal plant which could normalize

the function of beta cells of pancreas. Further research on medicinal plants in this

direction is being conducted in our laboratory in Quaid-i-Azam University, Islamabad.


111

4.6 IMMUNOMODULATORY STUDIES

The immune system plays a vital role in the defense against infections. Its integrity

and efficiency is important during chemotherapeutic intervention for the treatment of

many diseases (Ishizuka et al., 1995). Modulation of the immune response to alleviate

disease then has been of interest for many researchers. In traditional medicine different

plant parts are believed to have specific medicinal properties including the ability to

stimulate the body’s disease-fighting mechanisms (Craig, 1999; Jones, 1996). The plant-

derived immunomodulators thus have tremendous future potential for developing new

pharmaceutical products (Amirghofran, 2000). The practice of the use of medicinal plant

to strengthen the immune system is relatively slow. However, this important component

for health improvement has been realized and medicinal plants were analyzed for this

purpose. The immunomodulatory studies for the selected medicinal plant in this

experiment were carried out for the very first time.

After comparing with the standard drug Ibuprofen, majority of the plant used in the

study exhibit highly significant potential immunomodulatory effect. Geranium collinum,

Artemisia vulgaris, Boerhavia procumbens, Capsella bursa-pastoris, Clematis grata,

Rubia cordifolia, and Persicaria barbata showed the significant immunomodulatory

effect while Impatiens edgeworthi, Geranium wallichianum and Berberis

lycium displayed the non-significant activities. Among the significant plants all exhibit

the IC50 lower than that of the standard drug IC50 i.e., 11.8±1.9.

In short, it can be concluded that no previous record was found about the

immunomodulatory effect of all the medicinal plants studied. This study will be helpful

to recognize the medicinal plants showing significant activity as immunomodulator.

4.7 ANTI-CANCER ACTIVITY

Cancer is a group of diseases characterized by uncontrolled growth and spread of

abnormal cells (Hornberget al., 2006), if the spread is not controlled, it can result in


112

death. Prostate cancer is the most frequently daignosed malignancy in men and second

leading cause of cancer related drug (Cooperberget al., 2004). The majority of Prostate

cancers are regarded as adenocarcinomas characterized by uncontrolled proliferation of

malignant tumor cell. Various models were used to study prostate cancer including

established cell line derived from metastatic human prostate cancers, two of the most

common used cell lines are LNCaP (Horoszewicz et al., 1983; 1980) and PC3 (Kaighn et

al., 1979). Lung cancer accounts for more deaths than any other cancer in both male and

female, accounting for about 28% of all cancer deaths in 2012 (Annon, 2012)

Conventional cancer therapies involve surgery, radiotherapy and chemotherapy in

majority (DeVita et al., 2001). An alternative source of anticancer drug is natural

products, which frequently seems to be more effective and/or less toxic. In view of the

enormous biodiversity of the planet, a promising future for natural products seems far

more likely than for compounds achievable by synthesis (Ma and Wang, 2009).

Moreover, natural products are even better for metabolic functioning and with fewer side

effects on human body.

Chemosensitivity testing in microplates has been used for in-vitro anticancer drug

screening. The MTT assay was used to determine the efficacy of selected medicinal

plants against PC-3 human cell line, which determines mitochondrial activity (Mosmann,

1983). This assay was considered to be sensitive, reproducible and rapid. The

development of Sulforhodamine B (SRB) protein staining assay developed by Skehanet

al., (1990) was adopted for routine use in National Cancer Institute (NCI) for in-vitro

anti-cancer measurement (Kerkvliet, 1990; Monks et al., 1991). The SRB assay was used

to determine the anticancer efficacy of the plants against human prostate adenocarcinoma

cell line (LNCaP-1) and human lung cancer cell line (LU-1).

All the medicinal plants were screened for their potential anticancer activity

against human prostate cancer cell line (PC 3) by using MTT assay. Doxorubicin, a

refrence drug was used a positive control which produce the IC50 value 2.8±0.12 μg/ml.

After comparing all the plants result only three plants produced the significant results


113

Geranium collinum with IC50 value 12.796±0.22 μg/ml, Impatiens edgeworthi with IC50

value 14.1883±0.275 μg/ml and Rubiacordifoliawith IC50 value 20.09±0.395 μg/ml.

While Berberis lycium with IC50 value 24.149±0.981 μg/ml was regarded to have

moderate activity. Rest of the plants tested to have non-significant activity (IC50 value >

30 μg/ml). It was known that none of the plant was earlier tested for their anticancer

potential against human prostate cancer cell line (PC 3) after a thorough search of

previously published data.

Sulforhodamine B assay was performed to evaluate the cytotoxicity for three best

medicinal plants (include Geranium collinum, Persicaria barbata and Geranium

wallichianum) based on the cumulative previous result against the Human lung

carcinoma (LU-1) and human prostate adenocarcinoma (LNCaP-1). Vinblastine (a

reference drug) was used as positive control. Geranium collinum was considered to

present the significant result against the LU-1 with IC50 value 15 μg/ml, while other both

were regarded as non-significant showed IC50 value >50 μg/ml. In-case of LNCaP-1,

Geranium collinum and Geranium wallichianum produced the significant results with

IC50 value at the border line i.e., IC50>25 µg/ml. Persicaria barbata showed the non-

significant results.

Overall, it can be summarized that Geranium collinum is the only medicinal

plants among all tested displayed the best results against all three cell line used in the

study. Hence, it can be proved as a good source of anticancer drugs in future. Previously,

it was not known about this plant. Hence, this study is a new addition in the already

available knowledge of herbal agents of anticancer drugs. As herbal drug can constitute a

broad component for effective control and the treatment of cancer in future. It has been

known from ethnomedicinal sources in Pakistan that Fagonia cretica and Berginia ciliata

(rhizome powder) were reported to be utilized for treatment of melagnant tumor and

blood cancer. Similarly a well-known drug Taxol has been prepared from Taxus

wallichiana and Catharanthus roseus as source of anticancerous alkaloids. Several other

plant based drugs used for the treatment of cancer are being used as mentioned in

introduction part of this thesis.

5. REFRENCES

Chapter 5 References

Abbasi, A. M., Khan, M. A., Ahmad, M., & Zafar, M. (2011). Medicinal plant

biodiversity of lesser Himalayas-Pakistan. Springer.

Abbasi, A. M., Khan, M. A., Ahmad, M., Qureshi, R., Arshad, M., Jahan, S. & Sultana,

S. (2010). Ethnobotanical study of wound healing herbs among the tribal

communities in Northern Himalaya Ranges District Abbottabad, Pakistan.Pak J

Bot, 6, 3747-3753.

Abbasi, M. A., Rubab, K., Rehman, A., Riaz, T., Shahzadi, T., Khalid, M., & Ajaib, M.

(2012). In vitro assessment of relief to oxidative stress by different fractions of

Boerhavia procumbens. Pakistan journal of pharmaceutical sciences, 25(2), 357.

Abbink J. (1995). Medicinal and Ritual Plants of the Ethiopian Southwest: An Account

of Recent Research. Indigenous Knowledge and Development Monitor. 3(2):6-8.

Adnan, M., & Hölscher, D. (2011). Medicinal plants in old-growth, degraded and re-

growth forests of NW Pakistan. Forest Ecology and Management, 261(11), 2105-

2114.

Adnan, M., & Hölscher, D. (2010). Medicinal plant abundance in degraded and

reforested sites in Northwest Pakistan. Mountain Research and

Development,30(1), 25-32.

Afolayan, A.J. (2003): Extracts from the shoots of Arctotis artotoides inhibit the growth

of bacteria and fungi. Pharmceutical Biology. 41: 22-25.

