ISOLATION OF BIOACTIVE SECONDARY METABOLITES AND ...

ISOLATION OF BIOACTIVE SECONDARY

METABOLITES AND PHARMACOLOGICAL STUDIES

OF VIOLA SERPENS WALL

By

RUKHSANA Ph.D

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR

2017

ISOLATION OF BIOACTIVE SECONDARY

METABOLITES AND PHARMACOLOGICAL STUDIES

OF VIOLA SERPENS WALL

Thesis submitted to the Department of Pharmacy, University of Peshawar,

Peshawar, Pakistan in partial fulfillment for the Degree of

DOCTOR OF PHILOSOPHY

IN

PHARMACEUTICAL SCIENCES

FEBRUARY, 2017

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR

APPROVAL SHEET

A Thesis presented by Rukhsana entitled “Isolation of Bioactive Secondary

Metabolites and Pharmacological Studies of Viola Serpens Wall” to the

Department of Pharmacy, University of Peshawar in partial fulfillment for the award

of the Degree of Ph.D in Pharmaceutical Sciences.

We, the undersigned have examined this thesis and do hereby approve it for the

award of Ph.D Degree.

External Examiner: _________________________________

Supervisor: ______________________________

PROF. DR. MUHAMMAD SAEED

Chairman,

Department of Pharmacy,

University of Peshawar.

Co-supervisor: ______________________________

DR. MANZOOR AHMAD

Associate Professor,

Department of Chemistry,

University of Malakand.

II DDeeddiiccaatteedd

mmyy tthhiiss hhuummbbllee eeffffoorrtt ttoo mmyy bbeelloovveedd

PPaarreennttss && FFaammiillyy

i

ACKNOWLEDGEMENT

In the name of Almighty Allah, the most merciful and beneficent, Who gave me the

courage and ability for the better understanding and completion of my PhD project. I

bow my head before Allah for His greatness, Who provided me strength and courage

to accomplish a useful and beneficial work for the benefit of mankind.

With great honor and extreme happy feelings I pay my homage and debt to my

research supervisor, Prof. Dr. Muhammad Saeed, Chairman, Department of

Pharmacy, University of Peshawar. His broad vision, advice, encouragement and co-

operation helped and guided me for the completion of my Ph.D programme and

dissertation.

I am also extremely indebted to my co-supervisor Dr. Manzoor Ahmad, Associate

Professor, Department of Chemistry, University of Malakand. His sincere help,

guidance, provision of required resources for the accomplishment of the major part of

my research work.

I would like to thank Prof. Dr. Zafar Iqbal, Meritorious professor, Tamgh-e-

Imtiaz, Department of Pharmacy, University of Peshawar and Prof. Dr. Fazal

Subhan for their support and encouragement throughout my work. I am also grateful

to all the teaching faculty of the department for their support and cooperation.

I am very thankful to Professor, Prof. Dr. Haroon Khan, Abdul Wali Khan

University Mardan, for his sincerity, guidance, co-operation and encouragement at

every stage of my PhD work. I also acknowledge the guidance and support of

Mr. Ikran Illahi, Assistant Professor and Chairman, Department of Zoology

University of Malakand.

ii

I am very grateful to Mr. Atta-ur-Rehman, Institute for Natural Product Discovery,

Universiti Teknologi, MARA Puncak Alam Selangor D.E., Malaysia for the

spectroscopic studies of the isolated compounds. I am also very thankful to the staff

of PCSIR Laboratories especially Ms. Farah Gul and Mr. Yaqoob for their co-

operation and guidance in conducting anti-inflammatory activity of V. serpens.

Thanks to Dr. Nuzhat Sultana from Khyber Medical College Peshawar and Mr.

Mohammad Shahid, PhD scholar, for their help regarding the interpretation of histo-

pathological slides. I am also very thankful to Dr. Umer Sadique Khattak,

Chairman, Department of Animal Health Sciences, Agriculture University Peshawar

and Mr. Sajjad Ali Shah, research assistant and PhD scholar Department of Animal

Health Sciences, Agriculture University Peshawar for their facilitations and guidance

in preparing the histopathological slides and taking photographs by using camera

fitted microscope in the hepatoprotective and nephroprotective activities.

I wish to pay my sincere appreciation to my lab fellows Ms. Attiqa Naz, Miss

Samreen Pervez, Miss Noor-ul-Aain, Mr. Naveed and Mr. Asif Jan for their help,

support and collective team work.

At the end I pay my regards and duly acknowledge the co-operation, guidance and

support of my parents, husband, sisters-in-law, brothers and sisters who encouraged

and enabled me to fulfill this task

.

Rukhsana

iii

TABLE OF CONTENTS

S.No. Topic Page No.

Acknowledgement i

List of Figures vii

List of Tables ix

Abbreviations xi

Abstract xiii

Chapter – 1

1. INTRODUCTION 1

1.1 General Introduction 1

1.2 Traditionally Used Medicinal Plants in Pakistan 3

1.3 Importance of Herbal Medicines in Different Traditions 4

1.3.1 Chinese Traditional Medicine 4

1.3.2 Japanese Traditional Medicine 5

1.3.3 Indian Traditional Medicine 5

1.4 Violaceae Family 5

1.5 Literature Survey of Genus Viola 6

1.6 Plant Introduction 12

1.6.1 Viola serpens Wall 12

1.6.2 Local Names 12

1.6.3 Morphology 12

1.6.4 Classification (Taxonomical position of V.serpens) 13

1.6.5 Geographical Distribution of V.serpens 13

1.6.5.1 World Wide Distribution 13

1.6.5.2 Distribution In Pakistan 14

1.7 Plant Distribution 15

1.8 Ethno medicinal Uses 16

1.9 Phytochemical Investigations 16

1.9.1 Nutritive Values 16

1.9.2 Essential and Fixed Oils 16

1.10 Isolated Compounds from Genus Viola 17

1.11 Pharmacological Studies 17

1.11.1 In vivo Biological Activities 17

1.11.1.1 Antibacterial Activity 17

1.11.1.2 Anti-fungal Activity 19

1.11.1.3 Antiprotozoal Activity 20

iv


1.11.1.4 Cytotoxic Activity 20

1.11.1.5 Haemolytic Activity 20

1.11.1.6 Antiplasmodial Activity 20

1.11.1.7 Anti-malarial Activity 21

1.11.1.8 Anthelmintic activity 21

1.11.1.9 Antioxidant Activity 22

1.11.1.10 Anti-T.B Activity 22

1.11.1.11 Treatment of Jaundice 23

1.11.1.12 Urease Inhibitory Activity 23

1.11.1.13 Anti-HIV Effect 23

1.11.1.14 Insecticidal Activity 24

1.11.2 In vitro Biological Activities 24

1.11.2.1 Acute Toxicity 24

1.11.2.2 Antinociceptive Activity 24

1.11.2.3 Anti-Inflammatory Activity 25

1.11.2.4 Antipyratic Activity 25

1.11.2.5 Gastrointestinal Motility 26

1.11.2.6 Laxative Effect 26

1.11.2.7 Hepatoprotective Activity 27

1.11.2.8 Diuretic Activity 27

1.11.2.9 Anxiolytic Activity 27

1.11.2.10 Muscle Relaxant 28

1.11.2.11 Sedative-Hypnotic Effect 28

1.11.2.12 Anesthetic Effect 28

1.11.2.13 Uterotonic Effect 29

1.11.2.14 Anti-neurotensive 29

1.11.2.15 Anti-cancer Activity 29

1.11.2.16 Anti-hypertensive Effect 30

1.11.2.17 Anti-dyslipidemic Effect 30

1.11.2.18 Expectorant and Anti-tussive Effect 30

1.12 Aims and Objectives 33

Chapter – 2

2. EXPERIMENTAL 34

2.1 General Experimental Condition 34

2.2 Spectroscopic Technique 34

2.3 Physical Constants 35

2.4 Column Chromatography (CC) 35

2.5 Thin Layer Chromatography (TLC) 35

2.6 Drugs and Reagents 35

2.7 Plant Materials 37

v


2.7.1 Extraction and Fractionation 37

2.7.2 Isolation and Purification 40

2.8 Experimental Data of New Compounds from Viola serpens 42

2.8.1 Commulin-A (1) 42

2.8.2 Commulin- B (2) 42

2.8.3 Commulin- C (3) 43

2.9 Experimental Data of Known Compounds From Viola serpens 43

2.9.1 5-Hydroxy-7-methoxy flavone (tectochrysine) (4) 43

2.9.2 4́, 5-Dihydroxy-7-methoxy-6, 8-dimethylflavone (Sideroxylin)(5) 44

2.9.3 2,5-Dihydroxy-4-methoxybenzophenone (Cearoin) (6) 44

2.10 In-vivo Biological Activities 45

2.10.1 Experimental Animals 45

2.10.2 Acute Toxicity 45

2.10.3 Analgesic Activity 46

2.10.3.1 Acetic Acid Induced Writhing 46

2.10.3.2 Formalin Test 46

2.10.4 Anti-inflammatory Activity 47

2.10.4.1 Carrageenan Induced Paw Edema 47

2.10.4.2 Histamine Induced Paw Oedema 48

2.10.4.3 Xylene Induced Ear Edema 48

2.10.5 Larvicidal Bioassay 49

2.10.6 Nephroprotective and Hepatoprotective Activities 50

2.10.6.1 Animals Used 50

2.10.6.2 Animals Grouping and Dosing 50

2.10.6.3 Chemicals Used 51

2.10.6.4 Histopathology 51

2.10.6.5 Hematological and Serological profile of infected Rabbits 54

2.10.6.6 Statistical analysis 56

2.10.6.7 Collection and analysis of urine 56

2.11 In-vitro Biological Activities 58

2.11.1 Anti-oxidant Activity 58

2.11.1.1 Superoxide Anion Radical Scavenging Assay 58

2.11.1.2 DPPH Radical Scavenging Activity 58

2.11.2 Antibacterial Assay 59

2.12 Enzyme Inhibition 60

2.12.1 Chemicals Required for Anticholine Esterase 60

2.12.2 Acetylcholinesterase Inhibition 60

vi


Chapter – 3

3. RESULTS AND DISCUSSION 61

3.1 Biological Activities 61

3.1.1 In-vitro Biological Activities 61

3.1.1.1 Antimicrobial Activity 61

3.1.2 Effect of Crude extract/Fraction of V.serpens in DPPH free

Radical

66

3.1.3 Effect of Crude Extract/ Fractions of V.serpens in Larvicidal

Effect

68

3.1.4 Effect of Crude Extract/ Fractions of V.serpens in Acetyl

Cholinesterase Assay

70

3.2 In-vivo Biological Activities 72

3.2.1 Acute Toxicity 72

3.2.2 Hepatoprtotective and Nephroprotective Effects of Crude

Extract/Fractions of V. serpens

72

3.2.2.1 Hepatoprotectve Effect 72

3.2.2.2 Nephroprotective Effect of V.serpens Crude Extract and its

Subsequent Fraction

77

3.2.2.3 Antinociceptive Activity 84

3.2.2.4 Anti-inflammatory Activity 94

3.3 Isolated Compounds 112

3.3.1 New Compound From Viola serpens 112

3.3.1.1 Commulin-A (1) 112

3.3.1.2 Commulin-B (2) 114

3.3.1.3 Commulin-C (3) 117

3.3.2 Known Compounds from Viola serpens 120

3.3.2.1 5-Hydroxy-7-methoxy flavone (tectochrysine) (4) 120

3.3.2.2 4́, 5-Dihydroxy-7-methoxy-6, 8-dimethylflavone (Sideroxylin) (5) 121

3.3.2.3 2, 5-Dihydroxy-4-methoxybenzophenone (Cearoin) (6) 122

CONCLUSION 124

REFERENCES 126

vii

LIST OF TABLES

S.No. Title Page No.

1.1 Viola species list, native to Pakistan and their worldwide distribution,

parts used chemical constituents and medicinal uses

6

1.2 List of other viola spp. of the family Violaceae, their geographical

distributions, Medicinal uses and part/s used mostly

9

1.3 List of some various other species of viola and their geographic

distributions

10

2.1 List of Drugs / Chemicals used with their Sources 36

3.1 Antimicrobial Activity of the Crude Extract along with the

Subsequent Fractions of Viola serpens

64

3.2 Antimicrobial activity of the Isolated Compounds from V. serpens 64

3.3 DPPH Scavenging Activity of Crude extract/Fractions of V.serpens

and Zones of Inhibition are Given in mm

67

3.4 Anti-oxidant Activities of Pure Isolated Compounds 1–6 from

V.serpens whole Plant

67

3.5 Larvicidal effect of the crude extract along with the subsequent

fractions of V.serpens against Aedes aegypti and Culex

quinquefasciatus species of mosquitoes

69

3.6 The Enzyme Inhibition effect of the Crude Extract and the

subsequent Fractions of V.serpens against the Enzyme Acetylcholine

Esterase

71

3.7 Acute Toxicity of the Crude Extract along with the Fractions of V.

serpens

72

3.8 Effects of the Crude Extracts/Fractions of V.serpens Wall on the

Liver Related Parameters (AST, ALT and ALP) in the Rabbits

Models

75

3.9 Effect of Crude Extract/ fractions of V.serpens Wall. on the Kidney’s

functions and Clearance in the Rabbits Models

81

3.10 The Effect of Crude Extract/Fractions of V.serpens in Acetic Acid

Induced Writhing Tests in Mice (i.p)

85

3.11 Effect of the crude/ fractions of V. serpens in formalin induced pains

for analgesia test in mice at doses of 100, 200 and 300 mg/kg, i.p

91

3.12 Anti-inflammatory effect against carrageenan and Histamine

induced paw edema in mice for V.serpens crude extract

105

viii

S.No. Title Page No.


induced paw edema in mice for V.serpens n- hexane fraction

106

3.14 Anti-inflammatory effect of chloroform fraction of V.serpens

in carrageenan and Histamine induced paw edema in mice

107

3.15 Anti-inflammatory effect of Ethyl acetate fraction of V.serpens

in carrageenan and Histamine induced paw edema in mice

108


induced paw edema in mice for V.serpens aqueous fraction

109

3.17 Effect of the crude extract along with the subsequent fractions of

V.serpens on xylene induced ear edema in mice

110

3.18 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-A (1)

in CDCl3

114

3.19 1H- (400 MHz.) and 13C-NMR (125 MHz) Data of Commulin-B (2)

in CDCl3

117

3.20 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-C (3)

in CDCl3

119

ix

LIST OF FIGURES

S. No. Title Page No.

1.1 Percentage of Medicinal Plants In Undeveloped and Developed

Countries

3

1.2 Illustration of Viola serpens specie of the Genus Viola 31

1.3 Flower of V. serpens species of the Genus Viola 32

1.4 Seeds of V. serpens species of the Genus Viola 32

2.1 Scheme of plant extraction and fractionation 39

2.2 Scheme representing the isolation of pure compounds using Ethyl

acetate fraction

41

3.1 % inhibition of the tested bacteria against the Crude extract/

fractions of V. serpens. Where CHCl3 represents Chloroform,

ETA represents Ethyl acetate and H2O represents the Aqueous

fraction

65

3.2 Liver photomicrographs of the rabbits treated with paracetamol,

crude extract and n-Hexane fractions of V. serpens at doses of 150

and 300 mg/kg (H&E, 100X and 400X)

77

3.2.1 Normal saline treated liver showing normal architecture of central

vein (CV), sinusoidal spaces (small arrows), hepatocytes (large

arrows) with a centrally placed nucleus and foamy cytoplasm.

(100X H&E).

77

3.2.2 Liver showing accumulation of lymphocytes (small arrows)

around the central vein (CV), fatty changes (small arrow head)

and focal area of necrosis (asterisk) with paracetamol (100X

H&E).

77

3.2.3 Liver showing regeneration, containing normal liver plates (large

arrows) along central vein (CV) with n-hexane 150 mg/kg b.w.

(H&E).

77

3.2.4 Liver showing normal appearance of central vein (CV) and plates

of hepatocytes (large arrows) with n-hexane 300 mg/kg b.w.

(100X H&E).

77

3.2.5 Liver showing hexagonal hepatocytes (large arrows) with

prominent cell borders (small arrows), nuclei (arrow heads) with

nuclear clearing and prominent nucleoli with crude extract at a

dose of 150 mg/kg b.w. (400X H&E).

77

3.2.6 Liver showing regeneration of hepatocytes (large arrows) with

congestion of sinusoids (asterisks) containing red blood cells

(small arrows) with crude extract at a dose of 300 mg/kg b.w.

(400X H&E).

77

x


3.3 Photomicrogrphs of the Kidneys of Rabbits Treated with

Paracetamol and plant Extract/ Fractions at Different Doses (H&E)

83

3.3.1 Photomicrograph (100X H&E) of a section of kidney from a rabbit

treated with normal saline showing normal histological appearance

of renal cortex. The cortex contains renal corpuscles (large arrows)

embedded among proximal (arrow heads) and distal (asterisk)

convoluted tubules.

83


rat treated with PCM showing necrosis of cuboidal epithelial cells

(large arrows) of proximal convoluted tubules with exfoliation of

their brush border. The lumen (asterisk) of tubules contains

numerous cellular casts (small arrows).

83

3.3.3 Photomicrograph (100X H&E) of a kidney section from a rabbit

treated with n-hexane soluble fraction 150 mg/kg showing normal

histo-architecture of distal convoluted tubules with wider lumen

(asterisk) and lined by cuboidal epithelial cells (arrow heads).

Numerous loop of Henle tubules are also visible (large arrows).

83


treated with n-hexane soluble fraction 300 mg/kg showing normal

renal corpuscles (large arrows) with mild dilatation of proximal

(arrow heads) and distal (asterisk) convoluted tubules

83

3.3.5 Photomicrograph ((100X H&E)) of a section of kidney from a rabbit

treated with chloroform soluble fraction 150 mg/kg showing normal

renal corpuscles (large arrows), proximal (arrow heads) and distal

(asterisk) convoluted tubules

83


treated with ethyl acetate soluble fraction 150 mg/kg showing

normal renal corpuscles (large arrows) with mild dilatation of

proximal (arrow heads) and distal (asterisk) convoluted tubules.

83

3.3.7 Photomicrograph ((100X H&E)) of a section of kidney from a rabbit

treated with chloroform soluble fraction 300 mg/kg showing normal

renal corpuscles (large arrows) and proximal convoluted tubules

(arrow heads). The distal convoluted tubules (asterisk) exhibited

mild tubular necrosis of the cuboidal epithelial cells

84


rat treated with ethyl acetate soluble fraction 300 mg/kg showing

normal proximal convoluted tubules (large arrows) with numerous

loop of Henle tubules (asterisk). The interlobular blood vessels

(arrow heads) among the renal tubules exhibited mild congestion

with red blood cells.

84

xi


3.3.9 Photomicrograph (100X H&E) of a section of kidney from a rat

treated with aqueous soluble fraction 300 mg/kg showing normal

renal corpuscles (large arrows). The renal tubules exhibited

dilatation (arrow heads) with exfoliation of the brush border lining

the proximal convoluted tubules into their lumen.

84

3.3.10 Photomicrograph (100X H&E) of a section of kidney from a rat

treated with aqueous soluble fraction showing mild congestion of the

renal corpuscles (large arrows) with severe dilatation of the renal

tubules (asterisk). Numerous cellular casts (arrow head) is also

visible in the lumen of renal tubules.

84

3.4 Anti-nociceptive effect of crude extract of V. serpens in Acetic acid

induced writhing test.

87

3.5 Anti-nociceptive effect of n-hexane Fraction of V. serpens in Acetic

Acid induced writhing test.

87

3.6 Anti-Nociceptive Effect of Chloroform Fraction of V. serpens in

Acetic Acid induced writhing test.

88

3.7 Antinociceptive Effect of Ethyl Acetate Fraction of V. serpens in

Acetic Acid induced writhing test.

88

3.8 Anti-Nociceptive Effect of Aqueous Fraction of V. serpens in Acetic

Acid induced writhing test.

89

3.9 Antinociceptive Effects of Formalin Induced Pain in Mice of the

Crude Extract of V.serpens

94

3.10 Antinociceptive effects of Formalin Induced Pain in Mice of the

Hexane Fraction of V.serpens

94


Chloroform Fraction of V.serpens

95


Ethyl Acetate Fraction of V.serpens

95


Aqueous Fraction of V.serpens

96

3.14 Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on

Carrageenan Induced paw Edema

102

3.15 Anti-Inflammatory Effect (%) of the n-Hexane Fraction V. serpens

on Carrageenan Induced paw Edema

102

3.16 Anti-Inflammatory Effect (%) of the chloroform Fraction V. serpens

on Carrageenan Induced paw Edema

103

3.17 Anti-Inflammatory Effect (%) of the Ethyl Acetate Fraction V.

serpens on Carrageenan Induced paw Edema

103

xii


3.18 Anti-Inflammatory Effect (%) of the Aqueous Fraction V. serpens on

Carrageenan Induced paw Edema

104

3.19 Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on

Histamine Induced paw Edema

104

3.20 Anti-Inflammatory Effect (%) of the n-Hexane Fraction V. serpens

on Histamine Induced paw Edema

105

3.21 Anti-Inflammatory Effect (%) of the chloroform Fraction V. serpens

on Histamine Induced paw Edema

105

3.22 Anti-Inflammatory Effect (%) of the Ethyl Acetate Fraction V.

serpens on Histamine Induced paw Edema

106

3.23 Anti-Inflammatory Effect (%) of the Aqueous Fraction V. serpens on

Histamine Induced paw Edema

106

3.24 Percent inhibition of Xylene induced ear edema in mice at different

doses of the crude extract and fractions of V. serpens

111

3.25 Commulin-A (1) 112

3.26 Key HMBC interaction in Compound 1 114

3.27 Commulin-B (2) 115

3.28 Key HMBC interaction in Commulin-B (2) 116

3.29 Commulin-C (3) 118

3.30 Key HMBC interaction in Commulin-C (3) 119

3.31 Structure of compound Tectochrysine (4) 120

3.32 Structure of compound Sideroxyline (5) 121

3.33 Structure of compound Cearoin (6) 122

xiii

LIST OF ABBREVIATIONS

ACh: Acetylcholine

ACh-E: Acetylcholine esterase

AIDS: Acquired Immune Deficiency Syndrome

ALP: Alkaline phosphatase

ALT: alanine aminotransferase

AST: aspartate aminotransferase

A.parasiticum: Amoebidium parasiticum

C. albicans: Candida albicans

CC: Column Chromatography

CHCl3: Chloroform

C.littoralis: Congregibacter littoralis

Co-A: Coenzyme A

COSY: Correlation Spectroscopy

COX: Cyclo-oxygenase

DPPH 2, 2-diphenyl-1-picryl hydrazyl

BHT Dibutylhydroxy toluene

DMSO: Dimethyl Sulfoxide

DNA: Deoxyribonucleic Acid

EDTA: Ethylene Diamine Tetra Acetic acid

EI-MS: Electron Ionization Mass Spectrometry

ELISA: Enzyme Linked Immunosorbent Assay

E. coli: Escherichia coli

ETA: Ethyl acetate

FAB-MS: Fast Atom Bombardment Mass Spectrum

xiv

GC: Gas Chromatography

GCMS: Gas Chromatography Mass Spectrometry

HMBC: Heteronuclear Multiple Bond Coherence

HSQC Heteronuclear single quantum coherence spectroscopy

IC50: Concentration causing 50% Inhibition

IR: Infra red

KP: Khyber Pakhtunkhwa

K. pneumonia: Klebsiella pneumonia

LD50: Median Lethal Dose

NIH: National Institute of Health

NBT nitroblue tetrazolium chloride

NMR: Nuclear Magnetic Resonance

PCM: Paracetamol

P. postuma: Hemidesmus indicus

P.aeruginosa: Pseudomonas aeruginosa

RSA Radical scavenging assay

SGOT: Serum Glutamic Oxaloacetic Transaminase

SGPT: Serum Glutamate Pyruvate Transaminase

S.aureous: Staphylococcus aureus

S. typhi: Salmonella typhi

TLC: Thin Layer Chromatography

NOESY: Nuclear Overhauser Effect Spectroscopy

UV: Ultra Violet

VLC: Vacuum Liquid Chromatography

WHO: World Health Organization

xv

µg: Micro gram

µL: Micro Litre

xvi

ABSTRACT

Viola serpens which belongs to the family Violaceae has been reported to have many

folkloric uses including use as antipyretic, laxative, emollient, expectorant, purgative,

diuretic, demulcent, diaphoretic, anti-asthmatic and anti-cancer. The current

investigation was carried out to evaluate the crude methanolic extract and various

fractions of Viola serpens for its antioxidant, antimicrobial, enzyme inhibiting

potential, larvicidal activities using using in vitro assays and for antinociceptive, anti-

inflammatory activities, hepatoprotective and nephroprotective effects using in-vivo

studies. Furthermore, bioactive secondary metabolites were also isolated and

characterized. The results showed that the crude methanolic extract and various

fractions including chloroform, ethyl acetate fraction, n-hexane fraction and aqueous

fractions possessed significant antimicrobial activities against S. typhi and E. coli, B.

subtilis, S. flexeneri and P. aerogenes.

The compounds (1-6) isolated from the plant were also tested for activity against

different spp. of Gram positive and Gram negative bacteria except the compound

commulin-C. Against S. typhi, sideroxyline showed maximum zone of inhibition (18

mm) followed by the other tested compounds, each with 17 mm zone of inhibition.

Against the P. aerogenes, tectochrysine and cearoin proved more effective followed

by sideroxylin and commulin-B. S. flexeneri proved more susceptible to the

compounds tectochrysine and cearoin (17 mm) followed by commulin-A, commulin-

B but sideroxylin was inactive against it. S. aureus was susceptible to all four

mentioned isolated compounds except commulin-B and cearoin. Commulin-B and

tectochrysine were effective against E. coli.

xvii

Viola serpens crude extract, fractions and pure compounds also showed significant

free radical scavenging activity in DPPH free radical scavenging assays.

Concentration-dependent scavenging effect is showed by the crude methanolic

extract, n-hexane and chloroform soluble fractions against DPPH with maximum

activity of 67.99%, 75.98 % and 79.00 %. In pure form the isolated compounds

commulin-A, commulin-B and commulin-C showed effective scavenging activity

with the percent values 78.05 %, 89.45% and 78.05% with IC50 value 201 ppm, 98.15

ppm and 168 ppm respectively. Larvicidal effect of the plant was tested against the A.

aegypti and C. quinquefasciatus species. The ethyl acetate soluble fraction caused

maximum percent inhibition followed by the chloroform and the crude methanolic

extract at 600 ppm with values of 89.91 %, 85.21 % and 59.67 % respectively.

