Isolation of Bioactive Secondary Metabolites and Pharmacological Studies of Viola Serpens Wall

Home , Viola adunca, Viola arvensis, Viola betonicifolia, Viola biflora, Viola canadensis, Viola hederacea

ISOLATION OF BIOACTIVE SECONDARY METABOLITES AND PHARMACOLOGICAL STUDIES OF VIOLA SERPENS WALL

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.

I Dedicated my this humble effort to my beloved Parents & Family 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.

i 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 histopathological 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

ii 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

iii S.No. Topic Page No. 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

iv S.No. Topic Page No. 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

v S.No. Topic Page No. 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

vi LIST OF TABLES

S.No. Title Page No.

1.1 Viola species list, native to Pakistan and their worldwide distribution, 6 parts used chemical constituents and medicinal uses 1.2 List of other viola spp. of the family Violaceae, their geographical 9 distributions, Medicinal uses and part/s used mostly 1.3 List of some various other species of viola and their geographic 10 distributions 2.1 List of Drugs / Chemicals used with their Sources 36 3.1 Antimicrobial Activity of the Crude Extract along with the 64 Subsequent Fractions of Viola serpens 3.2 Antimicrobial activity of the Isolated Compounds from V. serpens 64 3.3 DPPH Scavenging Activity of Crude extract/Fractions of V.serpens 67 and Zones of Inhibition are Given in mm 3.4 Anti-oxidant Activities of Pure Isolated Compounds 1–6 from 67 V.serpens whole Plant 3.5 Larvicidal effect of the crude extract along with the subsequent 69 fractions of V.serpens against Aedes aegypti and Culex quinquefasciatus species of mosquitoes 3.6 The Enzyme Inhibition effect of the Crude Extract and the 71 subsequent Fractions of V.serpens against the Enzyme Acetylcholine Esterase 3.7 Acute Toxicity of the Crude Extract along with the Fractions of V. 72 serpens 3.8 Effects of the Crude Extracts/Fractions of V.serpens Wall on the 75 Liver Related Parameters (AST, ALT and ALP) in the Rabbits Models 3.9 Effect of Crude Extract/ fractions of V.serpens Wall. on the Kidney’s 81 functions and Clearance in the Rabbits Models 3.10 The Effect of Crude Extract/Fractions of V.serpens in Acetic Acid 85 Induced Writhing Tests in Mice (i.p) 3.11 Effect of the crude/ fractions of V. serpens in formalin induced pains 91 for analgesia test in mice at doses of 100, 200 and 300 mg/kg, i.p 3.12 Anti-inflammatory effect against carrageenan and Histamine 105 induced paw edema in mice for V.serpens crude extract

vii S.No. Title Page No.

3.13 Anti-inflammatory effect against carrageenan and Histamine 106 induced paw edema in mice for V.serpens n- hexane fraction 3.14 Anti-inflammatory effect of chloroform fraction of V.serpens 107 in carrageenan and Histamine induced paw edema in mice 3.15 Anti-inflammatory effect of Ethyl acetate fraction of V.serpens 108 in carrageenan and Histamine induced paw edema in mice 3.16 Anti-inflammatory effect against carrageenan and Histamine 109 induced paw edema in mice for V.serpens aqueous fraction 3.17 Effect of the crude extract along with the subsequent fractions of 110 V.serpens on xylene induced ear edema in mice 3.18 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-A (1) 114 in CDCl3 3.19 1H- (400 MHz.) and 13C-NMR (125 MHz) Data of Commulin-B (2) 117 in CDCl3 3.20 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-C (3) 119 in CDCl3

viii 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, 65 ETA represents Ethyl acetate and H2O represents the Aqueous fraction 3.2 Liver photomicrographs of the rabbits treated with paracetamol, 77 crude extract and n-Hexane fractions of V. serpens at doses of 150 and 300 mg/kg (H&E, 100X and 400X) 3.2.1 Normal saline treated liver showing normal architecture of central 77 vein (CV), sinusoidal spaces (small arrows), hepatocytes (large arrows) with a centrally placed nucleus and foamy cytoplasm. (100X H&E). 3.2.2 Liver showing accumulation of lymphocytes (small arrows) 77 around the central vein (CV), fatty changes (small arrow head) and focal area of necrosis (asterisk) with paracetamol (100X H&E). 3.2.3 Liver showing regeneration, containing normal liver plates (large 77 arrows) along central vein (CV) with n-hexane 150 mg/kg b.w. (H&E). 3.2.4 Liver showing normal appearance of central vein (CV) and plates 77 of hepatocytes (large arrows) with n-hexane 300 mg/kg b.w. (100X H&E). 3.2.5 Liver showing hexagonal hepatocytes (large arrows) with 77 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). 3.2.6 Liver showing regeneration of hepatocytes (large arrows) with 77 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).

S. No. Title Page No. 3.3 Photomicrogrphs of the Kidneys of Rabbits Treated with 83 Paracetamol and plant Extract/ Fractions at Different Doses (H&E) 3.3.1 Photomicrograph (100X H&E) of a section of kidney from a rabbit 83 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. 3.3.2 Photomicrograph (100X H&E) of a section of kidney from a rabbit 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). 3.3.3 Photomicrograph (100X H&E) of a kidney section from a rabbit 83 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). 3.3.4 Photomicrograph (100X H&E) of a section of kidney from a rabbit 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 3.3.5 Photomicrograph ((100X H&E)) of a section of kidney from a rabbit 83 treated with chloroform soluble fraction 150 mg/kg showing normal renal corpuscles (large arrows), proximal (arrow heads) and distal (asterisk) convoluted tubules 3.3.6 Photomicrograph (100X H&E) of a section of kidney from a rabbit 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. 3.3.7 Photomicrograph ((100X H&E)) of a section of kidney from a rabbit 84 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 3.3.8 Photomicrograph (100X H&E) of a section of kidney from a rabbit 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.

x S. No. Title Page No. 3.3.9 Photomicrograph (100X H&E) of a section of kidney from a rat 84 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. 3.3.10 Photomicrograph (100X H&E) of a section of kidney from a rat 84 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. 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 89 Acid induced writhing test. 3.9 Antinociceptive Effects of Formalin Induced Pain in Mice of the 94 Crude Extract of V.serpens 3.10 Antinociceptive effects of Formalin Induced Pain in Mice of the 94 Hexane Fraction of V.serpens 3.11 Antinociceptive effects of Formalin Induced Pain in Mice of the 95 Chloroform Fraction of V.serpens 3.12 Antinociceptive effects of Formalin Induced Pain in Mice of the 95 Ethyl Acetate Fraction of V.serpens 3.13 Antinociceptive effects of Formalin Induced Pain in Mice of the 96 Aqueous Fraction of V.serpens 3.14 Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on 102 Carrageenan Induced paw Edema 3.15 Anti-Inflammatory Effect (%) of the n-Hexane Fraction V. serpens 102 on Carrageenan Induced paw Edema 3.16 Anti-Inflammatory Effect (%) of the chloroform Fraction V. serpens 103 on Carrageenan Induced paw Edema 3.17 Anti-Inflammatory Effect (%) of the Ethyl Acetate Fraction V. 103 serpens on Carrageenan Induced paw Edema

S. No. Title Page No. 3.18 Anti-Inflammatory Effect (%) of the Aqueous Fraction V. serpens on 104 Carrageenan Induced paw Edema 3.19 Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on 104 Histamine Induced paw Edema 3.20 Anti-Inflammatory Effect (%) of the n-Hexane Fraction V. serpens 105 on Histamine Induced paw Edema 3.21 Anti-Inflammatory Effect (%) of the chloroform Fraction V. serpens 105 on Histamine Induced paw Edema 3.22 Anti-Inflammatory Effect (%) of the Ethyl Acetate Fraction V. 106 serpens on Histamine Induced paw Edema 3.23 Anti-Inflammatory Effect (%) of the Aqueous Fraction V. serpens on 106 Histamine Induced paw Edema 3.24 Percent inhibition of Xylene induced ear edema in mice at different 111 doses of the crude extract and fractions of V. serpens 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

xii 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

xiii 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

xiv µg: Micro gram

µL: Micro Litre

xv 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.

xvi 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-

xvii 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.

xviii 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.

xix Structures of the new compounds:

3' HO OCH 3' 3 2' 4' OCH 4' B 3 2' 1' B 5' 1' HO O 6' O 5' 8 1 HO 6' 8 1 7 10 2 A C 7 10 2 6 9 3 A C 5 6 9 3 4 5 4 H3C H3C O OH OH O

Commulin-A (1) Commulin-B (2)

H3CO 3' OCH 4' 3 2' B 1' 5' HO O 6' 8 1 10 2 7 A C 6 9 3 5 4 H3C

OH O

Figure-1 Commulin-C (3)

H HO H 3' 3' OCH3 2' 4' OCH3 2' 4' B 1' B 5' 1' 5' HO O 6' HO O 6' 8 1 8 1 7 10 2 10 2 A 7 6 H A 9 3 C 6 9 3 H 5 5 C 4 4 H3C H H3C H

OH O OH O

Figure-2: Key HMBC interactions in compound 1. Figure-2: Key HMBC interactions in compound 2.

H3CO 3' OCH3 2' 4' B 1' 5' HO O 6' 8 1 7 10 2 A 6 9 3 5 C 4 H3C H

OH O

Figure-2: Key HMBC interactions in compound 3.

xx Structures of the compounds isolated from V. serpens for the first time but already reported from the other sources:

' 3' 2 4' H3CO O 1' 5' 8 6' 7 9 2 6 10 3 5 4

OH O

Tectochrysine (4)

Figure 3.27: Structure of compound Tectochrysine (4).

CH3 2' 3' OH H3CO O 1' 4' 8 5' 7 9 2 6' 6 10 3 5 4 H3C OH O Sideroxyline (5)

Figure 3.28: Structure of compound Sideroxyline (5).

O OH

2' 1' 2 3' 1 3 A 4' B 4 5' 6' 6 5 OCH3 OH Cearoin (6)

Figure 3.29: Structure of compound Cearoin (6).

xxi Chapter – 1 INTRODUCTION

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

1 Chapter – 1 INTRODUCTION

(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

2 Chapter – 1 INTRODUCTION 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

3 Chapter – 1 INTRODUCTION 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).

4 Chapter – 1 INTRODUCTION

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).

5 Chapter – 1 INTRODUCTION

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 Part/s Geographical Chemical Constituents Uses name of the Used distribution specie Viola Whole Pakistan, 3-methoxydalbergione As astringent, antipyretic 1 betonicifolia plant Malaysia, (Muhammad et al 2014), 3- anticancer, diaphoretic & (Leaves, India, China, Methoxy dalbergion, purgative (Shinwari et al., roots, Nepal, Sri Undecanoic acid, 2,4- 2010a). In epilepsy, nervous flowers) Lanka,Burma, Dihydroxy, 5- disorders, for sinusitis, blood Japan, methoxycinnamic acid, 4- abnormalities, skin diseases, Australia Hydroxy coumarine, Beta- cough, cold, pharyngitis &Taiwan Sitosterol, Ursolic acid, (Bhatt and Negi et al., 2006) benzoic acid, Trihydroxy an astringent, for cooling benzoic acid (Muhammad effect, diuretic, laxative and et al., 2012a) purgative (Husain et al., 2008). For kidney diseases, bronchitis, pneumonia and boils (Husain et al., 2008). Urease inhibitor (Muhammad et al., 2014) Viola biflora Whole Europe, Protein [ vibi A-K, As antispasmodic, antiseptic, 2 plant Central Asia, (cycloviolacin O2, O9, varv cough, cold, diaphoretic, India, China, A, vitri A) (Burman et al., laxative emetic, antipyretic, Korea, 2010) intestinal pain, leucoderma, Pakistan Psoriasis and dermititis America & (Chandra et al 2015). Japan Viola Whole India, Alkaloid violin, methyl For Cold, cough, respiratory 3 canescens plant (oral) Pakistan, salicylate, quercitrin, problems, as antipyretic, Nepal and glycosides and saponins antimalarial, demulcent, Bhutan (Rana et al., 2010). astringent, diaphoretic, Alkaloid (emetine) malic purgative, febrifuge, anti acid, glucoside (viola cancerous, carminative, quercitrin). 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) Viola cinerea Whole Yemen (Kilian Triterpenoids (Tabba et al., Aphrodisiac (Marwat, 2008) 4 plant (oral) et al., 2004) 1989), cyclotide alkaloids specially Iran, Pakistan (Chen et al., 2005). roots and and Oman. Flavonoids (Vukics et al., leaves 2008). Caffeic and