Afonso V, Chamy R, Mitrovic D, Collin P, Lomri A (2007) Reactive oxygen species and

superoxide dismutases: role in jointdiseases. Joint Bone Spine 74:324–329

Ahmad, B., Ismail, M., Iqbal, Z., & Chaudhry, M. I. (2003). Biological Activities of

Geranium wallichianum. Asian Journal of Plant Sciences, 2(13), 971-973.

Ahmad, H. (2004). People and Plants-Pakistan: Capacity Building in Ethnobotany

Applied to Conservation and Sustainable Use of Plant Resources. Annual Report,

WWF Sahibzada Abdul Qayum Road University Town Peshawar.

Ahmad, H., (2003). Capacity building for cultivation and sustainable harvesting of

medicinal and aromatic plants. In: Ahmad, H., Khan, A.A. (Eds.), Proceedings of

114


International Workshop on Conservation and Sustainable uses of Medicinal and

Aromatic Plants in Pakistan. WWF-Pakistan, pp. 31–36.

Ahmad, M. S., & Ahmed, N. (2006). Antiglycation properties of aged garlic extract:

possible role in prevention of diabetic complications. The Journal of nutrition,

136(3), 796-799.

Ahmed N. (2005). Advanced glycation endproducts—role in pathology of diabetic

complications. Diabetes Research and Clinical Practice. 67:3–21.

Ahmed, E., Arshad, M., Ahmad, M., Saeed, M., & Ishaque, M. (2004).

Ethnopharmacological survey of some medicinally important plants of Galliyat

Areas of NWFP, Pakistan. Asian Journal of Plant Sciences, 3(4), 410-415.

Ahmed, S., & Shakeel, F. (2012). Voltammetric determination of antioxidant character in

Berberis lycium Royel, Zanthoxylum armatum and Morus nigra Linn plants.

Paistan. Journal Pharmceutical Sciences, 25(3), 501-507.

Ahmad, S. S., & Javed, S. (2007). Exploring the economic value of underutilized plant

species in Ayubia National Park. Pak. J. Bot, 39(5), 1435-1442.

Ahmed, E., M. Arshad, A. Saboor, R. Qureshi, G. Mustafa, S. Sadiq and S. K. Chaudhari. 2013.Ethnobotanical appraisal and medicinal use of plants in Patriata, New Murree, evidence from Pakistan. Journal of Ethnobiology and Ethnomedicine, 9:13

Ahua, K. M., Ioset, J. R., Ioset, K. N., Diallo, D., Mauël, J., & Hostettmann, K. (2007).

Antileishmanial activities associated with plants used in the Malian traditional

medicine. Journal of ethnopharmacology, 110(1), 99-104.

Alexiades M. 1996. Collecting ethnobotanical data. An introduction to basic concepts and

techniques. In Selected Guideline for ethnobotanical research: A Field Manual.

Edited by Alexiades M. USA Sheldon JW: The New York Botanical Garden;:53–

94.

Ali, S. I., & E. Nasir. (Ed.) (1970-2002). Flora of Pakistan. 01-215. Department of

Botany, University of Karachi.

115


Ali, S. I., & Qaiser, M. (1986). A phytogeographical analysis of the phanerogams of

Pakistan and Kashmir. Proceedings of the Royal Society of Edinburgh. Section B.

Biological Sciences, 89(1), 89-101.

Ali, H., & Qaiser, M. (2009). The ethnobotany of Chitral valley, Pakistan with particular

reference to medicinal plants. Pak. J. Bot, 41(4), 2009-2041.

Amirghofran, Z., Azadbakht, M., & Karimi, M. H. (2000). Evaluation of the

immunomodulatory effects of five herbal plants. Journal of ethnopharmacology,

72(1), 167-172.

Annon., (2012). American Cancer Society. Cancer Facts & Figures 2012. Atlanta.

Anon. (2004). Infectious Diseases Society of America. Statement of the IDSA concerning

‘Bioshield II: Responding to an ever-changing threat’. Alexandria, VA: IDSA;

Ardestani A, Yazdanparast R (2007) Cyperus rotundus suppresses AGE formation and

protein oxidation in a model of fructose-mediated protein glycoxidation. Int J Biol

Macromol. 41(5):572-578

Arshad, M. 1991. Ecology of Murree vole, Hyperacrius Wynnei Blandford, In Galliyat.

Hazara, Pakistan.PhD thesis. University of Peshawar.

Arshad, M., & Ahmad, M. (2005). Ethnobotanical Study of Galliyat for Botanical

Demography and Bio-Ecological Diversification. Ethnobotanical Leaflets, 1:21.

Ashraf, M., Hayat, M. Q., Jabeen, S., Shaheen, N., Khan, M. A., & Yasmin, G. (2010).

Artemisia L. species recognized by the local community of northern areas of

Pakistan as folk therapeutic plants. J Med Plants Res, 4, 112-119.

Aumeeruddy-Thomas, Y., Shinwari, Z. K., Ayaz, A., & Khan, A. A. (2004). Ethnobotany

and the management of fodder and fuelwood at Ayubia National Park, North West

Frontier Province, Pakistan. People and Plants working paper, (13).

Aumeeruddy, Y., Ayaz, A., Gillani, A., Jabeen, A. Z., & Jabeen, H. (1998). Detailed

workshop report: People and Plants workshop on applied ethnobotany at Ayubia

National Park. NWFP, Pakistan, 14-16.

116


Bafna, A., & Mishra, S. (2010). Antioxidant and immunomodulatory activity of the

alkaloidal fraction of Cissampelos pareira linn. Scientia pharmaceutica, 78(1), 21.

Balick, MJ, Cox PA. (1996). Plants, People and Culture: Science of Ethnobotany. New

York: Scientific American Library

Balick, M. J. & Cox, P. A. (1997).Plants, People, and Culture: the Science of

Ethnobotany (Scientific American Library, New York, U.S.A.

Baron, J. (1838). The Life of Edward Jenner. Colburn.

Baumann, S. B., Wozny, D. R., Kelly, S. K., & Meno, F. M. (1997). The electrical

conductivity of human cerebrospinal fluid at body temperature. Biomedical

Engineering, IEEE Transactions on, 44(3), 220-223.

Bazzaz, B. S., & Haririzadeh, G. (2003). Screening of Iranian plants for antimicrobial

activity. Pharmaceutical biology, 41(8), 573-583.

Belay,G., Y. Tariku, T. Kebede, A. Hymete, Y. Mekonnen. (2011).

Ethnopharmacological investigations of essential oils isolated from five Ethiopian

medicinal plants against eleven pathogenic bacterial strains.

Phytopharmacology2011, 1(5) 133-143

Bennett, B. C. (2007). Doctrine of signatures: an explanation of medicinal plant

discovery or dissemination of knowledge?. Economic Botany, 61(3), 246-255.

Benzie, I.F.F. and Strain, J.J., (1996). The ferric reducing ability of plasma (FRAP) as a

measure of ‘antioxidant power’: The FRAP Assay, Anal. Biochem., 239:70-76.

Bero, J., Hannaert, V., Chataigné, G., Hérent, M. F., & Quetin-Leclercq, J. (2011). In

vitro antitrypanosomal and antileishmanial activity of plants used in Benin in

traditional medicine and bio-guided fractionation of the most active extract.

Journal of ethnopharmacology, 137(2), 998-1002.

Beutler B (2004) Innate immunity: an overview. Molecular Immunology 40:845–859

Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nat., 26:

1199-1200.

117


Bonnefont-Rousselot D. (2002). Glucose and reactive oxygen species. Curr Opin Clin

Nutr Metab Care. 5:561–8.

Cai, Y., Luo, Q., Sun, M., & Corke, H. (2004a). Antioxidant activity and phenolic

compounds of 112 traditional Chinese medicinal plants associated with anticancer.

Life sciences, 74(17), 2157-2184.