Acetyl cholinesterase assay was conducted by using three different concentrations of

250, 500 and 1000 ppm. The chloroform soluble fraction caused maximum percent

inhibition followed by the ethyl acetate soluble fraction, crude extract and the aqueous

soluble fraction with 89, 70.5, 68.55 and 50.75 % at 1000 ppm and IC50 values of 149,

156, 245 and 989 ppm respectively.

The crude extract along with the subsequent fractions were found safe in the in vivo

acute toxicity screening. Viola serpens as an effective hepatoprotective and

nephroprotective plant, was proved by evaluating various blood and urine parameters

(AST, ALP, ALT, urine clearance, urea creatinine and serum creatinine) against the

Paracetamol (PCM) induced toxicity and as evident by histopathological studies.

Being a safe drug, V. serpnse proved to be significant antinociceptive and anti-

inflammatory agents by using two different protocols for analgesia (Acetic acid

induced writhing test and formalin induced nociception test) and three for anti-

xviii

inflammation (carrageenan and histamine induced paw edema and xylene induced ear

edema). The plant extract and fractions were used at three different doses (100, 200

and 300 mg/kg). The plant proved to be an effective antinociceptive and anti-

inflammatory drug following the peripheral pathway for these activities mostly in a

dose dependent manner.

The plant extract and the different fractions with the decreasing polarity produced

more attenuated antinociceptive effect in a dose dependent manner. The effect is more

significant in the crude extract followed by the n-hexane, ethyl acetate, chloroform

and aqueous soluble fractions. Peripheral pathway is followed by either due to

inhibiting/reducing the release of cyclooxygenase or lipoxygenase enzymes or may

involve the release of certain mediators.

The anti-inflammatory effect of the plant was measured by using the protocols of

carrageenan and histamine induced paw edemas and xylene induced ear edema. In

histamine induced paw edema the crude extract at doses of 200 and 300 mg/kg

showed more pronounced anti-inflammatory effects that reached the maximum in the

3rdh. The n-hexane soluble fraction at a dose of 200 mg/kg in the 3rd h of histamine

induction showed maximum anti-inflammatory effect. The chloroform and aqueous

soluble fractions showed significant effects at the doses of 200 and 300 mg/kg on the

3rd h of histamine induced edema. Different percent inhibition values were obtained in

case of each fraction. In xylene induced ear edema the crude extract and n-hexane

fraction showed a dose dependent significance (Max. values at 300 mg/kg). This was

followed by the chloroform, ethyl acetate and aqueous fraction at doses of 200 and

300 mg/kg with the maximum inhibition values of 51, 49 and 48.5 % respectively.

xix

The chloroform and ethyl acetate soluble fractions were analyzed by column

chromatography which resulted in the isolation of six pure compounds. Among the

six compounds, three were new (not reported before) and the other three were already

reported from the other sources but obtained for the first time from this plant (V.

serpens) source. Commulin-A, Commulin-B and Commulin-C were the new

compounds whereas, tectochrysine, Sideroxyline and Cearoin were the already

reported compounds. 1H-NMR, 13C-NMR, COSY, NOESY, HMBC, IR, UV, E1-MS

and HRE1-MS were the different techniques used for elucidating the structures of the

new compounds.

In conclusion, V. serpens showed significant antimicrobial, antioxidant activities,

antinociceptive, anti-inflammatory, hepatoprotective and nephroprotective activities.

The marked pharmacological and phytochemical studies suggested further detailed

studies to confirm their folk uses and isolation of compounds that can act as potent

drug in future.

xx

Structures of the new compounds:

O

OOH

H3C

HO

OCH3

Commulin-A (1)

A

B

C

12

34

56

78

9

10

1'

2'3'

4'

5'6' O

OOH

H3C

HO

OCH3

Commulin-B (2)

HO

A

B

C

12

34

56

7

8

9

10

1'

2'3'

4'

5'6'

O

OOH

H3C

HO

OCH3

H3CO

Commulin-C (3)

A

B

C

12

34

56

78

9

10

1'

2'3'

4'

5'6'

Figure-1

O

OOH

H3C

HO

OCH3

A

B

C

12

34

56

78

9

10

1'

2'3'

4'

5'6'

H

HO

H

Figure-2: Key HMBC interactions in compound 2.

H

O

OOH

H3C

HO

OCH3

A

B

C

12

34

56

78

9

10

1'

2'3'

4'

5'6'

H

H

H

O

OOH

H3C

HO

OCH3

A

B

C

12

34

56

78

9

10

1'

2'3'

4'

5'6'

H

H3CO



xxi

Structures of the compounds isolated from V. serpens for the first time but already

reported from the other sources:

O

OOH

H3CO

9 2

34

56

78

10

Tectochrysine (4)

1'

2' 3'4'

5'6'

Figure 3.27: Structure of compound Tectochrysine (4).

O

OOH

H3C

H3CO

CH3

OH1'

2

345

6

78

9

10

2' 3'

4'

5'6'

Sideroxyline (5)

Figure 3.28: Structure of compound Sideroxyline (5).

O

OH

OCH3

OH

12

3

456

1'2'3'

4'5' 6'

AB

Cearoin (6)

Figure 3.29: Structure of compound Cearoin (6).

Chapter – 1 INTRODUCTION

1

CHAPTER – 1

1-INTRODUCTION

1.1 GENERAL INRODUCTION

The medicinal plants are enriched with ingredients that can be used in the synthesis of

drugs. Medicinal plants have been used for the cure of different ailments by the

diverse communities of the world for over thousands of years (Samuelsson, 2004). It

would be better to estimate the age of medicinal plants, being used for medication by

correlating it with the age of human civilization (Ramawat et al., 2009). The role of

medicinal plants can not be neglected in development of human cultures around the

world. World population use medicinal plants (approximatly 80-90 %) in raw and

unrefined extracts forms (Wanzala et al., 2005; Duke., 1985). The increased yearly

demand of herbal medicines globally, and particularly in the developing countries

clarifies its importance (Mahady, 2001). The consumtion of medicinal plant is going

on increasing with the passage of time (Wagner, 2009). The wide range of medicinal

plants used for the basic health care throughout the world is due to the uneconomic

and inaccessibility to the modern medical health facilities. Nature has kept treasures

of biologically active compounds in medicinal plants which facilitate the health

conditions of the huminity. The knowledge about these ethnomedicines is transferring

from one generation to another (Clark Hufford., 1993).

In most developing countries, people of the rural areas for their basic health care

mostly depend on medicine obtained from plants. This medication is comparatively

safer and inexpensive than the pharmaceutical products (Iwu et al., 1999; Idu et al.,

2007; Mann et al., 2008; Ammara et al., 2009). Isolation of active compounds from

plants is used for specific actions and characterization. In the 19th century morphine

was isolated from opium which was used particularly for the CNS related actions


2

(Kinghorn, 2001; Samuelsson, 2004). Plants of the Himalayan region are of great

importance in herbal pharmaceutical industries. These plants are affected by various

significant climatic factors such as drought, mutagenic UV-radiation, harsh winds etc.

These factors have great influence on the active constituents of the plants and their

metabolites.

According to the 2009 botanical survey total number of plant species estimated are

250,000 to 350,000 out of which 35,000 species are used as medicinal plants for the

management of numerous ailments (Fabricant and Farnsworth, 2001). According to

the current survey, 15% of all the medicinal plants were used for phytochemical

investigations whereas, only 6% were screened for their biological activities

(Farnsworth et al., 1985; Farnsworth, 1966).

World Health Organization (WHO) reported that the use of medicinal plants is

increasing day by day. In underdeveloped countries, plants are used approximately

80% (Ernst., 2000). In developing countries the percentage in different countries is

different for example: Ethiopia (90%), Benin (80%), India (70%), Uganda (60%) and

Tanzania (60%). However, in the developed countries it is approximately 70% in

Canada, France 49%, Australia 48%, Japan 60-70%, USA 40%, and Belgium 31%.

According to a report, the budget of the world traditional medicines is about US$

60,000 million and US$ 5 trillion is supposed to be in the year 2050 (WHO, 2002). In

developed countries the use of traditional medicines has sharply expanded in the 20th

century (ESCOP, 1999; Blumenthal et al., 1998). The scientific validity of medicinal

plants has increased their importance and uses. WHO has emphasized greatly on the

scientific study of the native herbal plants remedies especially in the developing

countries (Rates, 2001). Various active ingredients have been isolated from the plants


3

species which are particularly used for treatment of a particular disease (Qamar et al.,

2010).

Figure 1.1: Percentages of Medicinal Plants in Undeveloped & Developed Countries

1.2 TRADITIONALLY USED MEDICINAL PLANTS IN PAKISTAN

Almighty Allah has gifted our country with the treasure of medicinal plants. Both

cultivated and wild plants are the carpets of Pakistan’s land which posses great

potentialities. On the bases of photogeography, Pakistan has been divided into four

distant regions (Inrano-Turanian, Himalayan, Sindh and Indo-Pak). Pakistan has

diverse climatic zones with biodiversity found in its different parts. Species of

medicinal plants in the vast Pakistan’s flora are about 6000 (Ahmed et al., 1999). Out

of the total medically potent plants, 70% are found in the specific areas of the country

whereas; the remaining 30% are obtained from the various localities (Shinwari,

2010a). In various regions of Pakistan, from centuries the knowledge about local

medicinal plants has been practiced by about 40,000 unregistered and registered

tabibs/hakims (Saeed al., 2011). Mainly transfer of knowledge from one generation to

another occurs either verbally or through personal experiences applied and adopted


4

for the basic health problems (Shinwari et al., 2010a; Bhardwaj and Ghakar., 2005).

In 1950’s for the basic health care conditions approximately 84 percent of the

Pakistan’s population especially of the rural areas relied on the traditional medicines

of their locality (Hocking., 1958, Ahmed et al., 1999). With the passage of time

advancements were made in the knowledge related to the medicinal plants (Balick et

al., 1996).

Research work on phytomedicines has been carried out in Pakistan in the light of

pharmacological screening based on their folkloric uses. Research is also going on

for the discovery of lead compounds from these plants by isolating active ingredients

through the application of various isolation techniques. This instrumental level

research work in the country has paved the way for finding out best economical and

safe treatments of various diseases.

1.3 IMPORTANCE OF HERBAL MEDICINES IN DIFFERENT

TRADITIONS

Herbal remidies serve as healing tools for the management of various diseases in local

areas as well as world wide. Some of the traditional roles of medicinal plants are

given below.

1.3.1 Chinese Traditional Medicines

Chinese herbal medicines are very old and most of its citizens rely on the traditional

medication. More than 50% of the Chinese of the rural areas for the basic health care,

use their traditional medicines because China has also been gifted with thousands of

medicinal plants. Out of twelve hundred medicinal plants, tabibs/hakims use five

hundred medicinal plants most commonly (Li., 2000) WTO (World Trade

Organization).


5

1.3.2 Japanese Traditional Medicines

Japanese traditional system was derived from the Chinese system. In the 19th century.

Japanies classified their native plants in their first traditional pharmacopeia (Saito et

al., 2000).

1.3.3 Indian Traditional Medicines

Ayurveda system of traditional medicines is about 5000 years old. It was first

practiced in India for finding out solutions to the various health related problems

(Morgan., 2002).

1.4 Violaceae Family

Viola is an important genus of the family Violaceae. It is medicinal plant of great

importance on the basis of both its photochemistry and pharmacology. Violaceae is

also known as Retrosepalaceae /Alsodeiace/ Leoniaceae (Mabberley., 1987; Perveen

et al, 2009). The family includes about 23 genera which are tropical and consists of

about 930 species (Burman., 2010). Approximately 111 species were identified in

China (Wang et al., 1991) and about 17 species are distributed in different localities of

Pakistan (Qaiser et al., 1985; Marcussen et al., 2010). Eastern Asian mountains are

said to be the important taxonomical and pharmacological hubs of viola (Ballard et

al., 1999).


6

1.5 LITERATURE SURVEY OF GENUS VIOLA

The genus is distributed through out the world in various parts of the globe. Some of

the species along with their part/s used, distribution, chemical constituents and uses

are presented in the Tables 1.1-1.3.

Table 1.1: Viola species list, native to Pakistan and their world wide distribution,

Viola parts used, chemical constituents and medicinal uses

S.No. Botanical

name of the

specie

Part/s

Used

Geographical

distribution

Chemical Constituents Uses

1 Viola

betonicifolia

Whole

plant

(Leaves,

roots,

flowers)

Pakistan,

Malaysia,

India, China,

Nepal, Sri

Lanka,Burma,

Japan,

Australia

&Taiwan

3-methoxydalbergione

(Muhammad et al 2014), 3-

Methoxy dalbergion,

Undecanoic acid, 2,4-

Dihydroxy, 5-

methoxycinnamic acid, 4-

Hydroxy coumarine, Beta-

Sitosterol, Ursolic acid,

benzoic acid, Trihydroxy

benzoic acid (Muhammad

et al., 2012a)

As astringent, antipyretic

anticancer, diaphoretic &

purgative (Shinwari et al.,

2010a). In epilepsy, nervous

disorders, for sinusitis, blood

abnormalities, skin diseases,

cough, cold, pharyngitis

(Bhatt and Negi et al., 2006)

an astringent, for cooling

effect, diuretic, laxative and

purgative (Husain et al.,

2008). For kidney diseases,

bronchitis, pneumonia and

boils (Husain et al., 2008).

Urease inhibitor (Muhammad

et al., 2014)

2 Viola biflora Whole

plant

Europe,

Central Asia,

India, China,

Korea,

Pakistan

America &

Japan

Protein [ vibi A-K,

(cycloviolacin O2, O9, varv

A, vitri A) (Burman et al.,

2010)

As antispasmodic, antiseptic,

cough, cold, diaphoretic,

laxative emetic, antipyretic,

intestinal pain, leucoderma,

Psoriasis and dermititis

(Chandra et al 2015).

3 Viola

canescens

Whole

plant (oral)

India,

Pakistan,

Nepal and

Bhutan

Alkaloid violin, methyl

salicylate, quercitrin,

glycosides and saponins

(Rana et al., 2010).

Alkaloid (emetine) malic

acid, glucoside (viola

quercitrin).

For Cold, cough, respiratory

problems, as antipyretic,

antimalarial, demulcent,

astringent, diaphoretic,

purgative, febrifuge, anti

cancerous, carminative,

demulcent, antimicrobial,

treating nervous disorders,

eczema, anti epileptic,

anticancer, anti rheumatic,

heart burn, boils and sore

throat (Hamayun et al., 2006;

Hussain et al., 2011; Rani et

al., 2013)

4 Viola cinerea Whole

plant (oral)

specially

roots and

leaves

Yemen (Kilian

et al., 2004)

Iran, Pakistan

and Oman.

Triterpenoids (Tabba et al.,

1989), cyclotide alkaloids

(Chen et al., 2005).

Flavonoids (Vukics et al.,

2008). Caffeic and

Aphrodisiac (Marwat, 2008)


7

salicylic acid (Toiu et al.,

2008)

5 Viola

falconeri

Flower and

roots

India and

Pakistan


1989) cyclotide alkaloids



2008). ). Caffeic and


2008).

Flower used for cold and

cough whereas, roots used

against jaundice (Saqib and

Sultan, 2005).

6 Viola

fedtschenkoan

a

Whole

plant

Central Asian

countries

including

Northern

Pakistan







2008).

Not reported

7 Viola

kashmiriana

Whole

plant

India,

Pakistan,

Kashmir and

Afghanistan







2008)

Applied on scores and ulcers,

heals swollen mouth and foot

disease in cattles. It also cure

bronchitis (Ishtiaq et al.,

2006)

8 Viola

kunawurensis

Whole

plant

Afghanistan,

Pakistan,

India, Nepal,

China,

Turkestan, and

Tibet.

Not reported Not reported

9 Viola

macroceras

Whole

plant

Pakistan,

Afghanistan

Not reported Not reported

10 Viola

makranica

Pakistan Not reported Anti-inflammatory and

analgesic.

11 Viola odorata Whole

plant

especially

leaves

Europe Asia

and North

Africa.

Glycoside, salicylic acid

and essential oils (Furfural

α-Terpinene, α Thujene,

para-methyl Anisole, β-

Phellandrene,

α-Pinene, Sabinene ,

Myrcene, δ-3-Carene, Z-β-

Ocimene, Benzyl alcohol,

γ-Terpinene,

Acetophenone, Z-Sabinene

hydrate , Methyl benzoate,

Linalool, Z-linalol oxide,

8-para-Menthatriene, ortho-

Menthatriene, Z-para-

menth-2-en-1-ol , 1-

Terpineol, Ethyl

benzoate,Terpinen-4 ol,

Geraniol, α- Terpineol,

Pulegone , δ-Elemene, α

Cubebene , Isoledene, α-

Copaene , β- Bournonen, β-

Cubebene , α Gurjunene ,Z-

Caryophyllene, β

Antifungal, antimicrobial, for

cold, respiratory problems

such as congestion, sore

throat and coughing. As a

laxative, sedative, analgesic,

expectorant in digestive

disorders blood cleansing,

Jaundice, and headache

(Hammami et al., 2011;

Gautam and Kuma., 2012;

Amiri et al., 2013). It is also

used for the treatment of

catarrh, chronic bronchial

asthma, upper respiratory

tract symptoms, cold,

rheumatism, oral mucosa

inflammation, hysteria,

nervous strain, antipyretic,

insomnia, headache and

diaphoretic (Fleming et al.,

1998; Zargari., 1997; Dhar et

al., 2002).


8

Duprezianene, α-Guaiene

(Hammami et al ., 2011;

Stuart, 1989).

12 Viola

reichenbachia

na

Whole

plant

Pakistan Unknown Being an important medicinal

plant it is used in headache,

fever, cough, asthma,

constipation, bleeding piles,

skin diseases and throat

cancer. Used also as

diaphoretic and demulcent

(Kumar and Digvijay, 2014)

13 Viola

rupestris

Whole

plant

Pakistan Not reported Unknown

14 Viola serpens Whole

plant

India,

Pakistan,

Banaladash,

Ceylon, Nepal,

China and Java

(Rahman and

Choudhary.,

2012)

Flavonoids, terpenoids,

reducing sugars, amino

acids and tannins, methyl

salicylate, alkaloid voiline

gum, mucilage, glycoside,

quercitrin and saponin

(kumar et al., 2015).

As demulcent, diaphoretic

diuretic, in fever, jaundice,

asthma, piles bleeding, throat

cancer, constipation, cold,

cough, dermatitis and

headache (Kumar and

Digvijay, 2014; Kumar et al.,

2015).

15 Viola stocksii Whole

plant

India,

Pakistan, Iran,

Yemen and

Afghanistan

(Chandra et al.,

2015).

Not reported Virility in sexual masculine

power

(Marwat et al., 2008).

16 Viola tricolor Areal parts

(flowers)

Pakistan,

Europe, Asia,

America,

Australia,

Germany,

Turkey and

Spain.

Flavonoides, quercetin,

luteolin and luteolin 7-

polysaccharides, phenolic

acids, volatile oil,

Carotenoids, anthocyanins ,

cyclotides, tocopherol,

triacyl glycerolsglucoside

and Proteins (Burman et al.,

2010). Violaxanthin, vitri

peptide A, varv peptide A

(Craik et al., 1999; Molnar

et al., 2004). Monoterpenes,

sesquiterpenes, shikimic

acid derivatives and

aliphatics. Volatile

components (bisabolone

oxide) and trans-β-

farnesene

(Anca et al., 2009). Violine,

sugar resin, mucilage and

salicylic acid (Ghorbani et

al., 2012).

Dermatitis (Chevallier., 1996;

ESCOP 2009). Cystitis,

bronchitis, expectorant, anti-

inflammatory, diuretic skin

conditions, and rheumatism

(Anca et al., 2009). As anti-

epilepsy, anti-asthmatic, in

heart problems, inflammation

of lungs and heart (Ghorbani

et al., 2012).

17 Viola

turkestanica

Whole

plant

Pakistan,

India, Nepal

and Butan.

Ephidrin As nasal drops and used

mostly in veterinary (Anca et

al., 2009).


9

Table 1.2: List of other Viola spp. of the family Violaceae, their geographical

distributions, Medicinal uses and part/s used mostly

S.No Botanical Names Geographic Distribution Medicinal uses

1 Viola arvensis

(whole plant)

Romania anti-inflammatory, expectorant, diuretic skin

conditioner, bronchitis, cystitis and rheumatism

(Anca et al., 2009)

2 Viola adunca

(whole plant

especially flowers)

Pacific Northwest Spring-flowering nectar source and as a larval

host (NRCS Plant Guide).

3 Viola Canadensis

(Roots)

North America. Roots decoction used for bladder’s pain.

(NAGPTHG, 2005).

4 Viola diffusa

(whole plant)

Japan, East Asia, South

China, & Philippines.

(Benecke et al., 1985; Zhou

et al., 2008).

Used for hepatitis treatment (Dai et al., 2015).

5 Viola hondoensis

(Whole Plant)

Korea and Japan (Hikosaka

et al., 2010)

As expectorant, anti-inflammatory, diuretic, for

bronchitis, eczema rheumatism, and skin

eruptions (Moon et al., 2004)

6 Viola japonica

(whole plant)

Korea, Eastern Asia, Taiwan,

Shikago, Japan and China

(Benecke et al., 1985).

As anti- inflammation, detoxifier, as blood cooler

and pain alleviator. Used in boils, abscesses,

ulcers, acute conjunctivitis, acute jaundice,

laryngitis, hepatitis and in various kinds of

poisonings. In chest and lungs troubles, as

expectorant and for the treatment of chronic

catarrhal accumulations. Crushed leaves applied

to the cuts, ulcers, swellings and wounds (Moon

et al., 2004).

7 Viola pedata

(whole plant)

South Kurile Islands, East

America, Siberia Japan,

Korea and Dongbei (Benecke

et al., 1985).

In headache, dysentery, colds coughs, boils, as

expectorant and for lubricating medicine

((Moerman, 1998; Native American Garden).

8 Viola pubescens

(whole plant)

Northen America Colds, cough, headache, dysentery and used for

boils. (National Audubon Society, 1979)

9 Viola vulgaris Japan, Korea, Dongbei and

Himalayan regions (Benecke

et al., 1985)

Used symptomatically in mild seborrhea skin

conditions.(Community herbal monograph)

10 Viola yedoensis

(whole plant)

Flavones, coumarins, fatty

acid, phenolic acids (Hong et

al., 2011). Polysaccharides,

flavonoides, luteolin,

quercetin, volatile oil,

phenolic acids, carotenoids,

cyclotides, anthocyanins ,

triacyl glycerols glucoside

and tocopherols (Assessment

report on Viola tricolor)

For relief of symptoms and conditioning of mild

seborrhoeic skin. (Community herbal

monograph).

It is also used as an anti-rheumatic and for

treating infections like mastitis, rhinitis and for

treating acute pyogens (Jun-Li et al., 2011).

11 Viola yazawana

(whole plant)

Japan, Korea, Dongbei and

Himalayas region. (Benecke

et al., 1985).

As an anticoagulant and antithrombotic (Kumar

et al., 2011).

12 Vi!ola hederacea

(flowers, leaves)

Australia and Melbourne Flowers eaten especially by the Victorian but the

exact use is unknown (David, 2005).


10

Table 1.3: List of other species of Viola and their geographic distributions

S.No. Botanical Names Geographical Distribution

1 Viola alliaraefolia Mexico, North America and Japan (Benecke et al., 1985).

2 Viola bissettii Korea, Japan and Himalayas region (Benecke et al., 1985).

3 Viola banksii East Australia (Benecke et al., 1985).

4 Viola blandaeformis America and Japan (Benecke et al., 1985).

5 Viola brevistipulata Mexico, North America and Japan (Benecke et al., 1985).

6 Viola Canadensis North America (Benecke et al., 1985).

7 Viola confuse Japan and Taiwan (Benecke et al., 1985).

8 Viola diffusa East Asia, South China, Philippines and Japan (Benecke et al.,

1985; Zhou et al., 2008).

9 Viola faureana Japan (Zhou et al., 2008).

10 Viola fedtschenkoana Northern Pakistan and Central Asia (Zhou et al., 2008).

11 Viola eizanensis Korea, Japan and China (Benecke et al., 1985).

12 Viola grypoceras Japan and Korean islands (Benecke et al., 1985).

13 Viola grayii Japan (Benecke et al., 1985).

14 Viola hirtipes China and Korea (Benecke et al., 1985).

15 Viola hondoensis Korea and China (Richard et al., 1985).

16 Viola hultenii Korea, Japan, Kurile Islands and Siberia (Richard et al., 1985).

17 Viola hederacea Australia and Melbourne (Richard et al., 1985).

18 Viola iwagawai Ryukyus, Yakushima and Okinawa (Richard et al., 1985)

19 Viola japonica Japan, China, Eastern Asia, Shikago, Taiwan and Korea (Richard

et al., 1985).

20 Viola kusanoana Japan, Soviet Union and North Korea (Richard et al., 1985).

21 Viola kitamiana Australia and Malaya. (Richard et al., 1985).

22 Viola keiskei Japan, Korea, Ussuri and Manchuria (Richard et al., 1985).

23 Viola maximowicziana Japan and china (Richard et al., 1985).

24 Viola langsdorffii Alaska, Northern Eastern Asia and North America (Nathorst et

al., 1883; Richard et al., 1985).

25 Viola lactiflora Korea, Japan, North China and Manchuria (Richard et al., 1985)

26 Viola maximowicziana Japan (Richard et al., 1985)

27 Viola mandschurica Japan, China and Taiwa (Richard et al., 1985).

28 Viola nanligensis China (Jin-Zhou et al., 2008)

29 Viola nagasawai China (Jin-Zhou et al., 2008)


11

30 Viola ovato-oblonga South Korea (Richard et al., 1985).

31 Viola obtuse Korea and Japan (Richard et al., 1985)

32 Viola orientalis Japan, Koria, China and Southern Soviet (Richard et al., 1985)

33 Viola pedata Southern Kurile Islands, Siberia, Korea, Japan and Dongbei,

Japan (Richard et al., 1985)

34 Viola palustris Europe and America (Nathorst et al., 1883).

34 Viola patrinii Canada (Richard et al., 1985).

35 Viola pubescens Headache treatment (leaves poultice), blood, cough, colds,

dysentery (infusion), boils (crushed root).