6 Chapter – 1 INTRODUCTION

salicylic acid (Toiu et al., 2008) Viola Flower and India and Triterpenoids (Tabba et al., Flower used for cold and 5 falconeri roots Pakistan 1989) cyclotide alkaloids cough whereas, roots used (Chen et al., 2005). against jaundice (Saqib and Flavonoids (Vukics et al., Sultan, 2005). 2008). ). Caffeic and salicylic acid (Toiu et al., 2008). Viola Whole Central Asian Triterpenoids (Tabba et al., Not reported 6 fedtschenkoan plant countries 1989) cyclotide alkaloids a including (Chen et al., 2005). Northern Flavonoids (Vukics et al., Pakistan 2008). ). Caffeic and salicylic acid (Toiu et al., 2008). Viola Whole India, Triterpenoids (Tabba et al., Applied on scores and ulcers, 7 kashmiriana plant Pakistan, 1989) cyclotide alkaloids heals swollen mouth and foot Kashmir and (Chen et al., 2005). disease in cattles. It also cure Afghanistan Flavonoids (Vukics et al., bronchitis (Ishtiaq et al., 2008). ). Caffeic and 2006) salicylic acid (Toiu et al., 2008) Viola Whole Afghanistan, Not reported Not reported 8 kunawurensis plant Pakistan, India, Nepal, China, Turkestan, and Tibet. Viola Whole Pakistan, Not reported Not reported 9 macroceras plant Afghanistan Viola Pakistan Not reported Anti-inflammatory and 10 makranica analgesic. Viola odorata Whole Europe Asia Glycoside, salicylic acid Antifungal, antimicrobial, for 11 plant and North and essential oils (Furfural cold, respiratory problems especially Africa. α-Terpinene, α Thujene, such as congestion, sore leaves para-methyl Anisole, β- throat and coughing. As a Phellandrene, laxative, sedative, analgesic, expectorant in digestive α-Pinene, Sabinene , disorders blood cleansing, Myrcene, δ-3-Carene, Z-β- Jaundice, and headache Ocimene, Benzyl alcohol, (Hammami et al., 2011; γ-Terpinene, Gautam and Kuma., 2012; Acetophenone, Z-Sabinene Amiri et al., 2013). It is also hydrate , Methyl benzoate, used for the treatment of Linalool, Z-linalol oxide, catarrh, chronic bronchial 8-para-Menthatriene, ortho- asthma, upper respiratory Menthatriene, Z-para- tract symptoms, cold, menth-2-en-1-ol , 1- rheumatism, oral mucosa Terpineol, Ethyl inflammation, hysteria, benzoate,Terpinen-4 ol, nervous strain, antipyretic, Geraniol, α- Terpineol, insomnia, headache and Pulegone , δ-Elemene, α diaphoretic (Fleming et al., Cubebene , Isoledene, α- 1998; Zargari., 1997; Dhar et Copaene , β- Bournonen, β- al., 2002). Cubebene , α Gurjunene ,Z- Caryophyllene, β

7 Chapter – 1 INTRODUCTION

Duprezianene, α-Guaiene (Hammami et al ., 2011; Stuart, 1989). Viola Whole Pakistan Unknown Being an important medicinal 12 reichenbachia plant plant it is used in headache, na fever, cough, asthma, constipation, bleeding piles, skin diseases and throat cancer. Used also as diaphoretic and demulcent (Kumar and Digvijay, 2014) Viola Whole Pakistan Not reported Unknown 13 rupestris plant Viola serpens Whole India, Flavonoids, terpenoids, As demulcent, diaphoretic 14 plant Pakistan, reducing sugars, amino diuretic, in fever, jaundice, Banaladash, acids and tannins, methyl asthma, piles bleeding, throat Ceylon, Nepal, salicylate, alkaloid voiline cancer, constipation, cold, China and Java gum, mucilage, glycoside, cough, dermatitis and (Rahman and quercitrin and saponin headache (Kumar and Choudhary., (kumar et al., 2015). Digvijay, 2014; Kumar et al., 2012) 2015). Viola stocksii Whole India, Not reported Virility in sexual masculine 15 plant Pakistan, Iran, power Yemen and (Marwat et al., 2008). Afghanistan (Chandra et al., 2015). Viola tricolor Areal parts Pakistan, Flavonoides, quercetin, Dermatitis (Chevallier., 1996; 16 (flowers) Europe, Asia, luteolin and luteolin 7- ESCOP 2009). Cystitis, America, polysaccharides, phenolic bronchitis, expectorant, anti- Australia, acids, volatile oil, inflammatory, diuretic skin Germany, Carotenoids, anthocyanins , conditions, and rheumatism Turkey and cyclotides, tocopherol, (Anca et al., 2009). As anti- Spain. triacyl glycerolsglucoside epilepsy, anti-asthmatic, in and Proteins (Burman et al., heart problems, inflammation 2010). Violaxanthin, vitri of lungs and heart (Ghorbani peptide A, varv peptide A et al., 2012). (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). Viola Whole Pakistan, Ephidrin As nasal drops and used 17 turkestanica plant India, Nepal mostly in veterinary (Anca et and Butan. al., 2009).

8 Chapter – 1 INTRODUCTION

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 Romania anti-inflammatory, expectorant, diuretic skin (whole plant) conditioner, bronchitis, cystitis and rheumatism (Anca et al., 2009) 2 Viola adunca Pacific Northwest Spring-flowering nectar source and as a larval (whole plant host (NRCS Plant Guide). especially flowers) 3 Viola Canadensis North America. Roots decoction used for bladder’s pain. (Roots) (NAGPTHG, 2005). 4 Viola diffusa Japan, East Asia, South Used for hepatitis treatment (Dai et al., 2015). (whole plant) China, & Philippines. (Benecke et al., 1985; Zhou et al., 2008). 5 Viola hondoensis Korea and Japan (Hikosaka As expectorant, anti-inflammatory, diuretic, for (Whole Plant) et al., 2010) bronchitis, eczema rheumatism, and skin eruptions (Moon et al., 2004) 6 Viola japonica Korea, Eastern Asia, Taiwan, As anti- inflammation, detoxifier, as blood cooler (whole plant) Shikago, Japan and China and pain alleviator. Used in boils, abscesses, (Benecke et al., 1985). 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 South Kurile Islands, East In headache, dysentery, colds coughs, boils, as (whole plant) America, Siberia Japan, expectorant and for lubricating medicine Korea and Dongbei (Benecke ((Moerman, 1998; Native American Garden). et al., 1985). 8 Viola pubescens Northen America Colds, cough, headache, dysentery and used for (whole plant) boils. (National Audubon Society, 1979) 9 Viola vulgaris Japan, Korea, Dongbei and Used symptomatically in mild seborrhea skin Himalayan regions (Benecke conditions.(Community herbal monograph) et al., 1985) 10 Viola yedoensis Flavones, coumarins, fatty For relief of symptoms and conditioning of mild (whole plant) acid, phenolic acids (Hong et seborrhoeic skin. (Community herbal al., 2011). Polysaccharides, monograph). flavonoides, luteolin, It is also used as an anti-rheumatic and for quercetin, volatile oil, treating infections like mastitis, rhinitis and for phenolic acids, carotenoids, treating acute pyogens (Jun-Li et al., 2011). cyclotides, anthocyanins , triacyl glycerols glucoside and tocopherols (Assessment report on Viola tricolor) 11 Viola yazawana Japan, Korea, Dongbei and As an anticoagulant and antithrombotic (Kumar (whole plant) Himalayas region. (Benecke et al., 2011). et al., 1985). 12 Vi!ola hederacea Australia and Melbourne Flowers eaten especially by the Victorian but the (flowers, leaves) exact use is unknown (David, 2005).

9 Chapter – 1 INTRODUCTION

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)

10 Chapter – 1 INTRODUCTION

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)

11 Chapter – 1 INTRODUCTION

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.

12 Chapter – 1 INTRODUCTION

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.

13 Chapter – 1 INTRODUCTION

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).

14 Chapter – 1 INTRODUCTION

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.

15 Chapter – 1 INTRODUCTION

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,

16 Chapter – 1 INTRODUCTION 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

17 Chapter – 1 INTRODUCTION 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

18 Chapter – 1 INTRODUCTION 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

19 Chapter – 1 INTRODUCTION activity against C. albicans which showed satisfactory effect against the tested fungi

(Chen et al., 2005).

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

20 Chapter – 1 INTRODUCTION 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

21 Chapter – 1 INTRODUCTION 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.

22 Chapter – 1 INTRODUCTION 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.

23 Chapter – 1 INTRODUCTION

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

24 Chapter – 1 INTRODUCTION 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 antinociceptive 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

25 Chapter – 1 INTRODUCTION 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).

26 Chapter – 1 INTRODUCTION

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-

27 Chapter – 1 INTRODUCTION 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

28 Chapter – 1 INTRODUCTION 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).

29 Chapter – 1 INTRODUCTION

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

30 Chapter – 1 INTRODUCTION 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

31 Chapter – 1 INTRODUCTION

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

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

32 Chapter – 1 INTRODUCTION

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

33 Chapter – 2 EXPERIMENTAL

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)

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

2.3 PHYSICAL CONSTANTS

34 Chapter – 2 EXPERIMENTAL

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.

35 Chapter – 2 EXPERIMENTAL

Table 2.1: List of Drugs/Chemicals Drugs/ Chemicals Source Acetic acid Sigma Chemical Co, St Louis, MO, USA Acetyl choline esterase Acetyl thiocholine iodide Acid alcohol Merck Co (Darmstadt’s Germany) Amino alcohol Aspirin Reckitt Benckiser Pakistan Ascorbic acid China fooding Ltd. China. Acetyl choline esterase Sigma Chemical Co, USA Acetyl thiocholine iodide Brewer’s yeast Vahine Professional, France Sigma Chemical Co, Bismith nitrate 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 Sigma Chemical Co, 5,5 dithio-bis-nitro benzoic acid St Louis, MO, USA Ethyl acetate Eosin Merck Co Darmstadt Ltd., Germany Ethyl alcohol Sigma Chemical Co, Formalin St Louis, MO, USA Folin & Ciocalteu’s Merck, Millipore Corporation, Germany Galanthamine Merck, Darmstadt, Germany Hematoxyline 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 Merck Co (Darmstadt Germany) n-Hexane n-propylgallate Normal saline Santa Cruz Biotech, USA Sigma Chemical Co, Nutrient agar 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 Sigma Chemical Co, Potassium Iodide St Louis, MO, USA Riboflavin Alibaba Ltd. Pakistan. Silica gel Sigma Chemical Co, St Louis, MO, USA Silymarin Hisunny Chemicals, China Sod. Carbonate Merck, Darmstadt, Germany Sod. Pentobarbital Sulfuric acid Sigma Chemical Co, St Louis, MO, USA Tramadol Searle products Ltd. Pakistan Xylene Sigma Chemical Co, St Louis, MO, USA

36 Chapter – 2 EXPERIMENTAL

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.

37 Chapter – 2 EXPERIMENTAL

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.

38 Chapter – 2 EXPERIMENTAL

This whole process is presented in Figure 2.1.

Figure 2.1: Scheme of plant extraction and fractionation

39 Chapter – 2 EXPERIMENTAL

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).