Cai, Y., Sun, M., Xing, J., & Corke, H. (2004b). Antioxidant phenolic constituents in

roots of Rheum officinale and Rubia cordifolia: structure-radical scavenging

activity relationships. Journal of agricultural and food chemistry, 52(26), 7884-

7890.

Calne, R.Y.1960. The rejection of renal homografts. Inhibition in dogs by using 6-

ercaptopurine. Lancet, 1: 417

Cassady, J.M., Douros, J.D. (Eds.), 1980. Anticancer Agents Based on

Natural Product Models. Academic Press, New York.

Champion, H.G., S.K. Seth and G.M. Khattak. (1965). Forest Types of Pakistan. Pakistan

Forest Institute, Peshawar.

Chandra RK (1997). "Nutrition and the immune system: an introduction". The American

Journal of Clinical Nutrition66 (2): 460S–463S.

Chen F (1996) High cell density culture of microalgae in heterotrophic growth. Trends in

Biotechnology 14(11): 421–426

Chen, Y. F., Roan, H. Y., Lii, C. K., Huang, Y. C., & Wang, T. S. (2011). Relationship

between antioxidant and antiglycation ability of saponins, polyphenols, and

polysaccharides in Chinese herbal medicines used to treat diabetes. Journal of

Medicinal Plants Research, 5(11), 2322-2331.

Choi, S. Y., Jung, S. H., Lee, H. S., Park, K. W., Yun, B. S., & Lee, K. W. (2008).

Glycation inhibitory activity and the identification of an active compound in

Plantago asiatica extract. Phytotherapy Research, 22(3), 323-329.

Chopra, R.N., Nayer, S.L., Chopra, I.C., (1992). Glossary of Indian Medicinal Plants, 3rd

edn. Council of Scientific and Industrial Research, New Delhi, pp. 7–246.

118


Choudhary, M. I., Maher, S., Begum, A., Abbaskhan, A., Ali, S., & Khan, A. (2010).

Characterization and antiglycation activity of phenolic constituents from Viscum

album (European Mistletoe). Chemical and Pharmaceutical Bulletin, 58(7), 980-

982.

Clements Jr, J. S., & Peacock Jr, J. E. (1990). Amphotericin B revisited: reassessment of

toxicity. The American journal of medicine, 88(5N), 22N.

Coelho de Souza, G., A.P.S. Haas, G.L. Von Poser, E.E.S. Schapoval and E. Elisabetsky,

(2004). Ethnopharmacological studies of antimicrobial remedies in the south of

Brazil. J. Ethnopharmacol., 90: 135-43.

Cohen ML (1992). Epidemiology of drug resistance: implications for a post-antimicrobial

era. Science 257: 1050-1055.

Cooperberg MR, Park S, Carroll PR. (2004).Prostate cancer 2004: Insights from national

disease registries. Oncology (Williston Park) 18:1239–1247; discussion 48–50,

56–58.

Cotton CM. 1996. Ethnobotany Principles and Applications. Chichester, New York:John

Wiley and Sons.

Cowan MM. (1999). Plant products as antimicrobial agents. Clin Microbiol Rev.

12(4):564-82.

Cox, P.A., (2000). Will tribal knowledge survive the millennium? Science 287, 44–45.

Cragg, D. J. Newman, and K. M. Snader, (1997). “Natural roducts in drug discovery and

development,” Journal of Natural Products, vol. 60, no. 1, pp. 52–60.

Cragg, G. M., and M. R. Boyd. (1996). “Drug discovery and development at the

National Cancer Institute: the role of natural products of plant origin,” in

Medicinal Plant Resources of the Tropical Forest, M. J. Balick, E. Elisabetsky,

and S. A. Laird, Eds., pp. 101–136, Columbia University Press, New York, NY,

USA.

119


Cragg, G.M., Kingston, D.G.I., Newman, D.J. (Eds.), (2005). Anticancer Agents from

Natural Products. Brunner-Routledge Psychology Press, Taylor & Francis Group,

Boca Raton, FL.

Craig WJ (1999). Health-promoting properties of common herbs. Am J Clin Nutr, 70,

491S-9S.

Crespi, B. and Summers, K. (2005) Evolutionary biology of cancer. Trends Ecol. Evol.

20, 545–552

Croft SL and Yardley V. (2002). Chemotherapy of leishmaniasis.Curr. Pharm. 8: 319-

342.

Dar, M. E. I. (2003). Ethnobotonical uses of plants of Lawat district Muzaffarabad Azad

Jammu and Kashmir. Asian J Plant Sci, 2(9), 680-682.

Davies, C.R., Kaye, P., Croft, S.L. and Sundar, S. (2003). Leishmaniasis: new approaches

to disease control. Brit. Med. J., 326: 377-82.

Davis, J., (1994). Inactivation of antibiotic and the dissemination of resistance genes.

Science 264, 375–382.

Deak, T., & Beuchat, L. R. (1996). Handbook of food spoilage. New York, USA: CRC

Press.

DeVita, V.T., Hellman, S., Rosenberg, S.A. (2001). Cancer: Principles and practice of

Oncology, sixth ed. Lipponcott William & Wilkins, Philidelphia, PA, USA.

Eldeen, I.M.S., Elgorashi, E.E., Mulholland, D.A., Van Staden, J., (2006). Anolignan B:

a bioactive compound from the roots of Terminalia sericea. Journal of

Ethnopharmacology 103, 135–138.

Elisabetsky. (1990). Plants used as analgesics by Amazonian Capbocols. International

Journal of Crude Drug Research, 28: 309-320.

European Committee on Antimicrobial Susceptibility Testing. (2000). Determination of

minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution.

EUCAST Definitive Document E. Def 3.1. Clinical Microbiology and Infection 6,

509–515.

120


Fabricant DS, Farnsworth NR. (2001).The value of plants used in traditional medicine for

drugdiscovery. Environ Health Perspect.109, 69-75.

Farnsworth, N. R., Akerele, O., Bingel, A. S., Soejarto, D. D. & Guo, Z. (1985).

Medicinal plants in therapy. Bull World Health Organ 63, 965-81.

Farsi DA, Harris CS, Reid L, Bennett SA, Haddad PS, Martineau LC, Arnason JT (2008)

Inhibition of Non-enzymatic Glycation by Silk Extracts from a Mexican Land

Race and Modern Inbred Lines of Maize (Zea mays). Phytother. Res. 22:108-112

Fransworth, N.R. and D.D. Soejarto, (1991). Global importance of medicinal plants. In:

O., Akerele, V. Heywood and H. Synge, (Eds.): The conservation of medicinal

plants: proceedings of an international consultation 21-27 March 1988, Chiang

Mai, Thailand Cambridge University Press, Cambridge, 25-51.

Ganju,L., D. Karan, S. Chanda, K.K. Srivastava, R.C. Sawhney, W. Selvamurthy. 2003.

Immunomodulatory effects of agents of plant origin, Biomedicine &

Pharmacotherapy. 57(7):296-300,

García, M., Perera, W. H., Scull, R., & Monzote, L. (2011). Antileishmanial assessment

of leaf extracts from Pluchea carolinensis, Pluchea odorata and Pluchea rosea.

Asian Pacific Journal of Tropical Medicine, 4(10), 836-840.

Gerschman, R., Gilbert, D. L., Nye, S. W., Dwyer, P., & Fenn, W. O. (1954). Oxygen

poisoning and x-irradiation—A mechanism in common. Science, 119, 623–626.

Gilles, H.M. (Editor) (1999). Protozoal diseases. ARNOLD publisher, London, pp 413-

503

Gallis, H. A., Drew, R. H., & Pickard, W. W. (1990). Amphotericin B: 30 years of

clinical experience. Review of Infectious Diseases, 12(2), 308-329.