(National Audubon Society)

36 Viola phalaerocarpa Korea, Japan and China (Richard et al., 1985).

37 Viola repens Korea and Japan (Richard et al., 1985).

38 Viola rostrate Japan, North America, Asia and Georgia (Nathorst et al., 1883).

39 Viola raddeana Japan, Korea, Amur and Southern Manchuria (Richard et al.,

1985).

40 Viola rossii Japan, Korea and Himalayas region (Richard et al., 1985).

41 Viola sachalinensis Japan (Richard et al., 1985).

42 Viola shikokiana Japan, Korea, Himalyan region and Dongbei (Richard et al.,

1985).

43 Viola seiboldii Korea, Japan & china (Richard et al., 1985).

44 Viola selkirkii Afghanistan, India, Japan, North America, Sweden, Iran,

Greenland Norway, Russia, Caucasus, Siberia, Altai, Baikal,

Manchuria, Kamtschatka and British Columbiia. (Nathorst et al.,

1883).

44 Viola teshioensis Japan, china and korea (Richard et al., 1985).

45 Viola tashiroi Japan and china (Richard et al., 1985).

46 Viola tokubuchiana Japan (Richard et al., 1985).

47 Viola utchinensis Japan (Richard et al., 1985).

48 Viola verecunda Tasmania, Taiwan and New Zealand (Richard et al., 1985).

49 Viola variegata China, Japan (Richard et al., 1985).

50 Viola violacea Yakushim, Korea, Japan and Goto Islands (Richard et al., 1985).

51 Viola vaginata Korea, Japan, Himalayas region and Dongbei . (Richard et al.,

1985).

52 Viola yubariana Japan, Mexico and North America (Richard et al., 1985).

53 Viola yezoensis Japan, Hokkaido and Tokyo (Richard et al., 1985)


12

1.6 PLANT INTRODUCTION

1.6.1 Viola serpens Wall

V. serpens Wall. is the synonym of Viola canescens Wallich ex Roxburgh (Satish et

al., 2013; Masood et al., 2014). The common names are Smooth-Leaf White Violet,

Ghatte ghans and Huikhon in different languages (Chauhan et al., 2003). Its common

English name is Himalayan White Violet because it is mostly found in the Himalayan

region (Masood et al., 2014).

1.6.2 Local Names

V. serpens is known with different names in different localities of Pakistan and India.

Its Urdu name is Banafsha (Saqib et al., 2014; Ahmad and Habib., 2014; Ahmad et

al., 2012; Barkatullah et al., 2012; Ali et al., 2011; Hamayun et al., 2006; Hamayun et

al., 2005; Shinwari et al., 2000; Naain, 1999). In KPK, it is called as Banaqsha,

Banafsha or Savar Phal (Adnan and Hoscher., 2010) in Baltistan as Skoramindoq

(Hussain et al., 2014). In lesser Himalyas it is known as bamasha, Phulnaqsha or

sweet violet. Indians call it with the name Ratmundi or Vanaksha (Rana et al., 2014),

Vanafsha (Dua et al., 2011; Suyal et al., 2010). In Himachal Pardesh, it is called

Gugluphul (Kumar et al., 2013), Banaksha and Banfasa (Rani et al., 2013). In

Uttarakhand, it is commonly called Vanfsa (Rana et al., 2010). In Nepal it is locally

called Ghatteghaans (Adhikary et al., 2011).

1.6.3 Morphology

V. serpens being a perennial herb with tufted appearance. The plant occurs both in

cluster or solitary forms. There is no clear stem (short stem) and the leaves seemed to

be originated directly from the creeping roots. Leaves are long, narrower, ovate-lance

shaped and pointed, white thin hairs may or may not be present above, 5-7 veined.


13

Leaf-stalk and leaf blade are almost of the same length. Flowers are lilac or almost

white in color with 1-1.5 cm across. Normally the upper petals at the base bear hairs.

Stigma is beaked and having 3-lobs. Stipules are toothed and not fringed. Stem is light

green, straight, somewhat pubescent or glabrous. Fruits are in pods, which are pale

brown in color. Each pod contains many tiny blackish seeds. The plant odor is very

good and attractive.

1.6.4 Classification (Taxonomical position of V. serpens)

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Sub class: Dilleniidae

Super order: Dillenianae

Order Malpighiales

Family: Violaceae

Genus: Viola

Species: Serpens/ Pilosa.

Herbarium: Submitted in the Department of Botany, University of

Peshawar, Peshawar.

Voucher No: Bot. 20158 (PUP).

1.6.5 Geographical Distribution of V.serpens

1.6.5.1 World Wide Distribution

The world wide geographic distribution of V. serpens is in Pakistan, China, India, Sir

Lanka, Nepal, Java, Philippines, Thialand and Nagaland.


14

1.6.5.2 Distribution in Pakistan

In Pakistan V. serpens is distributed widely in Swat, Shangla, Buner, Chitral, Hazara,

Kaghan valley and Shogram (Barkatullah et al., 2012; Hamayun et al., 2006; Shinwari

et al., 2000). It is also found in Rawalpindi, Kotli Sattian, Azad Jammu Kashmir and

NeelumValley (Ahmal et al., 2012; Ahmad and Habib., 2014; Saqib et al., 2014).

Tropical and temperate zones are the hubs of this plant which is restricted only to the

hilly areas (Singh et al., 2005). In Pakistan, V. serpens is also found in the localities of

Shawal (NorthWaziristan), Parachinar (Kurrum Agency), Swat, Dara Adam Khel,

Teera (Orakzai Agency), Bajour, Razmak, Miran Shah (South Waziristan), Fizagut

and Kaalam (Shinwari et al., 2010a). In the temperate Himalayas Mountains the plant

exsists at about 2000 m of elevation (Kumar et al., 2013).


15

1.7 PLANT DESCRIPTION

They grow as perennial herbs, shrubs, and trees or treelets (Hekking., 1984,

Munzinger et al., 2003). Viola is the Violaceae largest genus, distributed greatly in the

northern hemisphere (Marcussen et al., 2012). Rhizomes of viola are somewhat

elongated but mostly short. Usually maximum height of the plant is about 4 inches or

they lack the short stem. Similarly the branched stem arises from the hairy rhizomes if

present (Muhammad et al., 2012), which ends with winged or non-winged leaves

which may be ovate-triangular stalked, cordate, crenate or serrate. Flowers are

zygomorphic, different colors in different species like pale, blue, white and violet.

Most of the species have violet color flowers due to which the family is also known as

violet family. Each flower arises singly from the axil of the leaves with a along stalk

comprises of unequal sized five petals. A spur is formed by the lower ones containing

nectar for attracting insects for pollination. (Clark and Trelawny., 1998). The number

of calyx is five which surround the petals of the flower. Three united carpels form a

compound pistil. Stamens number is also five, short filaments with coherent anthers

and form ring around the gynoecium. Fruits are inferior (hypergynous) to ovules.

Ovary is surrounded by the anthers which are connate, filaments are broad separated

and short, the lateral two ends resulting in the corolla spur formation. Stigma is lobed,

usually beaked or straight. The shape of the seeds pod (ovary) differs in different

species which may be pointed or triangular. Seeds are smooth, shiny with caruncle

(fleshy outgrowth) important for dispersion automatically by wind when the seed pod

bursts (Qaiser et al., 1985; Reznick and Voss, 2012; Clark and Trelawny., 1998).

Such flowers are known as cleistogamous flowers. The floral formula of the flower is

K5 Co5 S5 P (3). Ovary is sessile with curved style at base.


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1.8 ETHNOMEDICINAL USES

Various species of viola with their medicinal uses, geographic distribution, parts used

and chemical constituents after comprehensive literature survey have been enlisted in

the previous Tables 1.1 and 1.2.

1.9 PHYTOCHEMICAL INVESTIGATIONS

1.9.1 Nutritive Values

V. betonicifolia was investigated for various elemental studies. The study clarified the

presence of micronutrients Pb, Cu, Cr, Fe, Mn, Ni, Zn and macronutrients Na, K and

Ca in different parts of the plant. The concentration of elements was found in all parts

of the plant in all fractions with different percentage values. V. betonicifolia plant also

contains certain phytochemical nutrients such as carbohydrates, proteins, sterols and

triterpenoids, alkaloids, tannins and saponins (Muhammad et al., 2012).

The different percent elemental composition of V.odorata flowers contained elements

like carbon 14-26 %, Oxygen 42.39%, magnesium 0.9% (Bibi et al., 2006). The

studies confirm nutritional values of the genus.

1.9.2 Essential and Fixed Oils

The isolation of volatile oils from various species of Viola has been reported.

Composition of few species so far has been studied for essential oils. The analysis of

GC-MS of V.betinocifolia whole plant determined presence of about 53 fixed oils. V.

odorata GC-MS analysis reported existance of 23 volatile oils. Mostly volatile oils

are the derivatives of shikimic and aliphatic acids from the leaves. Essential oil in the

form of methyl salicylate has been reported from V. etrusca (Anca et al., 2009).

Essential oils reported from V. tricolor and V. arvensis are composed of aliphatic,


17

monoterpenes, sesqueterpenes and shikimic acid derivatives. The total reported

essential oils are 35 in number (Anca et al., 2009).

1.10 ISOLATED COMPOUNDS FROM GENUS VIOLA

The literature survey describes isolation of important pharmacologically active

compounds from various species of the genus. These isolated compounds belong to

various naturally occurring classes such as cyclotide alkaloids (Simonsen et al., 2005),

salicylic acid (Toiu et al., 2008), flavoniods (Vukics et al., 2008a; Vukics et al.,

2008b), derivatives of caffeinec acid (Toiu et al., 2008) and triterpenoids (Tabba et

al., 1989). Some of the secondary metabolites from various species of the genus are

presented in the Table 1.1.

1.11 PHARMACOLOGICAL STUDIES

Various pharmacological studies including in vivo and in vitro screening have been

carried out by different researchers in different eras.

1.11.1 In vivo Biological Activities

1.11.1.1 Antibacterial Activity

Crude aqueous methanolic extract of V. betonicifolia whole plant with the subsequent

fractions were applied to the antibacterial activity. The in vitro anti-bacteria bioassay

was conducted against Bacillus cereus, Escherichia coli, Staphylococcus aureus,

Enterobacter aerogenes, Proteus mirabilis, Salmonella typhi and Enterococcus fecalis

in which remarkable activity against E. coli and Salmonella typhi was observed

(Naveed et al., 2013b). V. odorata showed pronounced effect against the tested

microorganisms (Hassan and Naeem., 2014). The methanol soluble extract and its

subsequent fractions, the aqueous, acetone and petroleum ether soluble extracts

showed antimicrobial activity. The methanolic extract showed maximum inhibition


18

against Haemophilus influenzae (24 mm) and Streptococcus pneumoniae (19 mm).

Whereas, lowest inhibition observed against Pseudomonas aeruginosa (13 mm) was

caused by aqueous, acetone and petroleum ether in the descending order (Kinghorn et

al., 2007). V. odorata possessed about 5.2 % triterpene, saponins, ursolic acid in the

form of glycone and galacturonic acid or galactose (Rastogi., 1984). Some toxic

metabolites may be responsible for the significant antimicrobial activity. V. odorata

aqueous fraction (flowers) revealed effective antibacterial activities against S. aureus

E. coli and B. subtilis (Khatibi et al., 1989). The aerial part at a concentration 3, 2 and

1 mg/kg was effective against B. subtilis, S. aureus and E. coli (Ramezani et al.,

2012). Cyclotides (Cyclotide cycloviolacin O2) are peptides which are rich in small

disulfide, obtained from the dried areal plant of V. odorata. Oils of V. odorata

methanolic extract are also effective against microbes (Hammami et al., 2011). It

efficiently inhibits the growth of E. coli, K. pneumonia and P. aeruginosa but does

not show any effect against S. aureus (Pranting et al., 2010; Ashfaque Khan et al.,

2011). The range of MICs showed by the standard antibiotic erythromycin was 3.12

to 12.5 mg/ml whereas, MICs presented by V. odorata fractions against S.

pneumoniae, H. influenzae and S. pyogenes were similar to the standard (6.25 mg/ml).

Better MIC showed by the methanolic extract against S. aureus (3.12 mg/ml) and

least MIC (12.5 mg/ml) was recorded against P. aeruginosa. MICs of ethyl acetate

fraction for E. coli and K. pneumoniae were 10.l g/L and 5.5 lg/L respectively

(Gautam and Kumar., 2012). The use of V. odorata for treating respiratory infections

provides a rationale for future study (Khan et al., 2011; Salve et al., 2014).

Similarly, ethanolic extract of leaves of V. serpens Wall. was used (in vitro) against

bacterial diseases. The selected species of microbes were S. typhi, E. coli, S. aureus

and K. pneumonia. The result was that maximum antibacterial activity was shown by


19

the ethanolic extract of V. serpens. The zone of inhibition values of the plant extract

and different antibiotics were compared which proved to be more effective against the

microbes (Kumar et al., 2015). The isolated cyclotide (vhl-1) from the V. hederaceae

was also tested for the same activity against E.coli and S. aureus but the effect was

negative and showed no inhibition (Chen et al., 2005).

1.11.1.2 Anti-fungal Activity

The hydromethanolic extract/fractions of V. betonicifolia were subjected to antifungal

activity. In vitro antifungal bioassay of the plant was performed against Aspergillus

niger, Aspergillus parasiticus, Candida albicans, Juncus effuses, Saccharomyces

cerevisiae, Trichophyton rubrum and Fusarium solani. The tested samples including

crude aqueous methanolic extract, ethyl acetate and chloroform fractions showed to

be effective against the selected fungi except C. Albicans. V. betonicifolia plant is

used effectively as an antifungal source (Muhammad et al., 2013). Various fractions

of V. canescens with different solvents (acetone, ethanol, petroleum ether & water)

were tested for the antifungal activity. Ethanol, petroleum ether & water showed

intermediate antifungal activity whereas, a dose of 1000 mg/ml acetone extract

showed maximum inhibited zone. The extracts of ethanol as well as the petroleum

ether also showed highest MIC (Rawal et al., 2015). The antifungal activity of V.

odorata crude extract and its essential oil were also investigated against Botrytis

cinerea. Against the tested pathogenic fungi the applied oils obtained from the plant

also showed strong antifungal effect (Kumar et al., 2011). The whole plant of the

crude extract of V. tricolor was tested against C. albicans which showed mild

antifungal activity against fungal diseases/infections (Banaszezak et al., 2005). The

isolated cyclotide (vhl-1) from the V. hederaceae was also tested for the antifungal


20

activity against C. albicans which showed satisfactory effect against the tested fungi


1.11.1.3 Antiprotozoal Activity

Four different concentrations of V. canescens plant were tested for the anti-protozoal

activity. The extract of petroleum ether among the other extracts showed effectiveness

against Leishmania donovani and Trypanosoma cruzi (Dua et al. 2011).

1.11.1.4 Cytotoxic Activity

V. canescens was tested in the infected rats for the cytotoxic activity for the skeletal

myoblasts (L-6 cells) which proved to be non-cytotoxic (Dua et al., 2011).

1.11.1.5 Haemolytic Activity

Cytotoxic and haemolytic activities are also shown by the plant having cyclotides so

the species of genus Viola have been gifted by nature with these cyclic peptides which

showed cytotoxic and haemolytic activities (Gran., 1973; Salve et al., 2014).

1.11.1.6 Antiplasmodial Activity

V. canescens petroleum ether fraction showed effective results for the antiplasmodial

activity (Dua et al., 2011). In South Korea various species of viola family i.e V.

albida, V.acuminate, V. dissecta, V. grypoceras, V. hondoensis, V. japonica, V.

keiskei, V. ibokiana, V. lactiflora, V. mandshurica, V. takeshimana, V. tokubuchiana,

V. varigata, V. verecunda and V. websteri were used for antispasmodic activity. The

isolated compound, epi-oleanoic acid from petroleum ether is also an antispasmodic

compound with IC50 value 0.18 µg/mL (Moon et al., 2007). V. websteri tested against

plasmodium falciparum showed antispasmodic activity (Lee et al., 2009). Two

compounds named 6-(8’ Z-pentadecenyl)-salicylic acid and 6-(8’z, 14’ Z-

heptadecatrienyl)-salicylic acid isolated from V. websteri with petroleum ether solvent


21

showed outstanding antispasmodic activity against the strain of P. falciparum having

sensitivity against chloroquine (Lee et al., 2009; Moon et al., 2007).

1.11.1.7 Anti-malarial Activity

Petroleum ether fraction of V. canescens collected from Northwestern Himalaya

proved as an anti-malarial. The extract showed its inhibitory action in comparison

with the standard drug against the causative agent of malaria (P. falciparum) (Verma

et al., 2011).

1.11.1.8 Anthelmintic activity

V. betonicifolia was tested for nematocidal activity. Anthelmintic activity of the plant

extract along with the subsequent fractions was tested against worms of various

species which resulted in significant vermicidal activity. Ethyl acetate and chloroform

fractions proved to be more effective showing 42 and 58 min. death time against P.

posthuma, respectively. The same fractions mortality rates after 48 h against C.

littoralis were 66 and 62% respectively. Whereas, against H. indicus ethyl acetate and

chloroform fractions showed 49 and 57% mortality rates respectively. On the bases of

these results V. betonicifolia being a natural source can be significantly used for

anthelmintic activity (Naveed et al., 2012).

Cyclotide shows anthelmintic activity. Various cyclotides isolated from V. odorata

were cycloviolacin O14, cycloviolacin O2, cycloviolacin O13, cycloviolacin O15,

cycloviolacin O8, cycloviolacin O16 and cycloviolacin O3. In nematode larval

development assays these cyclotide showed about 18-fold higher potency as

compared to the kalata B1 prototypic cyclotide. Cycloviolacin O14 and cycloviolacin

O2 are more potent than kalata. Cycloviolacin O2 residues, lysine and glutamic acid

residues are the most effective anthelmintic cyclotide. The anthalmentic activity of


22

cyclotide e.g methylation results in six times decrease in its activity. Acetylation

masks the positive charge effectively and the anthalmentic activity is decreased by18-

fold. It is concluded that V. odorata is a significant vermicidal or anthelmintic in

nature (Wang et al., 2008; Muhammad et al., 2012).

1.11.1.9 Antioxidant Activity

The crude ethanolic extract of V. serpens was investigated for the antioxidant activity.

The presence of certain phytochemicals in the ethanolic extract of the V. serpens

showed that it is an effective antioxidant plant. Ascorbic acid a non-enzymatic

antioxidant and the antioxidants enzymes such as catalase, ascorbate oxidase

peroxidase are present in V. serpens (Kumar et al., 2011). DPPH, standard scavenger

was used for the evaluation of antioxidant potential of Viola tricolor (Vukics et al.,

2008; Nikolova et al., 2011). Viola odorata also proved a strong antioxidant natural

source. It was verified by using DPPH reducing power assay, hydrogen peroxide and

ferric thiocynate scavenging protocols. Phytochemical investigation of the flavonoids

and the total phenolic compounds measurement in the plant extracts scientifically

strengthen it as strong antioxidant source. (Ebrahimzadeh et al., 2010)

1.11.1.10 Anti-Tubercular Activity

Subsequent fractions of the crude ethanolic extract of V. odorata were investigated for

the anti-tubercular activity. T.B causing bacterial stains, M. tuberculosis H37Rv and

MDRTB were used in the study. Among various fractions the dichloromethane and n-

hexane showed significant activity against both the strains. Isolated compounds

salicin and phenyl alanine ethyl ester from V. odorata were tested purely against the

tested strains. It was concluded that V. odorata is a rich source of important chemical

constituent which are effectively used against the M. tuberculosis H37Rv and M.


23

avium strains. Thus this plant is a lead potential anti-TB drug (Hassan and Naeem.,

2014).

1.11.1.11 Treatment of Jaundice

Leaves extract of V. serpens was tested for the antibacterial activity against the

jaundice causing bacteria. Bacterial strains were isolated from patients suffering from

jaundice and treated against the crude methanolic extract which showed pronounced

effect against the particular strains. Crude methanolic extract was applied which

showed pronounced effect against jaundice. The scientific proof increases the plant

importance for its use as remedies in jaundice along with many other infectious

diseases (Kumar et al., 2015).

1.11.1.12 Urease Inhibitory Activity

3-methoxy dalbergion is an isolated compound from V. betonicifolia (Naveed et al.,

2014). The mechanistic study on this natural compound as a urease inhibitor was

carried out by linking docking studies along with the enzyme kinetics. Findings of the

study are that the new naturally isolated compound (3-Methoxydalbergione) proved to

be helpful in the urease linked diseases. Urease is a harmful compound which is

directly involved in infectious stones formation, encrustation pyelonephritis,

urolithiasis, ammonia, hepatic coma, hepatic encephalopathy and urinary catheter.

Moreover, it is the main cause of peptic and gastritis ulcers induced by Helicobacter

pylori (Naveed et al., 2014).

1.11.1.13 Anti-HIV Effect

From various species of viola more than hundred macrocyclic compounds have been

isolated, having three disulphide bonds known as cyclotides. Important five cyclotides

have been isolated from V. yedoesis, having significant effects against HIV activities.


24

Among these cycloviolacin Y5 is one of the most important cyclotide tested

effectively in anti-HIV in vitro XIT-based assay (Wang et al., 2008).

1.11.1.14 Insecticidal Activity

Cyclotides are plants peptides having cyclic structures, which possess activities like

insecticidal, anthalmentic and anti-HIV. More than 100 cyclotid have been isolated

from viola family. These compounds are more effective insecticidal (Wang et al.,

2009).

1.11.2 In vitro Biological Activities

1.11.2.1 Acute Toxicity

V. odorata was tested for the acute toxicity in in vivo animal’s model (rats). V.

odorata was scientifically proved as a safe drug even at higher dose (2000 mg/kg)

which can be used safely for clinical purpose (Vishala et al., 2009). In 24h assessment

time duration V. betonicifolia methanolic extract also proved safe at high dose (2000

mg/kg i.p.) and one of its isolated compound [4-hydrose of oxyl coumarin (4HC)] was

also showed to be a safe drug at high doses. Both the plant in the crude form or the

isolated compound can be safely used for clinical purpose (Naveed et al., 2012;

Naveed et al., 2013).

1.11.2.2 Antinociceptive Activity

Antinociceptive activity of V. canescens aqueous ethanolic extract showed significant

effect (Barkatullah et al., 2012). Crude aqueous methanolic extract and the fraction of

n-hexane of V. betonicifolia Sm. were tested for the same activity at three different

doses. Both the methanolic extract and the fraction showed strong dose-dependent

analgesic effect against the acetic acid induced writhing test model (Naveed et al.,

2012, Naveed et al., 2012). At a dose of 400 mg/kg body weight V. odorata proved an

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outstanding anti-nociceptive drug (acetic acid and tail immersion tests) (Antil et al.,

2011). It also proved to be a dose dependent effective analgesic natural source

(Barkatullah et al., 2012). The flowers of V. tricolor were investigated for the anti-

nociceptive effect of a gel containing extract. V. tricolor flowers were investigated for

thermal burn by UVB irradiation and for gel stability performance study which

provided scientific proof to the plant as a natural source of analgesia in the ultraviolet

induced burn, at a temperature below 25 ºC (Piana et al., 2013).

1.11.2.3 Anti-Inflammatory Activity

Clear anti-inflammatory observations were obtained by testing the plant of V.

betonicifolia (n-hexane fraction) in BALB/c mice, following two different protocols

(carrageenan and histamine-induced protocols). It was found that the aqueous

methanolic extract at different test doses showed significant anti-inflammatory

activity. This justifies its use as pain killer in traditional medicine (Naveed et al.,

2012). V. odorata (whole plant) also proved significant anti-inflammatory plant

(Chatterjee et al., 1991; Kaul., 1997). The flowers of V. tricolor were investigated for

the anti-inflammatory effect of a gel containing extract and induced thermal burn by

UVB irradiations for gel stability performance study. Conclusion of the study was that

V. tricolor showed significant anti-inflammatory effect (Piana et al., 2013).

1.11.2.4 Antipyretic Activity

Viola betonifolia crude aqueous methanolic extract/n-hexane fraction were both

analysed agaist the induced pyrexia. The in vivo study conclusion was that both of

these (crude extract and the n-hexane fraction) showed excellent antipyretic activity.

This study provides preciseness and solid reasons to the plant that shows antipyretic

activity (Naveed et al., 2012). In the induced pyrexia, V. betonicifolia at 300 mg/kg


26

body weight showed dose dependent (78.23%) antipyretic effect (Naveed et al.,

2012). V. odorata also is used effectively for antipyretic activity (Chatterjee et al.,

1991; Kaul., 1997).

1.11.2.5 Gastrointestinal Motility

V. canescens was tested for the gastrointestinal motility. The test was performed in

the in vivo model in mice. The findings of the study was that the plant purgative

activity. This investigation verifies its traditional use for this purpose (Vishala et al.,

2009).

1.11.2.6 Laxative Effect

The n-butanol and aqueous fractions of V. odorata in 200 and 400 mg/kg doses

proved better laxative effect in the animal model (rats) in in-vivo protocol (Vishala et

al., 2009). Traditionally V. canescens is used as an effective laxative in traditional

medicines in the crude form (Vishala et al., 2009). Both the screening procedures

were carried out either in vitro or in vivo, of the whole plant of V. betonicifolia crude

methanolic extract at different doses. Partially atropine-sensitive prokinetic in both

low and high doses 50 and 100 mg/kg respectively were used. Laxative effect in the

doses 30 and 100 mg/kg also showed significant effects. Isolated rabbit guinea-pig

ileum and jejunum showed dose-dependent contractions at 0.03-5 mg/mL and 0.01-

0.3 mg/mL respectively. More effective spasmodic effect of the crude methanolic

extract was observed in guinea-pig ileum as compared to the preparation of rabbit

jejunum (Naveed et al., 2013).


27

1.11.2.7 Hepatoprotective Activity

V. odorata is traditionally used against liver diseases. Scientific background was

given to the folk uses by paracetamol induced hepatotoxicity protocol. Plant’s

aqueous methanolic extract was tested at two different doses (250 and 500 mg/kg) in

mice (in vivo). The results showed that the increased levels of AST, ALT, ALP (liver

enzymes) and the total bilirubin were significantly reduced to the normal levels.

