PHYTOCHEMICAL AND PHARMACOLOGICAL PROFILING ...

PHYTOCHEMICAL AND PHARMACOLOGICAL PROFILING OF

Dysphania botrys L.

BY

MUHAMMAD NAEEM KHAN

A dissertation submitted to The University of Agriculture Peshawar, in partial

fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY AND

GENETIC ENGINEERING

INSTITUTE OF BIOTECHNOLOGY & GENETIC ENGINEERING

FACULTY OF CROP PRODUCTION SCIENCES

THE UNIVERSITY OF AGRICULTURE

PESHAWAR, PAKISTAN

AUGUST, 2018


Dysphania botrys L.

BY

MUHAMMAD NAEEM KHAN

A dissertation submitted to The University of Agriculture Peshawar, in partial

fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY AND

GENETIC ENGINEERING

APPROVED BY:

Chairman Supervisory Committee

Dr. Asad Jan

Associate Professor

Co-Supervisor for Research

Dr. Inamullah Khan

Assistant Professor

Pharmacy (UOP)

Member Major Field

Dr. Safdar Hussain Shah

Member Minor Field

Prof. Dr. Farhatullah

Chairman and Convener Board of Studies Prof. Dr. Iqbal Munir

Dean Faculty of Crop Production Sciences

Prof. Dr. Zahir Shah

Director Advanced Studies and Research Dr. Shahid Sattar

INSTITUTE OF BIOTECHNOLOGY & GENETIC ENGINEERING

FACULTY OF CROP PRODUCTION SCIENCES

THE UNIVERSITY OF AGRICULTURE

PESHAWAR, PAKISTAN

AUGUST, 2018

TABLE OF CONTENTS

S. No. Title Page No.

List of Tables .......................................................................................... i

List of Figures ....................................................................................... iii

Abbreviations ......................................................................................... iv

Acknowledgments .................................................................................. vii

Abstract .................................................................................................. ix

I INTRODUCTION .............................................................................. 1

II. REVIEW OF LITERATURE ............................................................ 13

III. MATERIALS AND METHODS ....................................................... 32

3.1 Plant collection and identification ....................................................... 32

3.2 Extraction of plant material .................................................................. 32

3.3 Fractionation procedure ........................................................................ 33

3.4 Phytochemical investigation ................................................................. 35

3.4.1 Quantitative analysis of phytochmeicals ............................................. 35

3.4.1.1 Stock solution ....................................................................................... 35

3.4.1.2 Test for crude alkaloids......................................................................... 35

3.4.1.3 Test for saponins ................................................................................... 35

3.4.1.4 Test for phenols .................................................................................... 35

3.4.1.5 Test for flavonoids ................................................................................ 35

3.4.1.6 Test for tannins ..................................................................................... 36

3.4.1.7 Test for sterols ...................................................................................... 36

3.4.2 Qualitative analysis of phytochemicals ............................................... 36

3.4.2.1 Determination of total phenol ............................................................... 36

3.4.2.2 Determination of total saponins ............................................................ 36

3.4.2.3 Determination of total flavonoids ......................................................... 37

3.4.2.4 Determination of total alkaloids ........................................................... 37

3.4.3 Proximate composition ........................................................................ 37

3.4.3.1 Moisture content ................................................................................... 37

3.4.3.2 Inorganic matter .................................................................................... 38

3.4.3.3 Crude lipid ............................................................................................ 38

3.4.3.4 Dietary fiber .......................................................................................... 38

3.4.3.5 Crude protein ........................................................................................ 39

3.4.3.6 Nitrogen˗free extract ............................................................................. 39

3.4.4 Minerals analysis .................................................................................. 39

3.5 In-vitro studies ...................................................................................... 41

3.5.1 Antimicrobial activity ........................................................................... 41

3.5.2 Strains and culture media ...................................................................... 41

3.5.3 Antibacterial activity............................................................................. 42

3.5.4 Antifungal activity ................................................................................ 42

3.5.5 Phytotoxic activity ................................................................................ 42

3.5.6 Antioxidant assay .................................................................................. 43

3.5.6.1 1, 1-diphenyl-2-picrylhidrazyl (DPPH)) radical scavenging

activity .................................................................................................. ..43

3.5.6.2 ABTS (2, 2˗azinobis˗3-ethylbenzothiozoline-6-sulfonic acid)

radical scavenging assay ....................................................................... 43

3.5.7 Lipoxygenase-inhibitory assay (LOX) ................................................. 44

3.6 In-vivo studies ....................................................................................... 44

3.6.1 Acute toxicity study .............................................................................. 44

3.6.2 Anti-inflammatory effect ...................................................................... 45

3.6.2.1 Carrageenan induced paw edema model ............................................. 45

3.6.2.2 Xylene˗induced ear edema.................................................................... 45

3.6.3 Analgesic effect .................................................................................... 46

3.6.3.1 Formalin test ........................................................................................ 46

3.6.3.2 Hot plate test ......................................................................................... 46

3.6.4 Antipyretic effect ................................................................................. 47

3.6.5 Anti-diarrheal effect .............................................................................. 47

3.6.6 Anti-diabetic effect ............................................................................... 47

3.6.7 Hepatoprotective effect ......................................................................... 48

3.6.7.1 Carbon tetra chloride (CCl4) induced hepatotoxicity Model ................ 48

3.6.7.2 Biochemical investigations ................................................................... 48

3.6.8 Sedative/hypnotic effect ...................................................................... 49

3.6.9 Anticonvulsant effect ........................................................................... 49

3.6.10 Antidepressant effect ........................................................................... 49

3.7 Statistical analysis ................................................................................. 50

IV RESULTS ............................................................................................ 51


4.1.1 Qualitative analysis of phytochemicals ................................................ 51

4.1.2 Quantitative analysis ............................................................................. 51

4.1.2.1 Total phenols......................................................................................... 51

4.1.2.2 Total alkaloids....................................................................................... 52

4.1.2.3 Total saponins ....................................................................................... 53

4.1.2.4 Total flavonoids ................................................................................... 53

4.1.3 Proximate composition ......................................................................... 54

4.1.4 Mineral analysis .................................................................................... 55

4.2 In-vitro activities ................................................................................... 55

4.2.1 Antibacterial activity............................................................................. 55

4.2.2 Antifungal activity ................................................................................ 56


4.2.4 Antioxidant activity .............................................................................. 58

4.2.4.1 DPPH radical scavenging activity ........................................................ 58

4.2.4.2 ABTS radical scavenging activity ........................................................ 59

4.2.5 Lipoxygenase-inhibitory assay ........................................................... 60

4.3 In-vivo pharmacological activities ........................................................ 60

4.3.1 Acute toxicity ........................................................................................ 60

4.3.2 Anti-inflammatory activity ................................................................... 61

4.3.2.1 Carrageenan˗induced paw edema model .............................................. 61

4.3.2.2 Xylene-induced ear edema.................................................................... 62

4.3.3 Analgesic activity ................................................................................. 63

4.3.3.1 Formalin test ......................................................................................... 63

4.3.3.2 Hot plate test ......................................................................................... 64

4.3.4 Antipyretic activity ............................................................................... 65

4.3.5 Antidiarrheal activity ............................................................................ 66

4.3.6 Anti-diabetic activity ............................................................................ 66

4.3.7 Hepativeprotective activity ................................................................... 67

4.3.8 Sedative/hypnotic activity..................................................................... 68

4.3.9 Anti-convulsant activity ........................................................................ 69

4.3.10 Antidepressant activity ......................................................................... 69

V. DISCUSSION ...................................................................................... 71


5.1.1 Qualitative and quantitative analysis of phytochemicals ...................... 71

5.1.2 Proximate composition: ........................................................................ 72

5.1.3 Mineral analysis .................................................................................... 73

5.2 In-vitro activities ................................................................................... 75

5.2.1 Antimicrobial activity ........................................................................... 75


5.2.3 Antioxidant activities ........................................................................... 78

5.2.4 Lipoxygenase activity ........................................................................... 79

5.3 In-vivo pharmacological activities ........................................................ 80

5.3.1 Acute toxicity ........................................................................................ 80

5.3.2 Anti-inflammatory activity ................................................................... 80

5.3.3 Analgesic activity ................................................................................. 82

5.3.4 Anti-pyretic activity .............................................................................. 83

5.3.5 Antidiarrheal activity ............................................................................ 85

5.3.6 Antidiabetic activity .............................................................................. 87

5.3.7 Hepativeprotective activity ................................................................... 89

5.3.8 Sedative-hypnotic activity .................................................................... 90

5.3.9 Anti-convulsant activity ........................................................................ 91

5.3.10 Antidepressant activity ........................................................................ 92

VI. SUMMARY ......................................................................................... 94

VII. CONCLUSIONS AND RECOMMENDATIONS ........................... 96

Conclusions ........................................................................................... 96

Recommendations ................................................................................. 97

LITERATURE CITED ...................................................................... 98

i

LIST OF TABLES

Table No. Title Page No.

3. 1. Conditions for operation of micro and macro minerals ......................................... 40

3.2. Strains of bacteria for antibacterial activity ........................................................... 41

3.3. Strains of fungi used for antifungal activity ........................................................... 43

4.1. Phytochemical of methanolic crude extract and solvents fractions of D.

botrys ...................................................................................................................... 51

4.2. Proximate composition (%) of D. botrys whole plant ............................................ 54

4.3. Mineral composition of whole plant of D. botrys .................................................. 55

4.4. Antibacterial activity of methanolic crude extract and solvent fractions

of D. botrys ............................................................................................................. 56

4.5. Antifungal activity of methanolic crude extract and solvent fractions of

D. botrys ................................................................................................................. 57

4.6. Phytotoxic activity of methanolic crude extract and solvent fractions of

D. botrys. ................................................................................................................ 58

4.7. DPPH radicals scavenging activity of methanolic crude extract and

solvent fractions of D. botrys ................................................................................. 59

4.8. ABTS radicals scavenging activity of methanolic extract and solvent

fractions of D. botrys .............................................................................................. 59

4.9. Lipoxygenase-inhibitory assay of methanolic crude extract and solvent

fraction of D. botrys ............................................................................................... 60

4.10. In-vivo acute toxicity of methanolic crude extract of D. botrys ............................. 60

4.11a. Anti˗inflammatory activity of methanolic crude extract of D. botrys on

carrageenan provoked mice paw edema ................................................................ 61

4.11b. Percent inhibition of carrageenan-induced paw edema by methanolic

crude extract of D. botrys ....................................................................................... 62

4.12a. Anti˗inflammatory effect of methanolic crude extract of D. botrys on

xylene˗induced ear edema in mice ......................................................................... 62

ii

4.12b. Percent inhibition of xylene-induced ear edema by methanolic crude

extract of D. botrys ................................................................................................. 63

4.13a. Analgesic effect of methanolic crude extract of D. botrys on formalin-

induced pain in rats ................................................................................................ 63

4.13b. Percent inhibition of formalin-induced pain by methanolic crude extract

of D. botrys ............................................................................................................. 64

4.14. Analgesic effect of methanolic crude extract of D. botrys on pain

induced by hot plate in mice .................................................................................. 64

4.15. Antipyretic effect of methanolic crude extract of D. botrys on brewer‟s

yeast induced pyrexia in rats .................................................................................. 65

4.16. Antidiarrheal effect of crude extract of D. botrys on castor oil-induced

diarrhea in rats ........................................................................................................ 66

4.17. Anti-diabetic activity of methanolic crude extract of D. botrys on

alloxane induced diabetes in mice. ......................................................................... 67

4.18. Hepativeprotective activity of methanolic crude extract of D. botrys

extract on CCl4 stimulated toxicity in rats ............................................................. 68

4.19. Sedative/hypnotic activity of methanolic crude extract of D. botrys on

thiopental induced hypnosis ................................................................................... 68

4.20. Anticonvulsant Effect of methanolic crude extract of D. botrys on PTZ-

induced convulsions in mice .................................................................................. 69

4.21. Antidepressant activity of crude extract D. botrys on the time of

immobility in forced swim test model in rats ......................................................... 70

iii

LIST OF FIGURES

Fig.No. Title Page No.

3.1. Herbarium specimen of D. botrys .......................................................................... 32

3.2. Scheme of extraction and fractionation process ..................................................... 34

4.1. Total phenols in methanolic crude extract and subsequent fractions ..................... 52

4.2. Total alkaloids in methanolic crude extract and subsequent fractions ................... 52

4.3. Total saponins in methanolic crude extract and subsequent fractions ................... 53

4.4. Total flavonoids in methanolic crude extract and subsequent fractions ................ 54

4.5. Antibacterial activity of Ethyl acetate fraction and crude extract of D.

botrys against ......................................................................................................... 56

4.6. Antifungal activity of crude extract and Ethyl acetate of D. botrys

against .................................................................................................................... 57

iv

ABBREVIATIONS

A. flavus Aspergillus flavus

A. niger Aspergillus niger

A.alternatum Acremonium alternatum

ABTS 2, 2-azinobis-3-ethylbenzothiozoline-6-sulfonic acid scavenging assay

ALP Alkaline phosphatase

ANOVA Analysis of variance

ATCC American type culture collection

B. subtilis Bacillus subtilis

C Centigrade

C. michiganesis Clavibacter michiganesis

Cm Centimeter

CuSO4 Copper Sulphate

DCMF Dichloromethane fraction

DMSO Dimethyle sulphoxide

DPPH 1, 1-diphenyl-2-picrylhydrazyl

E. coli Escherichia coli

EAF Ethyle acetate fraction

Etc et cetra

F. oxysporum Fusarium oxysporum

F. solani Fusarium solani

FST Forced swimming test

HCl Hydrochloric acid

HNO3 Nitric acid

hrs hours

HxF n-Hexane fraction

i.e that is

i.p Intraperitoneal

IC Inhibition concentration

K. pneumonia Klebsiella pneumonia

KOH Potassium Hydroxide

L. minor Lemna minor

LD Lethal dose

v

LOX Lipoxygenase assay

M Molar

M. pirimis Mucar pirimis

MCE Mthanolic crude extract

mg Milligram

MIC Minimum inhibitory concentration

min Minutes

ml Milliliter

mm Milimeter

mM Millimolar

n Number

N Normal

N/saline Normal saline

NaCl Sodium chloride

NaOH Sodium hydroxide

NO Nitric oxide

OD Optical density

P. aeruginosa Peseudomonas aeruginosa

P. vulgaris Proteus vulgaris

PDA Potato dextrose agar

PDB Potato dextrose broth

PEG Polyethylene Glycol

PTZ Pentylene tetrazole

R. communis Ricinus communis

R. oryzae Rhizopus oryzae

R.dominica Rhizopertha dominica

S. aureus Staphylococcus aureus

s.c Subcutinious

sec Second

SGOT Serum glutamic oxaloacetic transaminase

SGPT Serum glutamic pyruvic transaminase

TB Total bilirubin

UV Ultraviolet

µg Microgram

vi

µl Microliter

v/v Volume by volume

w/v Weight by volume

X. campestris Xanthomonas campestris

ZI Zone of inhibition

vii

ACKNOWLEDGEMENTS

All praises are to Almighty ALLAH, the most Merciful and Compassionate, the

only creator of the universe and source of all knowledge and wisdom, who blessed me

with health, thoughts, loving parents, wife and children, talented teachers, helping

friends and afforded me to complete this study. Countless salutations are upon the

Prophet Muhammad (PBUH), the gleam of guidance, faith and knowledge for the

humanity.

I wish to express deepest gratitude and profound regards to my kind Supervisor

Dr. Asad Jan, Associate Professor, IBGE, for his supervision, planning, execution

and scholarly ideas that beautified the scientific nature of the research work carried out.

He always encouraged me and kept my morale high by his suggestions, appreciation

and motivation. Without his precious guidance and support I would never be able to

complete my research.

I feel great pleasure in expressing my ineffable thanks to my encouraging,

inspirational and cool minded co-supervisor Dr. Inam Ullah Khan, Associate

Professor, Department of Pharmacy, University of Peshawar for his sense of devotion,

creativity, affectionate criticism and keen interest in my work; it was because of his

inspiring guidance and dynamic cooperation during entire study program that I could

complete this manuscript.

I tender my thanks to Professor Dr. Iqbal Munir, Director IBGE for his

administrative support and cooperation. Special thanks are extended to Professor Dr.

Safdar Hussain Shah for his kind attitude, valuable suggestions and help. I am also

thankful to Prof. Dr. Farhatullah, Dr. Aqib Iqbal, Dr. Shakoor and all the faculty

members of IBGE and UAP for their moral help and support. Also thanks to the

members of my supervisory committee for their cooperation and valuable suggestions

during my research work.

I would also like to acknowledge Department of Pharmacy, University of

Peshawar and Department of Pharmacognocy, Faculty of Pharmacy, University of

Karachi for providing me all technical and lab facilities.

I convey my thanks from the deepest of my heart to my very caring seniors and

friends Dr. Zahid Ullah (UOS), Dr. Zamarud Shah (USTB), Dr. Zafar Hashmi

(COMSATS), Dr. Abdul Wajid (PCSIR), Muhammad Imran, Abid, Hezbullah,

viii

Rameez and Medrar for their technical support and valuable suggestions during my

research work.

Words do not come out easy for me to mention the feelings of obligations

towards my affectionate parents. My father proved the ocean of Love, care for me in

which I saturated myself. Every aspect of my life is incomplete without him. I am most

earnestly thankful to My Mother for the strenuous efforts done by her in enabling me

to join the higher ideals of life. I am grateful to my parents, wife and sisters for their

financial and moral support, patience and prayers they had made for my success. May

ALLAH ALMIGHTY infuse me with the energy to fulfill their noble inspirations and

expectations and further edify my competence.

Muhammad Naeem Khan

ix


Dysphania botrys L.

Muhammad Naeem Khan and Asad Jan

Institute of Biotechnology & Genetic Engineering

Faculty of Crop Production Sciences

The University of Agriculture

Peshawar, Pakistan

August, 2018

ABSTRACT

Dysphania botrys L. is an annual herbaceous plant belongs to family Amaranthaceae,

native to Asia and Europe found in Pakistan, India and Iran. In the folk medicine D.

botrys has been utilized for the treatment of different ailments like asthma, cold,

influenza, head ach, liver and digestive problems and healing of wounds. The current

work was designed to evaluate methanolic crude extract (MCE) of D. botrys for

different in-vivo pharmacological activities and its various solvents fraction for

phytochemical analysis and different in-vitro activities, in order to provide scientific

authentication to its ethno-medicinal uses. Qualitative phytochemical study of MCE

and solvent fractions of D. botrys confirmed the presence of phenols, alkaloids,

flavonoids, sterols, tannins and saponins, however in n-hexane fraction (HxF) only

flavonoids and saponins were detected. In quantitative analysis, amongst all the

solvents, ethyl acetate fraction (EAF) had highest amount of phenol (27.4 mg/g),

flavonoids (15.5 mg/g) and alkaloids (3.14 mg/g), while MCE displayed maximum

amount of saponins (34.3 mg/g). In the proximate analysis, nitrogen-free extract were

present in higher amount (38.45 ± 0.83%) followed by protein (30.26 ± 0.72%) while

crude fibers were found least in amount (1.43 ± 0.53%). Among different minerals,

reasonable amount of calcium (3268 ± 0.53 μg/g), potassium (2873 ± 0.71 μg/g),

sodium (591 ± 0.23 μg/g) and iron (223 ± 0.46 μg/g) were found, while no cadmium

and chromium was detected. MCE and EAF displayed considerable antibacterial

activity against Xanthomonas campestris and Pseudomonas aeruginosa causing 12.6 ±

0.54 mm and 20.6 ± 0.53 mm zone of inhibition respectively which was analogous to

that of cefixime, used as standard drug. In case of antifungal activity MCE hindered the

growth of Fusarium oxysporum effectively, causing 19.3 ± 0.41 mm inhibition zone,

while effect of other solvents was low to moderate. Highest phytotoxic effect was

shown by MCE (1000 μg/ml) against the growth of Lemna minor, causing 70%

reduction in its growth. EAF exhibited maximum scavenging activity against 1, 1-

diphenyl-2-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethylbenzothiazoline-6-

sulfonic acid (ABTS) radicals causing 57.17 ± 0.49% and 72.46 ± 0.59% scavenging

activity respectively as compared to standard, ascorbic acid. The activity of

lipoxygenase (LOX) enzyme was inhibited effectively by EAF (64 ± 0.16%), while

HxF displayed least inhibiting effect (22 ± 0.21%). In the in-vivo pharmacological

activities of crude extract of D. botrys, acute toxicity analysis showed no sign of

mortality up to an amount of 2000 mg/kg. Crude extract (200 and 400 mg/kg) showed

considerable (p<0.05) antiinflammatory effect at early and late phase of

carrageenan˗stimulated paw edema while in case of xylene˗induced ear edema dosage

of 400 mg/kg was highly effective (p<0.01) in reducing ear inflammation (73.2%).

Dose of 200 mg/kg of plant extract displayed considerable (p<0.05) peripheral

x

analgesic activity at both phases of analgesia, causing 60.71% and 67% reduction in

severity of pain while in case of 400 mg/kg, its effect was highly significant (p<0.01)

causing 78.57% and 82.14% pain inhibition. In the central analgesic activity (hot plate

model) the effect of 400 mg/kg was highly significant (p<0.01) after 120 min of

assessment time interval. In the antipyretic assay, effect of 400 mg/kg of plant extract

was extremely significant (p<0.001) at all the assessment time intervals (1h-5h) and

was comparable to that of standard drug paracetamol in reducing body temperature to

normal, increased by brewer‟s yeast. In the antidiarrheal activity, plant extract of 400

mg/kg effectively (p<0.01) increased the latent period of diarrhea and caused a decline

in the total wet fecal frequency and mean weight of fecal drops as compared to control.

The elevated blood sugar induced by alloxane monohydrate in the anti-diabetic activity

was significantly reduced by crude extract (400 mg/kg), however its effect was highly

significant (p<0.01) at the 3rd

and 4th

hour of evaluation time. In the hepatoprotective

assay, MCE of plant at dosage of 400 mg/kg markedly (p<0.05) declined high level of

alkaline phosphatase (ALP) (179.22 ± 3.41 mg/dl) and total bilirubin (TB) (3.64 ± 0.13

mg/dl) while its effect was highly significant (p<0.01) in reducing the level of serum

glutamic pyruvic transaminase (SGPT) (31.2 ± 1.28 U/ml) and serum glutamic

oxaloacetic transaminase (SGOT) (48.31 ± 1.87 U/ml)) when compared to toxic

control. Plant extract (100 and 200) showed a significant (p<0.05) synergetic effect on

the thiopental induced hypnosis caused an early arrival of sleep and effectively

(p<0.01) prolonged the duration of sleep from 88.80 ± 1.91 min to 145.20 ± 1.76 min.

In the pentalyne tetrazol (PTZ) induced convulsion activity, plant extracts (200 and 400

mg/kg) effectively (p<0.05) delayed the onset of first clonus from 5.09 ± 0.22 min to

6.03 ± 0.28 min and 6.99 ± 0.07 min and prolonged the time of death from 9.72 ± 0.44

min to13.57 ± 0.6 min and 19.56 ± 0.15 min, respectively. The immobility time was

significantly (p<0.05) decreased by MCE of plant from 193.98 ± 1.35 seconds to 96.78

± 1.39 seconds, in the antidepressant activity.

Key words: Dysphania botrys, phytochemical analysis, antimicrobial activity,

antioxidant assay, in-vivo pharmacological activities

1

1. INTRODUCTION

Plants not merely give food and raw material to humans but also the source of

bioactive and medicinally important compounds. A plant is considered medicinal, when

it contains active molecules or substances that can be utilized for healing purposes or

act as precursor for making beneficial drugs. Phytotherapy also called phytomedicine,

herbal medicine or botanical medicine refer to any medicine that is obtained from

plants either in the crude or pure form having active ingredients or simply phytotherapy

is the utilization of plants and its products for therapeutic and medicinal purposes

against various ailments (Rates, 2001). The different parts of plants used in

phythotherapy include leaves, branches, roots, flowers, seeds, bark, berries or whole

plant. Medicinal plants are the back bone of folk medicinal system, which is based on

the utilization of medicinal plants and their derivatives. The vast diversity of medicinal

flora around the world provides a huge assets and infinite source for production of

herbal medicine. More than three forth of world population uses herbal medicine for

curing different diseases and relies upon it, because herbal medicine have no side effect

and can easily obtained from nature (Farnsworth, 1985).

Human had been used plants for preventing and curing diseases throughout the

history of mankind and analysis of fossils record shows, that the therapeutic use of

plants is as old as 60,000 years (Solecki and Shanider, 1975). Extensive studies have

indicated that phytomedicine represent the earliest form of medication. Documented

record about herbal medicine is approximately 5,000 years, dated back to Sumerians,

who explained different therapeutic uses of plants. However archeological

investigations have revealed that the use of phytomedicine dates back sixty to eighty

thousand years ago in Iraq and China respectively (Leroi Gourhan, 1975). Prehistoric

analysis have showed that medicine extracted from medicinal plants, either in crude or

pure form, represent the oldest method of medication. Archaeological investigations

have revealed that peoples in the ancient time had knowledge about therapeutic

properties of plants, since the practice of phytomedicine is as primal as human species

(Halberstein, 2005).