Gilani, S. A., Qureshi, R. A., & Farooq, U. (2001). Ethnobotanical studies of Ayubia

national park district Abbottabad, Pakistan. OnLine Journal of Biological

Sciences, 1(4), 284-286.

121


Gilani, S. A., Qureshi, R. A., & Gilani, S. J. (2006). Indigenous uses of some important

ethnomedicinal herbs of Ayubia National Park, Abbottabad,

Pakistan.Ethnobotanical Leaflets, 2006(1), 32.

Glugliano D, Cerriello A and Paolisso G. (1995). Diabetes mellitus, hypertension and

cardiovascular disease: which role for oxidative stress. Metabolism. 44:363-8.

González-Coloma, A., Reina, M., Sáenz, C., Lacret, R., Ruiz-Mesia, L., Arán, V. J., ... &

Martínez-Díaz, R. A. (2012). Antileishmanial, antitrypanosomal, and cytotoxic

screening of ethnopharmacologically selected Peruvian plants. Parasitology

research, 110(4), 1381-1392.

Govaerts, R., (2001). How many species of seed plant are there? Taxon 50, 1085–1090.

GraphPad Prism, (2007). version 5.01. GraphPad Software Inc.: San Diego, CA, USA.

Gugliucci A, Bastos DH, Schulze J, Souza MF (2009) Caffeic and chlorogenic acids in

Ilex paraguariensis extracts are the main inhibitors of AGE generation by

methylglyoxal in model proteins. Fitoterapia 80(6):339-344

Gupta, R., Bajpai, K. G., Johri, S., & Saxena, A. M. (2008). An overview of Indian novel

traditional medicinal plants with anti-diabetic potentials. African Journal of

Traditional, Complementary, and Alternative Medicines, 5(1), 1.

Gyamfi, M. A., Yonamine, M., & Aniya, Y. (1999). Free-radical scavenging action of

medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced

liver injuries. General pharmacology, 32(6), 661.

Habeck M. (2003). Diabetes treatments get sweet help from nature. Nat Med.9, 1228.

Halliwell, B. and Gutteridge, JMC. (1989). Free radical in biology and medicine. 2nd Ed.

Oxford university press. Oxford.

Halliwell, B., & Gutteridge, JMC. (1999). Free radicals in biology and medicine (3rd

ed.). Oxford University Press.

Halliwell, B.; Gutteridge, JMC. (1995). The definition and measurement of antioxidants

in biological systems. Free Radical Biol. Med.18:125–126.

Hamayun, M., Afzal, S., & Khan, M. A. (2006). Ethnopharmacology, indigenous

122


collection and preservation techniques of some frequently used medicinal plants

of Utror and Gabral, district Swat, Pakistan. African Journal of Traditional,

Complementary and Alternative Medicines, 3(2), 57-73.

Hamburger, M. & Hostettmann, K. (1991). Bioactivity of plants: the link between

phytochemistry and medicine. Phytochemistry 30, 3864-74.

Hamilton, A.C., (2004). Medicinal plants, conservation and livelihoods. Biodiversity and

Conservation 13, 1477–1517.

Harborne JB, Baxter H. (1999). The handbook of natural flavonoids, Vols 1 and 2.

Chichester, UK: John Wiley and Sons.

Harman, D. (1956). Aging—A theory based on free-radical and radiation-chemistry. J.

Gerontol., 11, 298–300.

Harshberger, J.W., (1896). The purposes of ethnobotany. Botanical Gazette 21, 146–154.

Hartwell, J.L., (1982). Plants Used Against Cancer. Quarterman, Lawrence, MA.

Havsteen B. (1983). Flavonoids, a class of natural products of high pharmacological

potency. Biochem Pharmacol. 32:1141–8.

Hill, A.F., (1952). Economic Botany. McGraw-Hill Book Company, INC., Tokyo.

Hocking, G. M. (1958). Pakistan Medicinal Plants 1.Qualitas Plantarum Et Material

Vegetables, 5: 145-153.

Hoffman, C. and Evans, A.C. (1911) The uses of spices as preservatives. Journal of

Indian Engineering and Chemistry3, 835–838.

Hornberg, J.J., F.J. Bruggeman, H.V. Westerhoff, J. Lankelma. (2006). Cancer: A system

biology disease. BioSystems. 83:81-90.

Horoszewicz JS, Leong SS, Chu TM, Wajsman ZL, Friedman M, Papsidero L, Kim U,

Chai LS, Kakati S, Arya SK, Sandberg AA.(1980). The LNCaP cell line—A new

model for studies on human prostatic carcinoma. Prog Clin Biol Res. 37:115–132.

123


Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA,

Murphy GP. (1983). LNCaP model of human prostatic carcinoma. Cancer Res.

43:1809–1818.

Hoyer DL, Kung HC, Smith BL. (2005). Natl. Vital. Stat. Rep. 53: 1-48.

Hunt JV, Bottoms MA, Mitchinson MJ. (1993). Oxidative alterations in the experimental

glycation model of diabetes mellitus are due to protein-glucose adduct oxidation.

Biochem J. 291:529–35.

Hussain F, Ilahi I. 1991. Ecology and Vegetation of Lesser Himalayas

Pakistan.Peshawar, Pakistan: Jadoon Printing Press; 1–185.

Hussain, K., Shahazad, A., & Zia-ul-Hussnain, S. (2008). An ethnobotanical survey of

important wild medicinal plants of Hattar district Haripur, Pakistan.

Ethnobotanical Leaflets, 2008(1), 5.

Hussain, M. (2003). Palynological and Ethnobotanical Studies of Chrysanthemum

leucanthemum L., from Gallies (Abbottabad), Pakistan. Hamdard Medicus, XLVI

(3): 13-14.

Ibrar, M., & Hussain, F. (2010). An ethnobotanical study on the usage of wild medicinal

herbs from Malana hills, Parachinar Kurram valley. International Journal of

Biology and Biotechnology, 7.

Ibrar, M., Hussain, F., & Sultan, A. (2007). Ethnobotanical studies on plant resources of

Ranyal hills, district Shangla, Pakistan. Pakistan Journal of Botany, 39(2), 329

Ishizuka M, Kawatsu M, Yamashita T, Ueno M, Takeuchi T (1995). Low molecular

weight immunomodulators produced by microorganisms. Int J Immunopharmacol,

17, 133-9.

Ismail, M., Hussain, J., Khan, A. U., Khan, A. L., Ali, L., Khan, F. U., & Lee, I. J.

(2012). Antibacterial, Antifungal, Cytotoxic, Phytotoxic, Insecticidal, and Enzyme

Inhibitory Activities of Geranium wallichianum. Evidence-Based Complementary

and Alternative Medicine, 2012.

124


Ivanova, D., Gerova, D., Chervenkov, T., & Yankova, T. (2005). Polyphenols and

antioxidant capacity of Bulgarian medicinal plants. Journal of

Ethnopharmacology, 96(1), 145-150.

Iwu, M.M. (2002): In: Threapeutic Agents from Ehtnomedicine. Ethnomedicine and

Drug Discovery. Iwu MM, Wootton JC (Eds) Elsevier Science, Amsterdam.

Jabeen, A. (1999). Ethnobotany of fodder species of Ayubia National Park (ANP)

Nathiagali; its conservation status and impacts on environment (M. Phil

thesis). Islamabad, Pakistan: Quid-I-Azam University.

Jan, A. K., Shah, M. R., Anis, I., & Marwat, I. K. (2009). In vitro antifungal and

antibacterial activities of extracts of Galium tricornutum subsp.

longipedunculatum. Journal of Enzyme Inhibition and Medicinal Chemistry, 24(1),

192-196.