Regeneration from hepatocellular necrosis and inflammation of the plant extract

treated groups with pure paracetamol was clear from the histopathological slides

(Qadir et al., 2014).

1.11.2.8 Diuretic Activity

The significant diuretic activity of the aqueous extract V. odorata’s aerial parts was

observed by adjusting two different oral doses, using in vivo animals model (Vishala

et al., 2009).

1.11.2.9 Anxiolytic Activity

The crude aqueous methanolic extract of V. betonicifolia was used for the in vivo

animal model. Results of the study were the dose dependent significant anxiolytic

activity of the plant extract. The test used for the activity was staircase test (Naveed et

al., 2013). The whole plant of Viola betonicifolia and its n-hexane fraction were tested

for sedation and different nervous disorders like muscle relaxant, antidepressant,

anxiolytic to assure the folk use. Various tests protocols were adopted for this purpose

i.e staircase test (anxiolytic activity) rota rod, traction test, chimney test and inclined

plane (muscle relaxant). Different doses of the crude and the n-hexane fractions gave

different results when administered to the test mice (i.p.). The fraction of n-hexane

was also monitored by using the forced swimming test for the effect of sleep-


28

induction activity and staircase test for the anxiolytic action. The fraction of n-hexane

as well the crude methanolic extract showed a noteworthy sleep inducing effect and

reduced locomotive activity. Sleep duration was increased in a dose-dependent mode.

The outcomes of the tests were that the n-hexane fraction showed sedative, muscle

relaxant, anxiolytic and for the assessment of various nervous disorders (Naveed et

al., 2013).

1.11.2.10 Muscle Relaxant

V. betonicifolia crude aqueous methanolic extract (100, 200 and 300 mg/kg) and its

isolate, 4-hydroxyl (20 and 30 mg/kg) showed significant muscle relaxant effects.

Thus crude aqueous methanolic extract had strong muscle relaxant and sedative-

hypnotic activities. Similarly, significant muscle relaxant activity was showed by 4-

hydroxyl coumarin. This gives a scientific proof to the folkloric uses of the plant as a

muscle relaxant (Naveed et al., 2013).

1.11.2.11 Sedative-Hypnotic Effect

In sedative-hypnotic activity the latency time was remarkably reduced and the

sleeping time was increased by the crude aqueous methanolic extract of V.

betonicifolia (Naveed et al., 2013). V. odorata was investigated for its sedative and

hypnotic effect in animal models (rats), adjusting different test doses. Dose dependent

sedation of the crude methanolic extract was proved from the study designed (Monadi

et al., 2013).

1.11.2.12 Anesthetic Effect

V. odorata was investigated for its pre-anesthetic effects. The results reveal the dose

dependency of the sedative effect in the plant extract. The extract at three different


29

doses (100, 200 and 400 mg/kg) were used in which only 400 mg/kg increased the

sedation effect (Monadi et al., 2013).

1.11.2.13 Uterotonic Effect

The presence of cyclotides in various species of violaceae family is also responsible

for the uerotonic activity. For example about 30 cyclotide have been isolated from V.

odorata and five important have been isolated from V. yedoesis (Schöpke et al.,

1993).

1.11.2.14 Anti-neurotensive

Naturally equipped plants with cyclotide are also rich sources of anti-neurotensive

effect. Most of Viola species are gifted with cyclotide so they are significantly used

for anti-neurotensive activity (Schöpke et al., 1993; Tam et al., 1999).

1.11.2.15 Anti-cancer Activity

V. odorata acetone extract possess chemo-preventive effect in the in vivo animal

model (Perwaiz and Sultana., 1998). Cycloviolacin O2 isolated from V. odorata,

showed cytotoxic effect against the ten different lines of the cancer cell which include

myeloma, leukemia, lymphoma & renal adenocarcinoma and small-cell lung cancer.

V. odorata proved as a more significant antitumor drug (Melo et al., 2011; Talib.,

2011). A cyclotide, Cycloviolacin O2, an isolate of V. odorata showed outstanding

antitumor activity. The cyclotides also showed positive results in the management of

breast cancer by using doxorubicin (presence/absence) by assay of cell proliferation

for the establishment of chemosensitization abilities (Gerlach et al., 2010). Cyclotides

of V. tricolor are toxic in nature (Tang et al., 2010). The study provides a legal

scientific background to the plant for its folkloric uses in treating throat, tongue breast

and lung cancers (Lindholm et al., 2002; Salve et al., 2014).

http://www.ncbi.nlm.nih.gov/pubmed/?term=de%20Melo%20JG%5Bauth%5D


30

1.11.2.16 Anti-hypertensive Effect

The leaves extract in crude form of V. odorata possess anti-hypertensive action by

using both the in vivo and in vitro protocols. This resulted in lowering of the mean

arterial blood pressure in vivo by using rats in the animal models. Guinea-pig isolated

atria were used in the in vitro antihypertensive protocol. This resulted in spontaneous

atria contraction by force and rate inhibition (Hasan et al., 2012). V. mandshurica

specie of Violaceae family also plays a vital role in antihypertensive activity.

Angiotensin-converting enzyme inhibitors (ACE) are responsible for blood vessels

contraction. The absorbance of V. mandshurica extract at 228 nm was maximum. For

ACE inhibition, captopril, a standard reagent was used. The roots extract showed

more effectiveness than petioles and leaves for this activity (Huh et al., 2015).

1.11.2.17 Anti-dyslipidemic Effect

V. odorata Linn. showed dyslipidemic effect when used in the in vivo and in vitro

assays. Tyloxapol-induced dyslipidemia protocol was followed. Significant reduction

in the total cholesterol lever in the dyslipidimic induced rats was caused by the plant

extract. So the conclusion was that the vasodilatation action of the plant extract was

mediated through various pathways such as its release from intracellular stores, Ca++

influx inhibition through Ca++ channels membrane, and NO mediated pathways,

resulted in the fall in blood pressure. Thus V. odorata is significantly used for

antidyslipidemic activity (Hasan et al., 2012).

1.11.2.18 Expectorant and Anti-tussive Effect

V. odorata different parts i.e roots, leaves and flowers were used for the expectorant

and anti-tussive activities. The active constituents responsible for the said activities

are alkaloids, salicylic acid, saponins, volatile oil and methyl ester, which give


31

scientific approval to the plant’s folk uses for this activity (Gairola et al., 2010;

Sellappan., 2015).

Figure 1.2: Illustration of V.serpens specie of the genus Viola


32

Figure 1.3: Flower of V. serpens specie of the genus Viola

Figure 1.4: Seeds of V. serpens specie of the genus Viola

http://en.wikipedia.org/wiki/File:Akkerviooltje_opengesprongen_doosvrucht.jpg


33

1.12 AIMS AND OBJECTIVES

To isolate medicinally important bioactive secondary metabolites from Viola

serpens.

To elucidate the structure of isolated compound(s) using various spectroscopic

techniques.

To evaluate the pharmacological activities (antioxidant, analgesic, acute

toxicity, anti-inflammatory, nephroprotective, hepatoprotective, enzyme

inhibition and larvicidal activity) of the crude extracts and fractions

.

Chapter – 2 EXPERIMENTAL

34

CHAPTER – 2

2. EXPERIMENTAL

2.1 GENERAL EXPERIMENTAL CONDITION

Chemical studies along with the different biological activities were carried out in the

Departments of Pharmacy and Chemistry, University of Malakand, Chakdara.

However, a part of the biological activities was performed in PCSIR laboratories,

Peshawar and Department of Animals Health Sciences, Agriculture University

Peshawar. Spectroscopic studies were performed in Atta-U-Rehman Institute for

Natural Product Discovery, Universiti Teknologi MARA Puncak Alam Selangor D.E.

Malaysia.

2.2 SPECTROSCOPIC TECHNIQUE

The isolated compounds were characterized by means of various spectroscopic

techniques including UV, IR, 1H and 13C-NMR, NOESY, COSY, HSQC

(Heteronuclear Single-Quantum Correlation) and HMBC. Fully automated, Hitachi

Spectrophotometer (model U-3200) was used for the UV spectra determination.

Spectrometer, model JASCO 302-A was used for the IR spectra on potassium

bromide (KBr) discs. The compounds low-resolution electrons impact spectra were

determined by means of mass spectrophotometer (model MAT311A) linked with

PDP11/34 system of computer. The 1H-NMR spectra of the compounds were

determined by using Nuclear magnetic resonance spectrometer by Bruker AM-300,

AM-400 and AMX-500. Internal reference TMS was used for the spectra at 300, 400,

or 500 MHz. Distortion less Enhancement by Polarization Transfer (DEPT)

experiments for the moieties CH, CH2 and CH3 at 90o and 135o were performed.

2.3 PHYSICAL CONSTANTS


35

The melting point instrument known as Gallenkamp electrothermal melting point

apparatus of the Model 5A-6797 (England) was used for the determination of the

melting points of the isolated compounds. Optical rotation of the compounds was also

recorded by using a digital polarimeter (JASCO DIP- 140).

2.4 COLUMN CHROMATOGRAPHY (CC).

In column chromatography (CC) silica gel was used. Isolation and purification, of the

compounds required silica gel 60 (Merck) on mesh size 230-270. The organic

solvents (n-hexane, chloroform, ethyl acetate and methanol) were used in column

chromatography as mobile phases

2.5 THIN LAYER CHROMATOGRAPHY (TLC)

Samples purification was insured by using thin layer chromatography technique

(TLC). F254 aluminum sheets, pre-coated Kiesel gel 60 (Merck) were used for TLC.

2.6 DRUGS AND REAGENTS

In various experiments different drugs and commercial grade chemicals were used.

Details of which are given in the Table 2.1. Different doses of crude methanolic

extract and the fractions were prepared in normal saline and distilled water for various

biological activities. These solvents were also used as negative control. Commercial

grade organic solvents (n-hexane, ethyl acetate, chloroform, n-butanol, and methanol)

were selected and used.


36

Table 2.1: List of Drugs/Chemicals Drugs/ Chemicals Source

Acetic acid

Acetyl choline esterase

Acetyl thiocholine iodide

Sigma Chemical Co, St Louis, MO, USA

Acid alcohol

Amino alcohol

Merck Co (Darmstadt’s Germany)

Aspirin Reckitt Benckiser Pakistan

Ascorbic acid China fooding Ltd. China.

Acetyl choline esterase

Acetyl thiocholine iodide

Sigma Chemical Co, USA

Brewer’s yeast Vahine Professional, France

Bismith nitrate Sigma Chemical Co,

St Louis, MO, USA

Chloroform Merck Co (Darmstadt Germany

Carrageenan Sigma Chemical Co, St Louis, MO, USA

Ceric sulphate Merck, Darmstadt, Germany

Diclofenac sodium Sigma Chemical Co, St Louis, MO, USA

Dragendorff's reagent Searle pharmaceuticals Pakistan Limited

DPPH Merck Millipore Corporation, Germany

5,5 dithio-bis-nitro benzoic acid Sigma Chemical Co,

St Louis, MO, USA

Ethyl acetate

Eosin

Ethyl alcohol Merck Co Darmstadt Ltd., Germany

Formalin Sigma Chemical Co,

St Louis, MO, USA

Folin & Ciocalteu’s Merck, Millipore Corporation, Germany

Galanthamine

Hematoxyline Merck, Darmstadt, Germany

H2SO4 Sigma Chemical Co,

St Louis, MO, USA

Imipenem Sigma Chemical Co,

St Louis, MO, USA

Ibuprofen Munawar pharma (pvt) Ltd. Pakistan.

Methanol

n-Butanol

n-Hexane

n-propylgallate

Merck Co (Darmstadt Germany)

Normal saline Santa Cruz Biotech, USA

Nutrient agar Sigma Chemical Co,

St Louis, MO, USA

Paracetamol Alfa Aesar - A Johnson Matthey Company

Paraffin China Shengtong petrochemical co. Ltd. China.

Phosphate buffer Santa Cruz Biotech, USA

Potassium Bromide

Potassium Iodide

Sigma Chemical Co,

St Louis, MO, USA

Riboflavin Alibaba Ltd. Pakistan.

Silica gel Sigma Chemical Co, St Louis, MO, USA

Silymarin Hisunny Chemicals, China

Sod. Carbonate

Sod. Pentobarbital Merck, Darmstadt, Germany

Sulfuric acid Sigma Chemical Co,

St Louis, MO, USA

Tramadol Searle products Ltd. Pakistan

Xylene Sigma Chemical Co,

St Louis, MO, USA


37

2.7 PLANT MATERIALS

The plant collection was done from District Shangla, (Village, Puran) Khyber

Pakhtunkhwa, Pakistan in the month of April, 2011. In order not to affect the flora of

the area, collection was done with the permission of Swat forest officer. Plant

specimen was identified by Professor Dr. Mohammad Ibrar, (Taxonomist)

Department of Botany, University of Peshawar. The plant specimen was deposited

with voucher number Bot. 20158 (PUP) kept in the herbarium of the same

Department. The whole plant (13 kg) was collected and shade dried at ambient

temperature.

2.7.1 Extraction and Fractionation

The plant was shade dried powdered (10 kg) and soaked in 90% organic solvent

methanol (25 L) for 10 days at a temperature of 25-30oC. The soaked plant was

vigorously stirred twice daily (morning and evening). After each three days and

finally after four days, colorless thin cloth was used to filter the aqueous-methanol

soluble residues which was filtered finally by Whatmann filter paper No. 1. After

each filtration the residue was soaked in the said solvents till the day 10. Through

rotary evaporator (Model: R-210, Buchi, Switzerland) the filtrate was dried by using a

heating bath (B-491) at 40-45oC fitted along with a re-circulating chiller (NESLAB

instruments). The total of 1.57 kg crude methanolic extract was obtained. 30 g of the

crude extract was separated for various biological activities whereas, the remaining

extract was fractionated by using a separating funnel of capacity 5 L. The crude

extract (1.37 kg) was dissolved in distilled water (1 L) and transferred into a

separating funnel along with 1.5 L n-hexane and shaken vigorously. Separating funnel

was kept on stand till the appearance of immiscible layers in which n-hexane

accumulated as an upper layer.


38

The procedure was repeated three times. The n-hexane soluble layer collected was

concentrated at low temperature (40-45oC) under reduced pressure. The n-hexane

fraction collected was 706 g. Chloroform was then added to the layer separated from

n-hexane layer in the separating funnel, vigorously shaken and kept for separating the

mixture into layers. As chloroform is a denser solvent so its fraction is collected as a

lower layer. The mixture (upper layer) was conducted for the acquisition of further

fractions which were carried out three times. The fraction of chloroform was collected

and concentrated under vacuum resulting into a semi solid mass of chloroform soluble

fraction (17 g). The same procedure was followed for the fractions of ethyl acetate

and n-butanol thus ethyl acetate (22.7 g), n-butanol (35 g) were finally obtained as

solid masses. The finally left fraction, after recovering the above mentioned soluble

fraction was concentrated and recovered as an aqueous soluble fraction (45 g). The

crude methanolic extract was subjected along with its five subsequent fractions for

isolation and different phytochemical and pharmacological activities.


39

This whole process is presented in Figure 2.1.

Figure 2.1: Scheme of plant extraction and fractionation


40

2.7.2 Isolation and Purification

The ethyl acetate (65 g) soluble fraction was subjected to fractions by means of VLC

over silica gel (1300 g). The elution was started from n-hexane, followed by

increasing polarity of n-hexane-chloroform gradients. Finally the column exhausted

by gradual increased in polarity of the mobile phase with methanol-chloroform (20%)

gradient that afforded five sub-fractions (FMC1-FMC5). Sub fraction FMC3 (100 mg),

was used in column chromatography on silica gel repeatedly and eluting with ethyl

acetate-n-hexane (20%). Finally, the compounds were separated through preparative

TLC, and checked for purity using TLC, yielded commulin-A (1), commulin-B (2)

and tectochrysine (4). Similarly, further purification of the sub-fraction FMC-5 was

done by using column chromatography on silica gel repeatedly by treatment with

ethyl acetate-n-hexane (30%) resulted in the isolation of pure compounds, Commulin-

C (3), sideroxylin (5) and (cearoin) (6).


41

.

using Silica gel (1300 g),

Et-Acetate fraction

VS (65 g)

RFCC, 3:7 Et.acetate:hexaneRepeated Flash Column Chromatography (RFCC), 2:8, Et-acetate:Hex

commulin-B

tectochrysine

FMC1 FMC2 FMC3

commulin-A

FMC4 FMC5

Commulin-C sideroxylin

cearoin

Figure 2.2: Scheme representing the isolation of pure compounds using Ethyl acetate

fraction.


42

2.8 EXPERIMENTAL DATA OF NEW COMPOUNDS FROM VIOLA

SERPENS

2.8.1 Commulin-A (1)

Physical State: Yellowish amorphous powder

Yield: 17.5 mg

Melting Point: 138-140oC

Solvent system used: Ethyl acetate and n-hexane (2:8)

Solubility: At room temperature soluble in methanol

[α] 30D : + 375.0o (c = 0.8, CHCl3)

UV activity: UV visible on TLC

IR max cm-1: 3450 (OH), 2968 1591, and 1463 cm-1 (aromatic CH), 1719

(saturated ketone), 1250-1383 (C - C)

EI-MS m/z: 298.084 [M]+ (C17H14O5, calcd.298.2940)

EI-MS m/z (Peak %): 298 (80), 267 (65), 250 (35), 235 (20), 218 (10), 141(6).

1H & 13C-NMR (300 & 100 MHz CDCl3): Details mentioned in

the Table 3.18.

2.8.2 Commulin- B (2)


Yield: 14.2 mg

Melting Point: 163-165oC

Solvent system used: Ethyl acetate and n-hexane (2:8)


[α] 30D : -166.7o (c = 1.2, CHCl3)


IR max cm-1: 3422 (OH), 2968 (aromatic CH), 1719 (saturated ketone), 1601

(aryl), 1659 (C = C), 1250-1383 (C - C)

EI-MS m/z: 314.068 [M]+ (C17H14O6, calcd. 314.2930)

EI-MS: m/z (Peak %): 314.068 (79), 283.06 (55), 266.06 (30), 251.03 (19),

234.03(11), 17.00 (8), 76.03 (5)

1H & 13C-NMR (300 & 100 MHz CDCl3): Details mentioned in the

Table 3.19


43

2.8.3 Commulin- C (3)


Yield: 11.5 mg

Melting Point: 1.15-1.33 oC

Solvent System: Ethyl acetate and n-Hexane at a ratio of 3:7


[α]30D: + 13.0o (c = 1.5, CHCl3)


IR max cm-1: 3560 (OH), 2968 (aromatic CH), 1717 (saturated ketone), 1628

(aryl)

FAB-MS m/z: 328.081 [M]+ (C18H16O6, calcd 328.32)

EI-MS: m/z (Peak %): 328.081 (89), 297.08 (64), 280.07 (35), 265.05 (21), 248.04

(17), 218.04 (9), 77.04 (5).

1H & 13C-NMR (300 & 100 MHz CDCl3): Details mentioned in the

Table 3.20

2.9 EXPERIMENTAL DATA OF KNOWN COMPOUNDS FROM VIOLA

SERPENS

2.9.1 5-Hydroxy-7-methoxy flavone (tectochrysine) (4)

Physical State: Colorless Solid

Yield: 13.1 mg

Melting Point: 162-168 oC

Solvent system used: Ethyl acetate and n-Hexane (3:7)


IR max cm-1: 3400, 3000, 1649, 1475, and 1460 cm-1

HR-EIMS m/z: 268.2013 [M]+ (calcd. for C16H12O4, 268.2011)

1H-NMR (500 MHz, CDCl3): δ 7.87 (1H, m, H-2′), 7.52 (1H, m, H-6′ & H-3′,), 6.65

(1H, s, H-3), 6.48 (1H, J = 2.2, H-6) δ 6.39 (1H, d, J = 2.2 Hz,

H-8), 3.94 (3H, s, OCH3)


44

2.9.2 4́, 5-Dihydroxy-7-methoxy-6, 8-dimethylflavone (Sideroxylin) (5)

Physical State: Yellow needles

Yield: 8.4 mg

Melting Point: 565.5°C

Solvent system used Ethyl acetate and n-Hexane (3:7)


IR max cm-1: 3500 and 1655 cm-1 (hydroxyl and ketonic)

HR-EIMS m/z: 312.0125 (calcd. for C18H16O5, 312.0123)

1H-NMR (500 MHz, CDCl3): δ 13.07 (1H, s, OH-5), 7.97 (1H, d, J = 8.8 Hz, H-2 &

H-6′), 6.87 (1H, s, H-3), 3.94 (3H, s, OCH3), 2.32 (3H, s, -CH3

ring A), 2.08 (3H, s, -CH3 ring A)

2.9.3 5-Dihydroxy-4-methoxybenzophenone (Cearoin) (6)

Physical state: Yellow amorphous powder

Yield: 12.3 mg

Melting Point: 182-189 C

Solvent system used: Ethyl acetate and n-Hexane (3:7)


IR max cm-1: 3448 (OH), 1743 (C = O) cm-1

HR-EIMS m/z: 244.1322 [M+1]+ (calcd. for C14H12O4, 244.1334)

1H-NMR (500 MHz, CDCl3): δ 11.95 (1H, s, OH-2), 8.89 (1H, s, OH-5), 7.61, 7.54

(m, aromatic protons, ring B), 6.88 (1H, s, H-6), 6.59 (1H, s, H-

3), 3.48 (3H, s, OCH3

.


45

2.10 IN-VIVO BIOLOGICAL ACTIVITIES

Different in-vivo biological activities of the crude methanolic extract/fractions (whole

plant) were performed by using different protocols.

2.10.1 Experimental Animals

BALB/C mice of both sexes, male and female were employed for the bio-assays. The

mice were bought from NIH (National Institution of Health) Islamabad, bred in the

animal house, Department of Pharmacy, University of Malakand. The animals were

kept at standard laboratory formula of 25oC temperature and at 12/12 h light/dark

cycle with free access to the food and water. Rules of the ethical committee were

followed before and after the experiments. Food and health, guidelines of the mice

were adjusted throughout the experiments according to the rules provided by the

institute of laboratory animal resources, Commission on life sciences, National

Research Council.

2.10.2 Acute Toxicity

The study was performed on crude methanolic extract (whole plant) at various doses

ranging from 1-2 g/kg body weight. The animals (mice) were uniformly grouped into

three, each of which contained six mice. The negative control group was treated with

distilled water (10 ml/kg dose) and the two remaining groups were given doses of the

crude methanolic extract (1mg/kg and 2mg/kg body weight). After the test doses

administration, 24 h observations of the animals were done. Observations of the first 4

h of the animals were for the effect of acute toxicity. Death/s, if occurred is identified

after 24 h (Araujo et al., 2014).


46

2.10.3 Analgesic Activity

Two different protocols were used to determine the mechanism(s) of the anti-

nociceptive effect of the plant V. serpens crude methanolic extract/ fractions.

2.10.3.1 Acetic Acid Induced Writhing

Crude methanolic extract and fractions of the whole plant were screened for analgesic

activity. BALB/C mice of both the sexes with body weights ranged from 18-22 gms

body weights. The animals were categorized into fourteen different groups (n=6).

Group I (negative control) and II (positive control) were treated with a dose of 10

ml/kg normal saline and 10 mg/kg Diclofenac sodium respectively. The supplied food

was withdrawn 2 h before starting the activity (Koster et al., 1959; Adzu et al., 2001;

Khan et al., 2010). Different fractions (n-hexane, Chloroform, Ethyl acetate, and

aqueous) along with the crude methanolic extract were administered to the remaining

groups, III to XIV in three different doses i.e. 100, 200 and 300 mg/kg body weight.

After 30 min of the previous treatments all the groups were administered equally with

1% acetic acid (i.p). After 5 min of acetic acid injection counting of the abdominal

writhes (constrictions) for 10 min duration was done (Collier et al., 1968). The

percentage of analgesic activity was calculated according to the designed formula

expressed below.

% Analgesic effect = 100 – No of Writhes in the Test Animals x 100

No of Writhes in Control Animal

2.10.3.2 Formalin Test

The formalin test was conducted according to the method of the previous study (Liu et

al., 2007). BALB/C mice of both the sexes (male and female) were selected in the

range of 18-22 g body weights. The animals were categorized into seven groups each

having six animals (n=6). Pains were induced in animals by injecting in the right hind


47

paw 0.05ml of 2.5% formalin (40% formaldehyde i.p). The Group I (control) and the

Group II (standard) received normal saline and standard drug (Diclofenac sodium) in

the dose of 10 mg/kg (i.p) respectively. Different fractions (n-hexane, chloroform,

ethyl acetate, and aqueous) along with the crude methanolic extract were administered

to the reset of the groups, III –XIV at doses of 100, 200 and 300 mg/kg (body weight

p.o) 60 min prior to the formalin injections. The pains indicators were the time spent

with responses of licking and biting of the injected paw. Measurement of responses

were done in two phases after injecting the formalin doses i.e for first 5 min (early

phase) and then the next 20 - 30 min (late phase).

2.10.4 Anti-inflammatory Activity

Crude methanolic extract along with different fractions were screened for anti-

inflammatory effect. The action of crude extract of the plant and its different fractions

were determined using three different protocols in order to make clear the mechanism

involved behind anti- inflammatory activity of the plant.

2.10.4.1 Carrageenan Induced Paw Edema

Crude methanolic extract along with its different fractions were incorporated for

judgment of anti-inflammatory effect in the plant. BALB/C mice of both sexes of

body weight 25-30 g were selected and divided into fourteen groups. Each group

included 6 mice (n=6). Group I and II were used as negative and positive control

respectively. Group I animals were treated with normal saline (10 ml/kg body weight)

whereas, the group II animals were treated with diclofenac sodium at a dose of 10

mg/kg body weight. The rest of the groups (III-XIV) were treated with the crude

methanolic extract and its various fractions (n-hexane, chloroform, ethyl acetate, and

aqueous), at different doses of 100, 200 and 300 mg/kg (body weight) respectively.


48

Each mouse after 30 min of treatment of the test samples dose, was treated with sub-

planter injection in right hind paw with 1% carrageenan. Anti-inflammatory effect

was measured with the help of plethysmometer (LE 7500 plan lab S.L) for 5 h i.e. at

0, 1st , 2nd , 3rd , 4th and 5th h (Collier et al., 1968). The below mentioned formula was

used for calculating the percent inhibition for edema.