40 Chapter – 2 EXPERIMENTAL

Et-Acetate fraction VS (65 g)

using Silica gel (1300 g),

FMC 1 FMC2 FMC3 FMC4 FMC5

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

Commulin-C sideroxylin commulin-A

tectochrysine commulin-B cearoin

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

41 Chapter – 2 EXPERIMENTAL

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 30 o [α] D : + 375.0 (c = 0.8, CHCl3) UV activity: UV visible on TLC -1 -1 IR max cm : 3450 (OH), 2968 1591, and 1463 cm (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). 1 13 H & C-NMR (300 & 100 MHz CDCl3): Details mentioned in the Table 3.18. 2.8.2 Commulin- B (2)

Physical State: Yellowish amorphous powder Yield: 14.2 mg Melting Point: 163-165oC Solvent system used: Ethyl acetate and n-hexane (2:8) Solubility: At room temperature soluble in methanol 30 o [α] D : -166.7 (c = 1.2, CHCl3) UV activity: UV visible on TLC

-1 IR max cm : 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) 1 13 H & C-NMR (300 & 100 MHz CDCl3): Details mentioned in the Table 3.19

42 Chapter – 2 EXPERIMENTAL

2.8.3 Commulin- C (3)

Physical State: Yellowish amorphous powder Yield: 11.5 mg Melting Point: 1.15-1.33 oC Solvent System: Ethyl acetate and n-Hexane at a ratio of 3:7 Solubility: At room temperature soluble in methanol 30 o [α] D: + 13.0 (c = 1.5, CHCl3) UV activity: UV visible on TLC

-1 IR max cm : 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). 1 13 H & C-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) Solubility: At room temperature soluble in methanol -1 -1 IR max cm : 3400, 3000, 1649, 1475, and 1460 cm

+ HR-EIMS m/z: 268.2013 [M] (calcd. for C16H12O4, 268.2011) 1 H-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)

43 Chapter – 2 EXPERIMENTAL

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) Solubility: At room temperature soluble in methanol -1 -1 IR max cm : 3500 and 1655 cm (hydroxyl and ketonic)

HR-EIMS m/z: 312.0125 (calcd. for C18H16O5, 312.0123) 1 H-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) Solubility: At room temperature soluble in methanol

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

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

44 Chapter – 2 EXPERIMENTAL

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).

45 Chapter – 2 EXPERIMENTAL

2.10.3 Analgesic Activity

Two different protocols were used to determine the mechanism(s) of the antinociceptive 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

46 Chapter – 2 EXPERIMENTAL 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.

47 Chapter – 2 EXPERIMENTAL

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

48 Chapter – 2 EXPERIMENTAL 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

49 Chapter – 2 EXPERIMENTAL

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

50 Chapter – 2 EXPERIMENTAL 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

51 Chapter – 2 EXPERIMENTAL

 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;

52 Chapter – 2 EXPERIMENTAL

 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 Ethyl alcohol 50% 1 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 Ethyl alcohol 75% 1 min Ethyl alcohol 100% 1 min Ethyl alcohol 100% 1 min Ethyl alcohol 100% 1 min

53 Chapter – 2 EXPERIMENTAL

 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.

54 Chapter – 2 EXPERIMENTAL

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

55 Chapter – 2 EXPERIMENTAL 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.

56 Chapter – 2 EXPERIMENTAL

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

57 Chapter – 2 EXPERIMENTAL 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

58 Chapter – 2 EXPERIMENTAL 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).

59 Chapter – 3 RESULTS & DISCUSSION

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

Chapter – 3 RESULTS & DISCUSSION 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.

Chapter – 3 RESULTS & DISCUSSION

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.

Chapter – 3 RESULTS & DISCUSSION

Table 3.1: Antimicrobial Activity of the Crude Extract along with the Subsequent Fractions of Viola serpens.

Zone of Inhibition (mm) Group Salmonella Escherichia Bacillus Shigella Staphylococcus Pseudomonas typhi coli subtilis flexneri aureus aeruginosa Imipenem 33 30 33 27 33 24 Crude 10 6 6 8 3 5 Extract n-Hexane - 2 - 2 - - Chloroform 21 18 16 5 2 -

Ethyl 13 14 7 8 3 - acetate

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

Zone of Inhibition (mm) Compound Staphylococcus Bacillus Shigella Escherichia Pseudomonas Salmonella aureus subtilis flexneri coli aeruginosa typhi Imipenem 33 33 27 30 24 25

1 (Commuline 24 15 12 _ _ 15 A)

2 (Commuline _ 15 10 10 13 17 B)

4 15 16 15 11 17 17 (Tectochrysine)

5 15 12 _ _ 16 18 (Sideroxyline)

6 _ 15 15 _ 17 17 (Cearoin) Data are presented as mean ± SEM of three independent assays.

Chapter – 3 RESULTS & DISCUSSION

40 40 B. subtilis E. coli

30 30

20 20

% inhibition % 10 10 % inhibition inhibition

0 0 Extract Hexane CHCl ETA H O Imipenem 3 2 Extract HexaneCHCl 3 ETA H2 O Imipenem

40 P.aerogenes 40 S.aureous

30 30

20 20

% inhibition % 10 10

% Inhibition Inhibition

0 0 Extract Hexane CHCl ETA H O Imipenem Extract HexaneCHCl ETA H O Imipenem 3 2 3 2

40 40 S.flexeneri S.typhi 30 30

20 20

% inhibition % 10 inhibition % 10

0 0

Extract Hexane CHCl3 ETA H2O imipenem Extract Hexane CHCl3 ETA H2O Imipenem

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.

Chapter – 3 RESULTS & DISCUSSION

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

Chapter – 3 RESULTS & DISCUSSION 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.

Chapter – 3 RESULTS & DISCUSSION

Table 3.3: DPPH Scavenging Activity of Crude extract/Fractions of V.serpens and Zones of Inhibition are Given in mm.

Concentrations Crude n-Hexane Chloroform Ethyl Aqueous BHT (ppm) extract acetate 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.

Chapter – 3 RESULTS & DISCUSSION

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,

Chapter – 3 RESULTS & DISCUSSION

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. 70

Chapter – 3 RESULTS & DISCUSSION

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

Chapter – 3 RESULTS & DISCUSSION 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

250 47.25±1.41 n-hexane 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

250 13.75±0.15 Aqueous 989 500 45±1.67 1000 50.75± 2.21

Chapter – 3 RESULTS & DISCUSSION

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 1000 - - Crude extract 1500 - - 2000 - - 1000 - - n-Hexane fraction 1500 - - 2000 - - 1000 - - Chloroform fraction 1500 - - 2000 - - 1000 - - Ethyl acetate fraction 1500 - - 2000 - - 1000 - - Aqueous fraction 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

Chapter – 3 RESULTS & DISCUSSION 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

Chapter – 3 RESULTS & DISCUSSION 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).

Chapter – 3 RESULTS & DISCUSSION

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

Groups Dose Liver-related parameters with % change values mg/kg 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*** *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.

Chapter – 3 RESULTS & DISCUSSION

Figure 3.2.1: Normal saline treated liver showing normal Figure 3.2.2: Liver showing accumulation of lymphocytes (small architecture of central vein (CV), sinusoidal spaces (small arrows) around the central vein (CV), fatty changes (small arrow arrows), hepatocytes (large arrows) with a centrally placed head) and focal area of necrosis (asterisk) with paracetamol nucleus and foamy cytoplasm. (100X H&E). (100X H&E).

Figure 3.2.3: Liver showing regeneration, containing normal Figure 3.2.4: Liver showing normal appearance of central vein liver plates (large arrows) along central vein (CV) with n-hexane (CV) and plates of hepatocytes (large arrows) with n-hexane 300 150 mg/kg b.w. (H&E). mg/kg b.w. (100X H&E).

Figure 3.2.5: Liver showing hexagonal hepatocytes (large Figure 3.2.6: Liver showing regeneration of hepatocytes (large arrows) with prominent cell borders (small arrows), nuclei (arrow arrows) with congestion of sinusoids (asterisks) containing red heads) with nuclear clearing and prominent nucleoli with crude blood cells (small arrows) with crude extract at a dose of 300 extract at a dose of 150 mg/kg b.w. (400X H&E). 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). 77

Chapter – 3 RESULTS & DISCUSSION

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).

Chapter – 3 RESULTS & DISCUSSION

 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

Chapter – 3 RESULTS & DISCUSSION

(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).

Chapter – 3 RESULTS & DISCUSSION

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

Chapter – 3 RESULTS & DISCUSSION

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 Kidney Related Parameters with % Change Values mg/kg 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

Chapter – 3 RESULTS & DISCUSSION

Figure 3.3.1: Photomicrograph (100X H&E) of a section of kidney Figure 3.3.2: Photomicrograph (100X H&E) of a section of from a rabbit treated with normal saline showing normal kidney from a rabbit treated with PCM showing necrosis of histological appearance of renal cortex. The cortex contains renal cuboidal epithelial cells (large arrows) of proximal convoluted corpuscles (large arrows) embedded among proximal (arrow tubules with exfoliation of their brush border. The lumen heads) and distal (asterisk) convoluted tubules. (asterisk) of tubules contains numerous cellular casts (small arrows).

Figure 3.3.3: Photomicrograph (100X H&E) of a kidney section Figure 3.3.4: Photomicrograph (100X H&E) of a section of from a rabbit treated with n-hexane soluble fraction 150 mg/kg kidney from a rabbit treated with n-hexane soluble fraction 300 showing normal histo-architecture of distal convoluted tubules mg/kg showing normal renal corpuscles (large arrows) with with wider lumen (asterisk) and lined by cuboidal epithelial cells mild dilatation of proximal (arrow heads) and distal (asterisk) (arrow heads). Numerous loop of Henle tubules are also visible convoluted tubules. (large arrows).

Figure 3.3.5: Photomicrograph ((100X H&E)) of a section of Figure 3.3.6:Photomicrograph (100X H&E) of a section of kidney from a rabbit treated with chloroform soluble fraction 150 kidney from a rabbit treated with ethyl acetate soluble fraction mg/kg showing normal renal corpuscles (large arrows), proximal 150 mg/kg showing normal renal corpuscles (large arrows) (arrow heads) and distal (asterisk) convoluted tubules. with mild dilatation of proximal (arrow heads) and distal (asterisk) convoluted tubules. 83

Chapter – 3 RESULTS & DISCUSSION

Figure 3.3.7: Photomicrograph ((100X H&E)) of a section of Figure 3.3.8: Photomicrograph (100X H&E) of a section of kidney from a rabbit treated with chloroform soluble fraction 300 kidney from a rabbit rat treated with ethyl acetate soluble mg/kg showing normal renal corpuscles (large arrows) and fraction 300 mg/kg showing normal proximal convoluted proximal convoluted tubules (arrow heads). The distal convoluted tubules (large arrows) with numerous loop of Henle tubules tubules (asterisk) exhibited mild tubular necrosis of the cuboidal (asterisk). The interlobular blood vessels (arrow heads) among epithelial cells. the renal tubules exhibited mild congestion with red blood cells.

Figure 3.3.9: Photomicrograph (100X H&E) of a section of kidney Figure 3.3.10: Photomicrograph (100X H&E) of a section of from a rat treated with aqueous soluble fraction 300 mg/kg kidney from a rat treated with aqueous soluble fraction showing normal renal corpuscles (large arrows). The renal tubules showing mild congestion of the renal corpuscles (large arrows) exhibited dilatation (arrow heads) with exfoliation of the brush with severe dilatation of the renal tubules (asterisk). Numerous border lining the proximal convoluted tubules into their lumen. 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).

Chapter – 3 RESULTS & DISCUSSION

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.