Greek civilization has made a great role in the development of herbal-

pharmaceutical science. Aristotle explained about five hundred crude drugs used

against various diseases (Oktay et al., 2003). Hippocrates regarded as the founder of

2

modern allopathic medicine, described approximately four hundred substances of plant

origin having therapeutic properties against various ailments (Sykiotis et al., 2006).

Theophrastus cited about five hundred crude herbal drugs and their medicinal

properties in his book. Claudius Galen Pergamum wrote three hundred books on

medicinal plants. He made herbal drugs employing various extraction methods called

Galenical and established the idea of pharmaceutical formulation to develop

therapeutically secure and efficient medicine (Newman et al., 2000; Buerky and Higby,

2007).

Chinese traditional prescription is considered one of the earliest systems of

medication based more than 85% on herbal products. Shen-Nong about five hundred

years ago described certain plants in the Chinese herbal medicine and because of his

efforts various plants were commonly used in the ancient China for health care

(Dharmananda, 2013). Before the time of Shen-Nong, few earliest Chinese books, such

as Shi-jing, Shang-Shu and Shing-Hai-Jing documented the use of plants as a remedy.

Shi-Jing not only explained the medicinal uses of plants but also described their habitat

and harvesting season. In this book eighty medicinal plants and ninety species of

insects with their therapeutic uses were recorded. Shan-Hai-Jing, the ancient Chinese

book documented nine plant species having nutritional value, forty five species having

therapeutic properties, six species having toxic effect for pests and animals and two

plants having noxious effect for human beings (Hu, 2008).

Ayurveda or Indian conventional medication is one of the ancient systems based

on natural products and considered mother of all therapies. The documented proof of

this system is available in the earliest literature for example, Atharva˗veda and

Rig˗veda about 5000 years BC. These books had the names of medicinal plants in the

form of poetry and transferred from generation to generation. (Mukherjee and Wahile,

2006).

Before the start of 19th

nineteenth century, phytomedicine were utilized in crude

form as infusion (herbal tea), tincture (alcoholic extract), syrup (sugar solution of

extract), and decoction (stem, root, flower, leaves or bark boiled extract) or consumed

externally as balms, ointments, essential oil and poultices (Newman et al., 2003).

However later on, scientist started the separation, purification and recognition of

therapeutically bioactive compounds from medicinal plants. This effort made it

3

possible to analyze and discover effective drugs which are even widely used in the

modern pharmaceutical products (Kong et al., 2003; Gupta et al., 2005).

According to botanical survey there are approximately 250, 000 to 350, 000

species of plant present over the earth. Among these, very less number of plants species

i-e 35, 000 to 70, 000 have been used for therapeutic purposes in different region of

world which form 14% to 28% of total plants (Jin˗Ming et al., 2003). These curative

plant derivatives are consumed generally in crude or partially processed shape, thus

require scientific authentication to ascertain their medicinal uses (De Smet, 2002;

Kinsel and Straus, 2003). Among all medicinal plants only 15% are phytochemically

investigated and only 6% have biological screening data, while the remaining medicinal

plants have no scientific data, thus have a great scope to search out for novel effective

pharmacological agents (Newman et al., 2000; Harvey, 2000 and Verpoort, 2000).

Majority population of the world consumes a large diversity of plants for

medicinal purposes, even though no scientific knowledge and information is present

about the efficiency and effectiveness of these medicinal plants. Regarding the

significance of drugs and bioactive compounds extracted from plants and lack of

scientific knowledge, it is necessary conduct and research work an organized manner

for herbal medicine and restorative plants (Schopene, 1983 and Awadeh et al., 2001).

Over the earlier decades, a huge attention has been given for identification and

extraction of bioactive compounds from plants, for pharmaceutical and dietary use (Ho

et al., 1992; Oktay et al., 2003; Wangensteen et al., 2004). Different plant parts for

example flowers, roots, stem and whole plant are used for obtaining bioactive

compounds which are consumed in herbal medicines for the treatment of several

ailments. Currently, because of development of advance scientific techniques and

equipments, herbal experts take great interest to explore new therapeutic agents from

bioactive metabolites of plants. These natural bioactive agents will serve infinite assets

for herbal pharmaceutical industry (Yakubu et al., 2007).

Plants having medicinal properties played very important role and contribution

in agriculture, pharmaceuticals and food industries. In spite of preeminence of synthetic

chemistry and modern techniques to determine and synthesize drugs, the role and

importance of phytochemicals for curing diseases and drug discovery is still remarkable

(Raskin et al., 2002). Due to modern synthetic techniques, pharmaceutical industries

4

have developed a large number of synthetic drugs for curing various diseases, however

the regular use of these drugs develop other chronic problems like microbe resistance

and side effects, effecting other activities of body. Compared to herbal drugs, synthetics

drugs are more expensive and ordinary peoples cannot afford it. The use of natural

products for health care is steadily growing and there is a shift in general trend from

synthetic to phytomedicine. More than four billion people are using phytomedicine as a

remedy against various ailments (Fabricant and Farnsworth, 2001). Phytotherapy has

been acknowledged by WHO as vital component for basic health care (Taylor, 2000).

In the modern scientific era the healing characteristics of medicinal plants have

been investigated throughout the world, because of their strong therapeutic efficiency,

antioxidant properties, no or less side effect and financial affordability. Medicinal

plants provide raw material to pharmaceutical industry on large scale to extract drugs in

pure form which are very efficient and economically viable. The significant therapeutic

role of medicinal plants, in curing chronic diseases, have been reported and proved by

scientific research (Martinez et al., 2008).

In case of cancer, researchers are expecting novel bioactive molecules having

anticancer properties which can be more effective as compared to synthetic drugs.

Flavonoids and other secondary metabolites extracted from various plants have

revealed considerable inhibitory effect on malignant cells (Jiangrong and Jiang 2007;

Zhao et al., 2007). Plants having hypoglycemic properties, offer a potential area for

herbal research to control and cure diabetes, affecting a huge number of peoples all

over the world (Renuka et al., 2009). Phytomedicine have been proved effective for

treating various cardiovascular disorders, which are the frequent reasons of death of

people all over the globe (Thippeswamy et al., 2009). Plants having hepativeprotective

properties have the ability to lower down the elevated level of enzymes in liver, in case

of viral infection and are the best alternative of synthetic drugs (Oshima et al., 1995;

Bhawna and Kumar, 2009). Plants of different taxonomic groups and regions have

shown antioxidant, antimicrobial, anti-inflammatory, antidiarrheal, antidepressant and

analgesic property (Ibrahim et al., 2006; Ali et al., 2008). Due to widespread interest

and application, medicines obtained from herb plays essential roles.

5

Economically medicinal plants play very important role because of their

widespread industrial uses. These include folk medicine, herbal tea and health care

products like neutraceuticals, phytopharmaceuticals, galenicals, semi-synthetic and

synthetic pharmaceuticals. Up to now, 125 different compounds extracted from curative

plants have been identified, screened and used in the pharmaceutical industry.

Medicinal plants are the valuable resources making foreign exchange particularly for

developing countries. Economically important phytochemicals and herbal medicine are

vinblastine, vincristine, taxol, colchicines, podophyllotoxine, qeuuinine, tinctures,

morphine, camptothecin, atropine, digitoxigenin, capsaicin, curcumin, capsaicin,

aspirins, codeine, artimisinin and ephedrine. Therapeutic plants contribute significant

role in the development of research in pharmaceutical industry which mainly focus on

the separation and screening of bioactive compounds or synthesis of semi-synthetic

products (Cordell, 2009).

The international market of plants derived natural products like phytochemicals,

phytopharmaceuticals, flavors, fragrances, color ingredients, exceed annually several

billion dollars. The trade of plants derived drugs and chemicals are increasing annually

with a rate of 6.4%. In United state of America (USA), less the 5% people used plant

derived products in 1991 but in 2004 this value increased to 50%. Net sale of herbal

products is three fourth of total sale in the market. The worth of plant derived

medicated confectioneries reached to 593 million US dollars (Patel, 2015; Tiwari,

2015). In 2014, there was an increase of 7%, in retail sale value of phytomedicine used

for cough, cold and different allergies. In USA, Japan and other developed countries of

world, the demand for neutraceuticals is more as compared to past and in 2010 its

market sale value was more than 80-250 billion US dollars, with a similar value in

Japan and Europe (Sapna, 2007).

Certain reports have revealed that the trade of phytomedicine in United

Kingdom (UK) has increased 43% in 1994-1998, having a worth of 50 million Euros,

while in 2003, this value reached to 60%. In Germany, more than 1500 medicinal

species belonging to 200 families have been therapeutically investigated and used in

different pharmaceutical products on industrial scale. The worth of plant derived

medicine in the market was 1.5 billion Euros. Currently, Poland, Germany and Bulgaria

are the main exporters of herbal medicine (Mensah et al., 2008).

6

The trade of healing plants and its extracts in South Africa is common and more

than 500 medicinal plant species are used on commercial scale. In Russia the sale of

phytomedicine in the market reached to an amount of 35.8 billion Russian rubles in

2014 (Shikov et al., 2012). Brazil contain nearly 55,000 indigenous species among

which 1200 are recognized as medicinal plants, while there are thousands of

undocumented species, utilized by the native people for healing purposes (Fabricant

and Farnsworth, 2001). The total sale of plant derived products in Brazil is 508 million

Brazilian reals (BRL) in 2014. According to a survey carried out by WHO, the worth of

herbal products in the current global market is 61 billion US dollar and it is estimated

that this value will exceed 5 trillion US dollar by 2050 (Venessa, 2015).

It has been calculated that roughly 50% of medicines used in clinical practices,

are originated from medicinal plants or its derivatives, because of having excellent

potential of therapeutic activities and less side effects (Sofowora, 1984; Cowan, 1999).

For pharmaceutical products plants are the primary source, due to certain features i-e

firstly plants manufacture a number of primary and secondary bioactive metabolites,

secondly they enable synthetic chemist to find out novel compounds or alter the

existing compounds into more valuable drugs, thus provide a platform for development

of a huge number of pharmaceuticals (Farnsworth, 1984). These bioactive compounds

or phytochemicals defend plants against various pathogens, pests or any environmental

stress. These compounds are organic in nature and can be categorized into primary and

secondary metabolite. Primary metabolite, include protein, carbohydrates, nucleic acid

and lipids which are fundamental bio molecules, necessary for development and

growth. Secondary metabolites synthesized by plants are flavonoids, alkaloids,

saponins, steroids, terpeniods, tannins, glycosides and volatile oils etc. These organic

compounds are pharmacologically active and can be employed as a medicine or drug

against different diseases. Among secondary metabolites, alkaloids have analgesic,

diuretic, anti-malarial, antispasmodic properties, terpeniods have anthelmintic, anti-

inflammatory, anti-malarial, anticancer, antiviral and antibacterial activities, flavonoids

and phenols have antibacterial and anti-allergic and antioxidant properties, saponins

have antiviral and anti-inflammatory activities and glycosides have antibacterial and

antifungal properties (Chopra and Doiphode, 2002; Maurya et al., 2008).

7

Pakistan is among one of the developing country where the usage of herbal

medication is extremely universal. The demand of phyto-medicine is rising with the

passage of time due to easy accessibility and economic viability. Across the country,

approximately 84% people use herbal products for their basic health care (Qureshi et

al., 2007). Herbal medicines are prescribed by herbal practitioners locally called Tabib

or Hakim (Saeed et al., 2011). Knowledge about therapeutic uses of plants is

transmitted from one generation to next generation in the folkloric system of medicine.

So these local practitioners have no scientific background, only have information about

medicinal plants got from their predecessors or based on past experience and traditions.

Therefore to provide logical background to herbal products, it is very necessary to

study safety, quality and efficiency of medicinal plants on scientific bases. It is

essential to evaluate pharmacological screening, invitro and invivo activities and finally

the extraction and purification of bioactive molecules (Schensul et al., 2006).

The biodiversity of Pakistan is very unique due to diverse environmental and

climatic conditions. Across the country approximately 6000 species of flowering plants

are present belonging to 1572 genera. Among these species, 30% are commonly found

everywhere throughout the country while 70% are endemic species found in particular

regions and districts. Pakistan is alienated into 4 main phyto-geographic regions.

Among these region, Irano-Turanian area contain 45% therapeutic plants, Himalayan

and Sindian regions contain 10% and 9% medicinal plants whereas the smallest amount

of therapeutic plants species, which is approximately 6%, are originated in the region

near the Indian boarder (Ali and Qaiser, 2009).

In Pakistan scientific research on plants having medicinal potential is carried

out mainly at academia or institutional levels. Such research activities include in-vivo

and in-vitro assays of different extracts of medicinal plants, phytochemical screening

and separation of therapeutically active compounds. These activities are carried out to

ascertain their traditional uses on scientific bases and to search out therapeutic activities

other than the folk uses of the plant. Such research activities have confirmed that most

plants contained active ingredients which help in controlling many disorders (Shinwari,

2010).

8

In recent decades there is a great focus on obtaining chemicals from plants

having significant antimicrobial and antioxidant activities. It is well established that

certain chemicals, produced by plants, having antimicrobial properties and are lethal for

bacteria and fungi (Harborne, 1988). The plants used these chemicals for their own

defense and these compounds belong to various groups of phytochemicals for example

phenols, flavonoids and iso-flavonoids etc. It is well documented that utilization of

antioxidant obtained from plants, reduce chances of cardiac disease and cancer

(Marchioli et al., 2001). These antioxidants decrease the chances of chronic diseases by

activating the antioxidants naturally occur inside the living organisms or by giving it

directly in diet (Stanner et al., 2004).

Dysphania botrys L. synonym Chenopodium botrys L. belongs to family

Amaranthaceae, having the English names sticky goosefoot, Ambrosia, Jerusalem oak

and feather geranium. Previously D. botrys was placed in the genus Chenopodium, but

due to recent taxonomic and phylogentic investigations, it was placed in a separate

genus, Dysphania. The transferring of fragrant species of Chenopodium to a separate

genus took place nearly in 2000 (Clemants and Mosyakin, 2003). In all the

contemporary phylogentic schemes, this updated circumscription was authenticated and

as in Flora of North America North of Mexico (Mosyakin and Clemants, 2002;

Mosyakin and Clemants, 2008) and Flora of China (Zhu et al., 2003), species were

reorganized between Chenopodium and Dysphania, due to modern molecular studies

(Kadereit et al., 2010; Fuentes-Bazan et al., 2012a).

D. botrys is an annual herb rising up to 0.6 m (2ft). It flowers from July to

October which are minute, green, numerous, terminal, small, panicle, frequently

reddening in fruiting phase, hermaphrodite and pollinated by wind. Its seeds are glossy

and black in appearance, round in shape having 0.5-0.75 mm diameter. It is an aromatic

herb having distinct odor and oak-like leaves (Kletter and Krichbaum, 2001). The

immature leaves of D. botrys appear like tiny versions of those of the oak (Watts,

2007). Stem have many erect branches enclosed with stalked glandular hairs.

D. botrys is native to Asia and Europe found in Pakistan, Africa, India, Turkey,

Australia, north Europe, Cyprus, and Southand North America (Seidemann, 2005). It

grows in cultivated fields, road sides in cities and villages and found on distressed soil

patch in grasslands and semi deserts, favoring sandy loose soil (Kletter and Krichbaum,

9

2001). It is cultivated sometime as garden plant, largely for its strongly scented foliage

and flowers, used in dried flower arrangements (Small, 2006). It can tolerate high

concentration of Cu and moderate amount of Zn, Fe and Mn and can grow in soil

contaminated with heavy metals (Yousefi et al., 2011). It accumulates heavy metals in

shoots and root (Nouri et al., 2009).

Species of Chenopodium have been utilized worldwide in herbal medicine for

curing different ailments. It is well known that C. album acts as tonic, diuretic, laxative,

and anthelmintic. From the immemorial time the C. ambrosioides is used against

intestinal parasites in South America. For the catarrh and humoral asthema D. botrys

has been utilized and is recognized as a good substitute for C. ambrosioides (Yadav et

al., 2007).

D. botrys has been utilized worldwide in herbal medicine for curing different

ailments. Extract of leaves and whole plant is used for the treatment of asthma, catarrh,

influenza, headache and different digestive problems (Quattrocchi, 2012). In Iran D.

botrys is used as a tonic and anticonvulsant agent for the cure of different nervous

convulsions and as an expectorant for treating cough and asthma (Zargari, 1993). In

Northern areas of Pakistan, an infusion is prepared from the whole plant of D. botrys,

having analgesic, diuretic and laxative properties, used as a remedy for headache, liver,

stomach and digestive ailments (Bano et al., 2014). In some areas of Kohistan, young

and fresh leaves are used as an antiseptic and for wounds healing (Hazart et al., 2011).

In Indian folk medicine D. botrys is well-known as diuretic, stimulant, carminative and

antispasmodic, used as a medicine for urinary, digestive, respiratory, liver and stomach

disorders (Khare, 2007). Its extract is utilized as a flavoring agent in soup of meat,

barley and cheese in some areas of India (Maksimovic, 2005). In Himalaya region of

Kashmir, extract is prepared from its seeds having vermicidal effect, used orally for

removal of tapeworms, particularly in children, however its seeds are comparatively

more toxic as compared to other parts (Kletter and Krichbaum, 2001).

In Ladakh it is used as vermicidal, laxative and diuretic agent in folk medicine.

In province of Uttar Pardesh, juice is extracted from its leaf which is used to remove

leeches from the nostrils of cattle and other domestic animals (Jain, 1984). In some

areas of western Indian Himalaya, fresh and green leaves and branches are used as

vegetable which also have analgesic effect and very effective to treat severe headache

10

(Sing, 2012). Inner bark of D. botrys is heated in boiled water and mix with sugar to

make sugar coated tablets used for treatment of tuberculosis (Watts, 2007).

In Southern Europe it is used as a remedy for humoral asthma and catarrh and

act as best alternative of C. ambrosioides (Yadav et al., 2007). In Germany, D. botrys

was cultivated as medicinal plant and used to repel moths (Hanelt, 2001). D. botrys

have characteristic odor and in the ancient time its leaves and branches were kept in

cloths and garments to repel clothes moths (Artschwager, 1996). In Spain, special tea is

prepared from its leaves and branches called „Te de valladolid‟, which is very effective

to cure cough and digestive ailments (Pardo de Santayana, 2005). In Serbian

conventional medicine, liquid extract is prepared from dried upper parts of plant having

antidiarrheic, antispasmodic, diuretic and carminative properties. Its dried parts also

used as a spice (Maksimovic, 2005).

D. botrys have characteristic odor due to presence of sesquiterpenes and

monoterpenes (Kletter and Krichbaum, 2001). Monoterpenes include fenchone,

camphor, linalool, δ˗3˗carene, nerol, menthone, pulegone, β˗pinene, thujone and

terpinol˗4, while sesquiterpenes consist of β˗elemene, β˗eudesmol and elemol

(Buchbauer et al., 1995). Other secondary metabolites present are alkaloids, flavonoids,

phenols, terpenoids, ascaridole etc present in varying amount depending upon its origin

and locality. Essential oil extracted from D. botrys is about 0.08-2%, however it has

been shown by a number of studies that its composition and amount is not fixed,

depending upon its origin and location it yield varying amount of oil (Yadav et al.,

2010; Morteza-semnani and Babaezhad, 2007). Various studies have shown that

ascaridole is the chief compound found in its essential oil (Rustembekova et al., 1975).

Ascaridole consist of dicyclic monoterpene having peroxide functional group

(Dembitsky et al., 2008). By heating ascaridole, isomerization occure forming

isoascaridole (Tisser and Young, 2014). It has been reported that ascaridole has

anthelmintic, anti-fungal, analgesic and sedative properties (Khare, 2007). It is a

powerful inhibitor for Leishmania amazonesis, Trypanosome curzi and Plasmodium

falciparum and tumor cell lines, inhibiting its in-vitro growth (Rai and Carpinella,

2006). Compounds like eudesmane, elemane, guaiane belong to sesquiterpenes and

chenopodic acid belong to terpeniods was extracted as component of essential oil

(Khare, 2007; De Pasual., 1980).

11

Various studies carried out on contents of flavonoids in D. botrys, led to the

separation of flavonoids which include; quercetins, chrysoeriol, hispidulin, flavones, 7-

methyleupatulin, 5-methylsalvigenin, salvigenin, jaceosidin and sinensetin (Kletter and

Krichbaum, 2001). Among alkaloids, betaine is present in prominent amount in all

parts of plant and has been isolated (Rustembekova et al., 1973). Investigation has been

carried out about the presence of phytoecdysteriods, which are analogues of steroid

hormones of invertebrates (Dinan et al., 1998).

D. botrys is natural growing wild plant, traditionally used by the rural and

endemic inhabitants of different regions of Pakistan for the curing of asthma, cough,

wounds, fever, pain, liver, respiratory, urinary and gastric complaints (Khare, 2007;

Hazart et al., 2011, and Bano et al., 2014). Outstanding therapeutic properties of D.

botrys has great potential and have fascinated the scientist to explore the active

compounds having different pharmacological properties. Therapeutic utilization and

health benefits of D. botrys are mainly based on legends and have no scientific

authentication, making it a superior contender to congregate documentation including

the phytochemical contents, in-vitro and in-vivo experiments using animal models

available in the scientific studies. The current work was planned to explore the

phytochemical composition and pharmacological potential of D. botrys in order to

present scientific validation to its traditional uses.

12

Objectives of study

1) To perform phytochemical investigations of the crude extract of whole plant

2) To explore in-vitro and in-vivo toxicity plant crude extract

3) To evaluate certain in-vivo activities, in order to provide scientific validation to

its folks use

4) To find out therapeutic uses other than folk uses for the whole plant by

performing targeted pharmacological screening of crude extract

13

II. REVIEW OF LITERATURE

Al-sayed et al. (1989) examined chemical composition of essential oil of C.

botrys found in Saudi Arabia. Rich amount of oil (2% v/w) were extracted from the

plants. GC, GC-MS and spectroscopic study of gross terpenoids components were

carried out. Among sesqui-terpenes α and β˗eudesmol were present in major amount.

Antimicrobial potential of essential oil was also performed.

Bedrossian et al. (2001) analyzed the chemical composition of essential oil of

C. botrys collected from California (central Sierra Nevada range). It was observed that

90 % compounds present in the essential oil belong to oxygenated sesquiterpeniods.

Among the identified compounds present in the oil α and β˗chenopodiol, eudesma˗3, 2-

dien˗6α˗ol, botrydiol, elemole, elemole acetate, γ˗eudesmol, α and β- eudesmol were

present 36%, 9.4%, 9%, 6.5%, 5.5%, 5.4% and 3.7% respectively. Two novel alcoholic

sesquiterpene, Guaia-3 9-dien-2-ol and eudesm-3-en 4α or 6α-diol were identified

present in the oil and their structure were established from the analysis of their single

crystal X-ray diffraction pattern.

Feizbakhsh et al. (2003) studied essential oil chemical composition of C. botrys

by GC/MS, isolated from two different sites in Iran. Among all the components of the

oil extracted from plants of different locations, thirty five and thirty components were

identified representing eighty one and seventy one percent respectively. Among all the

components of these two oils juniper camphor was (16 to 25%), elemol (13 to 14%)

and α-cadinol (8 to 11%) respectively.

Maksimovic et al. (2005) examined that the upper aerial part of C. botrys

contain essential oil, which after isolation (0.43%w/w) exhibited significant role against

selected strains of fungi and bacteria, viz. Bacillus subtilis, Aspergillus niger, Klebsiella

pneumonia, Candida albicans, Staphylococcus aureus, Escherichia coli, Pseudomonas

aeruginosa, Sarcina lutea, Salmonella enteridis and Shigella flexneri.

Nahar and Sarkar (2005) isolated novel phenolic glycoside named as cheo-

albuside from the methanolic extract of seeds of C. album. Its structure was determined

by using ultra violet light, mass spectroscopy and Nuclear magnetic resonance

14

spectroscopy while antioxidant effect was estimated by using DPPH assay. The new

compound showed significant radical scavenging activity as compared to standard.

Chalabian et al. (2006) analyzed essential oil of C. botrys extracted from its

aerial parts by using n-hexane solvent extraction and hyro-distillation methods. By

using n-hexane extraction method fourteen compounds were identified which forming

91% of total oil. Among these, major compounds were chenopodiol acetate and

eudesma˗3, 12˗dien˗6-ol present in 35% and 11% respectively. Oil extracted by hydro-

distillation method contained thirty four compounds of which twenty nine were

identified. Among these eudesmol, epi-murolol and cubenol present in 15%, 11% and

10% respectively were present as the main components. The antibacterial activity of oil

against Echerichia coli, Salmonella typhi and Shigella flexneri were studied.

Emamghoreishi and Heidari-Hamedani (2006) authenticated sedative/hypnotic

effect of aqueous extract, hydro-alcoholic and essential oil of Coriandrum sativum in

male abino rats, injected intraperitoneally, before 30 min of pentobarbital injection. It

was observed that aqueous extract, hydro-alcoholic extract and essential oil prolonged

sleeping time induced by pentobarbital, at different concentrations, but main active

constituents causing sedative/hypnotic effect were found in aqueous plant extract.

Tzakou et al. (2007) extracted oil from upper branches of C. botrys isolated

from different region of Greece and studied by using GC and GC/MS. In the extracted

essential oil, 54 compounds were identified forming 94% of total oil. Majority of

compounds belong to sesquiterpenes. Among these compounds elemol acetate,

elemole, botrydiol, α˗chenopodiol, β˗eudesmol and selina˗3˗11-dien˗6α˗ol were present

16%, 14%, 11%, 9%, 7% and 6% respectively.