Jang DS, Yoo NH, Kim NH, Lee YM, Kim CS, Kim J, Kim JH,Kim JS (2010) 3,5-Di-

caffeoyl-epi-quinic acid from the leaves and stems of Erigeron annuus inhibits

protein glycation, aldose reductase, and cataractogenesis. Biol Pharm Bull

33(2):329-333

Jemal A, Siegel R, Xu J, Ward E. (2010). Cancer statistic. CA Cancer J Clin

2010;60:277-300.

Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., & Forman, D. (2011). Global

cancer statistics. CA: a cancer journal for clinicians, 61(2), 69-90.

Joharapurkar, A. A., Zambad, S. P., Wanjari, M. M., & Umathe, S. N. (2003). In vivo

evaluation of antioxidant activity of alcoholic extract of Rubia cordifolia Linn. and

its influence on ethanol-induced immunosuppression. Indian journal of

pharmacology, 35(4), 232-236.

Jones FA (1996). Herbs-useful plants. Their role in history and today. Eur J

Gastroenterol Hepatol, 8, 1227-31.

Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW. (1979). Establishment and

characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol.

17:16–23.

125


Kala, C.P., (2005). Indigenous uses, population density, and conservation of threatened

medicinal plants in protected areas of the Indian Himalayas. Conservation Biology

19, 368–378.

Kanner, J., et al., (1994). Natural antioxidants in grapes and wines, J. Agric. Food.

Chem., 42:64-69.

Katlama, C., Regnier, B., Ben, S. N., Pichard, E., & Vachon, F. (1985). Toxicity of

Glucantime. A case]. In Annales de médecine interne 136(4):321.

Kenderick, K., (1989). Sri Lanka. In: Campbell, D.G., Hammond, H.D. (Eds.), Floristic

Inventory of Tropical Countries. The New York Botanical Garden, NY.

Kenneth JR and George CR (2004). An Introduction to Infectious Diseases. Sherris

Medical Microbiology: McGraw-Hill Companies Inc., 4th ed., PP 14-17.

Kerkvliet, G.J., (1990). Drug discovery screen adapts to changes. J. Natl. Cancer Inst. 82,

1087.

Khan, M. I. C. M., & Hanif, W. (2006). Ethno Veterinary Medicinal Uses of Plants from

S am ali ni Valley Dist. Bhimber,(Azad Kashmir) Pakistan. Asian Journal of Plant

Sciences, 5(2), 390-396.

Khan, S. W., & Khatoon, S. (2008). Ethnobotanical studies on some useful herbs of

Haramosh and Bugrote valleys in Gilgit, northern areas of Pakistan. Pakistan

Journal of Botany, 40(1), 43.

Khan SU, Hasan MU, Khan FK, Bari A. 2010. Climate classification of Pakistan.

Balwois Conference: Republic of Macedonia; pp:1–47.

Kim, W. Y., Kim, J. M., Han, S. B., Lee, S. K., Kim, N. D., Park, M. K., & Park, J. H.

(2000). Steaming of ginseng at high temperature enhances biological activity.

Journal of natural products, 63(12), 1702-1704.

Kovacic, P., & Jacintho, J. D. (2001). Mechanisms of carcinogenesis: Focus on oxidative

stress and electron transfer. Curr. Med. Chem., 8, 773–796.

Kumi‐Diaka, J. (2002). Chemosensitivity of human prostate cancer cells PC3 and LNCaP

to genistein isoflavone and β‐lapachone. Biology of the Cell, 94(1), 37-44.

126


Landis-Piwowar KR, Huo C, Chen D, Milacic V, Shi G, Chan TH, Dou QP (2007) A

Novel Prodrug of the Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate as a

Potential Anticancer Agent Canser Res. 67: 4303-4310

Lee, J. C., Kim, H. R., Kim, J., & Jang, Y. S. (2002). Antioxidant property of an ethanol

extractof the stem of Opuntia ficus-indica var. saboten. Journal of Agricultural and

Food Chemistry, 50(22), 6490-6496.

Lewington, A. (1990). Plants for People. Natural History Museum Publications, London.

Lima, M. I. S., Arruda, V. O., Alves, E. V. C., de Azevedo, A. P. S., Monteiro, S. G., &

Pereira, S. R. F. (2010). Genotoxic effects of the antileishmanial drug

glucantime®. Archives of toxicology, 84(3), 227-232.

Lin, J., Opoku, A.R., Geheeb-Keller, M., Hutchings, A.D., Terblanche, S.E., Jager, A.K.,

van Staden, J.(1999): Preliminary screening of some traditional zulu medicinal

plants for anti-inflammatory and antimicrobial activities. J Ethnopharmacol. 68:

267-274.

Linton, A. H. (1983). Theory of antibiotic inhibition zone formation, disc sensitivity

methods and MIC determinations. Antibiotics: Assessment of Antimicrobial

Activity and Resistance, 19-30.

Lipinski B. (2001). Pathophysiology of oxidative stress in diabetes mellitus. J Diabetes

Complications 15:203-10.

Ma, X., and Z. Wang. (2009). Anticancer drug discovery in the future: evolutionary

perspective. Review. Drug Discovery today. 14(23/24):1136-1142.

Maciel MAM, Pinto AC, Veiga Jr VF, Grynberg NF, Echevarria A. (2002). Medicinal

plants: the need for multidisciplinary scientific studies. Quim Nova. 25(3):429-38.

Mahomoodally MF, Gurib-Fakim A, Subratty AH. (2007). Effect of exogenous ATP on

Momordica charantia Linn. (Cucurbitaceae) induced inhibition of D-glucose, L-

tyrosine and fluid transport across rat everted intestinal sacs in vitro. J

Ethnopharmacol.110: 257-263.

127


Makare, N., Bodhankar, S., & Rangari, V. (2001). Immunomodulatory activity of

alcoholic extract ofMangifera indicaL. in mice. Journal of ethnopharmacology,

78(2), 133-137.

Mani, M.S., 1978. Ecology and Phytogeography of the High Altitude Plants of the

Northwest Himalaya. Oxford & IBH Publishing Company, New Delhi, IN.

Martindale, W.H. (1910) Essential oils in relation to their antiseptic powers as

determined by their carbolic coefficients. Perfumery and Essential Oil Research 1,

266–296.

Martin GJ. 1995. Ethnobotany: a methods manual London. UK: Chapman and Hall.

Matin, A., Khan, M. A., Ashraf, M., & Qureshi, R. A. (2001). Traditional use of herbs,

shrubs and trees of Shogran valley, Mansehra, Pakistan. Pakistan Journal of

Biological Sciences, 4(9), 1101-1107.

Matsuda, H., Wang, T., Managi, H. and Yoshikawa, M. 2003. Structural requirements of

flavonoids for inhibition of protein glycation and radical scavenging activities,

Bioorg Med Chem. 11: 5317 – 5323.

Mayer, G. 2006. "Immunology — Chapter One: Innate (non-specific) Immunity".

Microbiology and Immunology On-Line Textbook. USC School of Medicine.

Merlo, L. M., Pepper, J. W., Reid, B. J., & Maley, C. C. (2006). Cancer as an

evolutionary and ecological process. Nature Reviews Cancer, 6(12), 924-935

Mesaik MA, Ul-Haq Z, Murad S, Ismail Z, Abdullah NR, Gill HK, et al. 2006. Biological

and molecular docking studies on coagulin-H: Human IL-2 novel natural inhibitor.

Mol Immunol. 43: 1855-1163.

Miller, D. M., Buettner, G. R., & Aust, S. D. (1990). Transition metals as catalysts of

“autoxidation” reactions. Free Radic. Biol. Med., 8, 95–108.

Moller DE.2001. New drug targets for type 2 diabetes and the metabolic syndrome.

Nature.414, 821-827.

Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull, K., Vistica, D., Hose, C.,

Langley, J., Cronise, P., Vaigro-Wolff, A., Gray-Goodrich, M., Cambell, H.,

128


Mayo, J., Boyd, M., 1991. Feasibility of a high-flux anticancer drug screen using a

diverse panel of cultured human tumor cell lines. J. Natl. Cancer Inst. 83, 757.

Monzote, L., Garcia, M., Montalvo, A. M., Scull, R., Miranda, M., & Abreu, J. (2007). In

vitro activity of an essential oil against Leishmania donovani. Phytotherapy

Research, 21(11), 1055-1058.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:

Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55.

Mourice, M.I., Angela, R.D. and Chris, O.O. (1999). New antimicrobials of plant origin,

In:Perspectives on new crops and new uses. Janick, J. (Editor), ASHS press,

Alexanderia, pp. 457-62.

Murad, W., Ahmad, A., Gilani, S. A., & Khan, M. A. (2011). Indigenous knowledge and

folk use of medicinal plants by the tribal communities of Hazar Nao Forest,

Malakand District, North Pakistan. Journal of Medicinal Plants Research, 5(7),

1072-1086.

Nagai, T., Myoda, T., & Nagashima, T. (2005). Antioxidative activities of water extract

and ethanol extract from field horsetail (tsukushi) Equisetum arvense L. Food

chemistry, 91(3), 389-394.

Nascimento GGF, Locatelli J, Freitas PC, Silva GL. 2000. Antibacterial activity of plant

extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol.

31(1):247-56.

Nasir, E., & Ali, S.I. (1971-91) Flora of West Pakistan, No. 1-190. Pakistan Agric.

Research Council. Islamabad.

Nathan DM. 1993 Long-term complications of diabetes mellitus. N Engl J Med.

328:1676–85.

Newman, D.J., Cragg, G.M., Snader, K.M., 2003. Natural products as sources of new

drugs over the period 1981–2002. Journal of Natural Products 66, 1022–1037.

Nikaido, H., 1999. Microdermatology: Cell surface in the interaction of microbes with

the external world. J. Bacteriol., 181: 4-8.

129


Nowell, P.C. (1976) The clonal evolution of tumor cell populations. Science 194,23–28

Oboh, G., & Akindahunsi, A. A. (2004). Change in the ascorbic acid, total phenol and

antioxidant activity of sun-dried commonly consumed green leafy vegetables in

Nigeria. Nutrition and health, 18(1), 29-36.

Odjakova, M., Popova, E., Al Sharif, M., & Mironova, R. (2012). Plant-Derived Agents

with Anti-Glycation Activity. http://dx.doi.org/10.5772/48186.

Osadebe, P. O., & Omeje, E. O. (2009). Comparative acute toxicities and

immunomodulatory potentials of five Eastern Nigeria mistletoes. Journal of

ethnopharmacology, 126(2), 287-293.

Oubre AY, Carlson TJ, King SR, Reaven GM. 1997. From plant to patient: an ethno

medical approach to the identification of new drugs for the treatment of NIDDM.

Diabetologia. 40,614-617.

Parekh, J., Chanda, S. (2006): In vitro antimicrobial activities of extract of Launaea

procumbens Roxb.(Labiateae), Vitis vinifera (Vitaceae) and Cyperus rotundus

(Cyperaceae). Afr. J. Biomed. Res. 9: 89-93.

Parker, R. N., 1918. (Re-printed 1973) A forest flora of Punjab with Hazara and Dehli.

Govt. Publishing Press. Lahore.

Patil, V. V., Bhangale, S. C., & Patil, V. R. (2010). Studies on immunomodulatory

activity of Ficus carica. Int J Pharm Pharm Sci, 2(4), 97-99.

Peng X, Zheng Z, Cheung KW, Shan F, Ren GX, Chen SF, Wang M (2008) Inhibitory

effect of mung bean extract and its constituents vitexin and isovitexin on the

formation of advanced glycation endproducts. Food Chem. 106(2):457-481.

Piddock, K.J.V., Wise, R., 1989. Mechanisms of resistance to quinolones and clinical

perspective. Journal of Antimicrobial Chemotherapy 23, 475–483.

Pie & Manandhara (1987). The Percentage of Medicinal wild species in Himalayan

Ranges. Pakistan Journal of Botany. 10(6) : 65-74.

130


Pratt. D. E. 1992. Natural Antioxidants from Plant Material, Book Chapter in “Phenolic

Compounds in Food and Their Effects on Health II” pp 54–71. ACS Symposium

Series, Vol. 507.

Prieto, P., Pineda, M., & Aguilar, M. (1999). Spectrophotometric quantitation of

antioxidant capacity through the formation of a phosphomolybdenum complex:

specific application to the determination of vitamin E. Analytical biochemistry,

269(2), 337-341.

Priyadarsini, K. I., Maity, D. K., Naik, G. H., Kumar, M. S., Unnikrishnan, M. K., Satav,

J. G., & Mohan, H. (2003). Role of phenolic OH and methylene hydrogen on the

free radical reactions and antioxidant activity of curcumin. Free Radical Biology

and Medicine, 35(5), 475-484.

Qureshi R. A., and M.N. Choudhri. 1987. The endangered flora of Pakistan-Preliminary

report. Pakistan Systematics. 3(1):32-37.

Qureshi, R. A., Ghufran, M. A., Gilani, S. A., Sultana, K., & Ashraf, M. (2007a).

Ethnobotanical studies of selected medicinal plants of Sudhan Gali and Ganga

Chotti hills, district Bagh, Azad Kashmir. Pakistan Journal of Botany, 39(7), 2275-

2283.

Qureshi, R. A., Ghufran, M. A., Gilani, S. A., Yousaf, Z., Abbas, G., & Batool, A.

(2009). Indigenous medicinal plants used by local women in southern Himalayan

regions of Pakistan. Pakistan Journal of Botany, 41(1), 19-25.

Qureshi, R., & Raza Bhatti, G. (2008). Ethnobotany of plants used by the Thari people of

Nara Desert, Pakistan. Fitoterapia, 79(6), 468-473.

Rabe, T., Mulholland, D., Van Staden, J., 2002. Isolation and identification of

antibacterial compounds from Vernonia colorata leaves. Journal of

Ethnopharmacology 80, 91–94.

Rasooli, I., 2007. Food Preservation – A Biopreservative Approach Food Global Science

Books 1(2), 111–136.

Rates, S. M. 2001. Plants as source of drugs. Toxicon 39, 603-13.

131


Rauf, A., Baloch, M. K., Abbasi, F. M., Chattha, M. R., & Mahmood, T. Z. (2007).

Status, utilization and trade of Hazara areas healing plants of Pakistan. Journal of

Food Agriculture and Environment, 5(2), 236.

Ray, P. G., & Majumdar, S. K. (1976). Antimicrobial activity of some Indian plants.

Economic Botany, 30(4), 317-320.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).

Antioxidant activity applying an improved ABTS radical cation decolorization

assay. Free Radical Biology and Medicine, 26(9), 1231-1237.

Reddya L, Odhava B and Bhoolab KD. 2003. Natural products for cancer prevention: A

global perspective. Pharmacol. & Therap. 99, 1-13.

Res, A. J. M. (2011). A study on comparative pharmacological efficacy of Berberis

Lycium and Penicillin G. African Journal of Microbiology Research, 5(6), 725-

727.

Rice-Evans, C., & Miller, N. J. (1994). [241 Total antioxidant status in plasma and body

fluids. Methods in enzymology, 234, 279-293.

Ridnour, L. A., Isenberg, J. S., Espey, M. G., Thomas, D. D., Roberts, D. D., & Wink, D.

A. (2005). Nitric oxide regulates angiogenesis through a functional switch

involving thrombospondin-1. Proc. Natl. Acad. Sci. U.S.A., 102, 13147–13152.