% Inhibition = A–B / B x100

Where A and B represents edema volume of negative control, paw edema of tested

groups respectively.

2.10.4.2 Histamine Induced Paw Edema

The trial was adopted according to authentic protocol (Amann et al., 1995). The test

sample indomethacin and distilled water at doses of 10 mg/kg and 10 ml/kg

respectively were administered orally. Histamine (0.1 ml) was administered as sub-

plantar injection to right hand paw tissues after one hour to the test samples treatment.

After the histamine injection the paw thickness measurement was done for 3 h at a

regular interval of 30 min each. The % inhibition was calculated by using the formula.

Inhibition (%) = 100 x Value of Control Group – Value of the Test Sample

Value of Control Group

2.10.4.3 Xylene Induced Ear Edema

The xylene induced ear edema test was conducted by following the authentic

protocols (Dai et al., 1995; Amin et al., 2012). The positive- control group of mice

(BALB/C of both sex 25-30g body weights) was administered orally with Ibuprofen

(100 mg/kg). Plant crude extract and the fractions at doses of 100, 200 and 300 mg/kg

were used (p.o). The test animal after an hour, received 20 μl (0.02 mL) of xylene on

the right ear lobe at both posterior and anterior surfaces. The lobe of the left ear was

measured as control ones. Cervical dislocations of the treated mice were done after


49

one hour of the xylene injection. Using a cork borer circular ear section of 3 mm

diameter were taken from each ear of the killed mice and weighted. The ear edema

was calculated by taking out the percentage by comparing the weights of untreated

left ear with the treated one.

Inhibition (%) = 100 x Value of Controlled Group – Value of the Tested Sample

Value of Controlled Group

2.10.5 Larvicidal Bioassay

Crude extract of the whole plant along with its various fractions were tested for

larvicidal bioassay (Ikram et al., 2012). Larvae were collected from ponds water in a

plastic jar and kept in the laboratory conditions. The crude extract and its subsequent

fractions (n-hexane, chloroform, ethyl acetate, and aqueous) were subjected to 10,000

ppm stock solutions in distilled water. From the stock solution each 100 ml of 2000,

1500, 1000, 500, 100 and 50 ppm dilutions in 500 ml plastic jars were prepared

separately. Each jar was accordingly labeled and fed with 25 active larvae. Wide

mouthed glass dropper was used to transfer the calculated number of larvae to the

labeled plastic jar. Each jar, including the controlled one was also provided with a diet

of finely ground dog biscuits and brewer’s yeast in 2:3 ratios. The adjusted laboratory

conditions were 30 ± 2°C temperature and 70-75 relative humidity. After 24 h of

exposure, dead larvae numbers were counted. Cervical or siphon region was used for

the identification of dead larvae, as they fail to move after being prodded. The

experiment was repeated five times. Number of dead and live larvae (dead and live if

present) was identified and the species were confirmed.

Formula given below was used for Percentage mortality determination:

% Mortality = 100 – Number of Living Larvae in Test Sample x 100

Number of Living Larvae in Control


50

2.10.6 Nephroprotective and Hepatoprotective Activities

2.10.6.1 Animals Used

Sixty domestic local mature rabbits (Oryctolaguscuniculus) of both sexes were

purchased from local market. They were kept in a well ventilated and wide chambered

animal house at the University of Malakand, KP, Pakistan. The rabbits were fed on

chaw pellets along with fresh green vegetables, grasses and open access to fresh water

at libitum. Acclimatization of the animals was done for at least two weeks before

commencement of experiment.

2.10.6.2 Animals Grouping and Dosing

The rabbits were grouped into fifteen groups for eight days protocol (Ikram et al.,

2012; Gulati et al., 2012). Four rabbits were kept in each group. Two doses low (150

mg/kg) and high (300 mg/kg) were tested for each extract and fraction. Each group

was tagged separately for the purpose of identification. Group 1, administered with

normal saline, served as normal control, group 2 was treated with paracetamol only

(controlled group); group 3 served as standard control which was treated with

paracetamol from the first day followed by silymarin, a well-known standard

hepatoprotective drug. Groups 4, 5 received paracetamol followed by crude methnolic

extract at doses of 150 and 300 mg/kg body weight. The groups 6, 7 received

paracetamol followed by n-hexane fraction at doses of 150 and 300 mg/kg body

weight, groups 8, 9 received paracetamol followed by the fraction of ethyl acetate

(150 and 300 mg/kg). Group 10, 11 received paracetamol followed by the fraction of

chloroform at doses of 150 and 300 mg/kg body weight whereas, groups 12,13

received paracetamol followed by the fraction of n-butanol at doses of 150 and 300

mg/kg body weight. The groups 14 and 15 were treated with paracetamol followed by


51

aqueous fraction with same doses. The doses’ details were: paracetamol 1 g /kg body

weight (Sasidharan, 2012), silymarin 50 mg/kg body weight (Bak et al., 2012).

2.10.6.3 Chemicals used

ALT, AST, ALP and the serum levels were estimated by using commercially

available kits (purchased from AMP Diagnostics, Austria) on a UV visible light

spectrophotometer (Agilent 8453) and silymarin.

2.10.6.4 Histopathology

Examination of the dissected rabbit’s tissue from kidney and liver were collected and

stored in 10 % formalin solution. Standard protocol was adopted for processing the

samples (Bancroft and Gamble (2007).

Procedure for Histopathology

One centimeter of the kidney and liver were cut for tissue processing. After washing

with running tap water the samples were threaded and placed in water. The tissues

were washed in such a manner that they could not be damaged and washing was

continued overnight. Automatic processor of tissues (Tissue-Tek® Sakura, Japan) was

used for placing the tissues in ascending grade of alcohol for dehydration with

decreasing time period. Alcohols of various grades were used for tissues to be placed

for definite time as follows;

Dehydration

30% alcohol 3-4 hrs

50% alcohol 2 hrs

70% alcohol 2hrs

80% alcohol 1.5 hrs

95% alcohol 1.5 hrs

Absolute alcohol I 1 hr

Absolute alcohol II 1 hr


52

Clearing

Alcohol with Xylene 45 min

Xylene I 30 min

Xylene II 15 min

Impregnation

Paraffin, melted at 72ºC was used for tissue samples impregnation.

Paraffin I 2 hrs

Paraffin II 2hrs

Embedding

Blocks were made after tissues processing. Automatic tissue embedding assembly

(Tissue-Tek® TEC™ Sakura) was used for tissue blocks preparation. Tissues placed in

plastic cassettes were poured with molten paraffin for the preparation of blocks. Cold

plate of tissue blocks were shifted and allowed to dry.

Sectioning

By Microtome (Accu-Cut® SRM™ 200 Sakura) tissue blocks were sectioned, with

about 4-5 µm thickness. The folds were removed at 56oC by placing the obtained

sections in water bath (Sakura) which floated over the water surface. On slides

albumin was applied for proper cleaning and sticking of the sections to slides.

Sections were mounted over the slides and dried by keeping in oven (Daihan Lab

Tech Co., ltd) for 3-4 h.

Staining

Slides after final drying were placed for staining. Hematoxylin and Eosin (H & E)

staining of slides sections and automatic slide stainer (Tissue-Tek® DRS™ 2000

Sakura, Japan) was used. Standard staining protocol was followed;


53

Removal of Paraffin

Reagents Time period

Xylene 3 min

Xylene 3 min

Xylene 3 min

Removal of Xylene with Alcohol

Ethyl alcohol 100% 1 min

Ethyl alcohol 100% 1.30 min


Tap water 2 min

Distilled water 2 min

Principal Dye

Hematoxylin (Annexure-3) 6 min

Tap water 2 min

Decolorization

Acid alcohol (Annexure-4) 2 dips

Tap water 1 min

Mordanting the Tissue Sections

Amino alcohol 5 min

Water (tap) 1 min

100% Ethyl alcohol 1 min

100% Ethyl alcohol 1 min

Counter Staining

Eosin (Annexure-5) 1 min

Dehydration






54

Clearing

Xylene 1.30 min

Xylene 1 min

Xylene 1.30 min

Mounting of Cover Slip

Slides were cleaned properly after completion of staining process. DPX (Scharlau)

pouring and covering was done with great caution so as to avoid formation of bubbles

and form clear and neat slides.

2.10.6.5 Hematological and Serological profile of infected Rabbits

Blood samples were collected from rabbit in clean EDTA tubes. Serology required the

collection of 3 mL blood in tubes and allowed to clot. By centrifugation the blood for

10 min at 3000 rpm serum was collected in 1 mL Eppendorf tubes and was kept at

4°C until further use.

Serology

3 mL blood samples were collected in clean tubes, centrifuged for 10 min at 3000

rpm, 1mL Eppendorf tubes were used for serum separation. Serum glutamic pyruvate

transferase (SGPT), total serum proteins, albumin and globulin were measured by

using Biochemistry analyzer (PS-520 SHENZHEN PROCAN ELECTRONICS,

CHINA).

Serum Glutamic Pyruvate Transferase (SGPT)

Reagents R1 and R2 are two reagents in kit (Reactivos, GPL Barcelona, Spain) for

estimation of serum glutamic pyruvate transferase (SGPT). As per manufacturer

instruction solution was prepared by mixing 4 volumes of R1 and 1 volume of R2.


55

1mL of solution was then mixed with 100 µL of serum sample, incubated at 37 ºC for

1 min. In Automatic Biochemistry analyzer SGPT activity sample was loaded.

Total Serum Protein (TSP)

Estimation of total serum protein was done by using Kit (Reactivos, GPL Barcelona,

Spain) which contains a calibrator and reagent (R). Reading was obtained by mixing a

reagent (R) 1 mol with 25 µL of calibrator. Reagent (R) (1mL) was then mixed with

25 µL of serum sample and incubated at 37 ºC for 5 min. The sample was loaded in

an Automatic Biochemistry analyzer and result was recorded.

Serum Albumin

Estimation of serum albumin was used by using kit (Reactive, GPL Barcelona, Spain)

containing reagent (R) and a calibrator. Reading was obtained by using reagent (R) 1

mol mixed with 5 µL of calibrator. By incubation at 37 ºC for 5 min, 1mL of reagent

(R) was mixed with 5 µL of serum sample. The sample was loaded in an Automatic

Biochemistry analyzer and the result was recorded.

Serum isolation and assessment of some liver related serum enzymes and

kidney parameters

This is an 8 day protocol. On the 9th day the animals were dissected. Before twelve

hours from dissection, food was withdrawn and then anesthetized by chloroform

inhalation. Directly after dissection, blood was directly drawn with 21 Gauge needle

in 3 mL syringes from the heart chambers by cardiac puncture (Illahi et al., 2012).

The blood samples for haematological analysis were collected into EDTA (Ethylene

Diamine Tetra-acetic Acid-coated) tubes (K2-EDTA) with coagulant and kept at room

temperature for 1 hour. By centrifugation the blood for 5 min at 3000 rpm serum was

harvested and collected in Eppendorf (5702R, Germany) tubes and kept at –20˚C until


56

analyzed. The analyzed biochemical markers for hepatotoxicity were aspartate amino

transferase (AST), enzymatic activities of serum alanine amino transferase (ALT) and

alkaline phosphatase (ALP). Serum urea and creatinine tests were done for

nephrotoxicity. They were conducted as:

Determination of Glomerular Filtration Rate

The urea and creatinine clearance tests were used to estimate the glomerular filtration

rate.

Urea Clearance

The following formula was used for the urea clearance test:

GFR = [Urine urea x Urine volume]/Serum urea.

Creatinine clearance

The following formula was used for the creatinine clearance test:

GFR = [Serum creatinine x Urine volume]/Serum creatinine.

2.10.6.6 Statistical Analysis

One way ANOVA, Tukey Test of Post Hoc was applied on means of the data and

analyzed by using computer software SPSS 16.0.

2.10.6.7 Collection and analysis of urine

On the 9th day all the animals for collection of urine samples were kept in individual

cages. 24 h urine samples were collected. During this period the animals had free

access to drinking water. In graduated cylinder 24 h total urine volume in mL of each

rabbit was measured. The sample was analyzed for urinary creatinine and urinary urea

after storage at 4oC for one day. The parameters were estimated through COBAS

chemistry automation using Roche Diagnostic kits.


57

2.11 IN VITRO BIOLOGICAL ACTIVITIES

The crude aqueous methanolic extract along with the various fractions were subjected

for various in vitro biological activities of the whole parts of the V. serpens.

2.11.1 Anti-oxidant Activity

2.11.1.1 Superoxide Anion Radical Scavenging Assay

The activity was based on a system of riboflavin- light-NBT (Beauchamp and

Fridovich, 1971). The reaction mixture contains 0.5 mol of phosphate buffer (50 mM,

pH 7.6), 0.25 mol PMS (20 mM), 0.3 mol riboflavin (50 mM) and 0.1 mol NBT (0.5

mM), before1 mol sample solution addition. Start of reaction was taken by using

fluorescent lamp for illuminating the reaction mixture with different concentrations of

the methanolic extract.

Keeping ascorbic acid as a standard at 560 nm wavelength, the absorbance was

measured by incubating for a period of 20 min.

The following formula was used for calculating the generated superoxide anions.

Hydrogen Peroxide Scavenging Activity = (1-Absorbance of the Sample) x 100

Absorbance of Control

2.11.1.2 DPPH Radical Scavenging Activity

The antioxidant activity ( in vitro) of the crude methanolic extract and its fraction i.e

Ethyl acetate, chloroform, butanol and aqueous fractions were evaluated by using

DPPH (2, 2-diphenyl-1-picryl-hydrazyl) scavenging assay established procedures

(Bursal and Gulcin, 2011). In methanol 500 ppm of each extract was prepared from

25 mol stock solution. From each extract in separate test tubes the stock solution of a

5 mol solution each of 0, 20, 40, 60, 80 and 100 ppm was prepared. Triplicate of each

concentration was taken. Ascorbic acid was taken as a standard chemical. The same

procedure was repeated thrice. To each test tube DPPH of 1 mol was added to each


58

test tube. A control was set by the addition of 1 mol of DPPH to 5 mol of methanol in

a test tube. For 30 min in dark and at room temperature, the test tubes were incubated

and then the absorbance of each extract and fractions, standard and control was

measured at 517 nm by using UV spectrophotometer (1700 Shimadzu Japan). There is

inverse proportionality between the scavenging effect of the free radical and the

absorbance of the reaction mixture. DPPH free radical percent scavenging effect was

expressed as the antioxidant activity of the extracts and the standard. The formula

used was as follow:

Percent Radical Scavenging Activity = (Ac – As / Ac) × 100

Where; Ac is the absorbance of control As is the absorbance of sample.

Chemicals Used

Diphyneyl-1-picryl-hydrazyl (DPPH), ascorbic acid, Folin-Ciocalteu, sodium

carbonate (Na2CO3) and sodium pentabarbital were purchased from Sigma Co.

(USA). Methanol, n-Hexane, ethyl acetate, chloroform and n-butanol used for plant

extraction were of analytical grade and were purchased from Merck Co. (Darmstadt,

Germany).

2.11.2 Antibacterial Assay

All the isolated compounds were screened against the various strains of bacteria

including: Bacillus subtilis Escherichia coli, Pseudomonas aeruginosa, Salmonella

typhi, Staphylococcus aureus and Shigella flexneri. Antibacterial screening was done

by dissolving 3 mg of sample in 3 mol DMSO. Nutrient agar media in the molten

state (45 ml) was poured into sterilized Petri dishes and let for solidifying. Soft agar

(sterile) having 100 mol of test-organisms culture were poured in the wells bored at

certain distance through the 6-mm metallic sterile borer. Then each well was poured


59

with 100 L of the test sample and incubated at a temperature of 37ºC for 24 h. The

measurements of zones of inhibition give the test results. The broad spectrum

antibiotic, Imipenem used as a positive control drug. DMSO was used as a negative

control drug (Imran et al., 2014; Boyanova et al., 2005).

2.12 ENZYME INHIBITION

2.12.1 Chemicals Required for Anticholine Esterase

Phosphate buffer (pH 8), acetyl choline esterase, acetyl thiocholine iodide, 5, 5 dithio-

bis-nitro benzoic acid (DTNB), galanthamine (standard drug).

2.12.2 Acetylcholinesterase Inhibition

Acetylcholinesterase enzymatic activity was measured using an authentic protocol

(Ferreira A et al, 2006) 98 lL (50 mM) Tris–HCl buffer pH 8, 30/L sample and 7.5 lL

acetylcholinesterase solutions containing 0.26 U ml _1 were mixed well in a plate of

ELISA and incubate for 15 min. Subsequently, 22.5 /L of AchI 0.023 mg ml_1 and

142 lL of (3 mM) DTNB were added. At equilibrium point of the reaction the

absorbance at 405 nm was noted. Water was used as a control in the reaction in place

of the extract/ fractions. The 100% activity was obtained from the value of

absorbance. The % Inhibition was calculated using the formula mentioned below:

Inhibition (%) = 100 x Value of Controlled Group – Value of the tested Sample

Value of Controlled Group

Tests were conducted three times and Tris–HCl was used as a blank with buffer as a

substitute for the enzyme solution. The percentage of inhibition was plotted against

the concentration of the extract solution in order to obtain the value of 50% inhibition

(IC50).

Chapter – 3 RESULTS & DISCUSSION

61

CHAPTER – 3

3. RESULTS & DISCUSSION

3.1 BIOLOGICAL ACTIVITIES

3.1.1 In-vitro Biological Activities

3.1.1.1 Antimicrobial Activity

Antibacterial Activity of the Crude Extract and Subsequent Solvent Fractions of

V. serpens

The antibacterial effect of crude extract/fractions of V. serpens is presented in Table

3.1 and 3.2. The crude extract, chloroform and ethyl acetate soluble fractions of the

plant showed significant effects against all the tested bacteria whereas, the n-hexane

and aqueous soluble fractions did not show significant effects. The imipenem, a broad

spectrum antibiotic was used as a standard drug which showed marked effect against

all the tested bacteria. Maximum zones of inhibition were shown by the soluble

fraction of chloroform followed by ethyl acetate and the crude extract. Chloroform

soluble fraction showed maximum zone of inhibition (16 mm) against S. typhi

followed by ethyl acetate soluble fraction (13 mm), crude extract (10 mm) and the

aqueous soluble fraction (5 mm). The maximum activity against E. coli was observed

for the chloroform soluble fraction followed by ethyl acetate with 18 and 14 mm

zones of inhibition respectively. B. subtilis was most susceptible to the chloroform

soluble fraction having a value of 16 mm as its zone of inhibition. Both the

chloroform and the aqueous soluble fractions showed sensitivity against S. flexeneri

with the zone of inhibition of 8 mm. The n-hexane soluble fraction did not show any

effect against S. aureus. The aqueous soluble fraction followed by the crude extract

showed antibacterial activity against P. aeruginosa with the values of 6 and 5 mm

respectively as zones of inhibition. The results clearly demonstrate that the crude


62

extract of the plant as well as various fractions possessed significant antibacterial

effect.

Similarly, the antibacterial effect of the isolated compounds commulin-A (1),

commulin-B (2), tectochrysine (4), sideroxylin (5) and 2, 5-dihydroxy-4-

methoxybenzophenone (6) (cearoin)} is depicted in Table 3.2. All of the isolated

compounds except compound 3 (due to insufficient quantity) were screened against

Gram positive and Gram negative strains of bacteria (S. aureus, B. subtilis, E. coli, P.

aeruginosa, S. flexneri and S. typhi). The results showed that all the pure compounds

exhibited significant antibacterial effects against most of the selected bacteria when

compared with the standard broad spectrum antibiotic, imipenem. The maximum zone

of inhibition (18 mm) was shown against S. typhi, by sideroxylin followed by

commulin-B, tectochrysine and cearoin having the same zones of inhibition (17 mm).

When studied against P. aeruginosa, maximum inhibition was caused by the

compounds, tectochrysine (17 mm) and cearoin (17 mm) followed by the compounds

sideroxylin (16 mm) and then commulin-B (13 mm). Similarly, when tested against S.

flexeneri, the maximum zone of inhibition was induced by tectochrysine (15 mm) and

cearoin (15 mm) followed by commulin-A, commulin-B (12 and 10 mm

respectively). Whereas, sideroxylin was found inactive against S. flexeneri.

Commulin-A, tectochrysine and cearoin possessed antimicrobial effect against S.

aureus (24 mm, 15 mm and 15 mm respectively) while commulin-B and cearoin were

found inactive. Compounds commulin-B and tectochrysine showed no significant

inhibitory effects against E. coli whereas, rest of the compounds were totally inert

against this particular bacterium.


63

The antibacterial activity of V. serpens against various bacteria including both Gram

positive and Gram negative showed that extract/fractions as well as the isolated

compounds possessed marked activity. Currently various microbes/pathogens have

become more resistant and have developed certain mechanisms for their protection.

For instance, causing mutation in their own genes or acquire genes from other bacteria

to resist the anti-metabolic actions of the antimicrobial drugs. E. coli, Shigella,

Salmonella, S. aureus and P. aeruginosa causing various bacterial infections

(Goosney, et al., 1999; Daniel et al., 2012; Bhattacharya, et al., 2012; Reygaert,

2013). Most of the antibiotics in clinical practice are resistant to most of the tested

pathogens so there is need for more effective antimicrobial drugs.

As the plant extract/fractions and its isolated compounds showed marked activity

against most of the tested pathogens, therefore, it can be assumed that the

extract/fractions as well as its isolated compounds could be useful natural alternatives

against the infections caused by these resistant bacteria. In this connection, further

detail studies are required to ascertain its therapeutic potential, safety and clinical

uses.


64

Table 3.1: Antimicrobial Activity of the Crude Extract along with the

Subsequent Fractions of Viola serpens.

Zone of Inhibition (mm)

Group Salmonella

typhi

Escherichia

coli

Bacillus

subtilis

Shigella

flexneri

Staphylococcus

aureus

Pseudomonas

aeruginosa

Imipenem 33 30 33 27 33 24

Crude

Extract

10 6 6 8 3 5

n-Hexane - 2 - 2 - -

Chloroform 21 18 16 5 2 -

Ethyl

acetate

13 14 7 8 3 -

Aqueous 5 5 - 5 2 6

Data are presented as mean ± SEM of three independent assays.

Table 3.2 Antimicrobial activity of the Isolated Compounds from V. serpens

Compound Zone of Inhibition (mm)

Staphylococcus

aureus

Bacillus

subtilis

Shigella

flexneri

Escherichia

coli

Pseudomonas

aeruginosa

Salmonella

typhi

Imipenem 33 33 27 30 24 25

1 (Commuline

A)

24 15 12 _ _ 15

2 (Commuline

B)

_ 15 10 10 13 17

4

(Tectochrysine)

15 16 15 11 17 17

5

(Sideroxyline)

15 12 _ _ 16 18

6

(Cearoin)

_ 15 15 _ 17 17

Data are presented as mean ± SEM of three independent assays.


65

Extract Hexane CHCl3 ETA H2O Imipenem

0

10

20

30

40 P.aerogenes

% i

nh

ib

itio

n

Extract Hexane CHCl3 ETA H2O imipenem

0

10

20

30

40S.flexeneri

% i

nh

ib

itio

n


0

10

20

30

40S.typhi

% i

nh

ib

itio

n

Figures 3.1: % inhibition of the tested bacteria against the Crude extract/

fractions of V. serpens. Where CHCl3 represents Chloroform, ETA

represents Ethyl acetate and H2O represents the Aqueous fraction.


0

10

20

30

40B. subtilis

% i

nh

ib

itio

n

Hexane CHCl H 2 O Imipenem

0

10

20

30

40 E. coli

% i

nh

ibit

ion

inh

ibit

ion

Extract ETA 3

Extract Hexane CHCl ETA H 2 O Imipenem

0

10

20

30

40 S.aureous

% I

nh

ibit

ion

Inh

ibit

ion

3


66

3.1.2 Effect of Crude extract/Fractions of V. serpens in DPPH free Radical

Scavenging Assay

The free radical scavenging effect of crude/fractions of V. serpens at various

concentrations is shown in Table 3.3. The crude extract caused concentration

dependent scavenging effect against DDPH with maximum activity of 67.99% at 500

ppm and IC50 value 182 ppm. Upon fractionation, considerable change in effect was

observed. Only the n-hexane and chloroform soluble fractions were significant with

the dose dependent scavenging activity of 75.98 and 79.00 % at 500 ppm having IC50

164 ppm and 144 ppm respectively.

The compound (1-6) isolated from the plant were also investigated for the radical

scavenging activity at different concentrations. The compounds antioxidant activity is

presented in the Table 3.4. Maximum radical scavenging activity was showed by

commulin-C (78.05 %) with an IC50 value 168 ppm followed by commulin-B (89.45

%) and IC50 value was 98.15 ppm. It was then followed by the tested pure compound

commulin-A having 78.05 % as the values of its radical scavenging activity with IC50

201 ppm.

The DPPH free radical scavenging assay is a simple, economical and most commonly

used for the assessment of test articles (Marinova and Blatchvarov, 2011; Anwar et

al., 2009). The scavenging effect is either by the loss of proton, radical’s dismutation

and formation of chelate by donating hydrogen, resulting in the stable phenoxyl

radicals (Das and Pereira, 1990; De Gaulejac et al., 1989; Hatano et al., 1989; Nahak

and Sahu, 2010; Illahi et al., 2013). Free radical generation or oxidative stress has

been observed in various diseases such as inflammation, coronary heart, diabetes,

aging and various types of cancer (Valko et al., 2007; Maaz et al., 2010). Abundant


67

existence of phenols and polyphenols in the plant species are the signs of antioxidant

property which works through various mechanisms. The presence of hydroxyl groups

is responsible for the chemical structure of phenolic compounds for free radical

scavenging activity (Anu et al., 2011; Siddharthan et al., 2007). As V. serpens

contained various phenolic compounds (Anu et al., 2011) was responsible for the

antioxidant activity of the plant. Moreover, the isolated flavonoids 1-6 from the ethyl

acetate soluble fraction of the plant also showed marked scavenging effect against

DPPH. The study provides a strong scientific background to use the plant in diseases

related to oxidative stress.


68

Table 3.3: DPPH Scavenging Activity of Crude extract/Fractions of V.serpens

and Zones of Inhibition are Given in mm.