Ibironke and Ajiboye (2007) evaluated the effect of C. ambrosioides dried leaf

methanolic extract for anti-inflammatory and analgesic. Folrmalin and hot-plate test

were adopted for painkiller activities while for anti-inflammatory activity cotton pellet-

induced granuloma and carrageenan-induced paw edema models were used. Dose of

300-700 mg/kg showed considerable anti-nociceptive and anti-inflammatory effect.

Jabbar et al. (2007) investigated the anthelmintic assay of seed kernal

Caesalpinia crista and whole plant C. album against trichostrongylid (nematodes) of

15

sheep. Both the tested plants displayed time and dose dependent lethal effects against

worms, causing an increase in the rate of mortality and decline in hatching of eggs. C.

crista exhibited greater lethal effects as compared to C. album in eggs hatch test,

having minimum lethal concentration LC50 value of 0.134 mg/mL than LC50 value of

0.449 mg/mL respectively. In in-vivo study, significant decline in egg per gram of feces

was observed as 83.3% and 93.8% with C. album and C. crista aqueous methanolic

extract at 3 g/kg after 5 and 13 days post-treatment.

Nsimba et al. (2008) studied antioxidant potential of C. quinoa seeds different

extracts and their fractions by using FRAP (Ferric-reducing antioxidant power), DPPH

and β-carotene bleaching assays. Satisfactory antioxidant activity was shown by

different extracts of seeds and it was observed that the non-phenolic compounds, as

compared to phenolic compounds, exhibited major radical scavenging activity.

Shen et al. (2009) evaluated antidepressant potential of methanolic extract of

Bacopa monniera and its different fractions by using forced swimming test and tail

suspension test models in mice. It was observed that methanol extract, ethylene acetate

fraction and butyl alcohol fraction decreased significantly immobility time in both

forced swimming and tail suspension tests after five days consecutive oral

administration in mice, however the same doses did not show inhibitory effect in mice

against locomotor activity.

Chekem et al. (2010) examined in-vitro antifungal activities of C. ambrosioides

essential oil by broth micro dilution and well diffusion methods. The in-vitro assay of

essential oil against fungi was concentration dependent and the values of least

inhibitory concentrations ranged between 0.20 to 2 mg/ml. The antifungal effect was

assessed on stimulated rodent‟s vaginal candidiasis and it was found independent of

dose concentration.

Zapata-Sudo et al. (2010) evaluated effects of Dorstenia arifolia rhizome

methanolic extract on the activities of nervous system. Plant methanolic extract was

investigated for anticonvulsant, sedative and hypnotic effects by using pentylene

tetrazole induced convulsion, locomotor activity assessment and pentobarbital

stimulated sleeping time, respectively. Dosage of 50 mg/kg of methanolic extract

significantly declined locomotor activity and increased significantly the period of

16

pentobarbital induced sleeping time. In case of PTZ-stimulated convulsions significant

anticonvulsant activity was found in dosage dependent manner.

Dini et al. (2010) determined the amount of antioxidant compounds and their

scavenging ability of bitter and sweet seeds of C. quinoa, before and after cooking, by

using DPPH and FRAP assays. The bitter seeds exhibited greater antioxidant activity as

compared to sweet seeds. It was also observed that cooking caused considerable loss in

radical scavenging ability of bioactive compounds.

Gupta et al. (2010) examined anti-nociceptive and anti˗inflammatory effects of

methanolic extract of Murraya koenigii dried leaves in albino rats. Analgesic activity of

different doses of plant extract was determined by following formalin induced paw

licking method and hot plate method, while anti-inflammatory activity was evaluated

by following carrageenan-induced paw edema model. Plant extract displayed

significant decline in paw edema stimulated by carrageenan and percent increase in

pain and reaction time in formalin test and hot plate. The anti-nociceptive and anti-

inflammatory effects depend upon the dose of methanolic extract of plant as compared

to control and diclofenac sodium, as standard drug.

Hallal et al. (2010) investigated analgesic and antipyretic effect of C.

ambrosioides fresh leaf water extract by using acetic acid, hot plate and yeast tempted

pyrexia model in rats. Dose of 100 to 300 gm/kg of extract showed noticeable analgesic

effect in both acetic acid and hot plate models. Similarly significant inhibitory effect

was observed against pyrexia induced by yeast.

Pasko et al. (2010) determined the consequences of C. quinoa seeds in food on

certain biochemical factors and vital elements present in the serum of rodents fed with

elevated fructose. These include its effect on protein metabolism, glucose level, lipid

metabolism and certain elements like Mg, Ca, K, Na level. Fructose caused a notable

(p<0.05) decline in the level of low density lipo-protein (LDL) (42%) and alkaline

phosphatase activity (p<0.05, 21%) while amplified level of triglcerides (p<0.01, 80%).

The investigation of blood of rodents displayed that its seeds significantly declined

blood level of cholesterol (p<0.05, 26%), LDL level (p<0.008, 57%) and triglycerides

(p<0.05, 11%) as compared to untreated group. It also considerably declined blood

cholesterol (p<0.01, 10%) and level of protein (p<0.001, 16%). It also caused an

17

effective decline in the level of HDL (p<0.05, 15%) but its seed after addation stopped

its decreasing.

Amjid (2011) investigated pollens allergenicity in C. botrys and C. album.

Pollen grains were congregated from vicinity of Kandovan, Karaj and Tehran and by

mixing with phosphate-buffered saline (PH 7.4) extract were prepared. The pollen

extract of these plants were give to male guinea pigs after which tests of skin were

executed and measured based upon wheal diameter. The blood was taken directly from,

pollen extract treated; guinea pig sera and heart obtained from test samples were kept

for analysis at -20 oC. The allergenic sensitivity for C. botrys and C. album pollens

observed during skin prick test was with average wheal thickness of 4 cm and 2.5 cm

respectively. Blood analysis indicated an increase in the amount of IgE, in the number

of eosinophils and neutrophils after treatment with pollen extract as compare to control

group.

Foroghi et al. (2011) determined chemical composition of C. botrys by using

gas chromatography and spectrometry. It‟s essential oil antibacterial activities were

determined by employing agar well diffusion and agar disk methods. Macro broth tube

test was carryout to indicate MIC. The common compound found in essential oil of C.

botrys was α-eudesmol and it was studied that its oil having concentration 0.007 g/ml

caused deterrence in growth of Staphylococcus aureus and Escherchia coli.

Song et al. (2011) investigated anti-diabetic activity of C. ambrosiodes in mice

as test animal. Diabetes mellitus was induced by streptozocine (STZ) injection after

feeding for two with high-fat diet. Different concentrations of crude extract (100, 200

and 300 mg/kg) exhibited considerable hypoglycemic role as contrast to control.

Mahboubi et al. (2011) studied chemical ingredient of hydro-distilled essential

oil extracted from aerial branches of C. botrys by GC and GC-MS. By using micro

broth dilution and disc diffusion methods antimicrobial assay of essential oil was

analyzed against various kinds of microbes. Total of 43 components were identified

which form 98% of essential oil. Among these, major compounds were 2, 3-dehydro-4-

oxo-β-lonone and 7-epi-amiteol form 22.4% and 11.5% of total oil respectively. Strong

antimicrobial activity was shown by essential oil against Klebsiella pneumoniae,

Staphylococcus saprophyticus, Bacillus cerus, Streptococcus mutans, Staphylococcus

18

epidermidis, Salmonella typhimurium and Listeria monocytogenes. The growth of

Aspergillus was inhibited while Candida albican was less affected by essential oil of

plant.

Yousefei et al. (2011) inspected the consequences of different heavy metals on

development of anthers and pollen grains of C. botrys at various developmental stages.

As it is hyper accumulator for copper and fairly accumulator for manganese, iron, zinc

reported by early studies, the effects of heavy metals individually were determined on

the assembly growth and structure of anthers and pollen grains. This study was carried

out in the nearby area of copper and iron mine where the amount of heavy metals was

higher than the normal soil. C. botrys plants were grown on polluted and non-polluted

soil and were observed for structural and developmental studies. The study of

formation of anther in plants developed on polluted soil showed resemblance with that

of plants grown on non-polluted soil, however developmental stages of anther and

pollen were affected. The effects of heavy metals were stabilizing of tapetum layer,

rising in tapetm layer number, thickening of callose wall in microspores mother cell

stage, decreasing and varying size and shape of anther. Heavy metals also caused

decline in number of pollen in plants grown on heavy metals contaminated soil.

Hazrat et al. (2011) performed ethno botanical investigation of some vital

therapeutic plants in the area of Dir and Kohistan of Khyber Pakhtun Khwa (KPK),

North West province of Pakistan. During the study total of forty species belonging to

different families, including C. botrys, were found to be utilized by the local people as

folkloric medicine for curing various ailments.

Akuodor et al. (2011) studied leaf methanolic extract of Bombax buonopozense

for anti-diarrheal potential induced by castor oil, enteropooling and intestinal motility

assay in rodents. The effect of methanolic extract was dose dependent and significantly

decreased rate of feces, enteropooling and intestinal fluid motility. The LD50 value

calculated in mice for methanolic extract was larger than 5000 mg/kg.

Guo et al. (2011) investigated anticonvulsant, antidepressant and potential

bioactive components of Abelmoschus manihot ethanolic extract. It was observed that

plant extract defended mice against pentylenetetrazole induced clonic, convulsion and

mortality. Also caused decrease in immobility time in force swimming test in the tested

19

animals. The constituents detected in the ethanolic plant extract were hyperoside,

isoquercitrin, hibifolin, quercetin˗3˗о˗glucoside and quercetin.

Gesinski and Nowak (2011) studied the yield and content of amino acid in

protein obtained from the seeds of C. album and C. quinoa. It was observed that the

proportion of amino acid in the protein obtained from the seeds of C. quinoa was

greater than that of C. album. The biological value of C. quinoa protein, calculated with

essential amino acid index, was significantly greater than that of C. album.

Pal et al. (2011) studied acetone and methanol extracts of dried plant of C.

album against liver toxicity induced by paracetamol. Dosage of 200˗400 mg/kg

exhibited noteworthy hepatoprotective effect as compared to standard drug silymarin

and caused a remarkable decrease in the amount of enzymes like serum glutamate

transaminase, serum glutamate oxaloacetate. Also caused decline in the amount of

serum acid phosphatase, serum alkaline phosphatase and bilirubin. Plant methanol and

acetone extracts inhibited prominent amount of lipid perioxide and restored the normal

level of antioxidants. Hepativeprotective effect of acetone and methanol plants extracts

were also confirmed from histopathological investegations.

Nigam and Paarakh (2012) studied the anti-diarrheal potential of hydro

alcoholic extract of C. album aerial branches against diarrhea induced by castor oil

using rats as model animal. It was observed that in comparison of standard reference

drug i.e. loperamide (4 mg/kg), the potential of plant hydro alcoholic extract was dose

dependent and amount of extract from 200 to 400 mg/kg showed considerable anti-

diarrheal effect and caused notable decline in facial output and dropping frequency.

Amjid and Alizad (2012) studied the antibacterial effect of flower and leaf

ethanolic and methanolic extracts of C. album against Staphylococcus

aureus,Pseudomonas aeruginosa Escherichia coli and Bacillus cereus andby disc and

well diffusion method. It was noted that both methanol and ethanol extracts of flowers

and leaves did not show significant activity against the selected strains of bacteria.

Dwivedi and Singh (2012) evaluated antibacterial potential of Chenopodium

murale leaves methanol and aqueous extracts against important five pathogenic bacteria

i.e. Escherichia coli, Staphylococcus aureus, Pseudomonas aueruginosa, Salmonella

20

typhimurium and Proteus vulgaris by using disc diffusion method. Leaves methanol

extract showed significant degree of inhibition against S.aureus while leaves aqueous

extract ehibited strongest inhibition against P. aeruginosa.

Brend et al. (2012) determined the consequences of cooking and baking process

on ferric reducing ability of plasma antioxidant activity and total phenol and flavonoids

content of yellow and red C. quinoa plant seeds. Red quinoa seeds showed significant

antioxidant activity and notable amount of total phenols and flavonoids, as compared to

yellow quinoa plant seeds, thus might play significant role in the deterrence of chronic

ailment linked with free radicals.

Ahmad et al. (2012) inquired analgesic and spasmolytic effect of ethanol extract

of C. album and its, water, ethyl acetate, n-butanol and chloroform fractions. Crude

extract of plant showed dose dependent increase of smooth muscles relaxation, while

among the different fractions of C. album, n-butanol exhibited efficient relaxant

activity. Analgesic potential of plant crude extract was evaluated by following tail flick

method using mouse as model animal. Crude extract of 500 mg/kg dose exhibited

considerable analgesic activity.

Nayak et al. (2012) studied a range of extracts of above ground parts of C.

album for its hepativeprotective activity on liver toxicity tempted by CCl4 in rats. All

the exracts (ethyl acetate, ether and methanolic extracts) of aerial plants parts exhibited

significant hepativeprotective activity and caused considerable decrease in the elevated

amount of serum glutamic pyruic transaminase, serum glutamic oxaloacetic

transaminase, total cholesterol and bilirubin. It was observed from histopathological

study that among all the extracts, methanolic extract showed significant activity against

liver toxicity which was equivalent to silymarin used as standard drug.

Jain and Singhai (2012) studied radical inhibiting assay of leaves extract and

different fractions of C. album and their effects on liver against toxicity tempted by

carbon tetra chloride in model animals. It was observed that ethanol fraction was found

effectual than the other tested extracts and fractions against DPPH and superoxide free

radicals. The in-vivo investigation showed that ethanolic extract at concentration of

100, 200 and 400 mg/kg provide considerable protection against hazardous effects of

carbon tetra chloride and displayed significant hepative-protective activity.

21

Sousa et al. (2012) extracted Kielmeyera neglecta Saddi and C. ambrosioides

with diverse solvents like dichloromethane, hexane, ethyl acetate and ethanol. These

different extracts were evaluated for their action against brine shrimp, antimicrobial

potential and antifungal effect against Neurospora crassa cell wall. All the extracts of

C. ambrosioides exhibited inhibiting potential against brine shrimps which might be

due to cytotoxic effect against cancer cells, while that of K. neglecta only ethyl acetate

and ethanol extracts were effective. Hexane and dichloromethane extracts of C.

ambrosioides showed prominent antifungal activity against Candida krusei having

minimum inhibition concentration value of 100 μg/mL.

Gawik-Dziki et al. (2013) assessed anticancer and antioxidant effect of leaves

extract of C. quinoa through estimation of its phenolic constituents, analysis of its

phenolic compounds effects on cancer cells properties and assessment of in-vitro

antioxidant effects, bioavailability and bio accessibility. Significant amounts of rutin,

isorhamentin, kampferol, gallic acid, sinapinic and ferulic were studied in the leaf

extract. These compounds showed an inhibitory consequence on cellular ability for gap

junctional communication and proliferation of prostate cancer cell.

Song et al. (2013) explored in-vitro hypoglycemic consequences of crude

extract of C. ambrosioides, using mice as model animal. Mice were treated with

streptozocine to induce diabetes. Treatments of 100 to 300 mg/kg of plant crude extract

displayed considerable anti-diabetic effect as compared to control.

Aziz and Khan (2013) investigated sedative-hypnotic activities of Lycopus

europaeus methanolic extract on central nervous system by using thiopental induced

sleeping time and hole board methods. Diazepam was employed as standard reference

drug to which the activities of plant extract were compared. Plant extracts of 800 to

1000 mg/kg significantly showed sedative-hypnotic activities as compared to standard

drug.

Gqaza et al. (2013) analyzed and compared nutritional constituents of C. album

young body parts and mature leaves. The contents of vitamin, carbohydrates, protein,

fiber, amount of macro elements i.e. potassium, calcium, magnesium, amount of micro

elements i.e. zinc, iron, copper and amount of rare elements i.e. arsenic, chromium, tin,

in both young plant parts and mature leaves, were almost similar and no significant

22

difference was presents. It was inferred that both, young plant parts and mature leaves,

served as important source of essential dietary nutrients.

Dziki et al. (2013) studied in vitro anticancer and antioxidant study of leaves

extracts of C. quinoa. It was observed that notable amount of rutin, isorhamnetin,

kaempferol, gallic acid, sinapinic and ferulic were present in the C. quinoa leaf extract

having significant anti-carcinogenic, chemo-preventive and radical scavenging

properties.

Panday and Gupta (2014a) estimated nutritional constituents of C. album in

varying solvents (ether, methanol, petroleum, ethyl acetate, dichloromethane and

water). Nutritional investigation revealed that C. album can act as vital resource of

vigor, carbohydrate (glucose), proteins, beta˗carotene, ascorbic acid, micro amd macro

minerals i.e. iron, zinc, magnecium, calcium, potassium and soudium.

Andov et al. (2014) in Macedonia analyzed essential oil extracted from upper

branches of C. botrys isolated from five different sites and were studied using gas

chromatography (GC) and mass spectroscopy (MS). Total compounds identified were

seventy five, representing 90 to 91% of the oil. Its chemical composition has shown

that sesquiterpene components were present in rich amount i-e. (83%-87%) containing

seline-11-en-4α-ol (9.81%-13.5%), elemol acetat (9.88%-21.98%), elemol (5.57%-

9.49%) and selina˗4, 12˗dien-6α-ol (6.42%-9.71%) as major oxygen containing

sesquiterpenes. The components of oil present in lower amount were α-chenopodiol

(2.42%-5.43%), α-eudesmolacetat (3.24%-4.11%), α-chenopodiol-6-acetat (1.9%-

4.73%) and botrydiol (1.87-5.73%).

Karchegani et al. (2014) collected three ecotypes of C. botrys and analyzed its

chemical components by using GC/MS. The plants collected from Fars, Mazandaran

and Isfahan contained 19, 21, and 39 compounds respectively in upper parts which

were identified and analyzed. The plants collected from Fars contained α-pinene

(18.292%),Camphor (20.047%) and 1,8-Cineole (27.650%).The plants of Mazandaran

were consist of Camphor (10.509%), β-Myrcene (11.246%) and 1,8-Cineole (39.873%)

and the main chemical compounds present in plants of Isfahan were 1,8-Cineole

(10.823%), β-Mycene (11.250%) and Camphene (24.785%).

23

Saleem et al. (2014) studied hepativeprotective activity of C. murale aqueous

extract in mice used as model animal. It was observed that plant extract had a

significant hepativeprotective effect and caused inhibition of the paracetamol

stimulated high level of different enzymes in liver like aspartate transaminase, alanin

transaminase, alkaline phosphatase and total bilirubin. The notable hepativeprotective

activity of plant extract was confirmed from histopathalogical study and investigation

of phytoconstiuents.

Kumar et al. (2014) studied chemical composition and anthelmintic effect of C.

album leaves crude powder, methanol, diethyl ether and aqueous extract in different

concentrations. Phytochemical study of its water extract had revealed the existence of

triterpenes, sterols, resins, flavonoids, tannins, saponins and alkaloids. The anthelmintic

effect, at 0.5%, 1% and 2% concentrations of all extracts, was almost 100%. The

overall anthelmintic effects of crude and water extract was more effectual than

methanol and diethyl ether extracts.

Parkash and Patel (2014) investigated the effect of different concentration of C.

album leaves extracts against two gram positive bacteria B. subtilis and S aureus and

two gram positive bacteria E. coli and P. aeruginosa. Significant antibacterial activity

was shown by the leaves extract against all types of bacteria.

Panday and Gupta (2014b) evaluated antibacterial potential of C. album plant

extracts in dissimilar solvents i-e. ether, petroleum, methanol, ethyl acetate,

dichloromethane and water, against B. subtilis, E. coli, S. epidermidis and S. aureus.

Among all extracts, methanolic plant extract exhibited significant antibacterial effect

against all types of bacteria. The effect of extract of mixture of all solvents was found

to be significant stopping the growth of S. aureus.

Panday and Gupta (2014c) evaluated antioxidant potential of C. album, using

petroleum, dichloromethane, methanol, ether, ethyl acetate and water plant extracts in

equal proportion. Among all extracts of C. album, aqueous, methanol and petroleum

ether plant extracts exhibited significant antioxidant potential by using FRAP (ferric

reducing antioxidant assay) and ABTS assays.

24

Nedialkova et al. (2014) investigated pharmacognostic properties of C. foliosum

aerial parts and evaluated antioxidant properties of five flavonoids, isolated from plant

methanolic extract, by using DPPH and ABTS radical scavenging assays. It was

observed that plant extract had significant free radical scavenging properties and could

be used as neutraceuticals with antioxidant properties.

Yao et al. (2014) determined four saponins fractions by HPLC-MS, extracted

from the seeds of C. quinoa and investigated the anti-inflammatory effect of saponins

fractions on RAW 264.7 macrophages cells. They observed a significant decrease in

production of number of inflammatory mediators. The effect of fractions was dose

dependent, caused the inhibition of secretion of inflammatory cytokines including

tumor necrosis factor-α and interleukin-6.

Ullah and M. Ahmad (2014) determined hepativeprotective effect of ethanolic

extract of C. mural whole plant in rodents intoxicated by carbon tetrachloride. The

boosted level of serum markers like serum glutamic pyruvic transaminase, serum

glutamic oxaloacetic transaminase, alkaline phosphatase and total bilirubin induced by

carbon tetrachloride was effectively declined by plant extract, however the effect of

500 mg/kg of was highly significant (p<0.001). Histopathological investigation of liver

tissue further validated its liver defensive effects.

Moilo et al. (2014) explored the in-vitro and in-vivo anti-schistosomal effect of

extract of C. ambrosioides in mature worms. Different parts of plant (fruit, leaves, stem

and roots) were soaked and extracted using different solvents. Leaves and fruits crude

extract displayed remarkable effect (p<0.05) causing reduction in egg number. Among

the different solvent fractions, hydrated fraction of leaf and methanol fraction of fruit

caused 46% and 23% decline in the number of worms. The in-vitro observations

depicted that fruit methanol extract executed greater number of worms (adult) as

compared to leaf aqous extract. In case of fruit methanolic extract and leaf aqous

extract effect on amature worms of S. mansoni, the fruit methanolic extract showed

greater potential than leaf aqous extract. The mortality consequence of methanolic

extract of fruit and aqueous extract of were leaf were statistically comparable to

praziquantel.

25

Nowak et al. (2015) checked the antioxidant and cytotoxic abilities of bioactive

molecules taken out from various parts of four different plants belong to genus

Chenopodium. Greater amount of phenols was observed in seeds and herbaceous plants

while highest level of free polyphenols were found in seeds and roots of C. urbicum

(3.87 mg/g, 1.52 mg/g DW) respectively and extracts of C. album (3.36 mg/mg DW).

Among the different extracts analyzed, C. urbicum and C. rubrum had the highest

antioxidant activity. Significant anti-proliferative activity was shown, on the TOV-112

cell line, by the extract of seeds and herb of C. album and C. hybridum.

Parkash and Patel (2015) investigated the protective effect of extract of C.

album leaves on liver against the toxicity induced by CCl4 in rodents. They studied

SGPT, SGOT, alkaline phosphate, bilirubin and amount of protein in blood of various

treatment groups. The leaves extract of C. album caused considerable decline in the

level of mentioned serum enzymes and protein and its result was equivalent to that of

silymarin. Its defensive effect was also confirmed by histopathalogical examination of

cells of treated and control rodents.

Rahman et al. (2015) estimated the cytotoxic and anti-diarrheal activities of

Maranta arundinacea leaves methanolic extract in brine shrimp and rats respectively.

Cytotoxic activity was carried out by using lethality assay of brine shrimp while anti-

diarrheal activity was studied by following enteropooling assay, castor oil induced

assay and gastrointestinal motility assay in rodents, by using different concentration of

plant extract. Methanolic plant extract between 200 to 400 mg/kg significantly inhibited

diarrhea in all the three mentioned assays. It was also proved by cytotoxic assay that

highest does of plant extract was not harmful to mice.

Bahekar and Ranjana (2015) examined effect of ethanolic extract of leaves of

Manihot esculenta on diarrhea in rats used as model animal. The study was carried out

by using castor oil stimulated accumulation of liquid in intestine and charcoal passage

assay by using standard drug of Loperamide (5mg/kg) and atropine sulfate (5mg/kg)

respectively. Leaves ethanolic extract significantly decreased accumulation of intestinal

fluid and gastro-intestinal mobility in the test animals.

Socala et al. (2015) explored scientifically anticonvulsant, antidepressant and

anxiolytic activities of cultured Ganoderma lucidium water extract in mice. For

26

evaluation of anticonvulsant activity, timed intravenous pentylenetetrazole infusion,

maximal electroshock seizure (MES) and 6Hz psychomotor seizure models were used.

For anxiolytic and antidepressant effects elevated plus maze assay (EPM) and forced

swim test (FST) were evaluated. Plant water extract of 100 to 400 mg/kg increased

appreciably threshold values for psychomotor seizure in 6 Hz seizure test, similarly the

same amount of plant extract significantly decreased the time interval of immobility in

the forced swim test.