Sadeghi-Nejad, B., Saki, J., Khademvatan, S., & Nanaei, S. (2011). In vitro

antileishmanial activity of the medicinal plant—Satureja khuzestanica Jamzad. J

Med Plant Res, 5(24), 5912-5915.

Saghir, I. A., Awan, A. A., Majid, S., Khan, M. A., Qureshi, S. J., & Bano, S. (2001).

Ethnobotanical studies of Chikar and its allied areas of District Muzaffarabad.

Online J Biol Sci, 1, 1165-1170.

Sánchez-Moreno, C. (2002). Review: Methods used to evaluate the free radical

scavenging activity in foods and biological systems. Food Science and Technology

International, 8(3), 121-137.

132


Saqib, Z., & Sultan, A. (2005). Ethnobotany of Palas Valley, Pakistan. Ethnobotanical

Leaflets, 2005(1), 28.

Sawadogo, W. R., Le Douaron, G., Maciuk, A., Bories, C., Loiseau, P. M., Figadère, B.,

& Nacoulma, O. G. (2012). In vitro antileishmanial and antitrypanosomal activities

of five medicinal plants from Burkina Faso. Parasitology research, 1-5.

Schepetkin IA, Quinn MT (2006) Botanical polysaccharides: Macrophage

immunomodulation and therapeutic potential. Int Immunopharmacol 6(3): 317–

333.

Schippmann U, Leaman DJ, Cunningham AB. 2002. Impact of Cultivation and Gathering

of Medicinal Plants on Biodiversity: Global Trends and Issues. In (FAO)

Biodiversity and the ecosystem approach in agriculture, forestry and fisheries.

Satellite event on the occasion of the Ninth regular session of the commission on

genetic resources for food and agriculture. Rome 12 – 13 October, 2002 Inter

departmental working group on biological diversity for food and agriculture;

Rome.

Schultes, R. E. 1978. The kingdom of plants, p. 208.In W. A. R. Thomson (ed.),

Medicines from the Earth.McGraw-Hill Book Co., New York, N.Y.

Schultes, R.E., von Reis, S. (Eds.), 1995. Ethnobotany: Evolution of a Discipline.

Chapman & Hall, London, 414 pp.

Secretariat of the Convention on Biological Diversity, 2001. Handbook of the

Convention on Biological Diversity. Earthscan Publications Ltd., London/Sterling,

VA, Articles 15.1 and 8.j. pp. 3–26

(http://www.biodiv.org/convention/articles.asp).

Sell DR., & Monnier VM. 1989. Structure elucidation of a senescence cross-link from

human extracellular matrix. J Biol Chem. 264:21597–602.

Sell, S. (1987). Immunomodulation. Immunology Immunopathology and Immunity, 655-

83.

Shafique, M.C. 2003. Some aspects of bio-ecology of Ayubia National Park. PhD Thesis.

University of Karachi.

133


Shah, G. M., & Khan, M. A. (2006). Checklist of Medicinal Plants of Siran Valley,

Mansehra, Pakistan. Ethnobotanical leaflets, 2006(1), 6.

Shah, G. M., Ahmad, M., Arshad, M., Khan, M. A., Zafar, M., & Sultana, S. 2012.

Ethno-Phyto-veterinary medicines in Northern Pakistan. JAPS. 22(3):791-797

Shaheen, H., Qureshi, R. A., & Shinwari, Z. K. (2011). Structural Diversity, Vegetation

Dynamics and Anthropogenic Impact on Lesser Himalayan Subtropical Forests of

Bagh District, Kashmir. Pakistan Journal of Botany, 43(4), 1861-1866.

Shahwar, D., Ahmad, N., Ullah, S., & Raza, M. A. (2012). Antioxidant activities of the

selected plants from the family Euphorbiaceae, Lauraceae, Malvaceae and

Balsaminaceae. African Journal of Biotechnology, 9(7).

Sharma, U., Velpandian, T., Sharma, P., & Singh, S. (2009). Evaluation of anti-

leishmanial activity of selected Indian plants known to have antimicrobial

properties. Parasitology research, 105(5), 1287-1293.

Sher, H., Ahmad, A., Eleyemeni, M., Faiz-i-Hadi, S., & Sher, H. (2010). Impact of

nomadic grazing on medicinal plants diversity in Miandam, Swat-Pakistan

(preliminary results). Int. J. Biod. Conserv, 2(6), 146-154.

Shinwari, M.I. and M.A. Khan. 2000. Folk use of medicinal herbs of Margalla Hills

National Park, Islamabad, Pakistan. Journal of Ethno pharmacology, 69: 45-56.

Shinwari, Z.K. 2010. Medicinal Plants Research in Pakistan. Journ. Med. Pl. Res., 4(3):

161-176.

Shu, Y. Z. 1998. Recent natural products based drug development: a pharmaceutical

industry perspective. J Nat Prod 61, 1053-71.

Sies H. (ed.) Antioxidants in Disease, Mechanisms and Therapy, Academic Press, New

York, 1996.

Singh S, Sivakumar R. 2004. Challenges and new discoveries in the treatment of

leishmaniasis. J Infect Chemother.10: 307-315.

Singh, M., Srivastava, S., & Rawat, A. K. S. (2007). Antimicrobial activities of Indian

Berberis species. Fitoterapia, 78(7), 574-576.

134


Singha, U. K., Guru, P. Y., Sen, A. B., & Tandon, J. S. (1992). Antileishmanial activity

of traditional plants against Leishmania donovani in golden hamsters.

Pharmaceutical Biology, 30(4), 289-295

Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., & Boyd, M.

R. (1990). New colorimetric cytotoxicity assay for anticancer-drug screening.

Journal of the National Cancer Institute, 82(13), 1107-1112.

Stewart R.R. 1958. Catalogue of Plants of Rawalpindi. District superintendent, Govt

printing press, Punjab.

Sun Z, Peng X, Liu J, Fan K-W, Wang M, Chen F (2010) Inhibitory effect of microalgal

extracts on the formation of advanced glycation endproducts (AGEs). Food Chem.

120(1):261-267.

Tang, S. Y., Whiteman, M., Peng, Z. F., Jenner, A., Yong, E. L., & Halliwell, B. (2004).

Characterization of antioxidant and antiglycation properties and isolation of active

ingredients from traditional Chinese medicines. Free Radical Biology and

Medicine, 36(12), 1575-1587.

Tariku, Y., Hymete, A., Hailu, A., & Rohloff, J. (2011). In vitro evaluation of

antileishmanial activity and toxicity of essential oils of Artemisia absinthium and

Echinops kebericho. Chemistry & biodiversity, 8(4), 614-623.

Tempone, A. G., Sartorelli, P., Teixeira, D., Prado, F. O., Calixto, I. A., Lorenzi, H., &

Melhem, M. S. (2008). Brazilian flora extracts as source of novel antileishmanial

and antifungal compounds. Memórias do Instituto Oswaldo Cruz, 103(5), 443-449.

Thornalley PJ (2003) Use of aminoguanidine (Pimagedine) to prevent the formation of

advanced glycation endproducts. Arch Biochem Biophys 419(1): 31–40.

Tripathi, Y. B., Sharma, M., & Manickam, M. (1997). Rubiadin, a new antioxidant from

Rubia cordifolia. Indian journal of biochemistry & biophysics, 34(3), 302.

Tripathi, Y. B., Shukla, S., Sharma, M., & Shukla, V. K. (1995). Antioxidant property of

Rubia cordifolia extract and its comparison with vitamin E and parabenzoquinone.

Phytotherapy Research, 9(6), 440-443.

135


Tsao TF, Newman MG, Kwok YY, Horikoshi AK. 1982. Effect of Chinese and western

antimicrobial agents on selected oral bacteria. J Dent Res. 61:1103–6.