Chapter – 3 RESULTS & DISCUSSION

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±2.90 100 37.55±2.70* 43 Crude 200 25.80±2.90** 60.9 300 19.77±2.00** 70.05 100 39.40±2.50* 40.30 n- hexane 200 27.90±2.56** 59.09 300 21.50±1.9** 68.18 100 49.90±2.90 24.4 Chloroform 200 38.70±2.80* 42.42 300 31.50±1.70* 52.27 100 45.33±2.10 31.31 Ethyl acetate 200 37.12±2.75* 43.76 300 34.75±2.45* 50.37 100 55.50±2.90 15.9 Aqueous 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

Chapter – 3 RESULTS & DISCUSSION

1 0 0 C r u d e e x tr a c t * *

8 0 *

n o

t 6 0

c e

o * r

P 4 0

% 2 0

0 1 0 0 2 0 0 3 0 0 D ic lo

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 H e x a n e *

* *

8 0 *

n o

i 6 0 t

c *

t o

r * P

4 0 %

2 0

0 1 0 0 2 0 0 3 0 0 D ic lo

Figures 3.5: Antinociceptive Effect of n-hexane 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

Chapter – 3 RESULTS & DISCUSSION

1 0 0 *

C h lo r o f o r m * *

8 0

n *

o i

t 6 0

c e t *

r P

4 0 %

2 0

0 1 0 0 2 0 0 3 0 0 D ic lo

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 E t h y l a c e ta t e

* *

8 0

n o

i 6 0

t *

c e

t *

r P

4 0 %

2 0

0 1 0 0 2 0 0 3 0 0 D ic lo

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

Chapter – 3 RESULTS & DISCUSSION

1 0 0 A q u e o u s *

8 0

o *

i 6 0

t c

r P

4 0 %

2 0

0 1 0 0 2 0 0 3 0 0 D ic lo

Figures 3.8: Antinociceptive Effect of Aqueous 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

 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.

Chapter – 3 RESULTS & DISCUSSION

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

Chapter – 3 RESULTS & DISCUSSION 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).

Chapter – 3 RESULTS & DISCUSSION

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.

Chapter – 3 RESULTS & DISCUSSION

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 Late phase (0-5min) (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*** 100 60±1.36 50±2.19 n-hexane 200 53±1.79* 41±2.25* 300 42±2.24** 29±1.35** 100 62±2.45 51±2.50* Chloroform 200 56±2.50 38±2.35** 300 50±2.70* 29±2.50*** 100 60±2.36 44±2.50* Ethyl acetate 200 56±2.50 36±2.35** 300 51±2.10* 28±2.50*** 100 62±3.10 53±2.50 Aqueous 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.

Chapter – 3 RESULTS & DISCUSSION

Early Phase 80 Crude extract Late Phase

% Protection% 20

0 100 200 300 Tramadol

Figure 3.9: Antinociceptive Effects of Formalin Induced Pain in Mice of the Crude Extract of V.serpens

80 Early Phase n-Hexane Late Phase 60

% Protection% 20

0 100 200 300 Tramadol

Figure 3.10: Antinociceptive Effects of Formalin Induced Pain in Mice of the n- Hexane Soluble Fraction of V.serpens.

Chapter – 3 RESULTS & DISCUSSION

Early Phase Late Phase 50 Chloroform 40

% Protection % 10

0 100 200 300 Tramadol

Figure 3.11: Antinociceptive Effects of Formalin Induced Pain in Mice of the Chloroform Soluble Fraction of V.serpens.

80 Early Phase Ethyl acetate Late Phase 60

% Protection %

0 100 200 300 Tramadol

Figure 3.12: Antinociceptive Effects of Formalin Induced Pain in Mice of the Ethyl Acetate Soluble Fraction of V.serpens

Chapter – 3 RESULTS & DISCUSSION

80 Aqueous Early Phase Late Phase 60

% Protection % 20

0 100 200 300 Tramadol

Figure 3.13: Antinociceptive Effects of Formalin Induced Pain in Mice of the 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

Chapter – 3 RESULTS & DISCUSSION 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

Chapter – 3 RESULTS & DISCUSSION

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 98

Chapter – 3 RESULTS & DISCUSSION 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.

Chapter – 3 RESULTS & DISCUSSION

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).

100

Chapter – 3 RESULTS & DISCUSSION

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.

101

Chapter – 3 RESULTS & DISCUSSION

100 100/kgmg 200mg/kg 300mg/kg Diclofenac

*** 80 ***

***

** 60 ** * * * *

% inhibition % 40 * * *

0 1h 2h 3h 4h 5h

Figure 3.14: Anti-inflammatory Effect (%) of the Crude Extract of V. serpens on Carrageenan Induced Paw Edema

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

***

80 ***

** ***

** 60 **

* * *

% Inhibition % 40 * * *

0 1h 2h 3h 4h 5h

Figure 3.15: Anti-inflammatory Effect (%) of the n-Hexane Soluble Fraction of V. serpens on Carrageenan Induced Paw Edema

102

Chapter – 3 RESULTS & DISCUSSION

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

***

80 ***

***

** 60

40 % Inhibition % * * *

0 1h 2h 3h 4h 5h

Figure 3.16: Anti-Inflammatory Effect (%) of the Chloroform Soluble Fraction of V. serpens on Carrageenan Induced Paw Edema

100 100mg/kg 200/kgmg 300mg/kg Diclofanec

***

80 ***

***

** 60

40 % Inhibition % * * * *

0 1h 2h 3h 4h 5h

Figure 3.17: Anti-Inflammatory Effect (%) of the Ethyl Acetate Soluble Fraction of V. serpens on Carrageenan Induced Paw Edema

103

Chapter – 3 RESULTS & DISCUSSION

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

***

80 ***

***

** 60

% Inhibition % * *

0 1h 2h 3h 4h 5h

Figure 3.18: Anti-Inflammatory Effect (%) of the Aqueous Soluble Fraction of V. serpens on Carrageenan Induced Paw Edema

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

***

*** 80

***

60 ** **

* * 40 **

% Inhibition % *

0 1h 2h 3h 4h 5h

Figure 3.19: Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on Histamine Induced Paw Edema

104

Chapter – 3 RESULTS & DISCUSSION

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

***

*** 80

***

** 60 **

* 40 * * * % Inhibition % * * *

0 1h 2h 3h 4h 5h

Figure 3.20: Anti-Inflammatory Effect (%) of the n-Hexane Soluble Fraction of V. serpens on Histamine Induced Paw Edema

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

***

80 ***

***

** 60

40 % Inhibition % * * *

0 1h 2h 3h 4h 5h

Figure 3.21: Anti-Inflammatory Effect (%) of the Chloroform Soluble Fraction of V. serpens on Histamine Induced Paw Edema

105

Chapter – 3 RESULTS & DISCUSSION

100 100mg/kg 200mg/kg 300mg/kg Diclofenac

***

80 ***

** 60

% Inhibition *

0 1h 2h 3h 4h 5h

Figure 3.22: Anti-Inflammatory Effect (%) of the Ethyl Acetate Soluble Fraction of V. serpens on Histamine Induced Paw Edema

100 100 mg/kg 200mg/kg 300mg/kg Diclofenac

***

*** 80

***

** ** 60

40 * % Inhibition % * * 20

0 1h 2h 3h 4h 5h

Figure 3.23: Anti-Inflammatory Effect (%) of the Aqueous Soluble Fraction of V. serpens on Histamine Induced Paw Edema

106

Chapter – 3 RESULTS & DISCUSSION

Table 3.12: Anti-Inflammatory Effect of Crude Extract of V. serpens Against Carrageenan and Histamine Induced Paw Edema in Mice

Treatment Dose Normal Paw Size 0h 1h 2h 3h 4h 5h mg/kg (NPS) 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 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 Crude extract 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 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 Crude extract 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.

107

Chapter – 3 RESULTS & DISCUSSION

Table 3.13: Anti-Inflammatory Effect Against Carrageenan and Histamine Induced Paw Edema in Mice for V. serpens n- Hexane Soluble Fraction

Treatment Dose Normal Paw Size 0h 1h 2h 3h 4h 5h mg/kg (NPV)

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

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 n-Hexane 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

Anti-inflammatory effect against histamine induced paw edema

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 n-Hexane 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

108

Chapter – 3 RESULTS & DISCUSSION

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 0h 1h 2h 3h 4h 5h (NPS)

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

Anti-inflammatory effect against carrageenan induced paw edema

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 Chloroform 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

Anti-inflammatory effect against histamine induced paw edema

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 Chloroform 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

109

Chapter – 3 RESULTS & DISCUSSION

Table 3.15: Anti-Inflammatory Effect of Ethyl Acetate Soluble Fraction of V. serpens in 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

Anti-inflammatory effect against carrageenan induced paw edema 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 Ethyl Acetate 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

Anti-inflammatory effect against histamine induced paw edema

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 Ethyl Acetate 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

110

Chapter – 3 RESULTS & DISCUSSION

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 Anti-inflammatory effect against carrageenan induced paw edema

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 Aqueous 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

Anti-inflammatory effect against histamine induced paw edema 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 Aqueous 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

111

Chapter – 3 RESULTS & DISCUSSION

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 69.0 100 129.5±2.34 30.0 Crude extract 200 101.9±1.91 45.4 300 78.3±2.09 57.7 100 135±2.56 27.0 n-Hexane 200 105±4.01 43.0 300 83±2.11 55.0 100 137±4.12 25.9 Chloroform 200 112±2.78 39.5 300 90.5±3.11 51.0 100 133±2.34 28.0 Ethyl acetate 200 117±1.77 36.8 300 93.2±2.05 49.6 100 132±3.00 28.6 Aqueous 200 107±2.13 42.0 300 95.3±2.34 48.5

110

Chapter – 3 RESULTS & DISCUSSION

8 0 * 8 0 H e x a n e C ru d e e x tra c t *

6 0 * n 6 0 *

n o

e t

e 4 0 t

4 0 o

o r

r *

P *

% 2 0 2 0

0 0 1 0 0 2 0 0 3 0 0 D ic lo 1 0 0 2 0 0 3 0 0 D ic lo

8 0 8 0 * C h lo r o f o r m * E th y l a c e ta te

6 0 * 6 0

c c

* e e

t *

t 4 0 4 0

% % 2 0 2 0

0 0 1 0 0 2 0 0 3 0 0 D ic lo 1 0 0 2 0 0 3 0 0 D ic lo

8 0 A q u e o u s *

6 0 * n

c e

t 4 0

% 2 0

0 1 0 0 2 0 0 3 0 0 D ic lo

Figures 3.24: Percent Inhibition of Xylene Induced Ear Edema in Mice at Different Doses of the Crude Extract and Fractions of V. serpens

111

Chapter – 3 RESULTS & DISCUSSION

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

112

Chapter – 3 RESULTS & DISCUSSION 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)

113

Chapter – 3 RESULTS & DISCUSSION

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 114

Chapter – 3 RESULTS & DISCUSSION

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.

115

Chapter – 3 RESULTS & DISCUSSION

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)

116

Chapter – 3 RESULTS & DISCUSSION

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).

117

Chapter – 3 RESULTS & DISCUSSION

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.

118

Chapter – 3 RESULTS & DISCUSSION

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´

119

Chapter – 3 RESULTS & DISCUSSION

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.

' 3' 2 4' H3CO O 1' 5' 8 6' 7 9 2 6 10 3 5 4

OH O

Tectochrysine (4)

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.

120

Chapter – 3 RESULTS & DISCUSSION

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).

CH3 2' 3' OH H3CO O 1' 4' 8 5' 7 9 2 6' 6 10 3 5 4 H3C OH O 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

121

Chapter – 3 RESULTS & DISCUSSION 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

2' 1' 2 3' 1 3 A 4' B 4 5' 6' 6 5 OCH3 OH 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

122

Chapter – 3 RESULTS & DISCUSSION 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).

123

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

124 CONCLUSION 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.

125 REFERENCES

REFERENCES

Adhikary, P.; Roshan, K. C.; Kayastha, D.; Shresta, R.; Shresta, T.M. Phytochemical

screening and anti-microbial properties of medicinal plants of Dhunkharka

Community, Kavrepalan Chowk, Nepal. Inter. J. Pharma. Biol. Arch., 2011,

2(6), 1663-1667.

Adnan, M.; Hoscher, D. Medicinal plant abundance in degraded and reforested sites

in Northwest Pakistan. Mountain Res. Develop (MRD)., 2009, 30, 25–32.

Adzu, B.; Amos, S.; Wambebe, C.; Gamaniel, K. Anti-nociceptive activity of the

aqueous extract of Zizyphus spina-christi root bark. Fitoterapia., 2001, 72,

344–350.

Ahmad, K. S.; Habib, S. Indigenous knowledge of some medicinal plants of Himalaya

region, Dawarian village, Neelum valley, Azad Jammu and Kashmir, Pakistan.

Universal. J. Plant. Sci., 2014, 2(2), 40–47.