Calado et al. (2015) examined anti-inflammatory and anti-nociceptive effects of

hydrochloric crude extract (HCE) of C. ambrosioides in rodents by using osteoarthritis

model. In order to compare ascaridole (monoterpene), which is found in hydrochloric

crude extract, with NMDA receptors, molecular docking was carried out. After three

days of treatment, HCE caused a decline in the knee edema. The HCE5 exhibited lower

cellular infiltrate in synovium and cartilage and less intensity of allodynia from 3rd

day

and that of hyperalgesia from 7th

day up to day of treatment. The HCE5 and HEC50

treated groups showed improvement in forced walking. HCE was useful in the curing

of osteoarthritis because it reduced effectively synovial swelling and inflammation due

to severe pain.

Tang et al. (2015) conducted antioxidant assay, and characterization of betanins

and phenolics of three cultivar i-e black, white and red, of C. quinoa seeds. It was

surveyed that the amount of phenols were different in the three cultivars each having

radical scavenging properties, however the amount of phenols in the seeds of black

colored cultivar were greater having significant antioxidant activity.

Nowak et al. (2016) appraised antioxidant and cytotoxic abilities of lipid

soluble compounds of four Chenopodium species i.e. C. hybridium, C. album, C.

urbicum and Chenopodium rubrum. Cytoxic assays of the four plants extracts were

carried out against human lungs carcinoma A-549 and ovarian carcinoma TOV- 112D

and human fibroblast cell lines and it was observed that seed extract of C. hybridum

and C. album significant anti-proliferative activity on TOV-112 cell lines. The extract

of C. urbicum and C. rubrum showed significant antioxidant activity among the

extracts of other species.

27

Ajaib et al. (2016) evaluated the antibacterial potential of C. ambrosioides plant

parts different extracts against various strains of Staphylococcus aureus, Escherichia

coli, Pseudomonas aeruginosa, and Bacillus subtilis by using agar well diffusion

method. Bark petroleum ether extract displayed considerable inhibitory effect against

B. subtilis while the hydrated extract of all parts did not exhibited any lethal effect

against the selected strains.

Ozer et al. (2016) investigated the composition of phenol, enzymes inhibitory and

antioxidant assays of water and ethanol extracts of C. botrys. Amount of saponins, tannins

and flavonoids found in ethanolic were greater than present in water extract, however in

case of phenol, its amount was higher in the water extract. Plant extracts were also screened

for quantitative analysis of some selected compounds. Benzoic acid, among all compounds,

was found to be the most plentiful compound in both extracts. Antioxidant effect of

aqueous extract was greater as compared to ethanolic extract. Enzyme retarding effects of

both extract were also studied on α-glucosidase, α-amylase, tyrosinase, butyryl cholin

esterase (BChE) and Acetyl choline esterase (AChE). Its water extract displayed greater

inhibitory on Acetyl choline esterase (AChE), tyrosinase, α-amylase, and α-glucosidase

respectively. However the enzyme inhibitory activity of ethanolic extract on butyryl cholin

esterase (BChE) was greater than water extract.

Abdollahnejad et al. (2016) investigated the sedative-hypnotic potential of Aloe

vera hydrated extracts on rats. For the study of hypnotic effect of plant extract two tests

i.e. open field and loss of righting reflex, were employed. Sedative-hypnotic activities

of plant extract in the test animals were confirmed by investigation of

electroencephalographic (EEG) recordings, according to which there was a

simultaneous variation in rapid eye movement and non-rapid eye movement sleep in

similar with total prolonged sleeping time. It was confirmed from the investigation that

plant extract had sedative-hypnotic activities on electrical and functional actions of

brain.

Santiago et al. (2016a) evaluated antibacterial potential of essential oil of C.

ambrosioides against L. monocytogenes, S aureus, S. choleraesuis and E. coli by using

agar cavity diffusion method. Essential oil exhibited significant inhibitory effect against

all type of bacteria and rang of minimal inhibitory concentration was from 62.5-250 μl

ml-1

.

28

Santiago et al. (2016b) examined the antioxidant potential of C. ambrosioides

essential oil extracted through hydro-distillation technique. The radical inhibiting

consequence of essential oil was determined by checking the reduction of DPPH and by

oxidation of β-carotene-linoleic acid assay. Synthetic butylated hydroxyl-toluene was

for comparison in the same amount as that of essential oil. The antioxidant activity

exhibited by essential oil was with IC50 value equal to 455.7 μg ml-1

.

Ajaib et al. (2016) investigated antioxidant activity of C. ambrosioides bark and

fruit different extracts, by using ABTS, DPPH and metal chelating assays. It was

inferred that aqueous extract of fruit and bark displayed significant antioxidant activity

in ABTS and metal chelating assays while minimum activity in case of DPPH assay.

Antioxidant activity of petroleum ether was observed highest in case of DPPH assay.

Teware (2017) investigated antibacterial effect of C. album extract against

Proteus mirabilis and Streptococcus mutants. Ciprofloxacin was used as standard drug

to which the activity of plant extract was compared. It was observed that antibacterial

effect of plant extract was significant against both types of bacteria.

Zoufan et al. (2017) investigated heavy metal effects on the antibacterial

activity of C. murale and other plants methanolic and ethanolic extracts, growing in

polluted area near steel industry. The antibacterial activity was evaluated by using disc

diffusion method against S. aureus, B. subtilis, E. coli and P. aeruginosa. Significant

antibacterial potential was exhibited by methanolic and ethanolic extracts of C. murale

and other plants against P. aeruginosa and E. coli.

Rauf et al. (2017) analyzed phytotoxic, cytotoxic and antibacterial activities of

C. botrys, Teucrium stocksianum and Micromeria biflora found in Pakistan. Lemna

acquinoctialis based phytotoxic activity, Brine shrimp cytotoxic activity, and agar well-

diffusion method was used to carry out to evaluate phytotoxic, cytotoxic and

antibacterial activities respectively. Baccilus subtilis and Klebsiella pneumonia showed

noticeable venerability in response of aqueous and crude extract of the selected plants.

Significant cytotoxic and phytotoxic activities were exhibited by its methanolic and

aqueous extracts depending upon its concentration. Results obtained had shown that the

abovementioned plants have greater cytotoxic, pytotoxic and antibacterial effects which

29

might contribute an imperative role in research and advancement of novel therapeutic

agents.

Bojilov et al. (2017a) extracted volatile oil from the upper branches of C. botrys

by hydro-distillation and studied by GC-MS and GC-FID. C. botrys was collected from

six different location of Southern Bulgaria. Fifty three compounds were identified

found in the oil extracted from C. botrys of different regions. There was only

quantitative distinction in the chemical makeup of oil extracted from plants of different

location. Majority of identified components were belonging to oxygenated

sesquiterpenes (69% to 84%). The abundant components found in the oil were elemol

acetate (14%to 26%), elemol (10%to 18%), α-eudesmol (7%to 17%), juniper camphor

(3%to 11%), α-eudesmol acetate (5%to 6%), α-chenopodiol (4%to 6%). γ-costol was

the new compound identified for the first time, present in the essential oil of C. botrys.

Bojilv et al. (2017b) analyzed composition of flavonoids in C. botrys by using

different extraction and detection methods. For polyphenols primary extraction with

two dissimilar solvents, HPLC/PDA fingerprint profiling and Orbitrap UHPLC-MS/MS

detection were used. Study of fingerprint profile demonstrated that main components of

polyphenols were jaceosidin, hispidulin, nepetin and methoxylated flavones while

quercetin glycoside was present in least amount. For structural elucidation, ESI-MS

analysis was used for examination of fragmentation of compounds. Novel information

about methoxylated flavones fragmentation pathways was reported. Some components

like quercetin-o3-O-galactoside, rutin, eupatilin, nobiletin and nepetin were identified

for the first time in C. botrys polyphenols complex.

Ajayi et al. (2017) evaluated hydrated leaves extract of C. opulifolium for their

anti˗inflammatory and analgesic effects in model animals. It was presumed that dosage

of 100-400 mg/kg effectively reduced the intensity of pain both in the hot-plate and

writhing models, however displayed no effect on the locomotory assay in the treated

animals. The leaves extract notably (p<0.05) decrease (44.2%) paw edema stimulated

by egg albumin after 12 minute assessment time interval.

Ahmad et al. (2017) studied chemical composition, separation and recognition

of gallic acid and scopoletin in methanolic extract and subsequent fraction of n-

butanol, ethyl acetate, chloroform and petroleum ether of C. murale growing in Iraq.

30

The various spectroscopic and chromatographic analyses revealed the presence of

coumarin and galic acid. Gallic acid and scopoletin were found in the ethyl acetate

solvent fraction of C. murale.

Wu et al. (2017) explored anti-inflammatory and antioxidant assay of leaves

extract of C. quinoa by using water (cold), ethanol (95%), and methanol (50%). The

crude extracts of methanol and ethanol at concentration of 50 mg/mL exhibited

considerable scavenging effect against DPPH radicals causing 53% and 54% inhibition.

Mthanol and aqueous extracts having concentration of 10 mg/mL showed elevated

capacity of ferrous ion celating (26% and 29% respectively). Accumulation of nitric

oxide is considerably suppressed by extract of ethanol (95%) and methanol (50%) in

RAW (264.7) a cell, stimulated by LPS at concentration of 1 μg/mL and inhibitory

effect of nitric oxide synthesis was represented in dose (concentration) dependent

manner.

Oliveira et al. (2017) studied in-vitro effect of C. ambrosioides extract on cattle

ticks (Rhipicephalus microplus). Total of 125 females animals were selected and

divided into 5 groups on the basis of their weight, for the purpose to make sure that

females employed for the experiment had a uniform weight. The treatments consist of

C. ambrosioides (40 and 60%), ethanol, distilled water and amitraz (12%). It was

observed that groups treated with ethanol and distilled water, 88% and 92% of female

respectively sustained oviposition. While the effect of plant extract against the females

cattle ticks was extremely significant (p<0.001) and the dosage of 40 and 60 plant

extract, reduced the oviposition percentage up to 36 and 4 respectively.

Ren et al. (2017) studied lunasin content in C. quinoa and evaluated its radical

inhibiting and anti-inflammatory properties. The contents of lunasin in fifteen different

plant samples were found between 1.01× 10-30

g kg-10

to 4.89 × 10-30

g kg-1

plant dry

seed. There was a considerable (p<0.05) difference in the contents of lunasin among

various varieties of the similar region and the same variety of different regions. The

purified isolated lunasin showed lower radical scavenging activity against DPPH free

radicals, however exhibited strong activity against ABTS+

radicals. It also stopped the

synthesis of nitric oxide, interleukin-6 on lipo-polysaccharide induced paw 374.6

macrophages and tumor inducing factor- α by up to 44.8%, 33.5% and 39.9%

respectively.

31

Rios et al. (2017) determined the action of alcoholic aqueous crude extract of C.

ambrosioides and its hexane fraction on bacterial growth, phagocytes activation and

management of inflammatory responses by using model animals. It was noted that

hexane fraction stopped growth of bacteria and inflammatory responses by the

stimulation of phagocytes. However, alcoholic aqueous crude extract and hexane

fraction treatment caused an elevation in ex-vivo secretion of nitric oxide and hydrogen

peroxide by phagocytes. It also caused a decline in the serum level of pro-inflammatory

cytokine, showing a systematic inhibitory effect on inflammation.

Sayyedrostami et al. (2018) explored the effect of essential oils of C. botrys

leaves wound healing potential in rats. The animals were alienated randomly in to four

groups, each group containing six animals, including control group, test groups and

standard group. After treatment, the animals administrated with C. botrys caused a

reasonable (p<0.01) decline in the area of wound as compared to untreated, basal cream

and antibiotic (tetracycline) treated groups. Certain parameters like alignment of curing

tissue and formation of epithelial layer in plant administrated animals displayed a

notable increase in comparison with untreated animals. Its extract also narrowed the

surface area of wound and count of neutrophills, lymphocytes and elevated effectively

(p<0.01) the proportion of collagen and blood vessels number.

Kaur et al. (2018) determined the antibacterial consequences of diverse extracts

of C. album extracted from three unlike solvents i.e. chloroform, acetone and methanol.

Their detrimental effects were noted against B. subtilis, E. coli and L. bacillus, by

employing well diffusion method. The 100% concentration of various extracts of plant

exhibited highest potential against bacterial strains. It caused greatest inhibition activity

against E. coli and L. bacillus caused 19 mm zone of inhibition, however exhibited no

activity against B. subtilis.

32

III. MATERIAL AND METHODS

3.1 Plant collection and identification

The proposed study was conducted at Department of Pharmacy, University of

Peshawar and Department of Pharmacognocy, Faculty of Pharmacy, University of

Karachi, Pakistan. The specimen of D. botrys was collected from various parts of

District Swat, Khyber˗Pakhtunkhwa (KPK), particularly from marginal areas of river

Swat. The plant was identified by plant taxonomist Dr. Zahid Ullah, Center for Plant

Sciences and Biodiversity, University of Swat. Specimen of plant having voucher

number Swat000411 was deposited in herbarium of University of Swat for future

reference.

Fig. 3.1. Herbarium specimen of D. botrys

3.2 Extraction of plant material

Plant material was cleaned with tap water and then dried out in shade at normal

temperature for seventeen to twenty days time period. The dried plant material was

minced in a Willy mills after which, 9.2 kg of powdered plant material was obtained. It

was then mixed and extracted three times with 80% methanol for 72 hours at room

temperature with occasional shaking using maceration method. The combined filtrates

33

were concentrated at 40-45 °C using rotary vacuum evaporator (Buchi, Switzerland)

and the final residue formed was 790 g, which was the methanolic crude extract

(MCE). The crude extract of whole plant was alienated into two parts, 300 g were used

for in-vivo pharmacological activities while the remaining 490 g were added with water

(distilled) for further fractionation in various solvents i.e. n-hexane fraction (HxF),

dichloromethane fraction (DCMF) and ethyl acetate fraction (EAF) depending upon

their elevating polarity.

3.3 Fractionation procedure

Plant methanolic extract (490 g) was mixed with distilled water (1 liter) and

moved to separatory funnel for further fractionation. One liter solution of n-hexane was

poured to separatory funnel and mixed strongly. After putting the solution on a stand,

two layers were formed in which the outer layer was formed by hexane which was

alienated from the lower layer. This procedure was performed three times and the

pooled outer layers were condensed to 14 g n-hexane fraction, employing rotary

evaporator at 40 °C temperature. After partition of n-hexane fraction, the similar

method was pursued for DCM and EA solvents, yielded 31 g DCM and 34 g EA

fraction (Wagenen et al., 1993). Crude plant extract and the three solvent fractions were

utilized for phytochemical analysis and in-vitro activities.

34

Fig. 3.2. Scheme of extraction and fractionation process

35

3.4 Phytochemical investigation

3.4.1 Quantitative analysis of phytochmeicals

3.4.1.1 Stock solution

Stock solution was synthesized by dissolving 1 g of plant extract in 100 ml of

individual solvents. The prepared stock solution was utilized for testing of secondary

metabolites such as phenols, alkaloids, flavonoids, saponins, tannins and sterols by

following the standard protocols. All the chemicals used for the detection of active

biological molecules were of analytical grade.

3.4.1.2 Test for crude alkaloids

Alkaloids were detected in the plant extract by using Mayer‟s test. Dried plant

extract of 50 mg and 10 ml dilute HCl was mixed through regular stirring and was then

filtered. Two drops of Mayer‟s reagent were mixed to solution present in test tube.

Formation of white color precipitates proved alkaloid presence (positive). Mayer‟s

reagent employed in the test was of commercially grade (Tiwari et al, 2011).

3.4.1.3 Test for saponins

Saponins were detected by using Frothing test. Plant extract of 50 mg was

diluted with distilled water up to 20 mL. Then the solution was poured in graduated

cylinder and shake for 15 minutes. Formation of layer of foam indicated the presence of

saponins (Kokate, 1999).

3.4.1.4 Test for phenols

Plant samples of 500 mg were mixed with 5 ml of distal water. Aqueous filtrate

of each solvent was mixed with ferric chloride 2ml (5%) solution inside test tube.

Formation of green color designated existence of phenols (Sofowora, 1993).

3.4.1.5 Test for flavonoids

Flavonoids were detected by using Alkaline‟s reagent test. Plant extract and

fractions were mixed with solution of sodium hydroxide, formation of yellowish

36

(golden) color which becomes colorless by adding CH3COOH dilute solution, indicated

the presence of flavonoids.

3.4.1.6 Test for tannins

Ferric chloride test was used for tannins detection. 50 mg of each sample was

mixed with in 20 ml of deionized water. Then few drops of solution of ferric chloride

(0.1%) were mixed to each sample. Appearance of blue or black color confirms tannins

existence (Sofowora, 1993).

3.4.1.7 Test for sterols

Salkowski test was used for the confirmation of phytosterols. Chloroform

having volume of 2 ml was mixed with plant extract of 3 mg in test tube. The 2 ml of

pure H2SO4 was added to it. The formation of red color in the layer of chloroform after

trembling the solution for 5 minutes designated the existence of phyto-sterols (Tiwarei

et al., 2011).

3.4.2 Qualitative analysis of phytochemicals

3.4.2.1 Determination of total phenol

Total phenol contents in the methanolic extract and its consequent fractions

were examined following the protocol of Khan et al. (2008). 10 mg extract was mixed

with Folin˗Denis reagent (5 ml) and 20% sodium carbonate (10 ml). The solution was

diluted by using distal water by a factor of hundred. Solution was filtered and kept at

ambient temperature for 10 minutes. Spectronic 20 D (Milton Roy) was used for the

calculation of extract absorbance at 770 nm against blank. The total phenol

concentration in the plant crude extract and others fractions was examined by matching

with tannic acid constructed standard curve.

3.4.2.2 Determination of total saponins

Total saponins constituent in the plant extract and derived fractions were

determined by Khan et al. (2010) method. Test sample of 2 g was put in small beaker

and then 50 ml of petroleum ether was mixed and warmed on water bath for 5 minutes

37

up to 40 oC with usual shaking. The solution was cleaned and twice repeated the

process along with additional more ether (50 ml). It was further extracted on gentle

heating with methanol (5×48 ml). Then on water bath layer of methanol was

concentrated up to 25 ml after which 150 ml of dry acetone was mixed for saponins

precipitation. It was filtered and dehydrated to a constant weight at 90-100 oC by using

oven.

3.4.2.3 Determination of total flavonoids

To study the total amount of flavonoids, 10 g of CME and its fractions were

mixed with 80% methanol (10 ml). It was then filtered through filter paper i.e.

Whatman No. 42 and then the filtrate was put in crucible. Then using water bath it was

evaporated and then weighed (Boham and Kocipai, 1994).

3.4.2.4 Determination of total alkaloids

To examine the total contents of alkaloids in the plant extract and its solvent

fractions Khan et al. (2010) protocol was followed. 2 g of each sample was defatted by

dissolving in ether and warmed up to 40 oC with regular shaking for 5 minutes on water

bath. The solution was then acidified by treating with 100 ml acetic acid (20%) in

C2H5OH and kept for four hours. The final solution obtained was filtered through filter

paper and treated with NH4OH in order to increase its pH value up to 9 followed by

precipitation.

3.4.3 Proximate composition

Whole plant of D. botrys was utilized for proximate composition to investigate

moisture, crude protein, inorganic˗content (ash), ether-extract oil (lipid), fibers and

carbohydrates by employing protocol reported by AOAC (2005).

3.4.3.1 Moisture content

Grinded plant sample of 2 g was put in silica dish, formerly desiccated and

weighed. It was then heated in an oven at 100 oC for 2-3 hours. After heating, the

sample was cooled down in a desiccator and again noted its weight. The heating and

weighing of the sample was sustained until it attained a stable weight.

38

3.4.3.2 Inorganic matter

Crucible dish was cleaned and heated in oven then 2 g of plant sample was put

into crucible and weighed. By using burner flame, the sample was heated until charred

completely. Whitish gray residue was formed when the dish containing the sample was

placed approximately at 560 oC for 2 hours, in a muffle furnace.

3.4.3.3 Crude lipid

Plant sample of 3-5 g was taken in an extraction˗thimble employing Soxhlet‟s-

apparatus. It was kept in small unit and linked with a flask (200 ml). Ether was used for

extraction. After 4-5 minutes, siphoning would occur, when the process of extraction

continued for 4-5 hours. At the temperature of 105 oC the flask was dried, chilled and

again weighed at the end of process.

3.4.3.4 Dietary fiber

Plant sample of 2 g was put into a conical flask. Then mixed 200 ml (1.25%)

sulphuric acid and boiled the solution for 30 minutes, keeping the amount of solution at

steady level by pouring warm water. After boiling solution was removed, filtered and

rinsed with warm water. Then added 200 ml (1.25%) sodium hydroxide and heated for

further 30 minutes. After heating the sample was chilled and immediately filtered. The

insoluble filtrate was moved to sintered crucible, rinsed three times with diethyl ether,

and dried at 150 oC in an oven, till the time it gained a constant weight. Then for

incineration, crucible was kept at 560 oC for 1 hour in a muffle furnace.

Moisture % =Sample + dish Wt before drying sample after drying

Wt of sample x 100

crude lipid % = Wt of flask + lipid Wt of empty flask

Wt of sample x 100

39

3.4.3.5 Crude protein

The reported procedure of Kjeldahl (1983) was followed for determination of

crude protein. Crude protein in the plant sample was examined by following the

reported method of Kjedahl, having digestion system and distillation unit with titration.

The crude protein was determined by multiplying 6.25 factor with percent nitrogen of

sample.

Where N: Normality of acid, D: Dilution of sample, V: Volume after digestion of digest

and percent crude protein = 6.24 x % N

3.4.3.6 Nitrogen˗free extract

The sum of nitrogen˗free extract (carbohydrates) was estimated by subtracting

the amount of percentages of crude lipid, moisture, ash (inorganic matter), dietary

fibers and crude protein from hundred (James, 1995).

Nitrogen-free extract = 100 – (% moisture + % crude lipid + % ash + % dietary

fibers + % crude protein).

3.4.4 Minerals composition

Sample of 1 g was taken in conical flask and solution of 10 ml (67%) HNO3 was

mixed with it. The solution was kept at room temperature for 24 h after which 4 ml

(67%) of HClO4 was added to it. It was concentrated by heating on hot plate at 55 oC

until the formation of apparent solution having around 1 ml volume. After cooling the

solution double deionized/distilled water was added and then filtered through filter

paper (Whatman # 42). After that final solution of 100 ml was prepared by adding

deionized/distilled water which can be used as stock solution (Saeed et al., 2010). The

analysis of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb), zinc (Zn), iron (Fe)

and nickel (Ni) was carried by employing atomic absorption spectrophotometer

Nitrogen % = S B x N x 0.014 x D

Wt of sample x V x 100

40

(Polarized Zeiman-Hitachi 2000) while that of calcium (Ca), sodium (Na) and

potassium (K) was performed by employing flame-photometer (Jinway PFP7, UK).

The operational conditions maintained for each element on atomic absorption

spectrophotometer and flame photometer are depicted in Table 3.1. Material of

reference metals were purchased from Merck (Darmstadt, Germany). All the chemicals

used were of analytical grade.

Table 3. 1. Conditions for operation of micro and macro minerals

Micro mineral Lamp‟s current

(mA)

Wavelength (nm) Silts width (nm)

Cu 07.50 324.80 01.30

Cd 07.50 228.80 01.30

Cr 07.50 359.30 01.30

Fe 10.00 248.30 00.20

Zn 10.00 213.80 01.30

Ni 10.00 232.00 00.20

Pb 07.50 283.30 01.30

Macro˗minerals Filter-type

Ca Ca filter 422.70 00.70

Na Na filter 589.00 02.00

K K filter 766.50 00.20

41

3.5 In-vitro studies

The detail procedure of different in-vitro activities of crude methanolic extract

and solvent fractions are as follow.

3.5.1 Antimicrobial activity

3.5.2 Strains and culture media

Antibacterial activities of CME and various solvent fractions of D. botrys was

studied against gram-negative bacteria such as Klebsiella pneumonia (clinical isolate),

Peseudomonas aeruginosa (ATCC No. 9721), Escherichia coli (ATCCNo. 25922),

Xanthomonas campestris (ATCC No. 33913), Proteus vulgaris (ATCC No.6380) and

gram positive bacteria such as Staphylococcus aureus (ATCC No. 6538), Clavibacter

michiganesis(ATCC No. 10202) and Bacillus subtilis (clinical isolate) followed by

antifungal activities against Mucar pirimis (ATCC No. 52553), Aspergillus flavus

(ATCC No. 9643), Aspergillus niger (ATCC No. 6275 ), Fusarium solani (ATCC No.

11712) and Fusarium oxysporum (ATCC No. 42355). All the bacterial and fungal

strains were obtained from Department of Microbiology Quaid-e-Azam University

Islamabad, Pakistan. Strains of bacteria were cultured, and kept at 38 °C (on agar

slants) while the different colonies of fungi were inoculated and kept on PDA (potato

dextrose agar) at temperature of 28-30 °C. Bacterial and fungal stock cultures were kept

at 4 °C.