Tsuji-Naito K, Saeki H, Hamano M (2009) Inhibitory effects of Chrysanthemum species

extracts on formation of advaced glycation end products. Food Chem. 116(4):854-

859.

Tyler VE, (1997). Rational Phytotherapy, 3rd edition, Springer-Verlag,Berlin,

Heidelberg, New yolk.

Ueda-Nakamura, T., Mendonça-Filho, R. R., Morgado-Díaz, J. A., Korehisa Maza, P.,

Prado Dias Filho, B., Aparício Garcia Cortez, D., & Nakamura, C. V. (2006).

Antileishmanial activity of Eugenol-rich essential oil from Ocimum gratissimum.

Parasitology International, 55(2), 99-105.

Valko, M., Morris, H., Mazur, M., Rapta, P., & Bilton, R. F. (2001).Oxygen free radical

generating mechanisms in the colon: Do the semiquinones of Vitamin K play a

role in the aetiology of colon cancer? Biochim. Biophys. Acta, 1527, 161–166.

Valko, M., Rhodes, C. J., Moncol, J., Izakovic, M.,&Mazur,M. (2006). Free radicals,

metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact.,

160, 1–40.

Velioglu, Y.S., G. Mazza, L. Gao, B.D. Oomah. 1998. Antioxidant activity and total

phenolics in selected fruits, vegetables, and grain products. J. of Agric. and Food

Chem. 46:4113–4117.

Vinothapooshan, G., & Sundar, K. (2011). Immunomodulatory activity of various

extracts of Adhatoda vasica Linn. in experimental rats. Afr. J. Pharm. Pharmacol,

5(3), 306-310.

Vlassara H., and Palace MR. 2002. Diabetes and advanced glycation endproducts. J

Intern Med. 251:87–101.

Wang, H. Y. (2009). Studies on Antioxidant and Antiglycation properties of Several

Chinese Medicinal Herb Extracts.

136


Westh, H., Zinn, C.S., Rosdahl, V.T., Sarisa Study Group (2004): An international

multicenter study of antimicrobial consumption and resistance in Staphylococcus

aureus isolates from 15 hospitals in 14 countries. Microbial Drug Resistance 10:

169-176.

WHO (World Health Organization). 2011. The world medicines situation 2011.

Traditional Medicines: Global situation, issues and challenges. WHO press,

Geneva,

Switzerland.http://www.who.int/medicines/areas/policy/world_medicines_situatio

n/WMS_ch18_wTrad itionalMed.pdf; accessed on 9 Dec 2012.

WHO (World Health Organization). 2004. The Global Burden of Disease: Update.

Geneva: World Health Organization; 2008.

WHO (World Health Organization). 2002. World health report 2002: Reducing risks,

promoting healthy life. Geneva, World Health Organization, 30 October 2002.

ISBN 92 4 156207 2 ISSN 1020-3311, p. 248.

WHO (World Health Organization). 2001. General Guidelines forMethodologies on

Research and Evaluation of Traditional Medicine. WHO, Geneva, Switzerland, p.

1.

WHO/TDR, 2012, Leishmaniasis-disease information. URL:

http://www.who.int/tdr/diseases/leish/info/en/

Wojdyło, A., Oszmiański, J., & Czemerys, R. (2007). Antioxidant activity and phenolic

compounds in 32 selected herbs. Food Chemistry, 105(3), 940-949.

Xi, M., Hai, C., Tang, H., Chen, M., Fang, K., & Liang, X. (2008). Antioxidant and

antiglycation properties of total saponins extracted from traditional Chinese

medicine used to treat diabetes mellitus. Phytotherapy Research, 22(2), 228-237.

Yang B, Zhao MM, Shi J, Cheng GP, Ruenroengklin N, Jiang YM (2008). Variations in

water-soluble saccharides and phenols in longan fruit pericarp after drying.

Journal of Food Process Engineering 31(1): 66-77.

137


Zabihullah, Q., Rashid, A., & Akhtar, N. (2006). Ethnobotanical survey of Kot Manzary

Baba valley, Malakand Agency, Pakistan'. Pakistan Journal of Plant Science,

12(2), 115-121.

Zhai, L.; Chen, M.; Blom, J.; Teander, T.G.; Christensen, S.B.; Karazmi, A. 1999.The

antileishmanial acitivity of novel oxygenated chalcones and their mechanism of

action. Antimicrob. Agents Chemother. 43, 793–803.

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ANNEXES

139

ANNEX 1

ETHNOBOTANY QUESTIONAIRE FOR FIELD VISIT

1 General:

Village: _______________________________.

Name of the respondent: _________________________________________.

Age Group

a) Young (25 and below): ______.

b) Middle age (26–50): ______.

c) Old age (51 and above): _____.

Level of Education:

Primary: ______. Secondary: ____. Intermediate: ______. Higher: ______.

Occupation: _________________________.

Sex a) Male: ________. b) Female: _________.

2 About Medicinal Plants

What way of treatment you like:

a) Through medicinal plants ______________________________.

b) Allopathic drugs ______________________________________.

You go to Hakeem or a Doctor for your treatment? _____________________.

Do you use/collect any medicinal plant?

(a) Yes: ________. (b) No: ________.

If yes then which species from which locality? _______________________.

How many people of your family collect medicinal plants? _______________.

How many different species of medicinal plants you know? ______________.

How many people of your village use these medicinal plants? ____________.

Which illness/wound is treated with the help of which medicinal plants?

_______________________________________________________________.

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How and which part of medicinal plant you collect?

________________________________________________________.

How do you use medicinal plants? __________________________________.

The plants, which you use, are present in your village or not? _______________.

In your family who know best about medicinal plants? ___________________.

Do you keep medicinal plants in your home for your need or not?

___________________________.

In your village the use of medicinal plants is increasing or decreasing?

___________________________.

Do you cultivate medicinal plants? __________________________________.

From whom you learnt the indigenous knowledge of medicinal plants?

_______________________________________________________________.

General Remarks

___________________________________________________________

____________________________________________________________

_________________________________________________________

____________________________________________________________

141

ANNEX 2

1. DPPH Radical Scavenging Activity

1.1 Regression line equation used for the calculation of IC50 in case of DPPH Radical

Scavenging Activity at Human Body Temperature 37 ℃ after 15 minutes. All the

values represent the means of three replicates

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Key: Geranium collinum (GC), Persicaria barbata (PB), Impatiens edgeworthii (IE),

Rubia cordifolia (RC), Clematis grata (CG), Geranium wallichianum (GW), Berberis

lycium (BL), Artemisia vulgaris(AV), Boerhavia procumbens (BP), Capsella bursa-

pastoris (CBp).

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Scavenging Activity at Human Body Temperature 37 ℃ after 60 minutes. All the

values represent the means of three replicates

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pastoris (CBp).

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Scavenging Activity at Human Body Temperature 37 ℃ after 120 minutes. All

the values represent the means of three replicates

146




pastoris (CBp).

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Scavenging Activity at Room Temperature 25 ℃ after 15 minutes. All the values

represent the means of three replicates

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pastoris (CBp).

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150




pastoris (CBp).

151




152




pastoris (CBp).

153

ANNEX 3

ABTS Radical Cation Activity

Regression line equation used for the calculation of IC50 in case of ABTS Radical cation

Activity. All the values represent the means of three replicates

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pastoris (CBp).

155

ANNEX 4

Hydroxyl Radical Scavenging Activity

Regression line equation used for the calculation of IC50 in case of Hydroxyl Radical

scavenging Activity. All the values represent the means of three replicates

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pastoris (CBp).

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ANNEX 5

Phosphomolybdinum Activity

Regression line equation used for the calculation of IC50 in case of phosphomolybdinum

assay. All the values represent the means of three replicates

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pastoris (CBp).

159

ANNEX 6

Climatic Information (2010-11) of the Study Area

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