Ahmad, K. S.; Qureshi, R.; Hameed, M.; Ahmad, F.; Nawaz, T. Conservation

assessment and medicinal importance of some plants resources from Sharda,

Neelum valley, Azad Jammu and Kashmir, Pakistan. Int. J. Agric. Biol., 2012,

14(6), 997–1000.

Ahmed, H. Issues regarding the medicinal plants of Pakistan. Udyana Today., 1999,

6(3), 6-7.

Ali, H.; Sannai, J.; Sher, H.; Rashid, A. Ethnobotanical profile of some plant

resources in Malamjabba valley of Swat, Pakistan. J. Med. Plants. Res., 2011,

5(18), 4676-4687.

Amann, R.; Schuligoi, R.; Lanz, I.; Donnerer, J. Histamine-induced edema in the rat

paw- Effect of capsaicin denervation and a CGRP receptor antagonist. Eur. J.

Pharmacol., 1995, 279(2), 227-231.

126 REFERENCES

Amin, A.M.; Ishtiaque, A.; Chowdhury, K.M.M.; Mahbub.; Sattar, M.M.; Shahriar,

M.; Kuddus, M.R.; Rashid, M. A. Anti-inflammatory and analgesic activities

of Asteracantha longifolia, Nees. Bangl. Pharma. J., 2012, 15(2), 171-176.

Ammara, H.; Salma R.; Farah, D.; Shahid, M. Antimicrobial activity of some plant

extracts having hepatoprotective effects. J. Med. Plant Res., 2009, 3(1), 020-

023.

Anca, T.; Philippe, V.; Ilioara, O.; Mircea, T. Composition of essential oils of Viola

tricolor and V. Arvensis from Romania. Chem. Nat. Comp., 2009, 45(1), 91-

92.

Andrikopoulos, N.K.; Kaliora, A.C.; Assimopolou, N.A.; Papapeorgiou, V.P.

Biological activity of some naturally occurring resins, gums and pigments

against in vitro LDL oxidation. Phytother. Res., 2003, 17(5), 501–507.

Antil, V.; Kumar, P.; Kannappan, N.; Diwan, A.; Saini, P.; Singh, S.; evaluation of

analgesic activity of viola odorata aerial parts in rats. J. Nat. Pharma., 2011, 2(1),

24-27.

Antonio, M.A.; Souza, B.A.R. Oral antiinflammatory and antiulcerogenic activities of

a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia

(Turneraceae). J Ethnopharmacol., 1998, 61(3), 215-228.

Anu, K.; Chauhan, P.K.; Bhardwaj, V.S.; Ramesh, K.; Ankur, T. In vitro antioxidant

& phytochemical investigations of ethanolic extracts of Viola serpens &

Morus nigra. J. Chem. Pharm. Res., 2011, 3(4), 166-171.

Anwar, F.; Ali, M.; Hussain, A.I.; Shahid, M. Antioxidant and antimicrobial activities

of essential oil and extracts of fennel (Foeniculum vulgare Mill.) seeds from

Pakistan. Flav. Frag. J. 2009, 24, 170-176.

127 REFERENCES

Araujo, R. C.; Neves, F. A.; Formagio, A. S.; Kassuya, C. A.; Stefanello, M. E.;

Souza, V. V.; Pavan, F. R.; Croda, J. Evaluation of the anti-mycobacterium

tuberculosis activity and in vivo acute toxicity of Annona sylvatic. BMC

Compl. Alt. Med. 2014, 14(209), 1-10.

Assessment report on Viola tricolor L. and/or subspecies Viola arvensis Murray

(Gaud) and Viola vulgaris Koch (Oborny), herba cum flore. Europ. Med.

Agency Sci. Med. Health., 2010, 1-19.

Atta, A.H.; Alkofahi, A. Antinociceptive and antiinflammatory effects of some

Jordanian medicinal plant extract. J. Ethnopharmacol., 1998, 60(2), 117-124.

Bahekar, S.; Kale, R. Herbal plants used for the treatment of malaria- A Literature

Review. J. Pharmacog. Phytochem., 2013, 1(6), 141-146.

Bak, M. J.; Jun, M.; Jeong, W.S. Antioxidant and hepatoprotective effects of the red

ginseng essential oil in H2O2-treated HepG2 cells and CCl4-treated mice. Int. J.

Mol. Sci., 2012, 13, 2314-2330.

Balick, M. J.; Cox, P.A. Plants, people and culture: The science of Ethnobotany. New

York, USA, Book review. J.Nat. Prod. 1997, 60 (4). 228-429.

Ballard, H. E.; Sytsma, K. J.; Kowal, R. R. Shrinking the Violets: Phylogenetic

relationships of infrageneric groups in Viola (Violaceae) based on internal

transcribed spacer DNA sequences. Syst. Bot., 1999, 23(4), 439-458.

Banaszczak, W. E.; Bylka, W.; Matlawska, I.; Goslinska, O.; Muszynski, Z.

Antimicrobial activity of Viola tricolor herb. Fitoterapia 2005, 76(5), 458-

561.

Bancroft, J.D.; Gamble, M. Theory and practice of histological techniques. Churchill

Livingstone. London, New York & Sydney, 2007, 5, 377-694.

128 REFERENCES

Barkatullah, M.; Ibrar, N.; Ali, Muhammad, N.; Ehsan, M,; In-vitro pharmacological

study and preliminary phytochemical profile of Viola canescens Wall. Ex

Roxb. Afr. J. Pharma. Pharmacol., 2012, 6(15), 1142–1146.

Beauchamp, C.; Fridovich, I. Superoxide dismutase: improved assays and an assay

applicable to acrylamide gels. Anal. Biochem., 1971, 44, 276-287.

Benecke, C.; Clif.; Helen, L. L.; Joe Moodie Seattle, J.M.; Mercer Island, M.

Washington bulletin of the American rock garden society., 1985, 43(2), 59-

106.

Benjamin, M.N. Outline of veterinary Clinical Pathology. University press. Iowa.,

1978, 229-232.

Beris H. Antioxidant effects a basis of drug selection. Indian Drugs., 1991, 42, 569-

605.

Bernard, V. UV spectral determination of 5-hydroxy-and 5-hydroxy-3-methoxy

flavones with mono-(4), di-(3, 4), tri-(3, 4, 5)-substituted B ring. J.

Phytochem., 1983, 22(10), 2107-2145.

Bhardwaj, S.; Ghakar, S.K.; Ethnomedicinal plants used by the tribals of Mizoram to

cure cut and wound. Ind. J. Trad. Knowled., 2005, 4(1), 75-80.

Bhatt, V. P.; Negi, G. C. S. Ethnomedicinal plant resources of Jaunsari tribe of

Garhwal Himalaya, Uttaranchal. Indian J. Tradit. Knowl., 2006, 5(3), 331-

335.

Bhattacharya, D.; Sugunan, A.P.; Bhattacharjee, H.; Thamizhmani, R.; Sayi, D.S.;

Thanasekaran, K.; Manimunda, S.P.; Ghosh, A.R.; Bharadwaj, A.P.;

Seyinghania. M.; Roy, S. Antimicrobial resistance in Shigella - rapid increase

& widening of spectrum in Andaman Islands, India. Indian. J. Med. Res.,

2012, 135(3), 365-370.

129 REFERENCES

Bibi, S.; Dastagir, G.; Hussain, F.; Sanaullah, P. Elemental composition of Viola

odorata Linn. Pak. J. Plant. Sci., 2006, 2, 141-143.

Blumenthal, M., Busse, W. R., Goldberg, A., Gruenwald, J., Hall, T., Riggins, C. W.

& Rister, R. S. The Complete German Commission E. Monographs:

Therapeutic Guide to Herbal Medicines. Austin, TX: American Botanical

Council; Boston: Integrative Medicine Communication; 1998.

Boyanova, L.; Gergova, G.; Nikolov, R.; Derejian, S.; Lazarova, E.; Katsarov, N.;

Mitov, I.; Krastev, Z. Activity of Bulgarian propolis against 94 Helicobacter

pylori strains in vitro by agar- well diffusion, agar dilution and disc diffusion

methods. J. med. Microbiol., 2005, 54(5), 481-483.

Boyd, E.M.; Bereckzky, G.M. Liver necrosis from Paracetamol. Brit. J. Pharmacol.,

1966, 26, 606-614.

Brufani, M.; Marta, M.; Pomponi, M. Anticholinesterase activity of a new carbamate,

heptylphysostigmine, in view of its use in patients with Alzheimer-type

dementia. Eur. J. Biochem., 1986, 157, 115-120.

Burman, R. Distribution and Chemical diversity of cyclotides from violaceae

UPPSALA University., 2010.

Bursal, E.; Gülçin. I. Polyphenol contents and in vitro antioxidant activities of

lyophilised aqueous extract of kiwifruit (Actinidia deliciosa). Food. Res. Int.,

2011, 44, 1482-1489.

Chandrasekharan, N.V.; Daı, H.; Roos, K.L.T.; Evanson, N.K.; Tomsik, J.; Elton,

T.S.; Simmons, D.L. COX 3, a cyclooxygenase-1 variant inhibited by

acetaminophen and other analgesic/antipyretic drugs: cloning, structure and

expression. Proc. Natl. Acad. of Sci. USA., 2002, 99, 13926–13931.

Chatterjee, T. K. Medicinal plant with antifertility property. Herb. Option., 1997, 77.

130 REFERENCES

Chauhan, J, S.; Sultan, M.; Srivastava, S. K. Two new glycoflavones from the roots of

Phyllanthus niruri. Planta Medica., 1977, 32(7), 217-222.

Chauhan, N.S. Important medicinal and aromatic plants of Himachal Perdesh. 2003,

129, 442-443.

Chen, B.; Colgrave, M.L.; Daly, N.L.; Rosengren, K.J.; Gustafson, K.R.; Craik, D.J.

Isolation and characterization of novel cyclotides from Viola hederaceae:

Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed

cyclotide. J. Biol. Chem., 2005, 280(23), 22395-22405.

Chevallier, A. The Encyclopedia of Medicinal Plants Dorling Kindersley. London,

1996.

Choi, E.M. Antinociceptive and antiinflammatory activities of pine (Pinus densiflora)

pollen extract. Phytother. Res., 2007, 21, 471–475.

Clark, A.M.; Hufford, C.D. Discco and development of novel prototype antibiotics for

opportunistic infections related to the acquired immunodeficiency syndrome.

In: human medical agents from plants. American Chem. Soc., 1993, 534, 228-

241.

Clark, L. J., Trelawny, J. Wild Flowers of the Pacific Northwest. Harbor, Madeira

Park. 1998.

Clark, R.; Thompson, R.P.H.; Borirackanyavat, V.; Widoop, B.; Davidson, A.R.;

Goulding, R.; Williams, R. Hepatic damage and death from Overdose of

paracetamol. Lancet. 1973, 1, 66-69.

Collier, H. O. J.; Dinneen, L.C.; Johnson, C.A.; Schneider, C. Formalin test in mice, a

useful technique for evaluating mild analgesia. Br. J Pharmacol. Chemother.,

1968, 32(2), 295–310.

131 REFERENCES

Craik, D. J. Discovery and applications of the plant cyclotides. Toxicone., 2010,

56(7), 1092-1102.

Cuman, R.K.; Bersani-Amadio, C.A.; Fortes, Z.B. Influence of Type 2 diabetes on the

inflammatory response in rats. Inflamm. Res., 2001, 50(9), 460-465.

Dai, J.J.; Tao, H.M.; Min, Q.X.; Zhu,Q.H. Anti-hepatitis B virus activities of

friedelolactones from Viola diffusa Ging. Phytomed., 2015. 22(7-8), 724-729.

Dai, Y.; Liu, L. H.; Kou, J. P. Anti-inflammatory effect of aqueous extract of Wu-

HU-Tang. China. Pharma. Uni., 1995, 6, 362-364.

Daniel, A.T.; Zhao, S.; Tong, E.; Ayers, S.; Singh, A.; Mary, J.; Bartholomew,

McDermott. P.F. Antimicrobial drug resistance in Escherichia coli from

humans and food animals, United States, 1950–2002. Emerg. Infect. Dis.,

2012, 18 (5), 741-749.