Table 3.2. Strains of bacteria for antibacterial activity

Bacterial species Gram-Type Detail

Klebsiella pneumonia Nigative Clinicle-isolate, University of Peshawar

Peseudomonas aeruginosa Nigative ATCC No. 9721

Escherichia coli Negative ATCC No. 25922

Xanthomonas campestris Negative ATCC No. 33913

Proteus vulgaris Negative ATCC No.6380

Staphylococcus aureus Positive ATCC No. 6538

Clavibacter michiganesis Positive ATCC No. 10202

Bacillus subtilis Positive Clinical isolate, University of Peshawar

42

Table 3.3. Strains of fungi used for antifungal activity

Fungal Species Detail

Mucar pirimis ATCC No. 52553

Aspergillus flavus ATCC No. 9643

Aspergillus niger ATCC No. 6275

Fusarium solani ATCC No. 11712

Fusarium oxysporum ATCC No. 42355

3.5.3 Antibacterial activity

Antibacterial potential of plant extract was studied by using the protocol of Rios

et al. (1988) with some amendment. In 1ml of dimethyl sulfoxide (DMSO), 10 mg of

whole plant extract and other solvent fractions were dissolved. Inoculation of bacterial

colonies was performed on sterile plates of agar by using sterilized cotton swabs in

order to attain homogeneous growth. Disc (sterile) was soaked with 20 μl of MCE and

other fractions, placed on the inoculated agar and incubated for 24 h at 37 oC. Cefixime

was used as reference standard antibacterial drug and zone of inhibition (ZI) was noted

in millimeters.

3.5.4 Antifungal activity

Antifungal effect of plant extract was assessed by following Mbaveng et al.

(2008) procedure. Sterilized potato dextrose (PDA) plates were inoculated with fungal

cultures. Sterile disc was soaked with 20 μl of plant extract and placed on the media. At

a temperature of 37 oC the plates were kept for 72 h and the same process was repeated

three times. Clotrimazole was as standard antifungal drug and zone of inhibition was

recorded in millimeter (mm). The process was repeated three times.

3.5.5 Phytotoxic activity

Pyhtotoxic effect of the plant MCE and various solvent fractions were

investigated according to Atta˗ur˗Rahmman (1991) method, employing L. minor plant.

Appropriate E-medium was prepared by dissolving different inorganic constituents in

100 ml distilled water having pH 5.5-6.5, which was adjusted by mixing solution of

potassium hydroxide. It was then autoclaved at 121 °C for fifteen minutes. Sample of

20 mg/ml were mixed in methanol, serving as a stock. For each concentration three

43

separate medium sized flasks were used. These flasks were inoculated with 10, 100 and

1000 μL and kept overnight in sterile environment to evaporate the methanol. An

amount of 20 ml of medium and L. minor plants (total plant used 10) each having three

fresh fronds, were put in each medium flask and were reserved at ambient temperature

in the growth room for seven days. Number of frond in each flask noted on the seventh

day and the inhibition percentage was examined by applying the formula as under.

3.5.6 Antioxidant assay

3.5.6.1 1, 1-diphenyl-2-picrylhidrazyl (DPPH)) radical scavenging activity

The inhibiting effect of MCE and various fractions of D. botrys against DPPH

free radicals were determined by employing Jain et al. (2008) procedure. Each fraction

was mixed in ethanol (2˗3 ml, 20˗100 μg/ml) and then mixed with 1 ml (0.1 mM)

solution of DPPH. Following an interval of 30 min, absorption was recorded by

spectrophotometer at 517 nm. In case of ascorbic acid, utilized as standard, the similar

method was pursued. The percent inhibiting potential was measured by applying the

formula as under.

Where, Ao represents absorbance value of control while AI represents absorbance value

of standard or samples.

3.5.6.2 ABTS (2,2˗azinobis˗3-ethylbenzothiozoline-6-sulfonic acid) radical scavenging

assay

Re et al. (1999) method was followed to explore the ABTS radical-scavenging

activity of the plant extract. The cation radical i.e ABTS+ was formed by reacting 5 ml

4.9 mM potassium persulfate (K2S2O8) with 5 ml of 14 mM solution of ABTS and kept

at normal temperature in dark for 16 hours. Prior to use, the concentration of solution

was lowered by adding ethanol in order to acquire an absorbance value of 0.700 ±

0.020 at 734 nm. The extract of plant having different concentrations was homogenized

with 1ml solution of ABTS and its absorbance was studied at wavelength of 734 nm.

For every analysis ethanol blank was run and with a gap of 6 minutes, the entire

44

measurement was performed. By incorporating 950 μl solution of ABTS with 50 μl of

Ascobic acid, standard group reaction mixture was obtained. Inhibition percentage of

ABTS was calculated by a formula as under.

% ABTS inhibiting activity = (Ao – AI) / Ao x 100

Where Ao represents the value of control and AI represents absorbance value of

standard or sample.

3.5.7 Lipoxygenase-inhibitory assay (LOX)

Yawer et al. (2007) spectrophotometric procedure was used for inhibition

activity of lipoxygenase. Reaction mixture having lipoxygenase solution in 0.1 M

phosphate buffer having pH 8 and inhibition solution (plant extract) was incubated at

25 oC for 10 minutes. By adding solution substrate, the reaction was started.

Absorbance was measured after 6 minutes at 234 nm. The standard inhibitor, Baicalein

was used in this study.

Lipoxygenase percent inhibition activity was measured as:

Inhibition (%) = (1-A/B) x 100

Where, A represents enzyme activity in the absence of inhibitor, B represents enzyme

activity along with inhibitors.

3.6 In-vivo studies

3.6.1 Acute toxicity study

Acute toxicity test for plant methanolic extract was conducted by using mice in

order to evaluate any probable toxicity. Animals were alienated into five different

groups, each consist of six mice (n=6). Group I animals administrated with saline water

(10 mg/kg, p.o), while animals of other test groups were given with increasing doses

(100, 500, 1000, 2000 mg/kg) of plant extract. Before treatment body weight of each

animal was determined. Animals of all groups were keenly monitored for any

unpleasant consequence or mortality for a period of 24 hours (Bruce, 1985).

45

3.6.2 Anti-inflammatory effect

3.6.2.1 Carrageenan induced paw edema model

The anti-inflammatory effect of plant extract was investigated by using BALB/c

mice having weight about 21-26 g. The procedure of Khan et al., (2009) was used

according to which the animals were separated into five random groups, each consist of

6 animals. Group I animals were given 10 mg/kg N/saline and considered as control,

whereas group II animals were given 5 mg/kg diclofenac sodium. The plant extract of

100 mg, 200 mg and 400 mg was injected intraperitonealy (i.p) to animals of test group

(group III-V) with increasing doses. Carrageenan (1%; 0.05 ml) was injected

subcutaneously after 30 minutes to the animals right hind paw in the sub plantar tissue.

By using Plethysmometer (Plan-labe. S.L LE-7600). anti-edematous activity was

determined for five hours (at 0, 1, 2, 3, 4, 5 hr) continuously.

Following equation was used to calculate edema % inhibition.

3.6.2.2 Xylene˗induced ear edema

Xylene˗tempted ear edema model was used by employing Okokon et al. (2010)

procedure with some modification, by using BALB/c mice having weight 21-26 g.

Animals were segregated into five random groups each consist of six animals. All the

animals were fasted for 24 hours and then were treated with 0.5 mg/kg dexamethasone,

10 ml/kg N/saline and varying doses of plant extract. After 30 minutes, inflammation

was motivated by applying xylene few drops on the interior right ear surface. Xylene

was remained there for 60 minutes, after that the animals were given light anesthesia

and then scarified. Both the ears were cut down and divided in spherical shape by a

cork borer having 7 mm diameter. After weighing the cut section, the inhibition

percentage of ear edema was calculated in reference with left ear which was without

xylene.

46

3.6.3 Analgesic effect

3.6.3.1 Formalin test

Dubuisson and Dennis (1977) procedure was used according to Tjolsen et al.

(1992) modified method. The selected animals (mice) were given diverse doses of plant

extract i.e. 100, 200 and 400 mg/kg i.p. and after an interval of 30 min, 0.05 ml of 2.5%

formaldehyde was injected into plantar surface of right hind paw of mice. The

behavioral responses were observed as mice walking (running) or can stand on treated

paw, paw partly elevated, treated paw overall elevations, biting or stinging of treated

paw. Animals treated with formalin tempted behavioral responses distinguished by two

different phases. Response of rats noted at the initial 0-10 min, was primary phase of

pain, while response noted between 15 and 30 min, was last stage of analgesia.

Diclofenac sodium (5 mg/kg subcutaneously) was utilized as standard reference drug.

3.6.3.2 Hot plate test

Hot plate or thermal nociception test was used to examine the analgesic effect

of plant extract. The animals were alienated into five random groups in which each

consist of six animals (n=6). Two hours before to initiate experiment the animals were

deprived of food. The rodents were pre-tested by placing them on hot plate (Havard

apparatus) at temperature of 55 ± 0.10 °C. All those animals were discarded displaying

latency time during pre-testing more than 15 seconds (Kang et al., 2008). Group I and

II were given 10 ml/kg N/saline and 20 mg/kg tramadol, respectively, while group III to

V were treated with different doses of plant extract. After thirty minutes the animals

were put on hot plate one by one to record latency time of nociceptive responses (paw

licking and flicking and jumping) in seconds. To avoid tissue damage 30 seconds cut-

off time was opted for each animal. Latency period at 0, 30, 90 and 120 min for each

group was noted.

The following equation was used to estimate percent analgesic potential:

47

3.6.4 Antipyretic effect

Antipyretic potential of plant extract was assessed by using yeast˗stimulated

pyrexia test previously reported by Al˗Ghamdi (2001). Pyrexia was stimulated in rats

by inserting subcutaneously 10 ml/kg 15% water solution of brewer‟s yeast (Sigma

Aldrich, France). Clinical digital thermometer was used, by inserting about 3-4 cm in

the rectum, to note the temperature of rectum of each rat before and after 24 h of yeast

injection (Hartmann, Germany). All those animals were rejected that showed raise in

body temperature lesser than 0.5 °C after twenty four hours of yeast administeration.

The selected rodents were segregated into five groups, each consist of six animals

(n=6). The group I treated with saline water (10 ml/kg), positive control group treated

with 100 mg/kg paracetamol and test groups treated with varying amount (100, 200 and

400 mg/kg) of plant extract (Vimala et al., 1998; Khan et al., 2009). Temperature of

rectum of each animal was noted at regular period of time (1h) after treatment. The

magnitude of antipyretic activity was inferred that how much it reduce the induced

pyrexia.

3.6.5 Anti-diarrheal effect

Anti-diarrheal effect of plant extract was evaluated in albino rats having 150-

200 g weight, by using Meite et al., (2009) protocol. Animals were deprived of food for

eighteen hours before treatment and were separated into five groups, each consist of six

animals (n=6). Group I was administrated with 10 ml/kg saline solution, while group II

(toxic control) was treated with 2 mg/kg loperamide. Group III, IV and V (test group)

animals were given diverse doses of plant extract i.e. 100, 200 and 400 mg/kg

respectively. Animals of all groups were administrated by oral route. After an interval

of 30 minutes of oral administration of plant extracts and standard drug, rats of all

groups were administrated orally with 2 ml of castor oil in order to stimulate diarrhea.

Different parameters like beginning of diarrhea, amount of wet feces and quantity of

fecal output was studied in the next 4 hours time period.

3.6.6 Anti-diabetic effect

For anti-diabetic activity of plant extract Kunnur et al., (2006) procedure was

used in rats. By injecting 150 mg/kg alloxan monohydrate (i.p) in selected rats, diabetes

was stimulated. The rats after treatment were maintained for seven days in an

environment in which food and water was available to it. Then the animals were fasted

48

for 10-12 hours on the 8th

day and sugar level of their blood were examined through

one touch glucometer (Lifescan, Johnson & Johnson, California, USA). Animals

having blood glucose level greater than 120 mg/dl were evaluated for further study and

were indiscriminately alienated into 5 groups each having six animals (n=6). Group I, II

and III was administrated with 100, 200 and 400 mg/kg of plant methanolic extract.

Group IV was administrated with 150 mg/kg metformin, while group V animals were

given 10 ml/kg normal saline. Samples of blood were taken from tail vein after fasting

overnight with time intervals of 0, 1, 2, 3 and 4 hours. One touch glucometer (Lifescan,

Johnson & Johnson, California, USA) was used for evaluation of blood sugar (glucose)

level.

3.6.7 Hepatoprotective effect

3.6.7.1 Carbon tetra chloride (CCl4) induced hepatotoxicity model

Hepatoprotective effect of plant extract was determined by carbon tetra chloride

(CCl4) provoked hepatotoxicity model in rats. Animals having weight 150-200 g were

alienated into 5 groups each having 6 rats. Group I animals were served orally with 5%

1 ml/kg of body weight, gum acacia suspension daily with a single dose for five

consecutive days along with 1 ml/kg liquid paraffin subcutaneously on the second and

third day. Animals of group II (toxic control) were treated with 5% 1 ml/kg gum acacia

suspension daily with a single dose for five consecutive days along with liquid paraffin

and CCl4 having 1:1 ratio on the second and third day. Group III (standard) were served

orally with 25 mg/kg silymarin. Group IV and V (test groups) animals were

administrated with different doses of plant extract (200 mg/kg and 400 mg/kg) for five

days. Group III-V animals were served with liquid paraffin and CCl4 with 1:1 ratio (2.5

ml/kg, s.c) on second and third day after silymarin and eupalitin glycoside. After 24

hours of last treatment rats were scarified. After collection and clotting of blood by

centrifugating at 3000 rpm for 16-20 minutes serum was separated in order to perform

other biochemical analysis.

3.6.7.2 Biochemical investigations

Gornall et al., (1949) procedure was used for total level of protein while

Mallory and Evenlyn. (1937) method was employed for total level of total bilirubin

(TB). For other biochemical analysis like serum glutamic oxaloacetic transaminase

(SGOT), serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase

49

(ALP), the method of Henry and Cannon (1974) and Bergemeyer (1974) was followed

respectively.

3.6.8 Sedative/hypnotic effect

Sedative/hypnotic effect of plant extract was examined by using Thiopental-

provoked sleep model in rodents. Animals were alienated into 5 groups each consist of

six animals (n=6) and diazepam was used as a reference standard drug. Group I animals

were given distilled water, group II were administrated (i.p) with 3 mg/kg diazepam,

while group III, IV and V animals were treated with different doses (50, 100 and 200

mg/kg) of methanolic plant extract. Thiopental (a sub-hypnotic dose) 60 mg/kg (i.p)

was injected after 30 minutes in all the groups. The hypnotic effect was noted for onset

of sleeping and duration of sleeping disappearance (latency) and reappearance

(duration) of righting reflex. Hypnotic sleeping time was considered to be time interval

between disappearance and reappearance of righting reflex (Williamson et al., 1996:

Herrera-Ruiz et al., 2007).

3.6.9 Anticonvulsant effect

Anticonvulsant effect of plant extract was determined by using Pentylene

tetrazole (PTZ) induced convulsion model in mice (21-26 g). Animals were segregated

into five groups, each consist of six animals (n=6). Group I animals were given saline

solution 10 ml/kg, animals of group II administrated with 2 mg/kg clonazepam (i.p.) as

a standard drug, while animals of test groups i.e. III, IV and V were administrated with

different amount of plant extract (100, 200 and 400 mg/kg). After an interval of 30

minutes of treatment, convulsion was induced in all groups animals by injecting PTZ

(90 mg/kg i.p.). After the administration of PTZ, animals belonging to all groups were

thoroughly observed for the onset of first clonus. Animal‟s mortality rate was noted and

then the obtained information were statistically evaluated and compared with standard

(Nasir et al., 2008).

3.6.10 Antidepressant effect

Antidepressant action of plant extract was carried out by using Forced

swimming test (FST) following Porsolt et al., (1978) protocol. The animals were

instigated to go ahead in a container having water up to 15 cm height and temperature

of 25 oC. Rodents were separated into five groups in which six animals per group (n=6)

were present. Group I animals (control) were treated 10 ml/kg sodium chloride

50

solution, animals of group II (standard control) treated with fluoxetine (30 mg/kg i.p.),

while animals of groups III, IV and V (test groups) were given different doses (50, 100

and 200 mg/kg) of plant extract. In over all time interval of 10 minute, time period of

immobility was observed for the final 6 minutes. Animals were assumed to be in state

of immobility when they become stationary, stop struggling and motionless floating in

water. The variation in time period of immobility was observed for each group.

3.7 Statistical analysis

Statistical significance was examined by ANOVA, followed by post˗hoc

analysis.

51

IV. RESULTS


4.1.1 Qualitative analysis of phytochemicals

D. botrys whole plant MCE and its three solvent fractions were studied for

different important phytochemical as shown in the Table 4.1. MCE displayed positive

results for alkaloids, phenols, flavonoids, saponins, tannins and sterols while in HxF

only phenols and flavonoids were detected. DCMF and EAF gave negative results for

tannins and sterols, respectively.

Table 4.1. Phytochemical of methanolic crude extractand solvents fractions of

D. botrys

Phytochemical Name of test MCE HxF DCMF EAF

Alkaloids Mayer‟s test + - + +

Phenols Ferric chloride test + + + +

Flavonoids Alkaline reagent test + + + +

Saponins Frothing test + - + +

Tanins Ferric chloride test + - - +

Sterols Salkowski test + - + -

MCE: Methanolic crude extract, HxF: Hexane fraction, DCMF: Dichloromethane

fraction and EAF: Ethyl acetate fraction.

(+) = detected, (-) = not detected

4.1.2 Quantitative analysis

4.1.2.1 Total Phenols

Reasonable amount of total phenolic compounds was observed in the crude

extract and solvent fraction of D. botrys. Highest concentration of phenols was found in

the EAF (27.4 mg/g) followed by MCE (21.4 mg/g), DCMF (19.2 mg/g) and HxF (13.5

mg/g) having the lowest phenols concentration (Figure 4.1).

52

Figure 4.1. Total phenols in methanolic crude extract and subsequent fractions

4.1.2.2 Total Alkaloids

Maximum amount of alkaloids were found in the EAF (3.14 mg/g) followed by

DCM (2.6 mg/g) and crude extract (2.2 mg/g) while least amount of alkaloids were

found in HxF (1.2 mg/g) as shown in Figure 4.2.

Figure 4.2. Total alkaloids in methanolic crude extract and subsequent

fractions

2.2

1.2

2.6

3.14

0

0.5

1

1.5

2

2.5

3

3.5

4

mg/

g o

f p

lan

t

Crude extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction

21.4

13.5

19.2

27.4

0

5

10

15

20

25

30

35

mg/

g o

f p

lan

t


53

4.1.2.3 Total saponins

Regarding sponins content the plant was found very rich and MCE proved the

richest source of saponins while hexane fraction contains least amounts of saponins.

Highest amount of saponins were recorded in the MCE (34.3 mg/g) followed by EAF,

DCMF and HxF having 28.6 mg/g, 14.3 mg/g and 11.8 mg/g respectively (Figure 4.3).

Figure 4.3. Total saponins in methanolic crude extract and subsequent fractions

4.1.2.4 Total Flavonoids

EAF was found very rich regarding the total amount of flavonoids, having 15.5

mg/g flavonoids while the HxF had 4.7 mg/g flavonoids, proved weakest source. In

MCE and EAF 12.4 mg/g and 11.2 mg/g flavonoids were found, respectively (Fig 4.4).

34.3

11.8

14.3

28.6

0

5

10

15

20

25

30

35

40

45

mg/

g o

f p

lan

t


54

Figure 4.4. Total flavonoids in methanolic crude extract and subsequent

fractions

4.1.3 Proximate composition

The proximate composition analysis includes moisture, ash, protein, fiber, fat

and carbohydrate of whole plant of D. botrys as presented in Table 4.2. It was observed

that the plant contain reasonable amount of life basic essential nutrients like nitrogen-

free extract (carbohydrates) (38.45%), protein (30.26%), fats (3.68%) while crude

fibers (1.43%) were present in the least amount. The content of moisture was (7.45%)

while the amount of ash which represents the inorganic matter was (18.73%).

Table 4.2. Proximate composition (%) of D. botrys whole plant

Content Percent composition

Moisture 7.45±0.32

Ash 18.73±0.21

Fats 3.68±0.45

Protein 30.2 ±0.72

Fiber 1.43±0.53

Carbohydrates 38.4 ±0.83

All the values were taken as mean and standard error for each replicate (n=3)

12.4

4.7

11.2

15.5

0

2

4

6

8

10

12

14

16

18

20

mg/

g o

f p

lan

t


55

4.1.4 Mineral analysis

Mineral composition analysis of D. botrys plant showed reasonable amount of

macronutrients like Ca, K and Na, having 3268 μg/g, 2673 μg/g and 591 μg/g

concentrations respectively. Our results also indicated that it is a good source of Fe and

Zn having 223 μg/g and 46.7 μg/g respectively. All the tested metals were present

within the permissible limit and no Cr and Cd were detected (Table 4.3).

Table 4.3. Mineral composition of whole plant of D. botrys

Whole plant powder Concentration (μg/g)

Ca 3268±0.53

K 2873±0.71

Na 591±0.23

Fe 223±0.46

Zn 46.7±0.32

Cu 8.3±0.48

Ni 1.2±0.16

Pb 0.4±0.13

Cr ND

Cd ND


4.2 In-vitro activities

4.2.1 Antibacterial activity

Antibacterial potential of D. botrys extract and its consequent fractions were

evaluated as depicted in Table 4.4. Maximum antibacterial effect was shown by MCE

and EAF followed by DCMF while HxF displayed least antibacterial activities. The

range of inhibition was compared with standard drug cefixime which is used as broad

spectrum antibiotic. CME of plant exhibited effective antibacterial activity against X.

campestris and E. coli causing 12.6 ± 0.54 and 10.7 ± 0.43 mm zone of inhibition

respectively. Among solvent fractions EAF proved the most effective with maximum

antibacterial potential efficiently inhibited the growth of P. aerugonosa and P. vulgaris,

causing 20.6 ± 0.53 mm and 9.8 ± 0.63 mm zone of inhibition respectively. HxF

demonstrated least antibacterial ability against all the used strains of bacteria.

56

Table 4.4. Antibacterial activity of methanolic crude extract and solvent

fractions of D. botrys

Bacterial strain MCE

(mm)

HxF

(mm)

DCMF

(mm)

EAF

(mm)

*Standard

(mm)

C. michiganesis 9.7±0.15 6.4±0.61 8.5±0.32 8.7±0.42 13.8±0.04

B. subtilis 11.8±0.26 7.3±0.24 13.7±0.06 17.4±0.34 22.6±0.15

P. Aerugonosa 15.3±0.18 14.2±0.52 17.5±0.13 20.6±0.53 24.7±0.32

K. pneumonia 13.6±0.41 5.8±0.03 8.7±0.51 14.3±0.31 26.3±0.12

S. aureus 10.5±0.31 10.3±0.43 8.6±0.63 13.8±0.24 18.5±0.21

E. coli 10.7±0.43 5.3±0.51 9.7±0.32 7.5±0.41 13.8±0.13

P. vulgaris 6.3±0.14 7.2±0.31 8.5±0.52 9.8±0.63 12.5±0.01

X. campestris 12.6±0.54 6.8±0.42 10.4±0.61 7.4±0.71 14.6±0.13


fraction and EAF: Ethyl acetate fraction


*Standard: Cefixime

Figure 4.5. Antibacterial activity of Ethyl acetate fraction and crude extract of

D. botrys against (a) P. aerugonosa (b) and X. campestris

4.2.2 Antifungal activity

Anti-fungal activity of plant extract and consequent fractions were assessed as

represented in the Table 4.5. MCE exhibited highest antifungal activity against F.

oxysporum followed by EAF, causing 19.3 ± 0.41 mm and 18.4 ± 0.33 mm zone of

inhibition, respectively. EAF considerably stopped the growth of F. solani causing

maximum zone of inhibition i.e. 12.5 ± 0.53 mm as compared to other solvents. Zone

of inhibition was compared with reference drug clotrimazole, a standard antifungal

57

drug. The growth inhibiting effect of MCE and other fractions were low to moderate

against other fungal strains, however, HxF showed no effect on the growth of A. flavus

and A. niger.

Table 4.5. Antifungal activity of methanolic crude extract and solvent fractions

of D. botrys

Fungal strains MCE

(mm)

HxF (mm) DCMF

(mm)

EAF

(mm)

*Standard

(mm)

A.flavus 3.7±0.35 0.00±0.00 3.2±0.71 7.3±0.42 15.6±0.14

A. niger 4.2±0.13 0.00±0.00 3.5±0.63 6.8±0.35 18.3±0.41

M.piriformis 13.5±0.21 7.8±0.41 5.3±0.32 12.4±0.02 22.4±0.23

F. solani 9.5±0.51 6.2±0.18 6.4±0.12 12.5±0.53 14.7±0.36

F. oxysporum 19.3±0.41 10.8±0.13 13.6±0.71 18.4±0.33 24.8±0.15




*Standard: Clotrimazol

Figure 4.6. Antifungal activity of crude extract and Ethyl acetate of D. botrys

against (a) F. oxysporum and (b) F. solani


Plant methanolic crude extract and subsequent fractions were evaluated against

Limna minor inorder to investigate its synergetic or antagonistic effect on its

development as depicted in Table 4.6. The growth inhibiting effect of plant extract and

various fractions were dose dependent and maximum inhibition of growth was

58

performed by high dose (1000 μg/ml) of plant sample as compared to low dose (10

μg/ml). MCE of D. botrys displayed highest inhibiting effect on the growth of L. minor

at diverse concentrations i.e. 10 μg/ml, 100 μg/ml and 100 μg/ml caused 26%, 50% and

70% inhibition, respectively, HxF showed lowest retarding effect i.e. 10 μg/ml, 100

μg/ml and 1000 μg/ml caused 13%, 16% and 26%, respectively. Phytotoxic effect of

DCMF and EAF was moderate.