Concentrations

(ppm)

Crude

extract

n-Hexane Chloroform Ethyl

acetate

Aqueous BHT

20 0.62±1.45 3.65±1.33 3.99±0.13 1.66±0.00 0.93±0.02 8.51±1.01

40 6.63±1.54 7.63±0.21 6.04±0.08 1.99±0.02 3.95±0.11 20.58±2.00

60 10.43±1.11 11.79±1.03 9.08±0.16 10.41±0.22 10.68±0.26 59.52±3.10

80 27.01±1.09 29.13±1.11 33.21±1.45 19.77±0.21 14.80±0.23 66.64±2.45

250 66.31±0.21 69.75±1.54 74.04±3.21 25.90±0.20 15.06±1.32 76.45±3.02

500 67.99±2.14 75.98±2.43 79.00±2.56 37.52±0.31 26.76±2.13 87.43±2.63

IC50 182±3.70 164±4.21 144.0±2.56 - - 54±2.15

Values are mean ± SEM of three independent readings. Control= Methanol, Standard

= BHT (Dibutylhydroxytoluene).

Table 3.4 Anti-oxidant Effects of the Isolated Compounds 1–6 from V.

serpens whole Plant

Compounds % RSA IC50 μM

1 (Commuline-A) 78.05 201±2.01

2 (Commuline-B) 89.45 98.15±1.26

3 (Commuline-C) 82.12 168±3.19

4 (Tectochrysin) NA NA

5 (Sideroxyline) NA NA

6 (Cearion) NA NA

n-Propyl gallate (standard) 90.13 106±1.45

Data is presented as mean ±SEM of three independent assays.


69

3.1.3 Effect of Crude Extract/ Fractions of V.serpens in Larvicidal Effect

The crude methanolic extract and fractions of V. serpens were investigated for

larvicidal activity against Aedes aegypti and Culex quinquefasciatus species of

mosquito (Tables 3.5). The crude extract showed dose dependent activity against A.

aegypti. The maximum effect (59.67 %) was showed by crude extract against the

larvae of A. aegypti at 600 ppm. Upon fractionation, changes in the overall activity

were observed. The maximum percent inhibition was caused by the ethyl acetate

(89.91%) fraction followed by the chloroform (85.21%) at the concentration of 600

ppm. The n-hexane and aqueous fractions showed insignificant effect against the

larvae A. aegypti at any concentration.

Similarly, the crude extract and various fractions of V. serpens were investigated

against larva of Culex quinquefasciatus at different dilutions (Table 3.5). The crude

methanolic extract was effective at a concentration of 600 ppm with percent mortality

51 and IC50 value 539 ppm. The mere significant effect was observed in the

chloroform fraction with maximum percent mortality at a concentration of 600 ppm

(53 %) with the IC50 value 500 ppm followed by the fraction of ethyl acetate. The

ethyl acetate fraction with LC50 value 510 ppm also showed more significant effect

(52.43%) at a concentration of 600 ppm. Rest of the fractions (n-hexane and aqueous)

showed no significant effects against C. quinquefasciatus.

Throughout, the world, insects born diseases are mainly responsible for serious

diseases which may lead to mortality (Pavela, 2009).Various serious diseases (Dengue

fever, malaria, Japanies encephalitis, yellow fever, angioedema and filariasis) are

mainly caused by mosquitoes which annually causes millions of deaths (Peng et al.,

1999). Various insecticidals available in the market with the names; Methoprene,


70

Temephos, Arosurf MSF, Agnique MMF (monomolecular films) Bonide, BVA2, and

Golden Bear-1111 (GB-1111) (oils) but all of these have some side effects either

direct on the human life/ aquatic animals/ environment (Larvicides for Mosquito

Control, United State Environmental protection agency 2000, 735-F-00-002). So there

was need for a safer natural larvicidal with no side effects. From the results it is clear

that the crude extract and the subsequent fractions of V. serpens showed significant

larvicidal activity against both the species of mosquitoes (A. aegypti and C.

quinquefasciatus). Phytochemicals like alkaloids, flavonoids, saponins, tannins and

steroids contribute mostly to the larvicidal activity (Pedro et al., 2014). V. serpens has

also been provided by nature with these phytochemical which may be responsible for

its strong larvicidal activity (Pratik et al., 2011). Further detailed studies in this

connection, are required for assuring the therapeutic potential, safety, economical

source and clinical uses of V. serpens.

Table 3.5: Larvicidal effect of the crude extract along with the subsequent

fractions of V. serpens against Aedes aegypti and Culex

quinquefasciatus specie of mosquitoes.

Extract/Fractions Aedes aegypti LC50

ppm Percent Mortility

10 ppm 100 ppm 200 ppm 400 ppm 600 ppm

Crude extract 33.13±1.21 37.88 47.69 55.19 59.67 325 ppm

n-Hexane 12.0±1.40 14.13 15.09 16.20 16.90 -

Chloroform 49.11±1.21 57.21 62.32 77.31 85.21 59 ppm

Ethyl acetate 43.21±1.03 59.19 64.32 72.34 89.91 88 ppm

n-Butanol 6.91±1.33 10.31 13.37 25.25 29.44 -

Aqueous 7.21±1.43 7.89 8.32 8.84 9.12 -

Culex quinquefasciatus

Percent Mortility

Crude extract 34.24 39.33 43.12 47.67 51 539

n-Hexane 10.10 12.70 14.56 16.89 19 -

Chloroform 22.61 31.05 44.51 49.15 53 500

Ethyl acetate 20.16 30.98 34.88 46.18 52.43 510

Aqueous 13.10 16.14 18.36 20.05 22 -

Data is presented as mean ±SEM of three independent assays.


71

3.1.4 Effect of Crude Extract/ Fractions of V.serpens in Acetyl Cholinesterase

Assay

The effects of crude extract/fractions of V. serpens against acetylcholine esterase are

presented in the Table 3.6. The effect was observed at three different concentrations

(250, 500 and 1000 ppm). The crude extract showed concentration dependent

inhibition on acetylcholine esterase enzyme with maximum activity (68.55%) at 1000

ppm and IC50 value of 245 ppm. When crude extract was fractionated, considerable

changes in inhibitory profile was noted. Among the fractions, chloroform was the

most effective and caused maximum inhibition of 89% at 1000 ppm and IC50 value of

149 ppm. It was followed by ethyl acetate with maximum activity (70.5%) at a

concentration of 1000 ppm IC50 value of 156 ppm. The aqueous fraction also induced

significant inhibition (50.75 %) at a concentration of 1000 ppm with IC50 value 989

ppm. However, the n-hexane fraction was unable to produce significant effect at

tested concentrations.

Acetylcholinesterase being a secretary protein and important cholinergic synaptic

element hydrolyzes and releases acetylcholine from the nerves’ terminals

(presynaptic) (Brufani et al., 1986). Acetylcholine plays a key role in cognitive

functions such as learning and memorization (Rusted et al., 2000). The inhibitory

effect of acetyl cholinesterase plays a vital role in the management of neurological

disorders like Alzheimer’s disease (Rhee et al., 2001), Parkinson’s disease, senile

dementia, myasthenia gravis and ataxia (Repchinsky, 2004; Rahman and Choudhary,

2001). Acetyl cholinesterase inhibitor available in market now a days are donepezil,

tacrin and rivastigmin etc with side effects even for mild type of Alzheimer’s disease

(Schneider, 2001). So there is need for safe and effective drug. Medicinal plants with


72

different geographical conditions are sources of acetyl cholinesterase inhibitors

(Mukherjeea et al., 2007).

As the crude extract/fractions of V. serpens demonstrated marked inhibitory effect

against the acetyl cholinesterase in vitro therefore, it can be assumed that the plant

could be an effective natural source of acetyl cholinesterase inhibitors. Moreover,

quercetin has been reported from the plant which exhibited marked cholinergic

activity (Park et al., 1996; Jung and Park, 2007). Thus, it could be partially

responsible for the current action of the plant.

Table 3.6: Enzyme Inhibition effect of the Crude Extract and the subsequent

Fractions of V. serpens against the Enzyme Acetylcholine Esterase

Compounds Concentrations (ppm) Activity % IC50 (ppm)

Crude Extract 250 50.51±2.14

245

500 57±2.52

1000 68.55±3.10

n-hexane 250 47.25±1.41

189

500 34±1.35

1000 47.25±1.81

Chloroform 250 68.75±2.46

149

500 82.5±3.71

1000 89±3.90

Ethyl acetate 250 67.5±2.31

156

500 65±1.63

1000 70.5±2.70

Aqueous 250 13.75±0.15

989

500 45±1.67

1000 50.75± 2.21


73

3.2 IN-VIVO BIOLOGICAL ACTIVITIES

3.2.1 Acute Toxicity

V. serpens Wall. crude extract along with the subsequent fractions (n- hexane,

chloroform, ethyl acetate, n-butanol and aqueous) tested for acute toxicity at different

doses (1000, 1500 and 2000 mg/kg, i.p.) proved a safe herbal medicine. The mice

were safe and behaved normal when observed in the first 4 h and no death occurred

after 24 h. Assessment bioassay time period is represented in the Table 3.7.

Table 3.7: Acute Toxicity of the Crude Extract along with the Fractions of V.

serpens

Extract/Fraction Doses (mg/kg) Gross effect after 4h Mortality rate

after 24 h

Crude extract 1000

1500

2000

-

-

-

-

-

-

n-Hexane fraction

1000

1500

2000

-

-

-

-

-

-

Chloroform fraction 1000

1500

2000

-

-

-

-

-

-

Ethyl acetate fraction 1000

1500

2000

-

-

-

-

-

-

Aqueous fraction 1000

1500

2000

-

-

-

-

-

-

3.2.2 Hepatoprtotective and Nephroprotective Effects of Crude Extract/

Fractions of V. serpens

3.2.2.1 Hepatoprotective Effect

The hepatoprotective effects of the crude extract and subsequent fractions of V.

serpens are given in the Table 3.8 and Figures 3.2. Different blood parameters (ALT,

AST and ALP) along with the histopathological slides of the kidneys were selected.

The results showed that the ALT values noted in the groups of rabbits treated with


74

paracetamol alone showed a significant increase (six folds) than the values noted in

the normal saline treated animals. Silymarin, a standard hepatoprotective drug has

reduced the ALT value by two folds than the paracetamol value. Crude extract along

with all the fractions caused a greater reduction in the value as compared with the

paracetamol value. Chloroform soluble fraction at a dose of 150 mg/kg and ethyl

acetate soluble fraction at a dose of 300 mg/kg showed pronounced effects. Whereas,

the high doses of the crude extract and n-hexane soluble fraction showed no

significant effects. There was marked reduction in the AST values of the crude extract

along with all the fractions at both the low and high doses in comparison with the

paracetamol values. However, chloroform at low (150 mg/kg) and high (300 mg/kg)

doses, ethyl acetate and n-butanol at high doses (300 mg/kg) showed less significant

effect of AST values. Rest of the fractions in both the doses showed similar values to

the standard drug silymarin.

Similarly, there was a marked reduction in the ALP values of all the fractions along

with the crude extract at the doses of 150 and 300 mg/kg in comparison with the

paracetamol value. The ALP results showed to be even more pronounced than the

silymarin. The values are closer to the values of normal saline and silymarin. The

arrangement is given in the decreasing order of their effectiveness i.e silymarin >

aqueous fraction > n-butanol > chloroform > crude extract > ethyl acetate > n-hexane.

Effects of Histopathological Analysis

Histological sections of the liver of the rabbits treated with saline solution showed

normal tissue architecture with a centrally placed nucleus and foamy cytoplasm of

hepatocytes (Figures 3.2.1). No vascular disturbance was noted in the arterial and


75

venous system. The sinusoidal spaces were neither enlarged nor reduced but of

normal sizes.

The hepatocytes of the rabbits treated with paracetamol alone showed cellular

swelling and vacuolation (Figure 3.2.2). The rounded and sharply demarked

boundaries of the vacuoles were suggesting fatty changes. The sinusoidal spaces were

significantly decreased due to increased cell sizes. No vascular changes such as

congestion or hemorrhages were noted.

The crude extract of the plant showed a significant reduction in the paracetamol

induced damage to hepatocytes (Figure 3.2.3). Amelioration in the toxic effects of

paracetamol on the hepatocytes was noted in rabbit which were given crude extract at

both low (150 mg/kg) and high (300 mg/kg) doses (Figure 3.2.4). The protective

effects were more pronounced at a higher dose.

The rabbits treated with n-hexane extract of the plant showed a protective effect

against paracetamol mediated damage to hepatocytes. However, the higher doses of

plant extract exhibited minimal protection as noted in the lower dosed group.

Likewise, plant material extracted with chloroform, ethyl acetate and n-butanol

showed a lesser decrease in liver lesions at higher doses than the lower doses.

However, liver histology indicated a significant improvement in the group of rabbits

given aqueous plant extract at a higher dose than the lower dose (150 mg/kg).


76

Table 3.8: Effects of the Crude Extracts/Fractions of V. serpens Wall on the

Liver Related Parameters (AST, ALT and ALP) in the Rabbits

Models

*P<0.05, **P<0.01 ***P<0.001 when compared with PCM treated group % change =

Extract Treatment Value - Paracetamol Toxic Value/Test Sample Value X 100.

Groups Dose

mg/kg

Liver-related parameters with % change values

ALT AST ALP

Normal saline 1 mL/kg 20 ± 4.6 29.8±6.0 30.3 ± 4.3

Paracetamol Control 1000 129 ± 5.3 75.3 ± 18.8 185 ± 7.8

Standard Silymarin 50 65 ± 2.8*** 40 ± 6.9*** 81 ± 7.2

Crude extract 150 76 ± 9*** 65 ± 18.3*** 71.8 ± 10.4***

300 103 ± 1.8 47 ± 8.25*** 66.3 ± 3.1***

n-hexane 150 53 ±7.53*** 44 ± 8.3*** 89 ± 11.7***

300 96 ± 6.3 45 ± 2.6*** 80.3 ± 9.5***

Chloroform 150 27 ± 7.9*** 67.3 ± 4.9** 50 ± 8.6***

300 68 ± 8.2*** 82 ±15.4** 70 ± 9.5***

Ethyl Acetate 150 66 ± 1.9*** 66 ± 4.39*** 85 ± 16.3***

300 47 ± 4.039*** 83 ± 2.5 * 62 ± 6.7***

n-Butanol 150 68 ±14.3*** 67.3 ± 1.5** 44.3 ± 4.5***

300 73 ± 3.4*** 65 ± 1.96*** 45 ± 33.3***

Aqueous 150 75 ± 6.79*** 48 ± 1.9*** 39.5 ± 2.4***

300 62 ± 2.2*** 45.5 ± 5.3*** 34 ± 3.1***


77

Figure 3.2.1: Normal saline treated liver showing normal architecture of central vein (CV), sinusoidal spaces (small

arrows), hepatocytes (large arrows) with a centrally placed

nucleus and foamy cytoplasm. (100X H&E).

Figure 3.2.2: Liver showing accumulation of lymphocytes (small arrows) around the central vein (CV), fatty changes (small arrow

head) and focal area of necrosis (asterisk) with paracetamol

(100X H&E).

Figure 3.2.3: Liver showing regeneration, containing normal

liver plates (large arrows) along central vein (CV) with n-hexane

150 mg/kg b.w. (H&E).

Figure 3.2.4: Liver showing normal appearance of central vein

(CV) and plates of hepatocytes (large arrows) with n-hexane 300

mg/kg b.w. (100X H&E).

Figure 3.2.5: Liver showing hexagonal hepatocytes (large

arrows) with prominent cell borders (small arrows), nuclei (arrow

heads) with nuclear clearing and prominent nucleoli with crude

extract at a dose of 150 mg/kg b.w. (400X H&E).

Figure 3.2.6: Liver showing regeneration of hepatocytes (large

arrows) with congestion of sinusoids (asterisks) containing red

blood cells (small arrows) with crude extract at a dose of 300

mg/kg b.w. (400X H&E).

Figures 3.2.1-3.2.2 Liver photomicrographs of the rabbits treated with

paracetamol, crude extract and n-Hexane fraction of V.serpens at

doses of 150 and 300 mg/kg (H&E, 100X and 400X).


78

3.2.2.2 Nephroprotective Effect of V. serpens Crude Extract and its Subsequent

Fraction

The nephroprotective effects of the crude extract/fractions of V. serpens from the

blood biomarkers and histopahtological slides are summarized in the Table 3.9 and

Figures 3.3 respectively.

In kidney related blood parameters, blood urea of the crude extract and some of the

fractions are insignificant in comparison with the paracetamol values. Whereas,

aqueous fraction, in both the low and high doses (150 and 300 mg/kg), crude extract

and chloroform in low doses (150 mg/kg), n-hexane, ethyl acetate and n-butanol in

high doses (300 mg/kg) are comparatively more effective than the paracetamol values

and closer to the values of normal saline.

Serum creatinine values of all the fractions along with the aqueous methanolic extract

are significant in comparison with the paracetamol and normal saline values.

Creatinine clearance value has been reduced to the low level than the normal value by

paracetamol dose at 1mg/kg body weight for 8 days. No significant effects except

chloroform soluble and aqueous soluble fractions at high doses (300 mg/kg) were

obtained. Aqueous soluble fraction at low dose (150 mg/kg) is also comparatively

significant. The creatinine clearance values of all the fractions along with aqueous

methanolic extracts at both (low and high) doses are closer to the values of normal

saline (nephrprotective).

The histological section of the kidneys of rabbit treated with saline solution showed

normal tissue structure with normally placed glomeruli and tubules. The size of

glomerular cells and urinary spaces were normal. The tubular epithelial cells were

normal in size and adhered to basement membranes. No vascular disturbance was

observed (Figure 3.3.1-3.3.2).


79

Effects of Histopathological Analysis

The histological sections of the kidneys of the rabbits treated with paracetamol alone

showed a wide spread signs of toxicities. The most obvious ones were degeneration

changes in tubules, where the tubular epithelial cells were swollen (most probably

hydropic change) with some clear fatty changes. The sloughing of tubular epithelial

cells from the basement membrane and accumulation in the tubular lumen was

another prominent lesion in the tubular cells. The glomeruli showed shrinkages and

increase urinary spaces. No histological observable difference was noted in the

sections.

The protective role of the plant materials extracted with methanol and chloroform

were obvious from kidneys histology. The groups of rabbit given n-hexane, ethyl

acetate and n-butanol factions showed inverse dose dependent relationship in the

kidneys histology. The improvements in the lesions were lesser in groups given

higher dose of plant extract as compared to lower dosed group. However, the aqueous

fraction showed a dose dependent response.

Exposure of liver and kidneys to the drugs itself or its active metabolites results either

into direct toxicity or may get a chance of immunological reaction (Maaz et al., 2010).

Toxic metabolites are the results of about 62% of withdrawn drugs administration.

Paracetamol is a commonly used analgesic and antipyretic drug, results in acute

centrilobular necrosis and centrizonal heamorrhagic (Boyd and Bereckzky, 1966;

Clark et al., 1973). 90-95% PCM metabolism occurs through the liver and excreted

through kidneys (Temple and Himmel, 2002; Edwige et al., 2012). In body various

reactive radicals like hydroxyl radicals, hydrogen peroxide, superoxide anions, nitric

oxide, nascent oxygen and lipid oxides generation occur due to certain internal and

external factors resulting in disorders like hepatic ailment and kidneys disorders


80

(Beris et al., 1991; Malila et al., 2002; Yerra et al., 2005). In therapeutic doses of

PCM, only 5% of the drug was converted to N-acetyl-p-benzoquineimine (NAPQI), a

highly reactive cytochrome P450 mediated intermediate metabolite (Raucy et al.,

1989).Whereas, in toxic doses it is mostly oxidized by cytochrome P-450 enzymes to

highly reactive NAPQI (Kassem et al., 2013). Decreased glutathione store or

metabolites NAPQI covalently bond to vital proteins, hepatocyte membrane’s lipid

bilayer and raise the lipid peroxidation (McConnachie et al., 2007) responsible for

mediating liver and kidneys toxicity. Biochemical parameters (AST, ALT and ALP)

with increased levels better reflect the liver injury (Benjamin, 1978; Wittwer et al.,

1986; Edwards and Bouchier, 1991).

In the present study, the liver biomarkers, ALT, AST and ALP values were

significantly reduced and comparable to silymarin treated group in comparison with

the values of purely paracetamol intoxicated groups. This suggests the protection,

regeneration, and restoration of the cellular permeability of the plant extract and

fractions in the paracetamol intoxicated rabbit models. The mechanisms involved

behind this may be the free radical scavenging effect by intercepting the radicals

involved in paracetamol metabolism (microsomal enzymes). Antioxidants are agents

that can neutralize deleterious effects of free radicals. Exogenous support is taken for

keeping a balance between oxidants and antioxidants. Plants with antioxidant

properties are becoming more and more popular all over the world (Jayaprakash et al.,

2001). There is a strong relationship between the phenols and antioxidant activity

(Velioglu et al., 1998; Kahkonen et al., 1999; Javanmardi et al., 2003). The

antioxidant constituents and the phenolic compounds showed the potential to prevent

oxidative degradation of cellular components (Zhon and Zheng, 1991; Kahkonen et

al., 1999).


81

Phytochemically analysis showed that V. serpens contained antioxidant constituents

such as ascorbic acid, ascorbate oxidase, peroxidase and catalase (Vukics et al., 2009)

along with the phenolic contents which could be the reason behind its

hepatoprotective and nephroprotective effects against the paracetamol induced

hepatotoxicity and nephrotoxicity. Additionally, there was a linear positive correlation

between the total phenolic contents and antioxidant capacities of V. serpens

(Siddharthan et al., 2007). Moreover, one of the mechanisms in the hepatoprotection

and nephroprotection may be due to the phytochemicals presence like flavonoids,

glycosides, alkaloids, coumarins and tannins present in V. serpens plant (Pratik et al.,

2011). The scientific reports also indicated the hepatoprotective and nephroprotective

role of certain flavonoids, triterpenoids and steroids in toxicity (Garba et al., 2009).

Purely paracetamol treated rabbit groups histopathology showed cellular swelling and

vacuolation of the hepatocytes. Fatty changes with swollen vacuoles and decreased

sinusoidal spaces due to increased cell sizes have also been indicated. The histological

slides of crude extract of the plant both at low and high doses showed significant

recovery of the paracetamol-induced toxicity. The mentioned biochemical

constituents in the extract showed the presence and recovery of the toxified

hepatocytes which is dose dependent. The histopathology of rabbits treated with the

plant fractions showed protective effects. The effectiveness was more at low doses

than high doses whereas, the case was reversed in n-butanol.

The histological sections of the kidneys of the rabbits treated with paracetamol alone

showed a wide spread signs of toxicities like degeneration of the tubular epithelial

cells, swelling and fatty changes, shrunk glomeruli and increased urinary spaces. It is

clear from the histological slides of the groups treated with methanol (crude extract)

and chloroform along with the toxic paracetamol doses that the presence of certain


82

biochemical constituents in the plant extract/fraction, secure the kidneys against the

toxicity. The rabbit groups of n-hexane, ethyl acetate and n-butanol factions showed

inverse dose related relationship in the kidneys histopathology.

It is concluded from the present study that the crude extracts and different fractions of

V. serpens Wall. possess strong hepatoprotective and nephroprotective activities and

thus provided a scientific rationale for the uses of the plant in the treatment of liver

and kidney toxicities. In this regard, a further detailed study regarding the

phytochemistry and pharmacology is required to ascertain its chemical background.

Table 3.9: Effect of the Crude Extract/ Fractions of V. serpens Wall. on the

Kidney’s Function and Clearance in the Rabbits Models

Groups Dose

mg/kg

Kidney Related Parameters with % Change Values

Blood urea Serum Creatinine Creatinine

Clearance

Saline 1mL/kg 12.0 ± 2.6 0.3 ± 0.12 4.7 ± 2.8

PCM Control 1000 24.3 ± 2.3 1.5 ± 0.29 0.36 ± 1.3

Crude extract 150 15.3*** ± 1.3 0.6 ± 0.04*** 1.5** ± 0.29

300 21*± 2.5 0.5 ± 0.00 *** 1.35**± 0.26

n-hexane 150 25 ± 2.6 0.05 ± 0.03*** 1.1** ± 0.21

300 19.8**± 3 0.52 ± 0.02*** 1.36 ± 0.27

Chloroform 150 18.5**± 1.5 0.5 ± 0.11*** 2.0**± 0.6

300 22.3*± 2.3 0.4 ± 0.03*** 4.0 ± 0.9

Ethyl Acetate 150 24 ± 3.1 0.6 ± 0.12*** 0.84 ***± 0.18

300 19.8* ± 4.5 0.6 ± 0.06*** 0.93*** ± 0.24

n-Butanol 150 23.3 ± 3.1 0.62 ± 0.04*** 1.26**± 0.12

300 18.8**± 3.3 0.6 ± 0.04*** 1.5**± 0.4

Aqueous 150 17.7***± 2.0 0.7 ± 0.08*** 2.5* ± 0.59

300 14.3***± 2.0 0.5 ± 0.08*** 4.7 ± 1.0

*P<0.05, **P<0.01 ***P<0.001 when compared with PCM treated group

% change = Extract Treatment Value ˗ PCM Toxic Value/Test Sample Value X100


83

Figure 3.3.1: Photomicrograph (100X H&E) of a section of kidney

from a rabbit treated with normal saline showing normal

histological appearance of renal cortex. The cortex contains renal

corpuscles (large arrows) embedded among proximal (arrow

heads) and distal (asterisk) convoluted tubules.

Figure 3.3.2: Photomicrograph (100X H&E) of a section of

kidney from a rabbit treated with PCM showing necrosis of

cuboidal epithelial cells (large arrows) of proximal convoluted

tubules with exfoliation of their brush border. The lumen

(asterisk) of tubules contains numerous cellular casts (small

arrows).

Figure 3.3.3: Photomicrograph (100X H&E) of a kidney section

from a rabbit treated with n-hexane soluble fraction 150 mg/kg

showing normal histo-architecture of distal convoluted tubules

with wider lumen (asterisk) and lined by cuboidal epithelial cells

(arrow heads). Numerous loop of Henle tubules are also visible

(large arrows).


kidney from a rabbit treated with n-hexane soluble fraction 300

mg/kg showing normal renal corpuscles (large arrows) with

mild dilatation of proximal (arrow heads) and distal (asterisk)

convoluted tubules.