Das, T.S.; Pereira. N.P.; Das, T.A. Pereira Effects of flavonoids on thermal

autooxidation of palm oil: structure–activity relationship., J. Am. Oil Chem.

Soc., 1990, 67, 255–258.

Debral, L.; Taylor, M.K.; Tara, M.B.C.; Gordon, B.; Prasad, S.S.; Stanley, T.

Antimicrob Agent. Chemother., 1994, 1780-1787.

Deraedt, R.; Jouquey, S.; Delevallée, F.; Flahaut, M. Release of prostaglandin E and F

in an algogenic reaction and its inhibition. Eur J Pharmacol., 1980, 61(1), 7-

24.

Dhar, U.; Manjkhola, S.; Joshi M.; Bhatt, A., Bisht, A.K.; Joshi, M. Current status and

future strategy for development of medicinal plants sector in Uttaranchal.

India. J. Curr. Sci., 2002, 83, 956-64.

132 REFERENCES

Du, J.; Yu, Y.; Ke, Y.; Wang, C.; Zhum, L.; Qian, Z.M.; Ligustilide attenuates pain

behavior induced by acetic acid or formalin. J Ethnopharmacol., 2007, 112,

211–214.

Dua, V. K.; Verma, G.; Agarwal, D. D.; Kaiser, M.; Brun, R. Antiprotozoal activities

of traditional medicinal plants from the Garhwal region of North West

Himalaya, India. J. Ethnopharmacol., 2011, 136(1), 123–128.

Duarte, I.D; Nakamura, M.; Ferreira, S.H. Participation of the sympathetic system in

acetic acid-induced writhing in mice. Braz. J. Med. Biol. Res., 1988, 21(2),

341–343.

Dubuisson, D.; Dennis, S.G. The formalin test: a quantitative study of the analgesic

effects of morphi-ne, meperidine and brain stem stimulation in rats and cats.

Pain., 1977, 4(2), 161-174.

Duke, J.A.; Ayensu, E.S. Medicinal plants of China. Algonac, MI: Reference

Publications Inc., 1985, 1, 1131-1139.

Ebrahimzadeh, M. A.; Nabavi, S. M.; Nabavi, S. F.; Bahramian, S.; Bekhradnia, A. R.

Antioxidant and free radical scavenging activity of H. Officinalis L. Var.

Angustifolius, V. odorata, B. hyrcana and C. speciosum. Pak. J. Pharm. Sci.,

2010, 23(1), 29-34.

Edwards, C.R.W.; Bouchier, I.A.D. Davidson’s Principles and amp; Practice

Medicine Churchill Livingstone Press, UK. 1991, 130.

Edwige, Y.; Vouffo, Flaure M, Donfack, R.J.; Temdie, F.T.; Ngueguim, Jean, H.D.;

Desire, D.D.; Alain, B.D.; Theophile, D. Hepatho-nephroprotective and

antioxidant effect of stem bark of Allanblackia gabonensis aqueous extract

against acetaminophen-induced liver and kidney disorders in rats. J. Exp.

Integ. Med. 2012, 2(4), 337-344.

133 REFERENCES

Ernst, E. The role of Complementary and alternative Medicine. Brit. Med. J., 2000,

321, 1133-1135.

ESCOP (European Scientific Cooperative on Phytotherapy). ESCOP Monographs on

the Medicinal Uses of Plant Drugs, Exeter, UK. 1999.

Fabricant, D.S.; Farnsworth, N.R. The value of plants used in traditional medicine for

drug discovery. Environ. Health. Perspect., 2001, 109(1), 69-75.

Farnsworth, N.R. Biological and phytochemical screening of plants. J. Pharm. Sci.,

1966, 55(3), 225-276.

Farnsworth, N.R.; Akerele, O.; Bingel, A.S.; Soejarto, D. D.; guo, Z. Medicinal plants

in therapy. Bull.World. Health. Organ., 1985, 63(6), 965-981.

Ferreira, A.; Proenca. C.; Serralheiro, M. L. M.; Araujo, M. E. M. The in vitro

screening for acetylcholinesterase inhibition and antioxidant activity of

medicinal plants from Portugal. J. Ethnopharmacol., 2006, 108, 31–37.

Gairola, S.; Gupta, V.; Bansal, P.; Singh, R.; Maithani, M. Herbal antitussives and

expectorants – A Review. Inter. J. Pharma. Sci. Review and Res., 2010, 5(2),

5-9.

Garba, S.H.; Sambo, N.; Bala, U. The effect of the aqueous extract of Kohautia

grandifora on paracetamol induced liver damage in albino rats. Niger. J.

Physio. Sci. 2009, 24(1), 17 –23.

Gaulejac, D.; Glories, Y.; Vivas, N. Free radical scavenging effect of anthocyanins in

red wines food. Res. Int., 1999, 32, 327–333.

Gautam, S.S.; Kumar, N.S. The antibacterial and phytochemical aspects of Viola

odorata Linn. Extracts against respiratory tract pathogens. Proc. Nat. Acad.

Sci., India, Sect. B Biol. Sci., 2012, 82(4), 567–572.

134 REFERENCES

Gerlach SL.; Rathinakumar, R.; Chakravarty, G.; Goransson, U, Wimley WC. Anti-

cancer and chemosensitization abilitys of cycloviolacin O2 from Viola odorata

and psyle cyclotides from Psychotria leptothyrsa. Biopolymers., 2010. 94(5),

617-625.

Ghorbani, A.; Youssofabad, N.J.; Rakhshandeh, H. Effect of Viola tricolor on

pentobarbital-induced sleep in mice. Afr. J. Pharma. Pharmacol. 2012, 6(35),

2600-2606.

Goosney, D.L.; Knoechel, D. G.; Finlay, B.B. Enteropathogenic E. coli, Salmonella,

and Shigella: Masters of host cell cytoskeletal exploitation. Emerg. Infec. Dis.,

1999, 5 (2), 216-223.

Gran, L. Oxytoxic principles of Oldenlandia affinis. Lloydia. (Mechanism of action of

cytotoxic cyclotides: cycloviolacin O2 disrupts lipid membranes. J. Nat. Prod.

2007, 70(4), 643-7.

Grisebach, H.; Grambow, H.J. Biosynthesis of flavonoids- XV: occurance and

biosynthesis of flavonoids in Datisca cannabina. Phytochem., 1968, 7 (1), 51–

56.

Gulati, V.; Harding, I. H.; Palombo, E.A. Enzyme inhibitory and antioxidant activities

of traditional medicinal plants: Potential application in the management of

hyperglycemia. BMC Comp. Alter. Med., 2012, 12, 77.

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

Ethnobotanical profile of Utror and Gabral valleys, district Swat, Pakistan,”

Ethnobot. Leaflets., 2005

Hamayun, M.; Khan, S. A.; Sohn, E. Y.; Lee, I. Folk medicinal knowledge and

conservation status of some economically valued medicinal plants of District

Swat, Pakistan. Lyonia., 2006, 11(2), 101–113.

135 REFERENCES

Hammami, I.; Kamoun, N.; Rebai, A. Biocontrol of Botrytis cinerea with essential oil

and methanol extract of Viola odorata L. flowers. Arch. Appl. Sci. Res., 2011,

3(5), 44-51.

Hassan, F., Naeem, I. Biological activity of Viola odorata linn against Mycobacterium

tuberculosis. Int. J. Pharm. Bio. Sci., 2014, 5 (3), 61 – 69.

Hatano, T.; Edamatsu, R.; Hiramatsu, M.; Mori, A.; Fujita, A. Effects of the

interaction of tannins with co-existing substances. VI: effects of tannins and

related polyphenols on superoxide anion radical and on 1, 1-diphenyl-2-

picrylhydrazyl radical. Chem. Pharm. Bull., 1989, 37, 2016–2021.

Hekking, W. H. A. Studies on neotropical Violaceae—the genus Fusispermum. P. K.

Ned.C. Biol., 1984, 87(2), 121–130.

Herschman, H.R. Prostaglandin synthase 2. Biochem. Biophys. Acta., 1996, 1299,

125–140.

Hikosaka, K.; Kawauchi.; Kurosawa, T. Why Does Viola Hondoensis (Violaceae)

shed its winter leaves in spring Americ. J. Bot., 2010, 97(12), 1944–1950.

Hocking, G. M. Qualitas plantarum et material vegetables. Pak. Med. Plant., 1958, 5,

145-153.

Hong, J. L.; Zhou, H. Y.; Zhu, J.; Li, L.; Shu, P.; Ying, X.; Qin, X. Y.; Gang W, G.;

Lin, B. B.; Wang, G. K.; Wang, Q. In Comparative analysis of major

constituents in Viola yedoensis Makino and different species from the Genus

Viola by high performance liquid chromatography with chemometrics

methods. J. Med. Plant. Res., 2011, 5(21), 5230-5239.

Hughes, F.N. Compendium of Pharmaceutical Specialties, 1st ed. Toronto: Canadian

Pharma. Ass, 1960.

136 REFERENCES

Huh, M. K. Inhibition of angiotensin converting enzyme (ACE) by Viola

Mandshurica extraction. Eur. j. adv. Res. Biol. Life. Sci., 2015. 3(2), 5-11.

Husain, S.Z.; Malik, R.N.; Javaid, M.; Bibi, S. Ethonobotanical properties and uses of

medicinal plants of Morgah biodiversity park, Rawalpindi. Pak. J. Bot., 2008,

40 (50), 1897-1911.

Hussain, I.; Bano, A.; Ullah, F. Traditional drug therapies from various medicinal

plants of central karakoram national park, Gilgit-Baltistan, Pakistan. pak. J.

Bot., 2001, 43, 79-84.

Idu, M.; Omogbai, E. K. I.; Aghimien, G. E. I.; Amaechina, F.; Timothy, O.;

Omonigho S. E. Preliminary phytochemistry, antimicrobial properties and

acute toxicity of Stachytarpheta Jamaicensis (L.) Vahl. Leaves. Trends. Med.

Res., 2007, 2(4),193-198.

Ikram, I.; Asghar, K.; Mehboob, A.; Ubaid, U.; Jawad, A.; Murad, K. Effects of stone

dust exposure on some liver and kidney related serum parameters of stone

crush plant workers. J. Bio. Life Sci., 2012, 3, 211-219.

Ikram, I.; Imran, K.; Mohammad, T.; Izhar, A. Larvicidal activities of different parts

of Melia azedarach Linn. against Culex quinquefasciatus Say. (Diptera:

Culicidae). J. Basic Appl. Sci., 2012, 8, 23-28.

Ikram, I.; Samina, S.; Imran, K.; Izhar, A.In vitro antioxidant activities of four

medicinal plants on the basis of DPPH free radical scavenging. J. Pharma.

Sci., 2013, 26, 210-215.

Imran, M.; Ullah, F.; Sadiq, A.; Ayaz, M.; Ahmad, S.; Kamal, Z.; Zeb, A.

Investigation of total phenoliccontents, antibacterial, antifungal and

anthelmentic potential of crude methanolic extract, Subsequet fractions and

137 REFERENCES

crude saponins of Nonea micrantha Boiss and Reut. Pharmacol., 2014, 3, 26-

31.

Ishtiaq, D.M.; Khan, M. A.; Hanif, W. Ethno veterinary medicinal uses of plants from

Samahni valley Dist. Bhimber, Azad Kashmir. Pak. J. Plant. Sci., 2006, 5(2),

390-396.

Iwu, M. W., Duncan, D. R.; Okunji, C. O. New antimicrobials of plant origin. In:

Janick, J. (Ed.) Perspective on new crops and new uses. ASHS Press.

Alexandria, VA. 1999, 107-108.

Javanmardi, J.; Stushnff, C.; Locke, E.; Vivanco, J.M. Antioxidant activity and total

phenolic content of Iranian Ocimum accessions. Food Chem., 2003, 83, 547-

550.

Jayaprakash, G.K.; Singh, R.P.; Sakariah, K.K. Antioxidant activity of grape seed

(Vitis vinifera) extracts on peroxidation modles in vitro. J. Agric. Food. Chem.

2001, 73, 285-290.