Table 4.6. Phytotoxic activity of methanolic crude extract and solvent fractions

of D. botrys.

Sample Total fronds

No.

Sample

concentration

(μg/ml)

Survived

fronds No.

Died

fronds No.

%

Phytotoxic

effect

MCE

30 10 22 8 26

30 100 15 15 50

30 1000 9 21 70

HxF

30 10 26 4 13

30 100 25 5 16

30 1000 22 8 26

DCMF

30 10 23 7 23

30 100 18 12 40

30 1000 15 15 50

EAF

30 10 24 6 20

30 100 22 8 26

30 1000 18 12 40



4.2.4 Antioxidant activity

4.2.4.1 DPPH radical scavenging activity

The DPPH radical scavenging activity of D. botrys crude extract and subsequent

fractions were investigated as shown in the Table 4.7. The EAF at a concentration of

500 μg/ml showed highest scavenging activity (57.46 ± 0.49%) followed by MCE and

HxF (49.83 ± 0.92% and 41.63 ± 0.71%) respectively. DCMF exhibited the least

scavenging activity (37.52 ± 0.47) at all the tested concentration. The DPPH percent

scavenging activity of all the tested samples was as EAF>MCE>HxF>DCMF.

59

Table 4.7. DPPH radicals scavenging activity of methanolic crude extract and

solvent fractions of D. botrys

Solution % Scavenging effect Standard

Conc.

(μg/ml)

MCE HxF DCMF EAF Ascorbic acid

20 3.62±0.48 2.14±0.31 1.43±0.45 4.75±0.62 11.35±0.34

50 12.58±0.32 7.52±0.46 5.74±0.75 15.31±0.58 26.71±0.61

100 22.81±0.57 16.21±0.82 14.51±0.61 31.53±0.73 55.83±0.76

200 35.13±0.21 27.72±0.30 26.67±0.32 42.13±0.89 71.47±0.49

500 49.83±0.92 41.63±0.71 37.52±0.47 57.17±0.49 93.42±0.24

MCE: Methanolic crude extract, HxF: n˗Hexane fraction, DCMF: Dichloromethane



4.2.4.2 ABTS radical scavenging activity

The percent ABTS free radical scavenging activity of crude extract and solvent

fraction were calculated as summarized in Table 4.8. Among the tested samples

maximum scavenging activity was exhibited by EAF (72.17 ± 0.59%) while HxF

showed least (53.76 ± 0.57%) activities against ABTS free radicals at concentration of

500 μg/ml respectively. The scavenging activity of MCE was 61.54 ± 0.34%while that

of DCMF was 58.65 ± 0.47%. The ABTS radical scavenging activity of all the tested

samples was as EAF>MCE>DCMF>HxF.

Table 4.8. ABTS radicals scavenging activity of methanolic extract and solvent

fractions of D. botrys

Solution % Scavenging effect Standard

Conc

(μg/ml) MCE HxF DCMF EAF Ascorbic

acid

20 6.12±0.47 2.42±0.56 6.31±0.32 9.45±0.28 13.61±0.41

50 13.2±0.92 8.37±0.43 10.23±0.61 15.63±0.37 19.76±0.73

100 21.65±0.53 15.23±0.76 17.13±0.74 27.96±0.68 41.53±0.53

200 36.78±0.75 24.81±0.39 31.89±0.83 41.53±0.32 57.65±0.89

500 61.54±0.34 53.76±0.57 58.65±0.47 72.46±0.59 94.76±0.47




60

4.2.5 Lipoxygenase-inhibitory assay

The lipoxygenase-antagonistic potential of crude extract and different fractions

at a concentration of 100 mg/mL is summarized in the Table 4.8. The results indicated

that maximum retarding effect was shown by EAF causing 64% inhibition followed by

DCMF, inhibiting the activity of lipoxygenase enzymes by 58%. HxF displayed least

inhibiting effect and decreased its activity only 22%. The effect of MCE was moderate

reducing the activity of lipoxygenase enzymes by 43%.

Table 4.9. Lipoxygenase-inhibitory assay of methanolic crude extract and

solvent fraction of D. botrys

Sample (100 mg/mL) % Inhibition

MCE 43±0.84

HxF 22±0.21

DCMF 58±0.48

EAF 64±0.16

Baicalein 87±0.31




4.3 In-vivo pharmacological activities

4.3.1 Acute toxicity

Plant methanolic extracts of different concentrations were analyzed for acute

toxicity using model animals. It was observed that plant extract up to amount of 2000

mg/kg showed no lethal consequences on tested animals and all the animals remained

alive after 24 hrs of evaluation time (Table 4.10).

Table 4.10. In-vivo acute toxicity of methanolic crude extract of D. botrys

Treatment Dose

(mg/kg) No. of survived

animals (24 hrs)

No. of deceased

animals Dead (24

hrs)

Plant crude extract

100 All Nil

500 All Nil

1000 All Nil

2000 All Nil

Normal saline 10 All Nil

61

4.3.2 Anti-inflammatory activity

4.3.2.1 Carrageenan˗induced paw edema model

Our results indicated marked anti-inflammatory effect of methanolic extract of

D. botrys at all the tested doses in different assessment time intervals, against paw

edema induced by carrageenan in mice. The results of plant extract are illustrated and

compared with standard drug and control in Table 4.11a,b. Injection of carrageenan in

paw induced inflammation which enhanced slowly, getting highest size at 5th

hour of

injection. D. botrys extract at dose of 100 mg/kg exhibited anti-inflammatory activity

that became significant (p<0.05) at the last phase (after 5h) of inflammation. Anti-

inflammatory activity of 200 mg/kg and 400 mg/kg of plant extract was significant

(p<0.05) in both early and last phase of inflammation, and was comparable to that of

diclofenac sodium, used as standard drug. The overall anti-inflammatory activity of

plant extract was in concentration dependent manner and was more efficient in the last

phase as compared to early phase of inflammation.

Table 4.11a. Anti˗inflammatory activity of methanolic crude extract of D. botrys

on carrageenan provoked mice paw edema

Treatment Dose

(mg/kg)

Increase in paw volume (mm)

Early phase

(3 hours)

Late Phase

(5 hours)

Saline 10 5.73±1.09 5.81±1.21

Plant extract

100 4.89±1.27 4.63±1.89*

200 4.27±0.92* 3.62±1.09*

400 3.01±0.86* 2.97±0.98*

Diclofenac sodium 5 2.84±0.71** 2.41±0.67**

Values were taken as the mean and standard error from 6 replicates for each group.

*p<0.05, **p<0.01. Standard and every test group were compared with toxic group.

One-way ANOVA followed by Dunnet‟s test.

62

Table 4.11b. Percent inhibition of carrageenan-induced paw edema by

methanolic crude extract of D. botrys

Treatment Dose (mg/ kg)

% inhibitions of paw edema

Early phase

(3 hours)

Late Phase

(5 hours)

Saline 10 - -

Plant extract

100 14.65 20.31

200 25.47 37.69

400 47.46 48.88

Diclofenac sodium 5 50.43 58.51

4.3.2.2 Xylene-induced ear edema

All doses of D. botrys crude extract displayed considerable anti-edematous

effect in comparison to control as depicted in the Table 4.12a,b. Topical application of

plant crude extract (100, 200 and 400 mg/kg) inhibit significantly xylene-tempted ear

inflammation (p<0.05 and p<0.01). Its effect was dose dependent and greater action

was shown by amount of 400 mg/kg of plant extract. However, dexamethasone, used as

reference drug exhibited marked activity against inflammation of ear.

Table 4.12a. Anti˗inflammatory effect of methanolic crude extract of D. botrys on

xylene˗induced ear edema in mice

Treatment Dose (mg/kg) 15 min 60 min

Difference (mg) Difference (mg)

Saline 10 35.25±1.42 36.65±1.72

Plant extract

100 20.16±1.78* 14.42±1.73*

200 17.31±1.37* 13.39±1.16**

400 13.54±2.51* 10.72±1.09**

Dexamethasone 5 9.89±1.83** 8.24±1.61**




63

Table 4.12b. Percent inhibition of xylene-induced ear edema by methanolic crude

extract of D. botrys

Treatment Dose (mg/kg) % percent inhibition

15 min 60 min

Saline 10 - -

Plant extract

100 42.4 59.3

200 50.7 63.7

400 61.5 73.2

Dexamethasone 5 71.2 77.3

4.3.3 Analgesic activity

4.3.3.1 Formalin test

Plant extract of D. botrys displayed strong analgesic potential and the pain

induced by formalin was strongly attenuated (p<0.05) by the various dosages of plant,

however the analgesic effect of 400 mg/kg was extremely significant (p<0.01) and

comparable to that of standard drug. There was no major difference in the analgesic

potential of plant extract in the early and late phase of analgesia, however the pain

relieving potential of the plant extract was highly pronounced in the late phase after

formalin injection in comparison to early phase (Table 4.13a,b).

Table 4.13a. Analgesic effect of methanolic crude extract of D. botrys on

formalin-induced pain in rats

Treatment Dose (mg/kg)

Score of pain severity

Early phase

(0-10) min

Late Phase

(15-30) min

Saline 10 2.8±0.2 2.8±0.3

Plant extract

100 1.6±0.1* 1.5±0.2*

200 1.1±0.1* 0.9±0.1*

400 0.6±0.1** 0.5±0.3**

Diclofenac sodium 5 0.2±0.1** 0.2±0.2**




64

Table 4.13b. Percent inhibition of formalin-induced pain by methanolic crude

extract of D. botrys

Treatment Dose (mg/kg)

% inhibition

Early phase

(0-10) min

Late Phase

(15-30) min

Saline 10 - -

Plant extract

100 42.85 46

200 60.71 67

400 78.57 82.14

Diclofenac sodium 5 92.85 92.85

4.3.3.2 Hot plate test

Central analgesic potential of D. botrys extract was assessed as an elevation in

the latency time that was observed with a gap of time interval of 30 min starting from

0˗120 min, after the treatment of saline, different doses of plant extract and standard

drug, tramadol (Table 4.14). Dose of 400 mg/kg of plant extract exhibited significant

effect (p<0.05) after 30 min and 60 min (11.03 ± 0.56 and 11.98 ± 0.61, respectively)

and become more pronounced (p<0.01) after 120 min (12.97 ± 0.49). The standard

drug, tramadol, displayed highest analgesic potential (p<0.01) after 30 min of treatment

which persist till 120 min.

Table 4.14. Analgesic effect of methanolic crude extract of D. botrys on pain

induced by hot plate in mice

Traetment

Dose

(mg/kg)

Latency time of nociceptive responses (minutes)

0 30 60 90 120

Saline 10 8.24±0.23 8.49±0.19 8.68±0.33 8.70±0.31 8.77±0.49

Plant

extract

100 8.39±0.71 9.56±0.34 9.88±0.67 9.92±0.42 9.97±0.44

200 8.36±0.42 9.96±0.58 10.21±0.46 10.41±0.14 10.93±0.36

400 8.35±0.36 11.03±0.56* 11.98±0.61* 12.05±0.13* 12.97±0.49**

Tramadol 20 8.52±0.57 12.62±0.38** 15.78±0.52** 15.75±0.13** 15.63±0.34**




65

4.3.4 Antipyretic activity

The elevated body temperature induced by brewer‟s yeast was extensively declined by all the tested doses of plant extract, however the

effect of 200 and 400 mg/kg was highly significant (p<0.001) reducing the body temperature up to 37.02 oC and 36. 96

oC respectively, at the 5

th

hr of assessment time interval and was analogous to that of standard reference drug paracetamol (Table 4.15).

Table 4.15. Antipyretic effect of methanolic crude extract of D. botrys on brewer’s yeast induced pyrexia in rats

Treatment Dose

(mg/kg)

Temperature of rectum (oC)

Normal

Temperature

After 24 hrs After use of drug

1 hour 2 hours 3 hours 4 hours 5 hours

Saline 10 36.38±0.42 38.84±0.15 39.02±0.35 39.08±0.21 39.23±0.32 39.41±0.25 39.63±0.52

Plant extract

100 36.56±0.42 38.71±0.53 37.91±0.26* 37.51±0.41** 37.30±0.25** 37.27±0.61** 37.21±0.74**

200 36.87±0.63 38.80±0.83 37.62±0.41** 37.41±0.31** 37.19±0.37*** 37.14±0.42*** 37.02±0.74***

400 36.89±0.47 38.71±0.85 37.17±0.41*** 37.17±0.63*** 37.12±0.60*** 37.09±0.63*** 36.96±0.45***

Paractamol 150 36.62±0.28 38.95±0.27 37.18±0.14*** 37.03±0.37*** 36.72±0.36*** 36.45±0.43*** 36.18±0.45***

Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01, ***p<0.001. Standard and every test

group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.

66

4.3.5 Antidiarrheal activity

Our observation regarding antidiarrheal activity showed that methanolic extract

of plant have antidiarrheal effect in a dosage dependent manner and decreased the

episodes of diarrhea in rats, tempted by castor oil. Plant extract of 200 mg/kg and 400

mg/kg showed an overall vital (p<0.05 and p<0.01) antidiarrheal effects as compared to

control group. Both the doses of plant extract increased the latent period (118.25 ± 1.47

min and 241.5 ± 1.53 min) while caused a decline in total frequency of wet feces (4.13

± 0.81 and 2.84 ± 0.63) and mean weight of fecal drops (0.29 ± 0.07 g and 0.16 ± 0.05

g) upon administration of castor oil (Table 4.16).

Table 4.16. Antidiarrheal effect of crude extract of D. botrys on castor oil-

induced diarrhea in rats

Treatment Dose mg/kg Latent period (min) Total wet fecal

frequency

Mean weight of

fecal drops

Saline 10 64.27±1.29 7.83±0.74 1.3±0.12

Plant extract

100 72.41±1.79 5.89±1.03 0.43±0.09*

200 118.25±1.47* 4.13±0.81* 0.29±0.07**

400 241.5±1.53** 2.84±0.63** 0.16±0.05**

Lepromide 3 324.2±0.97** 2.19±0.94** 0.13±0.06**




4.3.6 Anti-diabetic activity

The effect of plant extract on the alloxane monohydrate induced diabetes in rats

is summarized in the Table 4.17. It was noted that the dosage of 400 mg/kg plant

extract effectively reduced elevated blood sugar level at all the assessment time

intervals however its effect was extremely significant (p<0.01) at the 3rd

and 4th

hour of

administration reducing blood sugar level up to 131.4 mg/dl and117.4 mg/dl

respectively and was equivalent to that of standard drug metformin. The other dosages

of 100 and 200 mg/kg showed considerable (p<0.05) effect in the last 2 hours of

assessment time interval.

67

Table 4.17. Anti-diabetic activity of methanolic crude extract of D. botrys on

alloxane induced diabetes in mice.

Treatment

Dose

mg/kg

(i.p)

Level of glucose in blood (mg/dl)

0 hour 1 hour 2 hour 3 hour 4 hour

Saline 10 168.7±0.73 169.0±0.54 169.6 ±0.62 169.2±0.98 169.5±0.59

Plant

extract

100 169.5±0.45 166.6±0.58 162.7±0.32 154.6±0.45* 143.3±0.87*

200 170.6±0.43 163.2±0.56 157.6±0.62* 148.4±0.41* 145.7±0.42*

400 171.2±0.51 157.2± 0.76* 148.2±0.43* 131.4±0.76** 117.4±0.43**

Metformin

150 169.9±0.41 141.4±0.53** 132.6±0.80*** 119.2±0.54*** 113.9±0.74***


*p<0.05, **p<0.01, ***p<0.001. Standard and every test group were compared with

toxic group. One-way ANOVA followed by Dunnet‟s test.

4.3.7 Hepativeprotective activity

Effect of methanolic extract of D. botrys on the elevated levels of serum

hepative specific markers like SGPT, SGOT, ALP and TB in CCl4 treated rats was

determined as shown in Table 4.18. Administration of CCl4 (2 mg/kg, s.c) considerably

(p<0.001) elevated the serum level of SGPT, SGOT, ALP and TB (63.7 ± 1.62 U/ml,

84.3 ± 1.58 U/ml, 251.8 ± 3.41 U/ml and 5.56 ± 0.22 mg/dl, respectively) in group II as

compared to control (group I). MCE of plant (400 mg/kg) caused a significant (p<0.01)

decline in the elevated level of SGPT and SGOT (31.2 ± 1.28 U/ml and 48.31 ± 1.87

U/ml) and that of ALP and TB (179.31 ± 3.41 U/ml and 3.64 ± 0.13 mg/dl) effectively

(p<0.05), however, its effect was less than silymarin, used as standard drug. While 200

mg/kg of methanolic plant extract have no prominent effect on the level of tested serum

markers.

68

Table 4.18. Hepativeprotective activity of methanolic crude extract of D. botrys

extract on CCl4 stimulated toxicity in rats

Treatment Dose

(mg/kg)

SGPT (U/ml) SGOT

(U/ml)

ALP (U/L) TB (mg/dl)

Saline Liquid

parafin

26.64±1.93 29.36±1.16 113±2.76 1.13±0.14

CCl4 2.5 63.7±1.62 84.3±1.58 251.8±3.41 5.56±0.22

Plant extract +

CCl4

200 52.82±1.72 72.9±2.48 236.1±2.89 4.57±0.15

400 31.2±1.28** 48.31±1.87** 179.31±3.41* 3.64±0.13*

Silymarin +

CCl4

25 24.47±1.89** 33.17±2.41** 147.22±2.04** 2.45±0.78**




TB: Total bilirubin, ALP: Alkaline phosphatase, SGOT: Serum glutamic oxaloacetic

and SGPT: Serum glutamic pyruvic transaminase.

4.3.8 Sedative/hypnotic activity

The synergetic effects of different doses of plant crude extracts on the thiopental

induced hypnoses were evaluated as summarized in Table 4.19. Plant extract of 100

mg/kg and 200 mg/kg significantly (p<0.05) induced an early onset of sleeping

reducing the time from 7.8 minutes to 6.41 and 5.93 minutes respectively. It also

effectively prolonged the time of sleeping from 88.80 minutes to 130.40 and 177.60

minutes respectively. The overall efficiency of plant extract was lesser than the

standard drug diazepam.

Table 4.19. Sedative/hypnotic activity of methanolic crude extract of D. botrys

on thiopental induced hypnosis

Treatment Dose

mg/k

Onset of sleeping

(m)

Sleeping duration

(m)

Saline 10 7.81±0.47 88.80±1.91

Plant extract

50 7.18±0.23 107.60±1.08

100 6.41±0.37* 130.40±1.69*

200 5.93±0.27* 145.20±1.76**

Diazepam 5 5.74±0.26* 177.60±1.44**




69

4.3.9 Anti-convulsant activity

The effect of plant extract on the convulsion induced by pentylenetetrazole was

studied as summarized in Table 4.20. Plant extract of 400 mg/kg dose notably (p<0.05)

deferred the arrival of first clonus from 5.09 to 6.99 minutes (1.90 minutes). It also

prolonged effectively (p<0.01) the duration of death from 9.72 to 19.56 minutes (9.84

minutes) and its effect was comparable to that of standard drug clonazepam. The other

doses of plant extract (100 and 200) also showed significant effect on increasing the

time of death.

Table 4.20. Anticonvulsant Effect of methanolic crude extract of D. botrys on

PTZ-induced convulsions in mice

Treatment Dose mg/kg Onset of first clonus

(min)

Time of death (min)

Saline 10 5.09±0.22 9.72±0.44

Plant extract

100 5.68±0.27 12.31±0.48*

200 6.03±0.28* 13.57±0.61*

400 6.99±0.07* 19.56±0.15*

Clonazepam 2 8.58±0.37** 23.84±0.62**




4.3.10 Antidepressant activity

The anti-depressant effects of different doses of plants extract using forced

swim test are summarized in Table 4.21. The result showed that amongst the variuos

tested doses of plant extract only 200 mg/kg of plant extract was effective and exhibited

considerable (p<0.05) anti-depressant effect decreasing the time of immobility from

193.98 to 96.78 seconds. The standard drug flouxetine exhibited highest anti-depressant

effect.

70

Table 4.21. Antidepressant activity of crude extract D. botrys on the time of

immobility in forced swim test model in rats

Treatment Dose (mg/kg) Immobility time (seconds)

Saline 10 193.98±1.35

Plant extract

50 139.8±1.52

100 145.38±1.16

200 96.78±1.42*

Fluoxetine 30 76.5±1.39**




71

V. DISCUSSION


5.1.1 Qualitative and quantitative analysis of phytochemicals

In order to study the biological assays of herbal derivatives it is necessary to

study their chemical composition. The active compounds that are naturally occurred in

the plants play very important role in their biological activities. Screening of different

secondary metabolites and their quantitative analysis in the crude plant extract and its

fraction exposed the pharmacological importance of D. botrys (Table 4.1). Crude

methanolic extract and its consequent fractions confirm presence of reasonable amount

of phenols. Their quantitative analyses revealed that maximum amount of phenols were

present in the EAF (27.4 mg/g) while HxF displayed least amount of phenol (13.5

mg/g) (Figure. 4.1). Our results regarding phenolics contents were slightly greater than

the quantity reported by Ozer et al. (2016) in the same plant which may be due to its

geographical location, soil and environmental condition. Phenolic compounds present

in the plant provide defense mechanism against reactive oxygen species, herbivores,

insects and microorganisms (Vaya et al., 1997).

The amount of alkaloids was maximum in the EAF (3.14 mg/g), however

relatively low as compared to other secondary metabolites (Figure 4.2). Alkaloids are

chemical compounds occurring naturally in nearly 20% of vascular plant species, most

commonly in herbaceous dicot plants and comparatively little in monocots and

gymnosperms. (Hegnauer et al., 1988). These active compounds have role in plant

protection against pathogens and herbivores. Isolated pure alkaloids and their

derivatives are employed all over the globe for medicinal purposes due to their

bactericidal, antispasmodic and analgesic properties. (Hartmann, 1991; Harborne,

1988). Among all the phytochemicals in the tested solvents, MCE exhibited highest

amount of saponins (34.3.41 mg/g) followed by EAF (28.6 mg/g) (Figure 4.3). Saponin

help in improving immunity system of animals against pathogens, decreasing blood

cholesterol level and decreasing the risk of intestinal cancer (Havsteen, 2002).

In case of total flavonoids, whole plant crude extract and its fraction had diverse

amount of total flavonoids concentration. EAF displayed highest concentration (15.5

mg/g) (Figure 4.4), which was in agreement with the observation of Panday and Gupta

72

(2014) who reported 15.68 mg/g flavonoids contents in C. album plant, belongs to the

same genus. Flavonoids have an imperative function in a broad range of biological

activities such as induction of cell apoptosis, reticence of cell-proliferation, enzyme

inhibition, antioxidant and antibacterial activities (Catoni et al., 2008; Cook and

Samman, 1996).

5.1.2 Proximate analysis:

In proximate composition analysis, the content of moisture was 7.45%, which is

very low from moisture content of C. album (84.8%) growing in Nigeria reported by

Adedapo et al. (2011), but however our results are comparable with the moisture

content of C. quinoa (11.2%) reported by Ogungbenle (2009). This low value of

moisture in the whole plant extract is extremely useful in increasing shelf life of herbal

drugs and decreases the chance of fungal and bacterial growth, which grows fast on

substances having high moisture contents as compared to low moisture containing

substances. The value of ash (18.73%) designates the existence of inorganic substances

in the plant extract and could be an excellent source of minerals (Table 4.2). Fats which

are also called triglycerides are the rich sources of energy are found in seeds and nuts of

plants and adipose tissue of animals. The fats found in animals are usually saturated

while in plants these are unsaturated and having low boiling and melting points and

very useful for human health. The value of fats contents (3.68%) was in the range as

reported by earlier literature in other members of this genus.

Reasonable amount of protein (30.26%) was observed which could contribute

the daily protein requirement and are good source of different amino acids (NRC,

1975). Proteins are the fundamental biological macro molecules having structural and

functional role inside living organisms. In our results 38.45% nitrogen free extract

(carbohydrates) was observed in the whole plant. Plants are the efficient sources of

carbohydrates and most of the carbohydrates are obtained from plant sources.

Carbohydrates are the most plentiful organic compound on earth and are the good and

efficient source of energy. Apart from energy source, carbohydrates perform vital

significant functions inside living organisms. In D. botrys the amount of crude fibers

was 1.43%, which is the main component of balance diet and help in the inhibition of

different chronic disorders such as cancer, blood pressure and diabetes. Inside intestine

fiber absorbs maximum amount of water, makes the stools softer and facilitate its flow

73

outside. Any one taking diet, having high amount of fiber, can alleviate hemorrhoids

and constipation (Bowman and Russell, 2001).

5.1.3 Mineral analysis

The amount of different metals analyzed in the whole plant of D. botrys may

account in the ethno-pharmacological use of herbal extract for the cure of many

diseases. The concentration range of different minerals in the plant may also helpful in

employing its doses within the permissible limits. Among the micro-nutrients copper is

involved in the ATP production, CO2 absorption, iron metabolism, connective tissue

formation and neurotransmitters formation and metabolism. The concentration of

copper found in the plant was 8.3 μg /g which is within the permissible limit of 10 μg/g

for plants.