Figure 3.3.5: Photomicrograph ((100X H&E)) of a section of

kidney from a rabbit treated with chloroform soluble fraction 150

mg/kg showing normal renal corpuscles (large arrows), proximal

(arrow heads) and distal (asterisk) convoluted tubules.

Figure 3.3.6:Photomicrograph (100X H&E) of a section of

kidney from a rabbit treated with ethyl acetate soluble fraction

150 mg/kg showing normal renal corpuscles (large arrows)

with mild dilatation of proximal (arrow heads) and distal

(asterisk) convoluted tubules.


84

Figure 3.3.7: Photomicrograph ((100X H&E)) of a section of

kidney from a rabbit treated with chloroform soluble fraction 300

mg/kg showing normal renal corpuscles (large arrows) and

proximal convoluted tubules (arrow heads). The distal convoluted

tubules (asterisk) exhibited mild tubular necrosis of the cuboidal

epithelial cells.


kidney from a rabbit rat treated with ethyl acetate soluble

fraction 300 mg/kg showing normal proximal convoluted

tubules (large arrows) with numerous loop of Henle tubules

(asterisk). The interlobular blood vessels (arrow heads) among

the renal tubules exhibited mild congestion with red blood

cells.

Figure 3.3.9: Photomicrograph (100X H&E) of a section of kidney

from a rat treated with aqueous soluble fraction 300 mg/kg

showing normal renal corpuscles (large arrows). The renal tubules

exhibited dilatation (arrow heads) with exfoliation of the brush

border lining the proximal convoluted tubules into their lumen.


kidney from a rat treated with aqueous soluble fraction

showing mild congestion of the renal corpuscles (large arrows)

with severe dilatation of the renal tubules (asterisk). Numerous

cellular casts (arrow head) is also visible in the lumen of renal

tubules.

Figure 3.3.1-3.3.10: Photomicrogrphs of the Kidneys of Rabbits Treated with

Paracetamol and Plant Extract/ Fractions at Different Doses

(H&E).


85

3.2.2.3 Antinociceptive Activity

Effect of Crude Extract/ Fractions of V. serpens Wall. in Acetic Acid Induced

Writhing Test

The results of crude extract/fraction of V. serpens in acetic acid induced writhing test

at various doses (100, 200 and 300 mg/kg i.p.) are shown in Table 3.10. The standard

drug diclofenac was used for comparison. The crude extract caused significant

attenuation of writhes induced by the injection of acetic acid in a dose dependent

manner with a more significant value of 19.77 with the inhibition of 70.05% reduction

in pain at 300 mg/kg i.p represented in the Figure 3.4. The crude extract upon

fractionation provoked different effects. The maximum effect is produced by the

soluble fraction of n-hexane in a dose dependent manner followed by the soluble

fraction of ethyl acetate. At a dose of 300 mg/kg maximum reduction in the numbers

of writhes were noted (21) with a percent reduction value of 68.8% represented in the

Figure 3.5. The chloroform soluble fraction also incorporated a significant effect in a

dose dependent manner with a significant value of 31.50 at a maximum dose of 300

mg/kg and at a percent reduction value of 50.37 % represented in the Figure 3.6. The

soluble fraction of ethyl acetate also produced a dose dependent analgesic effect with

the more significant value 34.75 at a dose of 300 mg/kg body weight with a percent

reduction value 50.37 % showed in the Figure 3.7. Whereas, the aqueous soluble

fraction does not produce significant antinociceptive effect at any test dose

represented in the Figure 3.8.


86

Table 3.10: The Effect of Crude Extract/Fractions of V. serpens in Acetic Acid

Induced Writhing Test in Mice (i.p)

Drugs Dose mg/kg No. of writhing (10min) % Protection

Saline 10 ml/kg 66±2.90

Crude 100 37.55±2.70* 43

200 25.80±2.90** 60.9

300 19.77±2.00** 70.05

n- hexane 100 39.40±2.50* 40.30

200 27.90±2.56** 59.09

300 21.50±1.9** 68.18

Chloroform 100 49.90±2.90 24.4

200 38.70±2.80* 42.42

300 31.50±1.70* 52.27

Ethyl acetate 100 45.33±2.10 31.31

200 37.12±2.75* 43.76

300 34.75±2.45* 50.37

Aqueous 100 55.50±2.90 15.9

200 50.12±3.10 24.1

300 45.27±2.90 31.8

Diclofenac 10 11.15±1.5 83.1

Values are reported as mean ±SEM for group of six mice. ANOVA followed by

Dunnett tests were used for data analysis. Significant and satisfactory values are

represented by asterisks from the control. *P<0.05 or **P<0.01


87

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

1 0 0

**

**

***

*

% P

ro

te

ctio

n

C r u d e e x tr a c t

Figures 3.4: Antinociceptive Effect of Extract of V. serpens in Acetic acid

Induced Writhing Test. Significant and satisfactory values are represented by

asterisks from the control. *P<0.05 or **P<0.01

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

1 0 0

**

*

**

*

**

*

% P

ro

te

ctio

n

H e x a n e

Figures 3.5: Antinociceptive Effect of n-hexane Soluble Fraction of V. serpens

in Acetic Acid Induced Writhing Test. Significant and satisfactory values are



88

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

1 0 0 **

*

*

*

% P

ro

te

ctio

n

C h lo r o f o r m

Figures 3.6: Anti-Nociceptive Effect of Chloroform Soluble Fraction of V.

serpens in Acetic Acid Induced Writhing test. Significant and satisfactory values

are represented by asterisks from the control. *P<0.05 or **P<0.01

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

1 0 0

*

**

*

% P

ro

te

ctio

n

E t h y l a c e ta t e

Figures 3.7: Antinociceptive Effect of Ethyl Acetate Soluble Fraction of V.

serpens in Acetic Acid Induced Writhing Test. Significant and satisfactory values

are represented by asterisks from the control. *P<0.05 or **P<0.01


89

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

1 0 0

*

**

***

% P

ro

te

ctio

n

A q u e o u s

Figures 3.8: Antinociceptive Effect of Aqueous Soluble Fraction of V. serpens in

Acetic Acid Induced Writhing Test. Significant and satisfactory values are


Effect of Crude Extract/ Fractions of V.serpens in Formalin Induced

Nociception Test

The effect of formalin induced nociception test in both phases is shown in Table 3.11

and Figures 3.9-3.13. In the initial phase (0-5 min) the crude extract showed dose

dependent antinociceptive effect. The maximum pain reduction (35%) was observed

at a dose of 300 mg/kg i.p. while in second phase (15-30 min) the crude extract

showed more pronounced pain relieving effect (64.8%) at a dose of 300 mg/kg i.p.

When the crude extract was subjected to fractions marked changes in effect were

observed. The fraction of n-hexane showed a dose dependent pain subsiding effect. In

both the early and late phases the more significant percent inhibitory effects found

were 38.23 % and 55.21 % respectively. This was followed by the chloroform fraction

which showed pain relieving effect more effectively in both the early and late phases.


90

The maximum percent inhibition of the fraction was 26.5% and 37.14% respectively

in the dose dependent manner. The Ethyl acetate fraction also showed significant

effect with 25.0 and 38.57% inhibitions in the early and late phases respectively. The

lowest antinociceptive effect among all the fractions of the plant extract was noted in

the aqueous fraction with the percent inhibition values of 23% and 25% in the early

and late phases respectively.

Acetic acid induced abdominal constriction/writhes is an assay used for the

measurement/determination of antinociceptive activity by the peripheral mechanism

(Du et al., 2007; Duarte et al., 1988). Writhes are define as the stereotypical response

of the mice/rats (serous membrane) resulting with the intraperitoneal administration of

the irritating agent in which the coordination of the motor activity is disturbed along

with the movement of the body and muscles (Zeashana et al., 2009) stressful

constriction of the abdominal cavity occur. As a result of acetic acid (pain inducer)

administration, release of pain mediators/ endogenous substance causes the increased

production of lipooxygenase as well as prostaglandin (PGE2 and PGE2α) in the

peritoneal fluid (Deraedt et al., 1980; Khan et al., 2010; Mbiantcha et al., 2011).

Capillary permeability is increased which ultimately causes the stimulation of

inflammatory pain through the peritoneal receptors (Collier et al., 1968; Choi 2007).

The reduction in the number of writhes indicates the antinociceptive activity. The

plant extract and its different fractions produced more attenuated antinociceptive

effect in dose dependent manners. The significance of the activity decreased with the

increased polarity of the solvents. Means that the crude extract showed more

attenuated analgesic effect whereas, the effect decreases as we go on increasing the

polarity i.e in n-hexane, ethyl acetate, chloroform and aqueous soluble fractions. In

the three test doses, 300 mg/kg (i.p) was more effective in all the fractions/crude


91

extract of the plant as compared with the low doses (100 & 200 mg/kg, i.p). The

peripheral pathway followed by the plant for this activity may be due to the

inhibition/hindrance of the local peritoneal receptors that may cause the

inhibition/reduction in the release of cyclooxygenase or lipoxgenase enzymes. The

release of certain mediators may also be involved in the analgesic activity of the plant.

Due to the pronounced antinociceptive effect, the plant may be effectively

recommended for clinical purpose.

In general, the acetic acid induced writhing test is nonspecific and therefore,

mechanistic approach was performed by using formalin induced paw licking test.

Formalin induced paw licking and flicking protocol is a suitable method for the

qualitative measurement of centrally acting analgesia (Dubuisson & Dennis 1977;

Tjolsen et al., 1992). Formalin induced nociception being a biphasic analgesic

behavioral protocol with the involvement of two clearly different stimuli. The

chemical released in the early phase (neurogenic phase) are the bradykinin and

substance P. The late inflammatory phase, involve the release of prostaglandins,

histamine, serotonin and bradykinin (Tjolsen et al., 1992). The plant extract/fractions

of V. serpens showed more pronounced antinociceptive effect in late phase as

compared to the early phase. The effect decreased with the increasing polarity of the

solvent in the subsequent manner. Means more significant effects were shown by the

n-hexane fraction followed by the chloroform, ethyl acetate and aqueous fraction in a

dose dependent manner. The effect of the plant being more significant in the second

phase indicates its similarity with the non-steroidal anti-inflammatory drug like

indomethacin and aspirin (Santos et al., 1994; Choi et al., 2001). Centrally acting dugs

like narcotic analgesics show effectiveness in both the early and late phases (Stai et

al., 1995; Santos et al., 1994).


92

The main constituents in the plant of V. serpens are alkaloids, saponins, tannins and

flavonoids (some also isolated in the present study). These compounds may be

responsible for the inhibition/decrease in mediators release like prostaglandins,

histamine, serotonin or bradykinin, responsible for the pain suppression in the late

phase of formalin induced pain. (Naveed et al., 2012; Naveed et al., 2012). The

outcome of the study is that the anti-nociceptive property of the plant was mediated

through the peripheral mechanism; augmented by interference of centrally acting pain

mediators. Thus, this study provided a scientific rationale for the traditional use of the

plant in different animal protocols.


93

Table 3.11: Effect of the Crude Extract/ Fractions of V. serpens in Formalin

Induced Pains for Analgesia Test in Mice at Doses of 100, 200 and

300 mg/kg, i.p

Drugs Dose mg/kg Early phase

(0-5min)

Late phase

(15-30 min)

Saline 10 ml/kg 68±1.79 93±2.30

Crude 100 59±2.95 50±2.00*

200 51±2.20* 42±2.25**

300 44±2.90** 33±1.70***

n-hexane

100 60±1.36 50±2.19

200 53±1.79* 41±2.25*

300 42±2.24** 29±1.35**

Chloroform

100 62±2.45 51±2.50*

200 56±2.50 38±2.35**

300 50±2.70* 29±2.50***

Ethyl acetate

100 60±2.36 44±2.50*

200 56±2.50 36±2.35**

300 51±2.10* 28±2.50***

Aqueous

100 62±3.10 53±2.50

200 57±2.90 51±2.35*

300 52±2.90* 46±2.50*

Tramadol 30 39±1.34 20±1.10**

Values are reported as mean ±SEM for group of six mice. ANOVA followed by

Dunnett tests were used for data analysis. Significant and satisfactory values are

represented by asterisks from the control. *P<0.05 or **P<0.01.


94

100 200 300 Tramadol

0

20

40

60

80Early PhaseLate PhaseCrude extract

% P

ro

tecti

on

Figure 3.9: Antinociceptive Effects of Formalin Induced Pain in Mice of the

Crude Extract of V.serpens

100 200 300 Tramadol

0

20

40

60

80 Early Phase

Late Phasen-Hexane

% P

ro

tecti

on

Figure 3.10: Antinociceptive Effects of Formalin Induced Pain in Mice of the n-

Hexane Soluble Fraction of V.serpens.


95

100 200 300 Tramadol0

10

20

30

40

50Early PhaseLate Phase

Chloroform%

Pro

tecti

on


Chloroform Soluble Fraction of V.serpens.

100 200 300 Tramadol0

20

40

60

80Early PhaseLate Phase Ethyl acetate

% P

ro

tecti

on


Ethyl Acetate Soluble Fraction of V.serpens


96

100 200 300 Tramadol0

20

40

60

80 Early PhaseLate Phase

Aqueous

% P

ro

tecti

on


Aqueous Soluble Fraction of V.serpens

3.2.2.4 Anti-inflammatory Activity

Effect of Crude Extract/Fractions of V. serpens on Paw Edema Induced by

Carrageenan

The effect of crude extract/fractions of V. serpens at various doses during different

assessment times is shown in Tables 3.12-3.16. It exhibited significant inhibition of

carrageenan induced paw edema only in the 3rd h of administration at 100 mg/kg i.p.

However, it showed marked anti-inflammatory effect after 2nd h of injection that

remained significant up to 5th h at a dose of 200 and 300 mg/kg i.p and the %

(percent) protection represented in the Figure 2.14.

The crude extract was then fractionated into various fractions which showed different

anti-inflammatory effects at different doses. The n-hexane soluble fraction showed

maximum anti-inflammatory effect against the carageenan induced paw edema at a


97

dose of 100 to 300 mg/kg i.p in the 2nd and 3rdh. Whereas, the effectiveness of the

carageenan induced anti-inflammatory effect remained till the 5th hour of injection.

The % protection is represented in the Figure 3.15. The chloroform and aqueous

soluble fractions showed significant effect only at a dose of 200 and 300 mg/kg in the

3rd h of induced inflammation and the significant % protections represented in the

Figure 3.16 and 3.18 respectively. Ethyl acetate soluble fraction showed anti-

inflammatory activity at a dose of 300 mg/kg in the 2nd h. Moreover, in the 3rd h both

the doses, 200 and 300 mg/kg were significant and the percent protection values

represented on the Figure 3.17.

Effect of Crude Extract/Fractions of V. serpens on Paw Edema Induced by

Histamine

The effect of the crude extract/fractions of V. serpens in histamine induced paw

edema at various doses (100, 200 and 300 mg/kg i.p) in various durations (1-5 h) is

presented in the Tables 3.12-3.16. The crude extract at doses of 200 and 300 mg/kg

showed more pronounced anti-inflammatory effects in the 2nd to the 5th hours of the

histamine induced edema. The significance level reached the maximum in the 3rd hour

and then decreased slowly till the 5th hour. The crude extract was then subjected to

various fractions, exhibiting different inhibitory effects showed in the Figure 3.19. In

fractions maximum anti-inflammatory effect against the histamine induced paw

edema was produced by the n-hexane soluble fraction at a dose of 200 mg/kg in the

3rd h with percent inhibition 46.70% represented in the Figure 3.20. The significant

anti-inflammatory effect started from the 2nd h and lasted till the 5th h of the edema

induction. On the other hand chloroform and aqueous soluble fractions showed

significant effects at the doses of 200 and 300 mg/kg on the 3rd h of histamine induced

edema with percent inhibition values of 31.48 and 34.60 % represented in the Figures


98

3.21 and 3.23 respectively. Whereas, the ethyl acetate soluble fraction was non-

significant in the three test doses at all the 5 mentioned test hours with the %

inhibition representation in the Figure 3.22.

Effect of Crude Extract/Fractions of V. serpens on Ear Edema Induced by

Xylene

Results of anti-inflammatory effect of V. serpens on xylene induced ear edema are

presented in the Table 3.17 and the percent inhibition in the Figures 3.24. The crude

extract/subsequent fractions of V. serpens were subjected for the anti-inflammatory

effect by using xylene induced ear edema protocol. Three test doses were selected

(100, 200 and 300 mg/kg Oral administration) for the anti-inflammatory effective

results determination. The crude extract showed maximum inhibitory effect (57.6 %)

at a dose of 300 mg/kg. The effect of the crude extract was significant in a dose

dependent manner. Upon treatment with different solvents the fractions obtained

showed different anti-inflammatory effects. The most effective and significant

fraction considered was the n-hexane which also showed significance in a dose

dependent manner with the maximum percent inhibition value of 55 % at 300 mg/kg.

This was followed by the chloroform and ethyl acetate soluble fractions and then by

the aqueous soluble fraction whose considerable effects were shown at doses of 200

and 300 mg/kg with the maximum inhibition values of 51, 49 and 48.5 %

respectively.

Inflammation being a complex process has direct association with pain which may

involve increase in: vascular permeability, cells migration (mononuclear and

granulocytes) and proliferation of granulomatous tissue. Anti inflammatory

compounds act through different mechanisms, either by blocking the pro-

inflammatory mediators (directly via enzyme like COX-2 inhibition) or enzyme


99

expression is decreased such as anti-inflammatory steroidal compounds or substrate

levels are decreased like reduction in the release of arachidonic acid. Immuno-

stimulation is also one of the mechanism i.e phagocytosis activation as well as

maturation of myeloid cells which ultimately response to the challenge of allergen

(Safaihy and Sailer, 1997). The plant extract/fractions of V. serpens demonstrated its

effectiveness against the induced inflammation protocols in carrageenan and

histamine paw edema and xylene ear edema.

Carrageenan being a choice of phlogistic agent is used for anti-inflammatory drugs

testing and having an extensive measurement of reproducibility (Winter, 1957). It is a

biphasic model with the early phase including 1–2 h, mediated mostly by the release

of serotonin, histamine and prostaglandins increased level. The late phase includes the

release of prostaglandin whereas, kinine releases in between the two phases (Antonio

and Souza, 1998; Zhou et al., 2008). The enzyme cyclooxygenase (COX) catalyses

the biosynthesis of prostaglandin metabolites (arachidonic acid) in the early phase

(Teather et al., 2002). COX-1 (constitutive form of COX) is involved in cellular

function (Herschman, 1996).COX-2 (inducible isoform) increases response to various

tissue inflammatory stimuli (Teather et al., 2002). COX-3 is determined in the heart

tissue and brain cortex (Chandrasekharan et al., 2002).

In carrageenan induced paw edema protocol the crude extract and n-hexane fractions

were effective against the inflammation challenge from the 2nd till the 5th h at 200 and

300 mg/kg. whereas the chloroform, aqueous and ethyl acetate fractions also showed

significant effects and reduced paw edema in the 3rd h at 300 mg/kg i.p. The crude

extract and the n-hexane fraction of V. serpens are effective in both the phases

whereas, rest of the fractions showed significant effects only in the late phase.


100

Histamine, a fundamental amine and mediator associated with inflammation and

allergic reactions, causes both increase in the vascular permeability and vasodilatation

(Rang et al., 2001; Linardi et al., 2002; Cuman et al., 2001). The lipoxygenase and

cyclooxygenase pathways are followed by the arachidonic acid metabolites.

Prostaglandin (PG) and prostaglandin E2 (PGE2) are mainly involved in the cause or

enhancement of the signs of cardinal inflammation. These enzymes of the arachidonic

acid provoke the inflammatory response (Young et al., 1984). The results of the

present study revealed that the two doses (200 and 300 mg/kg i.p) of V. serpens in the

crude as well as in the subsequent fractions suppressed the histamine induced edema

effectively which may be due to the presence of such compounds capable of resisting/

inhibiting the release of histamine, prostaglandins or mediators of the mast cells

(histamine, PG and 5-HT) (Rao et al., 2005).

The xylene-induced ear edema in mouse is a testing and investigating procedure for

acute anti-inflammatory activity response, resulting in severe vasodilatation and skin

edema (ear) (Atta and Alkofahi, 1998; Kim et al., 2007; Xiao-Jia et al., 2008). Xylene

tropical application on ear leads to an immediate mouse ear irritation resulting in the

fluid accumulation (edema formation) and acute response of inflammation (Okoli et

al., 2006). Anti-inflammatory steroidal and non-steroidal antiphlogistic agents are

evaluation by this method especially the ones inhibiting phospholipase A2 (Zaninir et

al., 1992). The results obtained from the study showed that the ear edema of the crude

extract as well as in the fractions subsided in a dose dependent manner (crude extract

and n-hexane). Whereas, in the other fractions significant effects were found only at

high doses (300 mg/kg). Thus, the effectiveness of V. serpens in the model suggests

that the plant extract and its fractions possibly act by inhibiting the enzyme

phospholipase A2 (PLA2) (Atta and Alkofahi, et al., 1998).


101

Phytochemically, different groups of compounds are reported to be present in Viola

species including triterpenoids, cyclotide, alkaloids and flavonoids. (Naveed et al.,

2012; Naveed et al., 2012). Triterpenoids are one of the important contributors of anti-

inflammatory activity (Safaihy and Sailer, 1997; Andrikopoulos et al., 2003). Along

with this the presence of inflammation sites in high concentration oxidant and free

radicals also contributes to the anti-inflammatory process and play an important role

in avoiding the process of inflammation (Salvemini et al., 1996). V. serpens also

contains various phenolic compounds (Anu et al., 2011) and possesses antioxidant

activity along with the triterpenes (anti-inflammatory compounds) which may be the

major contributors for its anti-inflammatory activity. In the present study the isolated

flavonoids 1-6 from the chloroform fraction of the plant also showed marked

scavenging effect against DPPH so this may also give a solid scientific background to

the plant as a strong anti-inflammatory agent. Moreover, further work in future is

required to be focused on this plant as an anti-inflammatory agent to make its use

more authentic and more common with the scientific knowledge.


102

1h 2h 3h 4h 5h0

20

40

60

80

100 100/kgmg 200mg/kg 300mg/kg Diclofenac

*

*

*

**

**

*** **

***

*

*

**

*

*

*

***

% i

nh

ibit

ion

Figure 3.14: Anti-inflammatory Effect (%) of the Crude Extract of V. serpens on

Carrageenan Induced Paw Edema

1h 2h 3h 4h 5h0

20

40

60

80

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

*

**

*

**

**

* **

*

**

*

***

**

*

* *

**

% I

nh

ibit

ion

Figure 3.15: Anti-inflammatory Effect (%) of the n-Hexane Soluble Fraction of

V. serpens on Carrageenan Induced Paw Edema


103

1h 2h 3h 4h 5h0

20

40

60

80

100100mg/kg 200mg/kg 300mg/kg Diclofenac

*

**

*

**

* **

*

**

*

*

% I

nh

ibit

ion

Figure 3.16: Anti-Inflammatory Effect (%) of the Chloroform Soluble Fraction of V.

serpens on Carrageenan Induced Paw Edema

1h 2h 3h 4h 5h0

20

40

60

80

100

100mg/kg 200/kgmg 300mg/kg Diclofanec

**

**

*

**

**

*

**

*

**

% I

nh

ibit

ion

Figure 3.17: Anti-Inflammatory Effect (%) of the Ethyl Acetate Soluble

Fraction of V. serpens on Carrageenan Induced Paw Edema


104

1h 2h 3h 4h 5h0

20

40

60

80

100


*

**

**

*

**

**

*

**

*

*

% I

nh

ibit

ion

Figure 3.18: Anti-Inflammatory Effect (%) of the Aqueous Soluble Fraction of

V. serpens on Carrageenan Induced Paw Edema

1h 2h 3h 4h 5h0

20

40

60

80


*

**

**

**

* *

**

*

**

*

***

**

*

**

**

*

**

*

% I

nh

ibit

ion

Figure 3.19: Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on

Histamine Induced Paw Edema


105

1h 2h 3h 4h 5h0

20

40

60

80


**

*

**

**

* *

**

**

**

*

* ** *

**

*

% I

nh

ibit

ion

Figure 3.20: Anti-Inflammatory Effect (%) of the n-Hexane Soluble Fraction of V.

serpens on Histamine Induced Paw Edema

1h 2h 3h 4h 5h0

20

40

60

80

100


*

**

**

* **

*

**

*

**

*

**

% I

nh

ibit

ion

Figure 3.21: Anti-Inflammatory Effect (%) of the Chloroform Soluble Fraction

of V. serpens on Histamine Induced Paw Edema


106

1h 2h 3h 4h 5h0

20

40

60

80


*

**

*** ******

**%

In

hib

itio

n

Figure 3.22: Anti-Inflammatory Effect (%) of the Ethyl Acetate Soluble Fraction of V. serpens on Histamine Induced Paw Edema

1h 2h 3h 4h 5h0

20

40

60

80

100100 mg/kg 200mg/kg 300mg/kg Diclofenac

***

**

**

**

***

***

*% I

nh

ibit

ion

Figure 3.23: Anti-Inflammatory Effect (%) of the Aqueous Soluble Fraction of V.

serpens on Histamine Induced Paw Edema


107

Table 3.12: Anti-Inflammatory Effect of Crude Extract of V. serpens Against Carrageenan and Histamine Induced Paw Edema

in Mice

Treatment Dose

mg/kg

Normal Paw Size

(NPS)

0h 1h 2h 3h 4h 5h

Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2092 ± 0.14

Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12

Anti-inflammatory effect against carrageenan induced paw edema

Crude extract

100 0.0915 ± 0.10 0.2134 ± 0.10 0.2018 ± 0.15 0.1803 ± 0.10 0.1408* ± 0.10 0.1572 ± 0.15 0.1590 ± 0.20

200 0.0965 ± 0.13 0.2050 ± 0.11 0.1700* ± 0.19 0.1495* ± 0.21 0.0901** ± 0.17 0.1130* ± 0.19 0.1230* ± 0.13

300 0.0970 ± 0.11 0.2178 ± 0.05 0.1670* ± 0.18 0.1233* ± 0.22 0.0702** ± 0.18 0.0830** ± 0.25 0.1003** ± 0.19

Anti-inflammatory effect against histamine induced paw edema

Crude extract

100 0.0970±0.05 0.2001±0.05 0.2005±0.19 0.1966±0.25 0.1850±0.25 0.1960±0.05 0.1990±0.35

200 0.0955 ± 0.10 0.2055 ± 0.20 0.1614* ± 0.15 0.1510* ± 0.20 0.0990** ± 0.23 0.1083* ± 0.25 0.11087* ± 0.10

300 0.0962 ± 0.15 0.2087 ± 0.30 0.1180* ± 0.55 0.1150* ± 0.10 0.0803** ± 0.20 0.0921** ± 0.30 0.1231** ± 0.30

Values are reported as mean ±SEM for group of six mice each for carrageenan and Histamine by applying ANOVA followed by Dunnett

tests for data analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01.