Jung, M.; Park, M. Acetylcholinesterase inhibition by flavonoids from Agrimonia

pilosa. Molecules., 2007, 12, 2130-2139.

Kahkonen, M.P.; Hopia, A.T.; Vuorela, H.J.; Rauha, J.P.; Pihlaja, K.; Kujala, T.S.;

Heinonem, M. Antioxidant activity of plant extracts containing phenolic

compounds. J. Agr. Food. Chem., 1999, 47(10), 3954-3962.

Kakishima, M.; Izumi, O.; Ono, Y. Graminicolous rust fungi (Uredinales) from

Pakistan.Cryptogam Flora Pak. 1993, 2, 181–186.

Kakishima, M.; Izumi, O.; Ono, Y. Rust fungi (Uredinales) of Pakistan collected in

1991.Cryptogam Flora Pak .1993, 2, 169-179.

Kassem, F.F.; Alqasoumi, S.I.; Sallam, S.M.; Bekhit, A.A.; Shaer, N.S.E.; Farrj, A.I.;

Abdel-Salam NA, Abdel-Kader MS: Evaluation of the hepatoprotective,

138 REFERENCES

nephroprotective and anti-malarial activities of different parts of Bauhinia

purpurae and Tipuana speciosa grown in Egypt. J Med Plants Res. 2013, 7,

1190-1200.

Kaul, M. K. Medicinal Plants of Kashmir and Ladkh. Indus Publishing Company F-5,

Tagore Garden, New Dehli. 1997.

Khan, H.; Saeed, M.; Gilani, A. U. H.; Khan, M.A.; Dar, A.; Khan, I. The

antinociceptive activity of Polygonatum verticillatum rhizomes in pain

models. J. Ethnopharmacol. 2010, 127(2), 521–527.

Khatibi, A.; Shah, A.H.; Ageel, A.M.; Ahmad, M.S.; Al-Yahya, M.A.; Tariq, M.

Saudi folk medicine: phytochemical and antimicrobial screening. Pak. J.

Pharm. Sci., 1989, 2(1), 29–34.

Kilian, N.; Hein, P.; Hubaishan, M.A. Further notes on the flora of the southern

coastal mountains of Yemen. Willdenowia., 2004, 34, 159-182.

Kim, H.D.; Cho, H.R.; Moon, S.B.; Shin, H.D.; Yang, K.J.; Park, B.R.; Jang, H.J.;

Kim, L.S.; Lee, H.S.; Ku, S.K. Effects of beta-glucan from Aureobasidium

pullulans on acute inflammation in mice. Arch. Pharma. Res., 2007, 30(3),

323–328.

Kinghorn A. D. Pharmacognosy in the 21st century. J. Pharm. Pharmacol., 2001, 53,

135–148.

Koster, R.; Anderson, M.; De Beer, J. Acetic acid for analgesic screening. Federal

Proceedings., 1959, 18, 412–417.

Kumar, A.; Kumari, M.; Mazumdar, R. S.; Dhewa, T. In-vitro antibacterial activity of

ethanolic extracts of Viola serpens and Morus nigra against pathogens isolated

from patients suffering from jaundice. World J. pharma. Res., 2015, 4(2), 889-

898.

139 REFERENCES

Kumar, P.; Digvijay. Assessment of genetic diversity of Viola serpens Wall. in

Himachal Pradesh using molecular markers. World. J. Pharma. Res., 2014,

3(2), 2716-2726.

Kumar, S.; Chand, G.; Sankhyan, P. Herbal folk remedies for curing various ailments

in lug valley of District Kullu, Himachal Pradesh (N. W. Himalaya). Int. J.

Ayurved Herb. Med., 2013, 3(5), 1308–1314.

Lee, S. J.; Park, W. H.; Moon, H. I. Bioassay-guided isolation of antiplasmodial

anacardic acids derivatives from the whole plants of Viola webster Hemsl.

Parasitol. Res., 2009, 104, 463–466.

Li, L. Opportunity and challenge of traditional Chinese medicine in face of the

entrance to WTO (World Trade Organization)]. Chin Inform trad Chin Med.,

2000, 7, 7–8.

Linardi, A.; Costa, S.K.P.; Silva G.R.D.; E. Antunes. Involvement of kinins, mast

cells, and sensory neurons in the plasma exudation and paw edema induced by

staphylococcal entrotoxin B in the mouse. Euro. J. Pharmacol. 2002, 399,

235-242.

Lindholm, P.; Göransson, U.; Johansson, S.; Claeson, P.; Gullbo, J.; Larsson, R.;

Bohlin, L. Anders Backlund Cyclotides: A novel type of Cytotoxic agents.

Mol. Cancer Therap., 2002, 1(6), 365-369.

Liu, Y.; Murakami, N.; Zhang, S.; Xu, T. Comparative anti-inflammatory

characterization of wild fruiting body, liquid-state fermentation, and solid-

state culture of Taiwanofungus camphoratus in microglia and the mechanism

of its action. Pharmazie., 2007, 62(9), 59-662.

Lounasmaa, M.; Puri, H. S.; Widen. C. J. Pholroglucinol derivatives of Callistemon

lanceulatus leaves. Phytochem., 1977, 16, 1851-1852.

140 REFERENCES

Maaz, A.; Bhatti. A.S.A.; Maryam, S.; Afzal, S.; Ahmad, M.; Gilani, A.N.

Hepatoprotective elevation of Butea monosperma against liver damage by

paracetamol in rabbit. Special. Edit. Annals. 2010, 16(1), 1-5.

Mabberley, D. I. The Plant Book. Camb. Univ. Press, Cambridge, New York. 1987.

Mabberley, D. The plant-book 3rd Edition. Cambridge University press, Cambridge,

New York. 2008.

Mahady, G.B. Global harmonization of herbal health claims. J. Nutr. 2001, 131(3s),

1120S-1123S.

Malila, N.; Virtamo, J.; Virtanen, M.; Pietinen, P.; Albanes, D.; Teppo, L. Dietary and

serum alpha-tocopherol, beta-carotene and retinol and risk for colorectal

cancer in male smokers. Eur. J. Clin. Nut. 2002, 56(7), 615- 621.

Mann, A.; Yahaya, Y.; Banso, A.; John, F. Phytochemical and antimicrobial activity

of Terminalia avicennioides extracts against some bacteria pathogens

associated with patients suffering from complicated respiratory tract diseases.

J. Med. Plant. Res., 2008, 2(5), 94-97.

Mann, C.; Staba, E.J. The chemistry, pharmacology, and commercial formulations of

chamomile in herbs, spices, and medicinal plants: recent advances in botany,

horticulture, and pharmacology. Oryx Press. Phoenix., 1986, 1, 235-280.

Marcussen, T.; Danihelka, K. J. J.; Ballard, H. E.; Blaxland, K.; Brysting, A.K.;

Oxelman, B. Inferring species networks from gene trees in high-polyploid

North American and Hawaiian Violets (Viola, Violaceae). Syst. Biol., 2012,

61(1), 107–126.

Marcussen, T.; Oxelman, B.; Skog, A.; Jakobsen, K.S. Evolution of plant RNA

polymerase IV/V genes: evidence of subneofunctionalization of duplicated

141 REFERENCES

NRPD2/NRPE2-like paralogs in Viola (Violaceae). BMC Evol. Bio. 2010, 10,

10-45.

Marinova, G.; B1atchvarov, V. Evaluation of the methods for determination ff the

free radical scavenging activity by DPPH. Bulgarian. J. Agri. Sci., 2011,

17(1), 11-24.

Marwat, S. K.; Khan, M.A.; Ahmad, M.; Zafar, M.; Rehman, F. Ethnophytomedicines

for treatment of various diseases in D. I. Khan district, Sarhad. J. Agric., 2008,

24, 305-316.

Masood, M.; Arshad, M.; Asif, S.; Chaudhari, S.K. Viola canescens: Herbal Wealth to

Be Conserved. J. Bot., 2014, 2014, 1-6.

Mbiantcha, M.; Kamanyi, A.; Tiponno, R.; Taponjou, A.; Watcho, P.; Nguelefack, T.

Evidence-based compl Alt Med: Ecam, 2011, 2011.

McConnachie, L.A.; Mohar, I.; Hudson, F.N.; Ware, C.B.; Ladiges, W.C.; Fernandez,

C.; Chatterton, K.S.; White, C.C.; Pierce, R.H.; Kavanagh, T.J. Glutamate

cysteine ligase modifier subunit deficiency and gender as determinants of

acetaminophen-inducedhepatotoxicity in mice. Toxicol. Sci. 2007, 99(2), 628-

636.

McConnachie, L.A.; Mohar,I.; Hudson, F.N.; Ware, C.B.; Fernandez, C.; Chatterton-

Kirchmeier, S.; White, C.C.; Pierce, R.H.; Kavanagh, T.J.; Glutamate cysteine

ligase modifier subunit deficiency and gender as determinants of

acetaminophen-inducedhepatotoxicity in mice. Toxicol. Sci. 2007, 99(2), 628-

636.

Melo, J. G.; Santos A.G.; Amorim, E.L.C.; Nascimentos, S.C.; Albuquerque, U.P.

Medicinal plants used as antitumour agents in Brazil: An ethnobotenical

approach. Evid. Based Compl. Alter. Med., 2011, 15, 8534–8542.

142 REFERENCES

Moerman, D. Very comprehensive but terse guide to the native uses of plants.

Excellent bibliography fully referenced to each plant, giving a pathway to

further information. Not for the casual reader. Native American Ethnobotany.

Timber Press. Oregon. 1998, 133, ISBN 0-88192-453-9.

Molnar, P.; Deli, J.; Zsifa, F.; Steck, A.; Pfander, H.; Toth, G. Preparation and (E/Z)-

isomerization of the diastereoisomers of Violaxanthin. Helvica Chemica Acta.,

2004, 87, 11-27.

Monadi, A.; Rezaie, A. Evaluation of sedative and pre-anesthetic effects of Viola

odorata Linn. extract compared with diazepam in rats. Bull. Env. Pharmacol.

Life Sci., 2013, 2(7), 125-131.

Moon, H. I.; Jung, J.C.; Lee, J. Antiplasmodic activity of triterpenoids isolated from

the whole plant of Viola genus from South Korea. Parasitol. Res., 2007,

100(3), 641-644.

Morgan, K. Medicine of the Gods: Basic. Principl. Ayurved. Med., 2002,

[http://www.compulink.co.uk/mandrake/ayurveda.htm.

Naveed, M., Saeed, M., Aleem, A., Khan, H. Ethnobotenical , phytochemical and

pharmacological studies of genus Viola. Pharmacol., 2012, 3(1) 214-226.

Naveed, M., Rehman, N.; Khan, H.; Saeed, M.; Gilani, A.H. Prokinetic and laxative

effects of the crude methanolic extract of Viola betonicifolia whole plant in

rodents. Comp. Alter. Med., 2013, 13, 70.

Naveed, M., Saeed, M.; Gilani, N.; Ikram-ul, H.; Khan, H. Analgesic and anti-

inflammatory profile of Viola betonicifolia whole plant. Tropica. J. Pharma.

Res., 2013, 11, 963-969.

143 REFERENCES

Naveed, M., Saeed, M.; Gilani, S.M.; Haq, I.; Khan, H. Analgesic and anti-

inflammatory profile of n-hexane fraction of Viola betonicifolia. Trop. J.

Pharm. Res., 2012, 11(6), 963-969.

Naveed, M., Saeed, M.; Khan, A.; Adhikari, A.; Wadood, A.; Khan, K.M.; Feo, V.D.

A New Urease Inhibitor from Viola betonicifolia. Mol., 2014, 19, 16770-

16778.

Naveed, M., Saeed, M.; Khan, H. Antipyretic, analgesic and anti-inflammatory

activity of Viola betonicifolia whole plant. Comp. Alter. Med., 2012, 12, 59.

Naveed, M., Saeed, M.; Khan, H.; Adhikari, A.; Khan, K.M. Muscle relaxant and

sedative-hypnotic activities of extract of Viola betonicifolia in animal models

supported by its isolated compound, 4-Hydroxy coumarin. J. Chem., 2013, 1-

Naveed, M., Saeed, M.; Khan, H.; Hassan, S.; Farah Gul. Evaluation of Viola

betonicifolia for its nutrition value. Pak. J. Pharm. Sci., 2012, 25(3), 639-644.