Prominent amount of iron (223 μg/g) was observed in the plant which further

increased the nutritional value of the plant. Among trace elements, iron occurs most

abundantly in human body. Optimal concentration of iron is necessary for survival of

plants, animals and microbes (Arredando and Nunez, 2005). It has reported by world

health organization that the world‟s 48% pregnant females and 46% kids are have

problem of anemia due to scarcity of iron. The daily optimum intake of iron

recommended by food and nutrition board is 8 mg, 18 mg and 27 mg per day for male,

female and pregnant women, respectively (IOM, 2001). The amount of iron in the

current plant was within the permissible limits (36-241 μg /g) and is therefore proved

an excellent source of iron.

Cadmium is considered to be one of the most phytotoxic because in plants it

disturbs various biochemical and physiological processes, leading to inhibition of

growth and cell death (Sandalio et al., 2001; Guo et al., 2009; Xu et al., 2009). It is

released mainly into the soil surface from industries and agricultural practices (Wagner,

1993) and has been rank seventh amongst top twenty toxins (Yang et al., 2004). The

permissible limit of cadmium in plant recommended by WHO (2005) is 0.03 mg/kg.

Our results revealed that no cadmium was detected in whole plant of D. botrys.

Lead is considered one of hazardous metals which enter human body through

different sources such as contaminated water, fruits and vegetables. Lead has no

metabolic function in the body and its intake in high amount may cause accumulation

74

inside body causing different noxious effects such as hepatic, cardiovascular and

digestive disorders. The permissible maximum value of lead for plants is 10 μg/g. Our

results displayed that whole plant powder of D. botrys had 0.4 μg/g of lead which fall

within the recommended range, which further authenticated that the plant can be used

safely for medicinal purposes.

Nickel occurred naturally in abundant amount in the earth crust and its scarcity

rarely occurs in different types of dietary products. Intoxication of nickel may cause

lung fibrosis, skin, heart and kidney problems. Nickel is stored inside pancreas and

have important role in the secretion of insulin. Its deficiency leads to malfunctioning of

liver (Denkhaues and Salnikow, 2002). For plant its acceptable concentration range is

1.5 μg/g. It is revealed by our results that the amount of nickle was 1.2 μg/g, which is

within the allowable concentration range.

Zinc is the crucial metal performing very important functions in living

organisms such as it act as antioxidant, anti-nociceptive, bone restorative agent and

significant for discharge of hormones and cell signaling. There are more than three

hundred proteins inside living organisms which are zinc dependent (Tapiero and Tew,

2003). The amount of zinc in our plant of study was 46.7 μg/g, slightly lower than the

the permissible range which is 50 μg/g for plants (Srivastava et al., 2006).

In the current study reasonable amount of calcium (3268 μg/g) was observed

which means that this plant can be used as an alternate source of calcium. Calcium have

important role in metabolic reactions and along with phosphorous have role in the

structure of teeth, bones and soft tissue (Shapiro and Heaney, 2003). Calcium played

very prominent role in controlling of neurons and muscles activity, vasodilatation and

vasoconstriction and glandular secretion. Deficiency of calcium causes weakness of

bones and muscles present in skeleton and deformity in beating of heart. Intoxication of

calcium causes kidney stone, constipation, nausea, loss of appetite, vomiting,

convulsion, abdominal pain, and sometime coma (Gallagher et al., 2012).

Potassium is important element present in all living organisms and its normal

concentration is important for regulating action potential and cell signaling in

electrically active cells. The cellular regulatory mechanisms include balance of signal

transduction, membrane potential, secretion of insulin and other hormones, regulation

of cell volume, immune system and vascular tone (Curran, 1998). The concentration of

75

potassium found in the whole plant of D. botrys was 2873 μg/g which increased the

nutritional value of the plant.

In our study, considerable amount of the amount of sodium was found 591μg/g

in the whole plant. Sodium is very essential nutrient for all living organisms performs

prominent role in different metabolic reactions. Normal amount of sodium is necessary

for growth and any deficiency in its amount may disturb the whole metabolic reactions

in all living organisms. Intracellular and extracellular movement of fluids also depends

upon the normal amount of sodium and any variation in its amount may alter its

balance and distribution (Morris et al., 2008).

It can be assumed on the basis of our observation that whole plant of D. botrys

is an incredible resource of essential nutrients which are necessary for regular physio-

chemical performance of healthy body. All of the tested metals were found within the

permissible range while cadmium and chromium was not detected (Table 4.3).

Therefore, the current observation further authenticated the folkloric use of the plant for

different ailments.

5.2 In-vitro activities

Plant methanolic crude extract and derived fractions were evaluated for diverse

in-vitro activities.

5.2.1 Antimicrobial activity

In recent decades, the irrational utilization of antimicrobial drugs has been

increased which lead to the growth and propagation of multi˗drug challenging types of

pathogenic microbes (WHO, 2001; Aibinue et al., 2003). Furthermore, there is an

increase in the rate of morbidity and mortality due to high cost and un˗availability of

latest antimicrobial drugs (Williams, 2000). So there is a great need to focus on

obtaining compounds from plants having significant antimicrobial activities. It is well

established that certain chemicals, produced by plants for their own defense, have

antimicrobial properties and are lethal for bacteria and fungi (Harborne, 1988). Thus

herbal crude extracts, various fractions and the extracted compounds offer efficient

source for the synthesis of antimicrobial drugs (Maloo et al., 2014). In this connection,

methanolic plant extract and its various fractions were screened for their antibacterial

and anti-fungal potential against different strains of bacteria and fungi.

76

Our observations indicate that MCE and EAF were more effective than DCMF

and HxF, in hampering the development of bacterial and fungal strains (Table 4.4 and

4.5). One of the probable reasons of highest antimicrobial potential exhibited by MCE

and EAF may be the existence of active resulting metabolites like alkaloids, phenols,

flavonoids, saponins, tannins and aromatic compounds (Bonjar et al., 2004). Such

compounds have the potential to restrain the growth of many human pathogenic

bacteria and fungi by attaching to proteins present at their surface, breaching peptide

bonds and altering their biochemical composition or by inhibiting the intake of existing

nutrient by microorganisms (Cowan, 1999).

In the present study, antibacterial and antifungal activities of crude extract and

subsequent fractions of D. botrys against pathogenic bacteria like C. michiganesis, P.

vulgaris and X. campestris and fungal strains such as A. flavus, A. niger, M. piriformis,

F. solani and F. oxysporum are described for the first time. C. michiganesis causes a

destructive disease of tomato called bacterial canker (Ark and Thompson, 1960). It

survives and lives in seeds, soil and has many alternative hosts (Thyr et al., 1975;

Moffet and Wood, 1984). Its growth was considerably hindered by MCE followed by

EAF causing 9.7 ± 0.15 and 8.7 ± 0.42 mm zone of inhibition. P. vulgaris is

widespread gram-negative bacteria commonly found in the environment and also

present inside human body as normal flora of gut. It is motile, rod shaped, non-sporing

and chemo-heterotrophic bacterium having diverse mode of propagation and

transmission (Herter and Broeck, 1911). It is considered the third most important

source of infections acquired from hospitals (Bahashwan and Shfey, 2013). Its growth

was effectively restrained by EAF and DCMF causing 9.8 ± 0.63 and 8.5 ± 0.52 mm of

zone of inhibition. X. campestris instigate citrus blight, rice blight and cabbage

infection or black rot around the globe (Britto et al., 2011). MCE exhibited highest

antibacterial activity against X. campestris followed by DCMF, causing 12.6 ± 0.54

mm and 10.4 ± 0.61 mm zone of inhibition respectively.

Fungal strains belonging to the genus of Aspergillus, have the ability to produce

toxic substances called mycotoxins which degrade food quality and therapeutic

potential of drugs extracted from plants (Gautam and Bhadauria, 2009). A. niger is

filamentous saprophytic fungus found in organic debris, earth and food products

causing stem and root rot of dracaena and sansevieria, cotton boll rot, discoloration of

dates, cashew kernel, dried prune, vanilla pods and figs (Bugno et al., 2006; Gautam

77

and Bhadauria, 2008; Bobbarala et al., 2009). While A. flavus causes infection in

maize, sorghum, wheat, rice, nuts producing a toxic substance called aflatoxins which

are mutagenic, carcinogenic and fatal fungal secondary metabolites (Zain, 2011; Hua,

2013; Kiswii et al., 2014). The growth of both these fungal strains was moderately

inhibited by EAF while HxF showed no effect on their growth. One of the probable

reasons for its resistance is that HxF did not contain sufficient amount of active

phtochemicals which retard its growth or might due to its complex structure.

F. solani is cosmopolitan in distribution and important plant pathogenic fungi

which commonly infects 111 different plants species belonging to 87 genera

(Kolattukudy and Gamble, 1995). Its growth was effectively retorted by EAF followed

by MCE. F. oxysporum is found in soil and causing different human and plant diseases

(Nelson et al., 1981). The antifungal activity of MCE and EAF against these two fungal

strains was comparable with that of standard drug which can be very useful for the

control and management of these pathogenic fungal strains. M. piriformis is a

pathogenic fungus found in soil which causes severe post-harvest losses in different

agriculture products. Fruits and vegetables such as tomatoes, pears, nectarines and

peaches which have high water content are more susceptible to it and are easily

attacked by this fungus (Moline and Kuti, 1984; Michailides and Spotts, 1990).

In conclusion, MCE and EAF exhibited considerable lethal effect against two

fungal strains which can be further exploited for the extraction of active metabolites

used for the control and management of these phyto-pathagenic fungi. However, the

overall effect of all the tested solvent against other strains was low to moderate.


In order to control weeds and to enhance crop production effectively, various

herbicides and pesticides are used, however, the severe use of synthetic chemicals

caused an increased risk of toxicity in the food chain and polluting the soil, water and

air (Roger et al., 1994, Pell et al., 1998, Aktar et al., 2009 and, Heap, 2014). Therefore

it is very necessary to search out alternative strategies for weed management, like

natural herbicides which are eco-friendly, easily biodegradable and more economical as

compared to synthetic herbicides. The phytotoxic effect of a plant extract or bioactive

constituents derived from it is very useful in assessing their herbicidal ability. L. minor

assay is an economical and simple method used for evaluating plants or their extracted

78

compounds for their inhibiting effects. L. minor is susceptible to majority of pollutants

and toxic substances, making it helpful in toxicity measurements (Ateeq˗ur˗Rehman et

al., 2009). Moreover, it has been noted that anti˗tumor substances reduce its

development while there are some compounds which enhance its growth, thus it is very

helpful for the screening of new plant development and growth inducers and fulfill the

current demand of less toxic, biodegradable natural herbicide (Lewis, 1995; Wang,

2007; Cayuela et al., 2007).

The current study was executed in order to explore and inspect the inhibiting

effect of plant extract and their various derived fractions against L. minor. Among all

the tested samples maximum phytotoxic effect was shown by MCE, causing maximum

inhibition of growth of L. minor followed by DCMF of the plant extract, showing that

active allelopathic constituents are present in maximum amount in these solvents as

compared to other fractions (Table 4.6). Some plants produce active secondary

metabolites which may act as allelochemicals for other plants, effecting the growth of

plants growing in its closed vicinity. These active compounds are released either by

exudation or by some other epidermal sceration (Patil and Magdum, 2011). Hussain et

al. (2010) studied phytotoxic effect of Roscoea nepalensis, Rumix dentatus, Rumix

hastatus, Polygonum persicaria, polygonum plebejum and Rheum australe. Ali et al.

(2009) evaluated growth inhibiting effect of Euphorbia wallichii root extract obtained

from different solvent. All of the mentioned plant extracts and their solvent fractions

showed considerable phytotoxic effect (55-100%) at higher concentration (1000 μg/

ml) as compared to low concentration (10 μg/ ml), displayed 25-60% inhibiting effect.

Our findings are in line with these previous observations, which further authenticat the

phytotoxic effect of D. botrys plant extract and solvent fractions.

5.2.3 Antioxidant activities

Free radicals which are known to cause numerous health problems have been

studied in order to avoid and control their detrimental effects. Antioxidants protect us

from negative effect of these free radicals by either scavenging free radicals or

shielding the antioxidant defense mechanisms (Umamaheswari and Chatterjee, 2008).

But most of the currently available free radicals scavengers are manmade and have

been alleged to cause harmful health effects (Barlow 1990; Sadiq et al. 2015). Due to

the unwilling side effects of artificial antioxidants there is a tendency to replace them

79

with antioxidants which occur naturally, having maximum efficiency and less negative

effects. Antioxidant based formulations of drugs are employed for the control and

prevention of numerous chronic diseases. Plants are the chief source of naturally

occurring antioxidants, produce large amount of secondary metabolites having anti-

oxidative properties.

The antioxidant potential of plant extract and solvent fraction can be evaluated

by employing various procedures but the frequently employed procedures are those to

produce free radicals and then deactivate these free radicals by substances having

antioxidant properties (Arnao et al., 2001). In the recent study, DPPH and ABTS

models were used for the assessment of radical scavenging activity. These are the

frequently and standard methods used for the evaluation of radical scavenging

potentials of plant extracts and antioxidants substances (Sanchez-Moreno et al., 1998).

In our observation EAF and MCE of the plant showed higher scavenging activities in

both models and were comparable to that of ascorbic acid, used as standard antioxidant

(Table 4.7 and 4.8). One of the probable reasons of higher scavenging activity is that

these plants extracts may contain bioactive phytochemicals that donate hydrogen to free

radicals reducing the potential damage (Jamshed et al., 2012). The phytochemical

composition of EAF and MCE also revealed that it contain maximum amount of

phenols and flavonoids which may be the other reason of their highest scavenging

activity. The overall scavenging activity of all the tested samples of plant extract was in

a dosage dependent manner and increased by increasing their concentration.

5.2.4 Lipoxygenase activity

It is reported in the literature that lipoxygenase enzymes have the ability to alter

linoleic, arachidonic and other various unsaturated fatty acid into bioactive metabolites

that have role in immune and inflammatory responses (Catalano and Procopio, 2005).

Lipoxygenases are the main enzymes causes the synthesis of leukotriens which have a

vital role in many inflammation causing ailments like asthma, arthritis, allergic

disorders and cancer (Rackova et al., 2007; Dobrian et al., 2011). The elevated levels

of leukotriens could be noted in case of psoriasis, colitis ulcerosa, asthma, rheumatoid

arthritis and allergic rhinitis (Schneide and Bucar, 2005). The synthesis of leukotriens

can be hindered through stopping lipoxygenase pathway and targeting it with inhibitors,

which can help in curing a number of human health disorders. It is suggested by

80

researchers that the inhibitors of lipoxygenase may help to plan pharmacologically and

biologically targeted curative strategies stopping lipoxygenase isoforms and their

bioactive metabolites which can be helpful in treatment of cancer (Pidgeon et al.,

2007).

The current study demonstrated considerable anti-lipoxygenase potential of D.

botrys crude extract and various solvent fractions (Table. 4.9). Among the crude extract

and solvent fractions, EAF exhibited maximum lipoxygenase activity as compared to

standard. Reactive oxygen species have a role in transmission of inflammation by

inducing the secretion of cytokines and by stimulation of enzymes such as lipoxygenase

from the inflamed tissues. Hence, plants having high antioxidant contents with high

antioxidant activity are proved to be helpful to neutralize reactions causing

inflammation. In our observation regarding the antagonistic potential against

lipoxygenase, EAF also displayed maximum activity which suggests that this fraction

must contained highest amount of active compounds, which could help in stopping the

function of lipoxygenase enzymes. The elevated lipoxgenase inhibitory activity might

also be due to considerable maximum phenolic, flavonoids, alkaloids, tannins and

essential oils (Santha et al., 1991; Manosroi et al., 2013). The phytochemical analysis

of crude extract and solvent fractions also displayed considerable amount of phenols,

flavonoids, alkaloids and saponins which might exert a combine synergetic effect,

inhibiting the function of lioxygenase enzyme.

5.3 In-vivo pharmacological activities

5.3.1 Acute toxicity

Plant crude extract of different concentrations were analyzed for acute toxicity

using model animal in order to investigate the minimum toxic limit of plant extract. It

was observed that plant extract up to dosage of 2000 mg/kg showed no toxic effect on

tested animals and all the animals remained alive after 24 hrs of evaluation time (Table

4.10). This observation confirm that the different doses of plant crude extract which are

used for different in-vivo activities were within the tolerable range and did not cause

lethal effect on the rodents used for the evaluation of these activities.

5.3.2 Anti-inflammatory activity

The anti-inflammatory effect of MCE of D. botrys was evaluated by employing

carrageenan induced paw edema model and xylene induced ear edema model in mice,

81

as model animals. Acute edema stimulated by carrageenan in mice paw is reputable

model to evaluate the anti-inflammatory potential of active natural plant constituents.

Injection of carrageenan at sub-plantar region stimulates local edema which increases

slowly. It causes maximum inflammation and almost hundred percent larger volume of

treated paw which steadily decreases with in 24 hour. The formation of carrageenan-

induced edema in mice paw is biphasic process, consist of initial or early phase and

second or late phase. The early phase (1-3 hr) is non˗phagocytic type of edema

followed by late phase, with enlarged edema development that persisted up to an

interval of 5 hrs (Rotelli et al., 2003 and Meckes et al., 2004). Different investigations

have been explained the participation of various mediators in different phases of edema

provoked by carrageenan. The initial phase of edema is attributed to the liberation of 5-

hydroxy-tryptamine, histamine, platelet activating factors, bradykinin and serotonin.

The late phase is ascribed by increased release of leukotriens and prostaglandins while

the connection between early and late phases is provided by kinins in the inflammatory

area (Nguemfo et al., 2007; Unnisa and Parveen, 2011).

It is inferred from our observations that crude extract of D. botrys displayed

considerable (p<0.05) anti˗inflammatory effect, acting effectively in a dose dependent

manner. Its effect was more pronounced and analogous to that of diclofenac sodium, in

the late phase of carrageenan induced assay of paw edema (Table 4.11a). Our

observations are in agreement with that of Vineger et al., (1969), Dirosa and

Willoughby (1971) who reported that, clinically anti-inflammatory drugs are more

efficient against the late phase of inflammation. Even though the exact mechanism of

action is yet to be established, it is possible that crude extract of D. botrys have

inhibitory effect on the synthesis of cyclo-oxigenase. Its effects seem similar to that of

non-steroidal anti-inflammatory drugs (NSAIDs) like indomethacin and diclofenac,

whose mechanism of action is stopping of cyclo-oxigenase enzyme, involved in the

production of cyclic endo-peroxides which again have role in the synthesis of

prostaglandins.

Xylene-stimulated model of ear edema is generally employed to assess the

intensity of vessels dilatation and extravasations of neurogenic inflammation. This

model is helpful in the assessment of steroidal and non-steroidal agents having anti-

inflammatory properties and has accurate predictive values in the selection of such

active substances (Kumawat et al., 2012; Sowemimo et al., 2013). Its topical

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application on the surface of ear induces vasodilatation and enhances the permeability

of vessels. It is associated with a neuro-modulator or neurotransmitter, substance P,

which is widely distributed in the central or peripheral nervous system having role in a

number of physiological processes (Junping et al., 2005). Release of this

neurotransmitter from sensory neurons induces vasodilatation and membrane

extravasations, signifying its role in neurogenic inflammation.

In our study, all the dosages of crude extract of D. botrys markedly decreased

the severity of ear edema in a dose-dependent manner and the anti-edematous activity

of 400 mg/kg was comparable to that of reference drug, dexamethasone (Table 4.12a).

The inhibition of ear inflammation indicated that crude extract of D. botrys alleviated

vasodilatation and plasma extravasations of neurogenic inflammation, which is

necessary in managing the initial phase of acute inflammation. The results of the

present study are in line with that of Ibironke and Ajiboye (2007) who evaluated the

anti-inflammatory ability of methanolic extract of C. ambrosioides (300 mg/kg) which

significantly inhibited the edematous effect, with increasing doses over time. The

present study has proved that crude extract of D. botrys have active principles

exhibiting anti-inflammatory properties, which authenticates the traditional use of this

plant as a remedy of pain and inflammation.

5.3.3 Analgesic activity

Analgesic assay of methanolic extract of D. botrys was evaluated by employing

chemical and thermal models of pain. The first test performed to investigate the

analgesic property of plant extract was formalin test (Table 4.13a). Formalin is used as

a chemical stimulant for inducing pain in animal behavioral studies, described by

Dubuisson and Dennis (1978). Formalin test is responsive to non-steroidal and mild

pain-relieving drugs. Pain induced by formalin consists of two different phases,

probably due to dissimilar types of pain mechanisms. The early phase of pain starts

immediately after formalin injection and remain for 5 minutes, due to direct stimulation

of nociceptors causing bradykinin and substance P release which further stimulate

afferent C fibers (Cui et al., 2004). The late phase initiates after 15-30 minutes of

formalin injection and remains up to 40 minutes. In this phase pain is induced due to

liberation of different pain mediators like histamine and prostaglandin (PG),

augmentation of cyclooxigenase COX and liberation of nitric oxide (Bars et al., 2001;

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Parada et al., 2001; Nakamoto et al., 2010). The biphasic mechanism of pain induction

of formalin test can be employed to expose the probable pathway involved in reducing

pain (Tjolsen et al., 1992). The action of various analgesic drugs differs in the early and

late phase of pain irritated by formalin. Certain drugs like opioids which act centrally,

exhibit good response and inhibit both phases of pain while other drugs like

acetylsalicylic acid, which act peripherally, stop prostaglandin (PG) synthesis and COX

activity, inhibit only late phase of pain (Hunskaar and Hole 1987; Shibata et al., 1989;

Yuri et al., 2004; Paschapur et al., 2009).

The analgesic potential of crude extract of D. botrys was also evaluated by hot-

plate test, a thermal˗nociception model which is considered one of the most frequent

tests used for the evaluation of centrally acting analgesics. Hot-plate induced pain in

experimental animals assesses the intricate response to acute nociceptive and non-

inflammatory input used for examining central nociceptive activity (Sabina et al.,

2009). Several drugs having peripheral analgesic activity, such as aspirin exhibits weak

activity against hot plat-induced pain. But other analgesics like morphine and ibuprofen

can reduce prostaglandin production through central inhibition of COX (cyclo-

oxygenase) or attach to specific opioid receptors in CNS, showing both peripheral and

central analgesic effects (Biorkman, 1995; Dolezal and Krsiak, 2002).

Regarding our observation of hot-plate assay, crude extract of D. botrys (400

mg/g) displayed significant analgesic result (p<0.01) after 60 min of treatment, while

the standard drug tramadol, which is similar to morphine in action (opioid agonist),

increased the threshold level of pain with in 30 min of treatment (Table 4.14). This

dissimilarity in the peak analgesic position could be elucidated by the variation in the

rate of metabolism or efficiency of active metabolites as the pain relieving potential of

tramadol is greater than crude extract of D. botrys (400 mg/g).

5.3.4 Anti-pyretic activity

Fever or pyrexia is a condition occurs due to abnormal elevation in body

temperature. It is induced by several endogenous inflammatory mediators and pyrogens

called cytokines like interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8),

tumor necroses factor-α, macrophage protein-1 and prostaglandins that are released by

activated mononuclear peripheral phagocytes and immune cells (Roth, 2006). Brewer's

yeast is commonly used exogenous pyrogen, which induces pyrexia in experimental

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animals. It binds to a specific immunological protein called lipo-polysaccharide binding

protein (LBP) (Ukwuani et al., 2012). Its binding causes the production and release of

different cytokines mentioned above which trigger arachidonic acid pathway and

finally the synthesis and liberation of prostaglandins E2 (PGE2) (Gege-adebayo et al.,

2013). Pyrexia induced by yeast is known as pathogenic fever (Aman and Patrick,

2011).

According to classical view, pyrexia is stimulated by inflammation causing

mediators (IL-1, IL-2, TNF-α etc), which are released by active peripheral mononuclear

macrophages and other immune cells (Zeisberger, 1999). These fever-provoking

cytokines are moved through definite carriers from blood to brain and enter the brain

through circum-ventricular organs, where they interact with their receptors present on

peri-vascular tissue or endothelial cells of brain (Banks et al., 1995; Schiltz and

Sawchenko, 2003; Roth et al., 2004; Matsumura and Kobayashi, 2004). This assumed

mechanism of induction of fever is commonly known as humoral hypothesis of fever

induction. The mentioned inflammatory mediators act on anterior hypothalamus

enhancing the release of PGE2 synthesized by cyclooxygenase (COX-2) causing an

increase in body temperature (Saper and Breder, 1994). Certain effective antipyretic

drugs like paracetamol works by inhibiting the effect of these pyrogenes on COX-2 and

PGE2 formation, in temperature neurons present in the anterior hypothalamus of brain

(Ashok et al., 2010).