108

Table 3.13: Anti-Inflammatory Effect Against Carrageenan and Histamine Induced Paw Edema in Mice for V. serpens n- Hexane

Soluble Fraction

Treatment Dose

mg/kg

Normal Paw Size

(NPV)

0h 1h 2h 3h 4h 5h

Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2092 ± 0.14

Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12


n-Hexane 100 0.0910 ± 0.20 0.2130 ± 0.10 0.2015 ± 0.05 0.1900 ± 0.20 0.1485* ± 0.20 0.1575 ± 0.10 0.1670 ± 0.15

200 0.0970 ± 0.15 0.2053 ± 0.10 0.1715* ± 0.20 0.1500* ± 0.24 0.0985** ± 0.19 0.1135* ± 0.20 0.1232* ±0.15

300 0.0970 ± 0.10 0.2180 ± 0.02 0.1672* ± 0.25 0.1235* ± 0.20 0.0785** ± 0.20 0.0930** ± 0.25 0.1003**±0.20


n-Hexane 100 0.0982±0.03 0.2091±0.15 0.2100±0.18 0.1963±0.19 0.1547±0.25 0.1700±0.12 0.1960±0.27

200 0.0895 ± 0.11 0.2119 ± 0.22 0.2021* ± 0.16 0.1506* ± 0.22 0.1052** ± 0.24 0.1285* ± 0.25 0.1330*±0.12

300 0.0901 ± 0.14 0.2067 ± 0.24 0.178`5* ± 0.25 0.1612* ± 0.11 0.1078** ± 0.15 0.1338** ± 0.30 0.1421**±0.26

Values are reported as mean ±SEM for group of six mice each for carrageenan and Histamine by applying ANOVA followed by Dunnett tests for data

analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01.


109

Table 3.14: Anti-Inflammatory Effect of Chloroform Soluble Fraction of V. serpens in Carrageenan and Histamine Induced

Paw Edema in Mice

Treatment Dose mg/kg Normal Paw Size

(NPS)

0h 1h 2h 3h 4h 5h

Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2090 ± 0.14

Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12


Chloroform 100 0.0917 ± 0.11 0.2135 ± 0.15 0.2020 ± 0.13 0.1976 ± 0.17 0.1610* ± 0.11 0.1669 ± 0.15 0.1699 ± 0.15

200 0.0972 ± 0.10 0.2001 ± 0.20 0.1811* ± 0.21 0.1702* ± 0.15 0.1433** ± 0.12 0.1692* ± 0.19 0.1734* ± 0.17

300 0.0999 ± 0.21 0.1788 ± 0.23 0.1627* ± 0.14 0.1601* ± 0.20 0.1386** ± 0.19 0.1630** ± 0.19 0.1688** ± 0.21


Chloroform 100 0.0964± 0.05 0.2112±0.05 0.2120±0.19 0.1989±0.25 0.16702±0.25 0.1761±0.05 0.1805±0.35

200 0.0955 ± 0.10 0.2050 ± 0.20 0.2003* ± 0.15 0.1770* ± 0.20 0.1483** ± 0.23 0.1709* ± 0.25 0.1790* ± 0.10

300 0.0962 ± 0.15 0.2085 ± 0.30 0.1831* ± 0.55 0.1697* ± 0.10 0.1432** ± 0.20 0.1670** ± 0.30 0.1699** ± 0.30


tests for data analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01.


110

Table 3.15: Anti-Inflammatory Effect of Ethyl Acetate Soluble Fraction of V. serpens in Carrageenan and Histamine Induced

Paw Edema in Mice


tests for data analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01

Treatment Dose mg/kg NPS 0h 1h 2h 3h 4h 5h

Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2090 ± 0.14

Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12


Ethyl Acetate

100 0.0888 ± 0.21 0.2100 ± 0.19 0.2029 ± 0.11 0.1991 ± 0.12 0.1665* ± 0.20 0.1865 ± 0.11 0.2011 ± 0.19

200 0.0972 ± 0.13 0.2021 ± 0.19 0.1811* ± 0.22 0.1732* ± 0.20 0.1500** ± 0.16 0.1692* ± 0.13 0.1734* ± 0.12

300 0.0892 ± 0.18 0.1868 ± 0.19 0.1699* ± 0.22 0.1598* ± 0.23 0.1416** ± 0.16 0.1689** ± 0.14 0.1723** ± 0.17


Ethyl Acetate

100 0.0932 ± 0.02 0.2109 ± 0.10 0.2021 ± 0.20 0.1980 ± 0.22 0.1773±0.10 0.1859 ± 0.12 0.1970 ± 0.33

200 0.09032 ± 0.13 0.2067 ± 0.18 0.1810* ± 0.22 0.1751* ± 0.25 0.1642 ± 0.18 0.1752 ± 0.22 0.1820 ± 0.09


111

Table 3.16: Anti-Inflammatory Effect of Aqueous Soluble Fraction of V. serpens against Carrageenan and Histamine Induced

Paw Edema in Mice

Treatment Dose mg/kg NPS 0h 1h 2h 3h 4h 5h

Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2090 ± 0.14

Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12


Aqueous 100 0.0864 ± 0.03 0.2092 ± 0.19 0.2101 ± 0.20 0.1833 ± 0.13 0.1571* ± 0.22 0.1782 ± 0.10 0.1968 ± 0.11

200 0.0921 ± 0.12 0.2090 ± 0.21 0.2152* ± 0.12 0.1705* ± 0.24 0.1423** ± 0.14 0.1680* ± 023 0.1902* ± 0.16

300 0.0906 ± 0.15 0.2099 ± 0.17 0.2012* ± 0.19 0.1643* ± 0.16 0.1230** ± 0.13 0.1598** ± 0.15 0.1860** ± 0.14


Aqueous

100 0.0961± 0.02 0.2009 ± 0.02 0.2013 ± 0.19 0.1993 ± 0.15 0.1603 ± 0.25 0.1803 ± 0.05 0.1995±0.30

200 0.0894 ± 0.13 0.2003 ± 0.23 0.1959 ± 0.15 0.1823± 0.21 0.1530 ± 0.23 0.1721 ± 0.21 0.1920 ± 0.20

300 0.0905 ± 0.13 0.2011 ± 0.32 0.1901 ± 0.55 0.1603 ± 0.14 0.1413 ± 0.20 0.1550 ± 0.24 0.1828 ± 0.28

Values are reported as mean ±SEM for group of six mice each for carrageenan and Histamine by applying ANOVA followed by Dunnett tests for

data analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01


110

Table 3.17: Effect of the Crude Extract/ Subsequent Fractions of V. serpens on

Xylene Induced Ear Edema in Mice

Group Doses (mg/kg) Ear weight (mg) Inhibition (%)

Control 10ml/kg 185

Ibuprofen 100 57.32±3.51 69.0

Crude extract

100 129.5±2.34 30.0

200 101.9±1.91 45.4

300 78.3±2.09 57.7

n-Hexane

100 135±2.56 27.0

200 105±4.01 43.0

300 83±2.11 55.0

Chloroform

100 137±4.12 25.9

200 112±2.78 39.5

300 90.5±3.11 51.0

Ethyl acetate

100 133±2.34 28.0

200 117±1.77 36.8

300 93.2±2.05 49.6

Aqueous

100 132±3.00 28.6

200 107±2.13 42.0

300 95.3±2.34 48.5


111

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

**

**

*

*

**

% P

ro

te

ctio

n

C ru d e e x tr a c t

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

**

*

**

*

% P

ro

te

ctio

n

C h lo r o f o r m

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

*

**

*

**

**

*

% P

ro

te

ctio

n

H e x a n e

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

*

**

*

**

% P

ro

te

ctio

n

E th y l a c e ta te

1 0 0 2 0 0 3 0 0 D ic lo

0

2 0

4 0

6 0

8 0

*

**

**

*

% P

ro

te

ctio

n

A q u e o u s

Figures 3.24: Percent Inhibition of Xylene Induced Ear Edema in Mice at Different

Doses of the Crude Extract and Fractions of V. serpens


112

3.3 ISOLATED COMPOUNDS

3.3.1 New Compound from Viola serpens

3.3.1.1 Commulin-A (1)

Commulin-A (1) was isolated from the methanolic extract of V. serpens as yellowish

amorphous powder (see section 2.7.2). The compound 1 was assigned molecular formula

C17H14O5 on the bases of ion peak at m/z 299 [M]+ in EI-MS and NMR spectral data. The

UV maxima at 340 and 264 nm indicated a flavonoid skeleton in compound 1 (Chauhan

et al., 1977). The IR spectrum revealed the presence of hydroxyl (3450 cm-1), conjugated

ester (1695 cm-1), conjugated carbonyl (1719 cm-1), and aromatic functionalities (2968,

1591, and 1463 cm-1).

Figure 3.25: Commulin-A (1)

The 1H-NMR spectrum of commulin-A (1) showed the presence of a methoxy, a methyl,

a methine and aromatic protons. In the downfield region of the spectrum a sharp singlet at

δ6.66was assigned to H-3methine proton. A broad singlet of one proton integration at δ

13.16 was assigned to the hydrogen-bonded hydroxyl group present at C-5. Similarly a

sharp signal of three proton integration was observed at δ 4.04 due to the presence of


113

methoxyl group in compound (1). The 1H-NMR spectrum also displayed multiplet in the

range of 7.04-8.01, were due aromatic protons

The 13C-NMR spectrum (Broad band decoupling (BB), Distortionless Enhancement

Polarization Transfer (DEPT)) (Table 3.18) showed seventeen signals, including one

methyl, one methoxy, six methines, and nine quaternary carbons. The downfield region

signals at 181.1 were assigned to the ketonic carbons of ring C. Similarly, signals at

158.5, 154.3, 153.4 and 147.7 were due to the C-2, C-5, C-7 and C-10 of the ring A

respectively. In the upfield region signals at 29.7 and 60.1 were assigned to methyl and

methoxyl group of the molecule.

Further structural assignments were made by using 2D-NMR experiments. In the HMBC

spectrum, the C-3 methine proton (δ 6.66) showed correlations with C-2 (δ 158.5), C-4 (δ

181.1) and C-9 (δ 111.3).The C-2′ methine proton showed correlations with C-1′ (δ

112.4) and C-3′ (δ 126.7). Similarly correlation of the C-4′ methine proton (δ 7.42) with

the C-3′ (δ 126.7), C-5′ (δ 126.5) and C-2′ (δ 122.3) were established from the same

HMBC spectrum. Moreover C-6′ (δ 7.62) methine proton exhibited interactions with C-1′

(δ 112.4) and C-5′ (δ 126.5). Also, C-6 (δ 1.57) methyl protons showed HMBC

interactions with C-5 (δ 154.3), C-7 (δ 153.4) and C-6 (δ 111.3). Similarly C-5 (δ 13.16)

hydroxyl proton showed HMBC interactions with C-4 (δ 181.1), C-9 (δ 111.3), and C-6

(δ 111.3). Thus on the basis all the spectroscopic data the structure of compound 1 was

deduced as flavone and having 8-methoxy, 6-methyl, 5, 7-dihydroxyflavone (Figure-3.21)


114

Figure 3.26: Key HMBC interaction in Compound 1

Table 3.18: 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-A (1) in

CDCl3

C.No. 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC

Correlations

2 - 158.5 C

3 6.66, s 94.2 CH 2, 4, 9

4 - 181.1 C

5 - 154.3 C

6 - 111.3 C

7 - 153.4 C

8 - 125.4 C

9 - 111.3 C

10 - 147.7 C

1´ - 112.4 C

2´ 8.01, d, 7.32 122.3 CH 1´, 2´, 3´, 4´

3´ 7.04, t, 7.38 126.7 CH

4´ 7.42, t, 6.98 128.2 CH

5´ 7.04, t, 7.38 126.7 CH

6´ 8.01, d, 7.32 122.3 CH 1´, 6´, 5´

CH3 1.57, s 29.7 ) CH3 5, 6, 7,

OCH3 4.04, s 60.1 CH3 10, 8, 7

3.3.1.2 Commulin-B (2)

Commulin-B (2) was isolated from the methanolic extract of V. serpens as yellow

amorphous powder (see section 2.7.2). The compound 2 had molecular formula

C17H14O6, as established on the basis of ion peak at m/z 314 [M+]+ in HREI-MS and


115

NMR spectral data .The HREI-MS exhibited an ion at m/z298 [M -17] + which resulted

from the loss of a hydroxyl group from the molecule. The HREI-MS also showed

fragments at m/z196, and 118 resulting from the retro Diels-Alders cleavage of the ring C

of compound 2 (Grisebach and Grambow, 1968). Similarly these ions confirmed the ring

A was substituted by two hydroxyl groups, one methyl and one methoxy group, while

ring B with one hydroxyl group. The IR spectrum of 2 included hydroxy (3422 cm-1),

conjugated ketone (1719 cm-1), and aromatic absorption (1601, 1500, 2968 cm-1).

Figure 3.27: Commulin-B (2)

The 1H-NMR spectral data of Commulin-B (2) showed the distinct resemblance with that

of compound 1 except for the absence of C-2′ methine proton in 1H-NMR and the

presence of additional quaternary signals at δ 154.3 in 13C-NMR spectrum (Table-3.19) of

compound 2, indicating the presence of a hydroxyl group at C-2′ in Commulin-B.

In the downfield region of the 1H-NMR spectrum, a sharp singlet at δ 6.54 was assigned

to H-3 methine proton. The 1H-NMR spectrum also displayed a singlet of three protons

integration at 4.04 due to the methoxyl group at C-8 position of ring A. Another singlet

of three proton integration at 1.29 was assigned to the methyl group of the same ring A.

A multiplet of four protons integration in the range of 7.07-7.9 was assigned to aromatic

methine protons of ring of ring B.


116

The 13C-NMR spectrum (Broad band decoupling (BB), Distortionless Enhancement

Polarization Transfer (DEPT) (Table 3.19) showed seventeen signals, including one

methyl, one methoxy, five methines, and ten quaternary carbons. The downfield region

signals at 179.8 were assigned to the ketonic carbons of ring C. Similarly, signals at

160.9, 154.3 and153.4 were due to the C-2, C-5, and C-7 of the ring A respectively. In the

upfield region signals at 29.4 and 60.9 were assigned to methyl and methoxyl group of

the molecule

In the HMBC spectrum, the C-3 methine proton (δ 6.54) showed correlations with C-2 (δ

160.9), C-4 (δ 179.8) and C-9 (δ 102.4). Similarly correlation of the C-4′ methine proton

(δ7.34) with the C-3′ (δ 125.5), C-5′ (δ 126.9) and C-2′ (154.3) were established from the

same HMBC spectrum. Moreover C-6′ (δ7.16) methine proton exhibited interactions with

C-1′ (δ 122.4) and C-5′ (δ 126.9). Thus structure of compound 2 was deduced as flavone

and having 8-methoxy, 6-methyl, 5, 7, 2′-trihydroxyflavone.

Figure 3.28: Key HMBC interaction in Commulin-B (2)


117

Table 3.19 1H- (400 MHz.) and 13C-NMR (125 MHz) Data of Commulin-B (2) in

CDCl3

C.No. 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC

Correlations

2 - 160.9 C

3 6.54, s 96.6 CH 2, 4, 9

4 - 179.8 C

5 - 154.3 C

6 - 110.8 C

7 - 153.4 C

8 - 123.2 C

9 - 102.4 C

10 - 147.7 C

1´ - 122.4 C

2´ - 154.3 C

3´ 7.07, d, 7.9 125.5 CH

4´ 7.34, t, 7.2 121.7 CH 2´, 3´, 5´

5´ 7.14, t, 7.1 126.9 CH

6´ 7.16, d, 6.8 111.3 CH 1´, 5´

CH3 1.29, s 29.4 CH3

OCH3 4.04 60.9 CH3

3.3.1.3 Commulin-C (3)

Commulin-C (3) was also isolated from the methanolic extract of V.serpens as an

amorphous powder. The compound 3 was assigned the formula C18H16O6 on the basis of

an ion peak at m/z 329 [M]+in FAB-MS and based on NMR spectral data. The FAB-MS

of Commulin-C (3) exhibited ion peak at m/z 297 which resulted from the loss of

methoxyl group from M+. The IR spectrum of compound 3 showed absorption bands

3560 (OH), 2968, 1610, 1515 (aromatic), 1717 (conjugated ketone).


118

Figure 3.29: Commulin-C (3)

The 1H-NMR spectral data of compound 3 indicated its resemblance with the compound

2 except for the presence of a methoxy group instead of hydroxyl group at C-2` in

compound 3. In the downfield region of the 1H-NMR spectrum, a sharp singlet at δ 6.54

was assigned to H-3 methine proton. The 1H-NMR spectrum also displayed two singlets

of three protons integration at 3.96 and 3.89 were due to the methoxyl groups of the

compound.

The 13C-NMR spectrum (BB, DEPT) (Table 3.20) showed eighteen signals, including one

methyl, two methoxy, five methines and ten quaternary carbons. In HMBC spectrum

(Figure 3.26), the C-3 methine proton (δ 6.54) showed correlations with C-2 (δ 161.5), C-

1′ (δ 123.2), C-4 (δ182.2), and C-9 (δ 102.4). The presence of the second methoxy group

at C-2′ (δ 158.1) was further confirmed by the HMBC correlations of methoxy protons (δ

3.89) with C-2′ (δ 158.1), C-1′ (δ 123.2), and C-3′ (δ 119.7).Thus on the basis of 1D and

2D NMR specral data the structure of compound 3 was deduced as flavone and having 2′,

8-dimethoxy, 6-methyl, 5, 7-dihydroxyflavone.


119

Figure 3.30: Key HMBC interaction in Commulin-C (3)

Table 3.20: 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-C (3) in

CDCl3

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC

Correlations

2 - 161.5 C

3 6.54, s 96.6 CH 2, 4, 9

4 - 182.2 C

5 - 155.9 C

6 - 110.8 C

7 - 155.9 C

8 - 123.2 C

9 - 102.4 C

10 - 149.8 C

1´ - 123.2 C

2´ - 158.1 C

3´ 7.03, d, 8.7 119.7 CH

4´ 7.45, t, 7.4 129.8 CH

5´ 7.09, t, 7.2 121.3 CH 5´, 6´, 4´

6´ 7.65, d, 7.9 130.8 CH 1´, 6´, 5´

CH3 1.57, s 29.3 CH3

OCH3 3.96, s 61.8 CH3

OCH3 3.89, s 56.5 CH3 1´, 2´, 3´


120

3.3.2 Known Compounds from Viola serpens

3.3.2.1 5-Hydroxy-7-methoxy flavone (tectochrysine) (4)

The compound 4 was isolated as colorless solid from the sub fraction FMC-3. The HREI-

MS showed the M+ ion peak at m/z corresponding to formula C16H13O4 (calcd. for

C16H13O4, 268.2011). The UV spectrum displayed 268.2013 the maxima at 325 and 267

nm. These values indicated that compound 4 is a flavone (Bernard, 1983). The IR

spectrum showed the absorption bands at 3400, 3000, 1649, 1475, and 1460 cm-1.

O

OOH

H3CO

9 2

34

56

78

10

Tectochrysine (4)

1'

2' 3'4'

5'6'

Figure 3.31: Structure of compound Tectochrysine (4)

The 1H-NMR spectrum showed in the downfield region a singlet at δ 6.65 corresponding

to the H-3 position, while two doublet at δ 6.48 (H, J = 2.2) and δ 6.39 (d, J = 2.2 Hz),

were assigned to H-6, and H-8. The multiplets at δ 7.87 and 7.52 were due to H-2′, H-6′,

H-3′, and H-4′, H-5′. A sharp singlet of three protons integration at δ 3.94 was assigned to

the methoxyl group present at ring A. The 13C-NMR spectrum (BB and DEPT) of

compound 4 corroborated the presence of one methyl, eight methine and seven quaternary

carbons. The signal at δ 165.6 corresponded to C-7, while the signal at δ 106.0 was due to

C-3. The signal at δ 60.3 was due to the presence of methoxyl group.


121

By comparison with the literature data, compound 4 was identified as 5-hydroxy-7-

methoxy flavone (4), previously reported from Boesenbergia pandurata plant (Debral et

al., 1994).

3.3.2.2 4́, 5-Dihydroxy-7-methoxy-6, 8-dimethylflavone (Sideroxylin) (5)

Compound 5 was isolated as yellow needles from the sub fraction FMC-5, showed the

UV absorption at max 330 and 275 nm. The IR spectrum displayed absorption bands at

3500 and 1655 cm-1 due to the presence of hydroxyl and ketonic functionalities. The EI-

MS showed the M+ at m/z 312. The molecular formula was confirmed as C18H16O5,

through M+ ion peak in HREI-MS at m/z 312.0125 (calcd. for C18H16O5, 312.0123).

O

OOH

H3C

H3CO

CH3

OH1'

2

345

6

78

9

10

2' 3'

4'

5'6'

Sideroxyline (5)

Figure 3.32: Structure of compound Sideroxyline (5)

The 1H-NMR spectrum showed a singlet at δ 6.87 corresponding to the H-3 position,

while the two doublets at δ 7.97 (J = 8.8 Hz) and 6.94 (J = 8.7 Hz) were assigned to H-2′,

H-6′, and H-3′, H-5′, respectively. A downfield one-proton singlet at δ 13.07 was assigned

to the hydroxyl group at C-5 position. The signal at δ 3.94 was assigned to methoxyl

group at C-7 position. Two singlets each of three protons integration at δ 2.08 and δ 2.32

were assigned to the methyl group present at C-6 and C-8 in ring A. The 13C-NMR

spectrum (BB and DEPT) of compound 5 corroborated the presence of three methyl, five

methine and ten quaternary carbons. The signal at 108.6 corresponded to C-8, while the


122

signal at 102.8 was due to C-3. In the up field region the signals at δ 60.3, 8.29, and 8.07

were due to the presence of one methoxyl and two methyl groups in ring A.

By comparison with the data published in literature, compound 5 was identified as 4′, 5-

dihydroxy-7-methoxy-6,8-dimethylflavone (5), previously reported from Eucalyptus

sideroxylone plant (Guimaraes et al., 1975).

3.3.2.3 2, 5-Dihydroxy-4-methoxybenzophenone (Cearoin) (6)

Compound 6 was isolated as yellow amorphous powder from sub fraction FMC-5 ethyl

acetate soluble part of crud methnol extract. The HREI-MS exhibited M+ at m/z 244.1322

corresponding to the molecular formula C14H12O4 (calcd. for C14H12O4, 244.1334). The

IR spectrum showed absorption bands at 3448 (OH), 1743 (C = O) cm-1.

O

OH

OCH3

OH

12

3

456

1'2'3'

4'5' 6'

AB

Cearoin (6)

Figure 3.33: Structure of compound Cearoin (6)

The 1H-NMR spectrum showed singlets at δ 6.88 and 6.59 corresponding to the H-6 and

H-3 position, while the multiplets at δ 7.61 and 7.54 were due to aromatic proton of ring

B, respectively. There were downfield signals at δ 11.95 and 8.89 due to hydroxyl group

at C-2 and C-5 positions. The 13C-NMR spectrum (BB and DEPT) of cearoin (6)

corroborated the presence of one methyl, seven methine, and six quaternary carbons. The

signal at δ 198.7 corresponded to carboxylic carbon, while the signal at δ 157.8 was due


123

to C-2 having the hydroxyl group. The signal at δ 60.3 was assigned to the methoxyl

carbon at C-4 position in ring A.

By comparison with the data published in literature, compound 6 was identified as 2, 5-

dihydroxy-4-methoxybenzophenone (cearoin) previously reported from Dalbergia

melanoxylon plant (Lounasmaa et al., 1977).

.

CONCLUSION

124

CONCLUSION

Over the years, medicinal plants have played historical role in new drug discovery.

For this reason, traditional uses of these plants need to be explored in well established

scientific paradigms. V. seprens, an important medicinal plant, is traditionally used for

the treatment of various ailments including jaundice, asthma, throat cancer, dermatitis

and constipation. These uses are purely based on empirical knowledge from

generations without any scientific rationale. In this regard, various in vivo and in vitro

pharmacological activities of V. serpens have been carried out in order to provide

scientific background to its folkloric uses.

The in vitro activities were included antimicrobial, DPPH free radical scavenging

assay, larvicidal and enzyme inhibition (acetylcholine esterase). The results showed

marked therapeutic potential of the crude extracts and subsequent solvent fractions in

various already reported tests. Similarly, the in vivo activities like acute toxicity,

antinociceptive, anti-inflammatory, hepatoprotective and nephroprotective were

carried in different recommended protocols. The animal based studies showed

profound effects in specific assays.

The column chromatography technique used led to the isolation of six compounds

including three new flavonoids. Among the six, three compounds were new (not

reported before) and the other three were already reported from the other sources but

first time from V. serpens. Commulin-A, Commuline-B and Commuline-C were the

new compounds whereas, tectochrysine, Sideroxylin and Cearoin were the already

reported compounds. The chemical structures of these isolated compounds were

elucidated using various spectroscopic techniques. When these isolated compounds

were tested for antibacterial activity against various pathogenic bacteria, most of them

CONCLUSION

125

were found susceptible. Additionally, when these isolated compounds were tested for

free radical scavenging effect against DPPH, they exhibited strong antioxidant action.

In short, we have provided scientific foundation to different traditional uses of this

plant in various in vitro and in vivo protocols. Similarly, the isolation of pure

secondary metabolites explored the molecular background of the plant. Keeping in

view the outstanding pharmacological activities of the crude extract and extracted

fractions, which were supported by the isolated compound, suggests further

mechanistic detail studies to discover new effective therapeutic agents for clinical

uses.

REFERENCES

126

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