In the current study, all the tested doses of plant crude extract showed a dose

dependent moderate to extreme antipyretic effect. Doses of 200 and 400 mg/kg of MCE

were highly significant in reducing the induced elevated body temperature of animals at

all the assessment time intervals and was analogous to effect of standard drug

paracetamol (Table 4.15). Substances having antipyretic potential have been reported to

suppress fever by stopping prostaglandin synthetase production, causing an obstruction

for the prostaglandin synthesis in brain or decreasing the synthesis of interleukins after

the formation of interferons. The plant MCE possibly caused a decline in the body

temperature by decreasing the amount of prostaglandins E2 in the temperature

controlling centre of brain via its reaction on cyclooxygenase or by raising the synthesis

of body’s individual substance having antipyretic properties i.e. arginine and

vasopressin (Okokon and Nwafor, 2010). Its antipyretic activity might also be

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attributed to active secondary metabolites like flavonoids, alkaloids and saponins which

are present in considerable amount in the plant extract might have a synergetic effect on

reducing the production of prostaglandin. These secondary metabolites suppress

peroxidation of arachidonic acid which reduces the level of prostaglandin causing a

decline in the intensity of pain and fever. Our results are in close concord with that of

Hallal et al. (2010) investigations, according to which dose of 100 and 300 mg/kg of C.

ambrosioides plant extract exhibited efficient antipyretic effect against the fever

induced by Brewer's yeast.

In conclusion, our investigation scientifically validated antipyretic activity D.

botrys, however additional exploration is required to segregate the active metabolites

which are involved in its antipyretic action and to examine the exact mechanisms of

their action.

5.3.5 Antidiarrheal activity

Castor oil induced diarrheal model was employed to authenticate antidiarrheal

potential of methanolic extract of D. botrys in selected animals. Diarrhea is a condition

of frequent and abnormal defecation of feces caused as a result of distorted motility of

water and electrolytes inside the intestinal tract. Castor oil has been commonly used for

stimulation of diarrhea in experimental animals because it is metabolized into active

molecule ricinoleic acid, which causes irritation and inflammation in the mucosal lining

of intestine (Vieira et al., 2000; Afroz et al., 2006). Numerous mechanisms have been

proposed which explain the diarrheal initiation potential of castor oil, including

inhibition of Na+

K+ ATPase activity in intestinal track, reduction in normal water

absorption (Capasso et al., 1994; Imam et al., 2012), motivation of mucosal cyclic

adenosine monophosphate (cAMP) mediated active secretion or activation of adenylate

cyclase (Pinto et al., 1992), stimulation of platelet activating factors and formation of

prostaglandin. The production of prostaglandin induces vasodilatation, contraction of

smooth muscle and secretion of mucus in small intestine. In both rodents and humans,

E series prostaglandin are considered good diarrheal agent (Mascolo et al., 1994).

In the current analysis the methanolic extract of plant showed antidiarrheal

potential in a dosage dependent manner and dose of 200 mg/kg and 400 mg/kg

markedly reduce the severity of diarrhea i-e increased latent period and decreased

frequency of total wet feces and mean weight of total wet feces, which is comparable to

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the standard drug loperamide (Table 4.16). It efficiently antagonized diarrhea induced

by castor oil and is one of most effective and common drug used for diarrheal

treatment. The consequences advocate that methanol extract of plant have antidiarrheal

potential, comparable to that of standard drug, but the exact active compound and its

mechanisms of action is unknown. The antidiarrheal activity of plant extract can be

anticipated from the mode of action castor oil that induces diarrhea i.e the plant extract

show counter activity to that of castor oil and bring the condition to normal. The extract

may increase electrolyte and water absorption or lower down the secretion of

electrolytes and fluid which cause a marked decrease in the surplus intestinal fluid and

frequency of diarrhea in a dosage dependent manner. The other possible mechanism of

antidiarrheal consequence of plant extract may be the reticence of ricinoleic acid effect

on prostaglandin E2 receptors. Blockage of prostaglandin receptors cause a significant

decrease in the secretary and intestinal motility effect of prostaglandins that are

produced due to the irritating activity of ricinolic acid derived from castor oil. The plant

extract may inhibit rapid peristalsis in the large intestine which causes slow movement

of feces through the intestine. The slow transit of feces also increases the chances of

greater fluid absorption from the feces, causing the stool drier that further slows its

passage. The antidiarrheal activity of plant extract may be due to its possible counter

effect on the generation or action of cyclic nucleotides i.e. cyclic guanosine

monophosphate and cyclic adenosine monophosphate. An elevation in either cyclic

guanosine monophosphate (cAMP) stimulates chloride ion (Cl-) secretion and at the

same time stops Na+/Cl

- absorption (Murek et al., 2010).

Phytochemical investigation of plant extract have revealed that it consist of

phenols, flavonoids, alkaloids, terpeniods, saponins, tanins and sterols which may play

a role in its antidiarrheal properties. Previous investigations have revealed that plant

derived active components like flavonoids, glycoside and tannins etc are the agents

having anti-dysentery and antidiarrheal properties (Palombo, 2005). It is also reported

that flavonoids have inhibitory effect on motility of fluid inside intestine (Mohammad

et al., 2009). Various in-vitro and in-vivo investigation have revealed that flavonoids

have the potential to restrain prostaglandin E2 stimulated intestinal secretion and

spasmogens tempted contraction. It also hinders release of autocoids and prostaglandin

(Dosso et al., 2011). Thus, flavonoids as the inhibitors of biosynthesis of prostaglandins

are considered to reduce intensity of diarrhea induced by castor oil (Brijesh, 2009).

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In conclusion, our investigation revealed that D. botrys extract contains

pharmacologically active substances, which are effective for management of diarrhea.

Further investigations are required to determine the active compounds and their

mechanisms of action responsible for the observed antidiarrheal effect.

5.3.6 Anti-diabetic activity

Diabetes has marked impact on human health, quality and life expectancy of

patients and health care expenditures (Ngugi et al., 2012).It is considered among one of

the chronic metabolic disorders and is allied with other disorders like hypertension and

obesity (Akah et al., 2011). Alloxane monohydrate is the common diabeto-genic

compound used for induction of diabetes in experimental animals, in order to estimate

the anti-diabetic prospective of different plant extracts and active compounds (Viana,

2004; Etuk, 2010). Alloxane monohydrate is organic compound derived from urea,

provokes diabetes by selective destruction of pancreatic beta-cells of islets of

Langerhans (Iranloye et al., 2011), which causes a massive decline in the synthesis and

release of insulin, making it biologically insufficient or unavailable and thus causes an

increase in blood sugar level. It confers its toxic effect on pancreatic beta cells through

blockage of glucokinase enzyme, formation of free radicals, oxidation of sulphydryl (-

SH) group and an instability in calcium homeostasis within the cell (Dunn, 1983;

Szkudelski 2001; Dhanesha, 2012). The basic mode of action involves selective

absorption of the compound because of its structural resemblance with glucose and

highly effective uptake mechanism of beta cells of pancreas (Lenzen, 2008).

In the present investigation, it was eminent that amount of 400 mg/kg

considerably reduced blood sugar level at all the assessment time interval, however its

effect was highly effective at the third and fourth hour of treatment and was analogous

to that of standard drug metformin (Table 4.17). Our investigations are in line with that

of Song et al., (2013) observations, in which it was reported that quantity of 300 mg/kg

plant crude extract of C. ambrosioides considerably declined elevated blood sugar

induced by alloxane monohydrate. Similarly, Kumar et al., (2015) analyzed that

methanolic leaf extract of C. album exhibited higher anti-diabetic potential than EAF at

various evaluation time intervals. It seemed from our study and the previous

investigations that D. botrys and the other related plants belonging to this family

contain some active metabolites, particularly in their crude extract which have anti-

diabetic potential and decreased the elevated level of sugar in blood to normal. The

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different apparent mechanisms through which MCE of plant brought the elevated sugar

level to normal might be its action to restore the damaged cells of islets of Langerhans,

increased the secretion of insulin (Bedoya et al., 1996; Shafighi and Amjad, 2013),

increased its sensitivity for absorption of glucose (Yolanda and Adriana, 2006) and

encouraged its conversion to glycogen. Its anti-diabetic potential might also be due to

its effect to restore abnormal absorption of carbohydrate in the small intestine and

facilitate uptake of glucose from blood by cells present in the peripheral regions

arbitrated by a glucose transporter GLUT-4, which depend upon the level of insulin

(Obatomi et al., 1994).

The restoring effect of plant extract on lowering blood sugar level might be due

to presence of active phytochemicals such as flavonoids, saponins, alkaloids, tannins,

sterols and terpeniods which have been allied with lowering elevated level of sugar in

blood (Middleton et al., 2000). The effectiveness of the mentioned active metabolites

against hyperglycemic condition has been reported by Venkatachalam et al., (2011) in

Lantana camara fruit ethanolic extract. Glauce et al. (2004) reported that flavonoids

caused a decline in the blood sugar level because of lipogenisis and elevated

transportation of sugar in lipocytes. Saponin extracted from Momordica charantia

caused an increase in the secretion of insulin and decrease in the high blood sugar level

in alloxane stimulated mice (Han and Wang, 2008). It has been reported by researchers

that alkaloids and its derivatives enhance pancreatic cells regeneration, reinstate insulin

secretion and restore the maximum sugar level to normal (Middleton et al., 2000).

Alkaloids extracted from Acanthus montanus leaves caused hypoglycemic effect at

varying doses of 100, 200 and 300 mg/kg of body weight in alloxane-stimulated

animals (Odoh and Ezugwu, 2012). Tannins isolated from plants exhibited anti-diabetic

action due to their inhibitory effect on alpha-glucosidase and alpha-amylase enzymes

(Kunyanga et al., 2011). It has been reported that terpenoids, extracted from plants, are

commonly used by hyperglycemic and hyper blood pressure patients because they

restore normal blood glucose level and decrease diastolic blood pressure (Piero et al.,

2015). The leaves of Emblica officinalis, which contain high amount of terpenoids, are

utilized for the curing of hyperglycemia (Treadway, 1994).

The current study had proved that D. botrys contains some active compounds

which might act independently or synergistically in promoting the hypoglycemic

potential of plant extract against the elevated sugar level induced by alloxane in rats.

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However further study is required to isolate the active phytochemicals which are

responsible for hypoglycemic activity and to investigate their mechanisms of action

responsible for the observed anti-diabetic activity.

5.3.7 Hepativeprotective activity

The preventive action of methanolic extract of D. botrys against damage of liver

provoked by CCl4 toxicity in rats was evaluated. CCl4 induced liver injury is one of the

most common method employed for the evaluation of hepatoprotective effects of

therapeutic plants and drugs in experimental model animals (Ahsan and Haque, 2009).

The effect of CCl4 on liver is similar to that of viral hepatitis (Rubinstein, 1962). It

cause toxicity in liver mainly because of its active metabolite, trichloromethyl free

radicals, which are formed due to specific type of cytochrome P-450 dependent mixed

oxidase enzyme (Lesiuk et al., 1999). These stimulated radicals react again with

oxygen and produce trichloro methyl peroxyl radicals. These radicals attack on lipids

present in the lipo-protein membrane of endoplasmic reticulum, causing peroxidation,

degradation and finally inducing cell death (Recknagel and Waller, 1989). Function of

liver can be assisted by evaluating the concentration of SGPT, SGOT ALP and TB,

which are normally present in elevated amount in cytoplasm. If there is some problem

in the liver, these enzymes leak out into blood stream, however its amount depend upon

the severity of liver damage (Nkosi et al., 2005).

In the present study when the animals treated with CCl4 alone for 24 h (group

II), caused oxidative stress and significant liver damage as revealed by elevated blood

level of SGPT, SGOT, ALP and TB as compared to animals of control group. It was

noted that methanol extract of D. botrys (400 mg/kg) extensively declined the increased

level of SGPT, SGOT, ALP and TB provoked by CCl4, indicating progress in the

functional condition of liver (Table 4.18). The improvement towards normal

histological architecture of liver and level of serum enzymes caused by D. botrys

methanolic extract (400 mg/kg) is similar to the hepatoprotective effect of silymarin, a

common drug having hepativeprotective properties. Similar notabl hepatoprotective

properties were shown by the 300 mg/kg, efficiently reduced the high level of these

enzymes in the blood (Parkash and Patel, 2005).

The hepatoprotective ability of plant extract might be due to the existence of

active metabolites and biomolecules like phenol, monoterpenes, carotiniods,

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glycosides, flavanoids, alkaloids and essential oil (Gupta and Misra, 2006). The active

bio-molecules present in the plant extract restore the damaged liver condition towards

normal by, inhibiting the activity of cytochrome P-450, stopping the process of lipid

peroxidation, stabilizing the membrane of liver and promoting the biosynthesis of

protein and glucoprotein. It is concluded from the result that the MCE of plant have

significant hepative-protective potential, depending upon its dose, however, the active

compounds which have hepativeprotective potential and the exact mechanism of its

action is unknown. Further studies are required to isolate the active components and

study its mode of action.

5.3.8 Sedative-hypnotic activity

The sedative-hypnotic prospective of methanolic extract of D. botrys was

estimated by using thiopental induced hypnosis and comparing with that of diazepam,

which was used as standard reference drug (Table 4.19). This is a classical model used

in behavioral pharmacology to investigate the sedative and hypnotic properties of

substances in the model animals. A number of reports revealed that the central nervous

system depressant barbiturates, such as thiopental, bind to barbiturates attaching sites

on gamma amino butyric acid GABAA receptors and stimulate GABA-mediated

increased polarization of post-synaptic neurons through allosteric alteration of GABAA

receptors (Fernandez et al., 2004).

The sedative-hypnotic and anxiolytic prospective of benzodiazepines (BDZs)

such as diazepam are typically credited to increase the potential of gamma amino

butyric acid (GABAA) (Yemitan and Salahdeen 2005). It unites to gamma sub-unit of

GABAA receptor, causing structural alteration and elevation in GABAA receptors

activity. Diazepam do not become an alternate for GABA, which attach at alpha (α)

sub-unit, but elevate the rate of channel opening actions which causes a raise in

chloride ion transmission and reticence of action-potential (Rang et al., 2003, Ali et al.,

2008). According to some reports the sedative-hypnotic potential of diazepam may be

because of direct instigation of glycine synapses inside brain (Snydar and Enna, 1975).

This may also elucidate the possible way of action of investigated plant sample, as it is

obvious from our findings, that sedative-hypnotic effect of plant samples was parallel

to that of diazepam. Our results demonstrated that plant methanolic extract of 100 mg

and 200 mg effectively induced early arrival of sleep and prolonged the duration of

hypnosis caused by thiopental as depicted in Table 4.19. It is Obvious from the current

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study that the MCE of plant contains some active substances which displayed

synergetic effect on thiopental action on the central nervous system, induced early and

prolonged hypnosis. It also showed a correlation between the sedative-hypnotic effect

of plant extract and that of diazepam.

The initial phytochemical assessment of the plant confirmed the existence of

phenols, flavonoids, alkaloids, tannins, sterols and saponins. There are many reports in

the scientific literature which confirmed that phenol, flavonoids, alkaloids and saponins

rich plants and their extracts possess effective sedative-hypnotic and anxiolytic

potential mediated through similarity (in-vitro) with benzodiazepine site of GABAergic

system (Trofimiuk et al., 2005; Awad and Ahmed 2009; Estrada-Reyes et al., 2010).

Besides, tannins are also involved in non-specific central nervous system depression

(Takahashi et al., 1986). It can be assumed that, along with other factors, the sedative-

hypnotic effect of plant methanolic extract may be due to the existence of these active

metabolites.

5.3.9 Anti-convulsant activity

Anti-convulsion potential of crude extract of D. botrys was examined in

experimental animals against PTZ-induced clonic seizures. PTZ is the common drug

used to induce convulsion in experimental animals due its interaction with ion channel

of GABAA receptors. GABA is the main inhibitory neurotransmitter present inside

brain and any reticence in its neurotransmission is considered to be the primary cause

of epilepsy (Silambujanaki et al., 2010). Increase in the GABAergic neurotransmission

is considered to inhibit seizers, while decrease in its transmission enhances seizers

(Amabeokua et al., 2007). The standard common antiepileptic and anticonvulsant drug

like phenobarbetone, diazepam and clonazepam are considered to enhance GABA-

induced opening of chloride ion channels on GABAA receptors causing more chloride

ions to enter the neurons which ultimately decreases the activity of neurons inside brain

(Mcddonald and Kelly, 1993; De-Sarro et al., 1999). Administration of moderate doses

of PTZ (90 mg/kg) provokes clonic seizures with severe negative neuro-chemical

effects such as decline in the level of GABA and subsequent reduction of inhibitory

responses, causing a condition which aggravates excitation (Walsh et al., 1999;

Eloqayli et al., 2003). Racine (1972) reported that there are five stages of epileptic

seizure or convulsion also called Racine-seizures score, according to which the first

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phase is ear facial trembling of model animal, second phase is spastic signal across the

body, third phase is cyclonic jerk, fourth phase is clonic-tonic seizures (it proceed into

the side position) and the final fifth phase is comprehensive clonic-tonic convulsions (it

proceed into the backward position).

In the current investigation, the consequence of 400 mg/kg of MCE was

comparable to that of standard drug clonazepam, radically delayed the onset of first

clonus and prolonged the duration of death effectively (Table 4.20). The resemblance

of plant extract with that of standard drug suggests that it might activate GABAA

receptors and enhance its inhibitory neurotransmission. Phytochemical investigation of

MCE of D. botrys showed the presence of considerable amount of active metabolites

such as saponins, flavonoids, steroids, tannins and alkaloids, among which various

phytochemicals have been observed to have activities effecting central nervous system.

Previously, flavonoids (Asl et al., 2007), alkaloids (Taesotikul et al., 1998), essential

oil (Dallmeier and Carlini, 1981), saponins, sterols are reported to have anticonvulsant

potential in experimental convulsion models such PTZ and MES (Chaohan et al., 1988

and Kastur et al., 2002). Different flavonoids extracted from plants have properties like

benzodiazepine, work in the nervous system and alter GABA-produced chloride flow in

different convulsion, sedation and anxiety models using experimental animals (Asl et

al., 2007).

In our observation the anti-convulsant activity shown by the plant extract might

be due to collective effect of its all active metabolites present in it, which need to be

isolated and investigate their proper mechanism of action. However our observation

confirmed the folkloric use for the treatment of convulsion and neurological disorders.

5.3.10 Antidepressant activity

The forced swim test (FST) is commonly employed for the evaluation of anti-

depressant prospective of drugs and herbal extracts in experimental animals. According

to this model, the extension in duration of mobility reflects anti-depressant effect, while

decrease in duration of mobility indicates neurological depression in the central

nervous system (Subarnas et al., 1993). The animal found motionless for longer

interval of time shows that it is in situation of exhaustion, sadness and fatigue are the

common indications of depression as also found in human (Aladeokin and Umukoro,

2011). There is close relationship between clinical efficiency of antidepressant drugs

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and their influence in FST, which is not found in any other behavioral model (Steru et

al., 1985; Porsolt, 1981).

The data obtained from our results showed that only higher dose (200 mg/kg) of

plant extract extensively condensed the immobility time interval and was analogous to

that of fluoxetine, used as standard antidepressant drug (Table 4.21). Fluoxetine reduce

the neurological depression by stopping nor-epinephrine (NE) re-uptake. Its effect in

the FST might be due to improved accessibility of neurotransmitters NE and serotonin

at post-synaptic site following reuptake inhibition (Pal and Dandiya, 1993). According

to primary hypothesis of depression projected 40 years ago, the common cause of

depression is the functional insufficiency of cerebral mono-aminergic transmitters like

dopamine (DA), serotonin (5-hydroxytryptamine) 5HT and NE which are situated at

synapses (Roiser et al., 2012). Certain studies have reported stabilizing effect of plant

extract through regularization of mono-aminergic level and stress parameters, which

provide evidence that the anti-depressant ability of plant extract might be due to

restoration of mono-aminergic neurotransmitters (Rai et al., 2003).

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VI. SUMMARY

Dysphania botrys is an annual herbaceous plant belongs to family

Amaranthaceae, native to Asia and Europe found in Pakistan, India and Iran.

Previously this plant belonged to genus Chenopodium but due some variation in

anatomic and taxonomic characteristics it was placed in a separate genus of Dysphania.

In conventional medicine, D. botrys has been utilized for the cure of diverse diseases

like cold and influenza, asthma, head ach, liver and digestive problems and healing of

wounds. The current work was designed to explore MCE and its various solvent

fractions for phytochemical analysis, various in-vitro and in-vivo pharmacological

activities (crude extract only) in order to furnish scientific authentication to its ethno-

medicinal uses. Qualitative phytochemical assessment of D. botrys proved the existence

of alkaloids, phenols, flavonoids, tannins, saponins, and sterols in the MCE while in

HxF only flavonoids and saponins were detected. Highest amount of phenol,

flavonoids, alkaloids were found in EAF while crude extract contained maximum

amount of saponins. In the proximate analysis, nitrogen-free extract were present in

higher amount followed by protein, inorganic matter, moisture and fats while crude

fibers were found least in amount. Among different minerals calcium was found in

maximum amount, followed by potassium, sodium, iron, zinc, copper, nickel and lead

while no cadmium and chromium were detected. MCE and EAF displayed considerable

antibacterial activity against X. Campestris and P. Aerugonosa respectively, while

exhibited moderate bactericidal action against other strains of bacteria. In case of

antifungal activity MCE hindered the growth of F. oxysporum effectively, while HxF

showed no effect on the growth of A. flavus and A. niger. Maximum phytotoxic effect

was shown by MCE, inhibiting the growth of L. minor, while HxF displayed least

effect on its growth. EAF exhibited maximum DPPH and ABTS scavenging activity

followed by MCE. EAF showed considerable antagonistic effect while HxF displayed

least inhibiting effect against lipoxygenase activity. In the in-vivo pharmacological

activities of MCE of D. botrys, toxicity test showed no sign of severe abnormality and

mortality up to a dose of 2000 mg/kg. Crude extract (200 and 400 mg/kg) showed

substantial (p<0.05) anti˗inflammatory action, both at the early and late phase of

carrageenan-induced paw edema while in case of xylene induced ear edema dose of

400 mg/kg was highly effective (p<0.01) in reducing ear inflammation. Dosage of 200

mg/kg of plant extract depicted peripheral analgesic activity at both phases of

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analgesia, causing considerable (p<0.05) diminution in severity of pain while dose of

400 mg/kg was highly significant (p<0.01) causing 78.57% and 82.14% pain

inhibition. In the central analgesic activity (hot plate test) the action of 400 mg/kg was

highly efficient (p<0.01) after 120 min of assessment time interval. In the brewer‟s

yeast-provoked pyrexia model the effect of 400 mg/kg of plant extract was extremely

significant (p<0.001) and was parallel to that of standard drug paracetamol in reducing

body temperature to normal, at all the assessment time intervals (1h-5h). Plant extract

(400 mg/kg) displayed substantial (p<0.01) antidiarrheal effect, increased the latent

period of diarrhea and caused a decline in the total wet fecal frequency and mean

weight of fecal drops as compared to control. The elevated blood sugar induced by

alloxane monohydrate in the anti-diabetic activity was significantly (p<0.05) reduced

by crude extract (400 mg/kg), however its effect was highly significant (p<0.01) after 4

h of evaluation time. In the hepatoprotective assay, plant extract at dosage of 400

mg/kg notably (p<0.05) declined elevated level of ALP and TB while its effect was

highly significant (p<0.01) reducing the level of SGPT and SGOT, when compared to

toxic control. Plant extract (100 and 200 mg/kg) demonstrated a remarkable (p<0.05)

synergetic effect on the thiopental induced hypnosis caused an early arrival of sleep and

effectively (p<0.01) prolonged the duration of sleep as compared to standard drug

diazepam. In the PTZ-induced convulsion activity, plant extracts (200 and 400 mg/kg)

effectively (p<0.05) postponed the initiation of first clonus and prolonged the time of

death as compared to control. In the antidepressant assay, amount of 200 mg/kg

extensively (p<0.05) decrease the immobility time as compared to control while the

other doses showed no significant effect.

96

VII. CONCLUSIONS AND RECOMMENDATIONS

Conclusions

1. Phytochemical investigation of the whole plant of D. botrys showed that it

contain considerable amount of phenol, flavonoids, alkaloids, saponins, nitrogen

free extract, protein, calcium, magnesium, sodium, iron and zinc while all the

tested metals were found within the permissible limit and no cadmium and

chromium was detected.

2. MCE and EAF showed maximum antibacterial, antifungal, DPPH and ABTS

radical scavenging and lipoxygenase activities while the other extracts showed

moderate effect.

3. MCE up to 2000 mg/kg was found safe and had no toxic effect after 24 h of

evaluation time.

4. MCE of plant showed significant pharmacological effects in model animals and

all the ethno-medicinal uses of the plant were scientifically authenticated.

5. MCE of 400 mg/kg of extract of plant displayed significant anti-analgesic,

anti˗inflammatory, antipyretic, anti-diabetic, antidiarrheal, anticonvulsant,

hypnotic and hepativeprotective effects in all the tested animals‟ models.

6. In-vitro activities like antifungal, lipoxygenase, phytotoxic activities and all the

in-vivo pharmacological activities of D. botrys were described for the first time.

97

Recommendations

1. The plant of D. botrys is an excellent source of a variety of nutrients and

noxious metals were found within tolerable range, so this plant may

possibly be utilized as a foodstuff supplement and also can be added in

silage and feed for domestic animals.

2. Further research is suggested to study phytochemical and

pharmacological consequence of crude extract and derived solvent

fractions of leaf, stem and roots individually.

3. Further investigation is necessary to separate the active compounds

having pharmacological properties and study its mechanism of action.

Once its mechanism of action is recognized then its effectiveness may

be enhanced by varying its configuration synthetically.

98

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