Naveed, M., Saeed, M.; Khan, H.; Qayum, M.; Barkatullah.; Badshah, A. Evaluation

of Viola betonicifolia for anthelmintic. Afric. J. Pharm. Pharmacol., 2012,

6(10), 698-701.

Naveed, M., Saeed, M.; Qayum, M.; Khan, H. Antimicrobial screening of Viola

betonicifolia. Mid-East. J. Scient. Res., 2013, 15(1), 55-60.

Mukherjeea, P. K.; Kumarb, V.; Malb, M.; Houghtona, P. J. Acetylcholinesterase inhibitors from plants.

Phytomed., 2007, 14, 289–300. Munzinger, J.K.; Ballard, H.E. Hekkingia (Violaceae), a new arborescent violet from

French Gui-ana with a key to genera in the family. Syst. Bot., 2003, 28, 345-

351.

Naain, S.C.A Handbook of medical and aromatic plants of Himachal Pardesh., 1999,

431-432.

144 REFERENCES

Nahak, G.; Sahu, R.K. In vitro antioxidant activity of Azaderachta indica and Melia

azedarach leaves by DPPH scavenging assay. J. American. Sci., 2010, 6(6),

123-128.

Nathorst, A.G. Polarforskningens Bidrag till Forntidens Växtgeografi. Stockholm.

Diagnoses plantarum novarum Asiati carum. (Bull, del’Acad. imp. deSt.

Petersbourg. Vol. Holm. Comparative studies of North American Violets.

discussed by Nathorst1). 1883, 23, 310.

National Audubon Society: Field Guide to North American Wildflowers, Eastern

Region National Audubon Society Alfred A. Knopf, New York 1979. ISBN:

0-394-50432-1.

Native American garden plants teaching herb garden (NAGPTG). The new England

unit of the herb society of America note: 2005.

Nikolova, M.; Evstatieva, L.; Nguyen, T. D. Screening of plant extracts for

antioxidant properties. Botanica serbica., 2011, 35(1), 43-48.

Okoli, C.O.; Akah, P.A.; Nwafor, S.V.; Anisiobi, A.I; Ibegbunam, I.N.; Erojikwe, O.

Anti-inflammatory activity of hexane leaf extract of Aspilia africana C.D.

Adams. J. Ethnopharmacol., 2006, 109(2), 219-225.

Pedro, M.; Gutierrez, J.; Aubrey, N.; Antepuesto,; Bryle A.L.; Eugenio, Maria F.L.

Larvicidal activity of selected plant extracts against the dengue vector Aedes

aegypti mosquito. Inte.r Res. J. Biol. Sci., 2014, 3(4), 23-32.

Park, C.H.; Kim, S.H.; Choi, W.; Lee, Y.J.; Kim, J.S.; Kang, S.S.; Suh, Y.H. Novel

anticholinesterase and antiamnestic activites of dehydroevodiamine, a

constituent of Evodia rutaecarpa. Planta. Med., 1996, 62(5), 405-409.

145 REFERENCES

Pavela, R. Larvicidal effects of some Euro-Asiatic plants against Culex

quinquefasciatus Say larvae (Diptera: Culicidae). Parasitol. Res., 2009;

105(3), 887–92.

Peng, Z.; Yang, J.; Wang, H.; Simons, F.E. Production and characterization of

monoclonal antibodies to two new mosquito Aedes aegypti salivary proteins.

Insect. Biochem. Mol. Biol. 1999, 29, 909–14.

Perveen, A.; Qaiser, M. Pollen flora of Pakistan.Violaceae. Pak. J. Bot., 2009, 41, 1-5.

Perwaiz, S.; Sultana, S. Antitumorigenic effect of crude extract of Viola odorata on

DMBA- induced two stage skin carcinogenesis in the Swiss albino mice. Asia.

Pacific. J. Pharmacol. 1998, 13(1), 43–50.

Pratik, A.; Roshan, K.C.; Deepika, K.; Dinesh, T.; Rajan, S.; Tirtha, M.S.; Rajendra,

G. Phytochemical screening and anti-microbial properties of medicinal plants

of Dhunkharka community, Kavrepalan chowk, Nepal. Int. J. Pharma. Bio.

Arch. 2011, 2, 1663-1667.

Qadir, M. I.; Ali, M.; Saleem M.; Hanif, M. Hepatoprotective activity of aqueous

methanolic extract of Viola odorata against paracetamol-induced liver injury

in mice. Bang. J. Pharmacol., 2014, 9, 198-202.

Qaiser, M.; Omer, S. Flora of Pakistan. Islamabad. Agric. Res. Council., 1985, 6, 28.

Qamar, Q. Z.; Anwar, M.; Dar, N. I.; Ali, U. Ethno-botanical study of wild medicinal

plants of Neelum Valley, Azad Jammu and Kashmir, Pakistan. Pak. J. Wildl.,

2010, 1(1), 25-30.

Rahman, A.U.; Choudhary, M.I. Bioactive natural products as a potential source of new pharmacophores a theory

of memory. Pure Appl. Chem. 2001, 73, 555–560. Rahman, M. M.; Saha, S. M.; Hosen, Z.; Paul, S.; Kader, S.; Rahim, Z. B.

Ethnomedicinal plants used against jaundice in Bangladesh and its economical

prospects. Bull. Pharma. Res., 2012, 2(2), 91-105.

146 REFERENCES

Ramawat, K.; Dass, S.; Mathur, M. The chemical diversity of bioactive molecules and

therapeutic potential of medicinal plants. Herbal Drugs: Ethnomed. Modern.

Med., 2009, 7-32.

Rana, C. S.; Sharma, A.; Kumar, N.; Dangwal, L. R.; Tiwari, J. K.

Ethnopharmacology of some important medicinal plants of Nanda Devi

National Park (NDNP) Uttarakhand, India. Nat. Sci., 2010, 8, 9-14.

Rana, P. K.; Kumar, P.; Singhal, V. K.; Rana, J. C. Uses of local plant biodiversity

among the tribal communities of Pangi valley of district Chamba in cold desert

Himalaya, India. The Sci. World. J., 2014, 2014, 1-15.

Rang, H.P.; Dale, M.M.; Ritter, J.M.; Pharmacology Guanabara-Koogan. Rio de

Janeiro., 2001, 691(4), 174–188.

Rani, S.; Rana, J. C.; Jeelani, S. M.; Gupta, R. C.; Kumari, S. Ethnobotanical notes on

30 medicinal polypetalous plants of district Kangra of Himachal Pradesh. J.

Med. Plants Res., 2013, 7(20), 1362-1369.

Rao, C.V.; Kartik, R.; Ojha, S.K.; Amresh, M and Rao, G.M.M. Antiinflammatory

and antinociceptive activity of stem juice powder of Tinospora cordifolia

Miers. in experimental animals. Hamdard Med., 2005, 48, 102-106.

Rastogi, R. P. Compendium of Indian medicinal plants. Cent. Drug. Res. Inst.,

Lucknow, 3, 1980-1984.

Rates, S.M.K. Plants as source of drugs. Toxicon., 2001, 39(5), 603-613.

Raucy, J.L.; Laskar, J.M.; Lieber, C.S.; Black, M. Acetaminophen activation by

human liver cytochromes P4502El and P4501A2. Arch Biochem Biophys.,

1989, 271, 270-283.

147 REFERENCES

Rawal, P.; Adhikari, R. S.; Tiwari, A. Antifungal activity of Viola canescens against

Fusarium oxysporum f. sp. Lycopersici. Int. J. Curr. Microbiol. App. Sci.,

2015, 4(5), 1025-1032.

Reygaert, W. C. Antimicrobial resistance mechanisms of Staphylococcus aureus.

Microbial pathogens and strategies for combating them: science, technology

Reznick, A. A.; Voss, E. G. Michigan Flora Online: Viola. Available at

http://michiganflora.net/genus.aspx?id=viola (modified). University Michigan.

Ann. Arbor. MI., 2012.

Rhee, I.K.; Meent, M.V.D.; Ingkaninan, K.; Verpoorte, R. Screening for acetylcholinesterase inhibitors from

Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining. J.

Chromatogr. A., 2001, 915, 217–223. Rusted, J.M.; Newhouse, P.A.; Levin, E.D. Nicotinic treatment for degenerative

neuropsychiatric disorders such as Alzheimer’s disease and Parkinson’s

disease. Behav. Brain. Res., 2000, 113, 121– 9.

Saeed, M.; Naveed, M.; Khan, H. Assessment of Heavy Metal Content of Branded

Pakistani Herbal Products. Trop. J. Pharm. Res., 2011, 10, 499-506.

Safaihy, H.; Sailer, E.R. Anti-inflammatory actions of pentacyclic triterpenes.

Planta Medica., 1997, 63, 487–493.

Saito, H. Regulation of herbal medicines in Japan. Pharmacol. Res., 2000, 41, 515–

519.

Salve,T.; Rathod,V.; Tike, S. K.; Kadam, R.; Khade, R. A review article on banafsha

(Viola odarata linn.) an international peer reviewed. Ayurved. j., 2014, 2, 1-8.

Salvemini, D., Wang, Z.Q., Bourdon, D.M., Stern, M.K., Currie, M.G., Man- ning,

P.T. Evidence of peroxynitrite involvement in the carrageenan- induced rat

paw edema. Europ. J. Pharmacol., 1996, 303, 217–220.

148 REFERENCES

Samuelsson, G. Pharmacognosy in the 21st Century. A Textbook of Pharmacognosy,

5th Swedish Pharmaceutical Press, Stockholm. J. Pharmacy. Pharmacol.

Drug. Nat. Origin., 2004, 53(2), 135-148.

Santos, A.; Filho, V.; Niero, R.;Viana, A.M.; Moreno, F.N.; Campos M.M.; Yunes,

R.A.; Calixto, J.B. Analgesic effects of callusculture extracts from selected

species of Phyllantus in mice. J. Pharm. Pharmacol., 1994. 46(9), 755-759.

Saqib, Z.; Mahmood, A.; Naseem, R.; Malik, A.; Mahmood, J.; Hussian, S.; Ahmad,

T. “Indigenous knowledge of medicinal plants in Kotli Sattian, Rawalpindi

district, Pakistan,” J. Ethnopharmacol., 2014, 151(2), 820–828.

Saqib, Z.; Sultan, A. Ethnobotany of Palas Valley, Pakistan. Ethnobot. Leaflets.,

2005.

Sasidharan, S.; Vijayarathna, S.; Jothy, S. L.; Ping, K.Y.; Latha, L.Y.

Hepatoprotective potential of Elaeis guineensis leaf against paracetamol

induced damage in mice: a serum analysis. Int. Proc. Chem. Biol. Environ.

Eng., 2012, 39, 231-234.

Schneider, L.J. Treatment of Alzheimer’s disease with cholinesterase inhibitors. Clin.

Geriatr. Med., 2001, 17, 337–339.

Schopke, T.; Hasan Agha, M.I.; Kraft, R.; Otto, A.; Hiller, K. Hämolytisch aktive

komponenten aus Viola tricolor L. und Viola arvensis Murray. Sci. Pharm.

1993, 61, 145–153.

Sellappan, D. M. Antitussive activity of certain herbs. Pharmacy practice. MIMS

Pharmacy., 2015, 21.

Shengji, P. Mountain culture and forest resource management of Himalayas. In: D.

W. Tiwari (Ed). Himalayan Ecosystem. Intel. Book Distributors, conservation.

New Delhi: Indus publishing company., 1992.

149 REFERENCES

Shinwari, M. I.; Khan, M. A. “Folk use of medicinal herbs of Margalla Hills National

Park, Islamabad,”. J. Ethnopharmacol., 2000, 69(1), 45–56.

Shinwari, M. I., “Botanical diversity in Pakistan; past present and future,”. World

Envir. Day., 2010, 85–104.

Shinwari, Z.K. Medicinal plant research in Pakistan. J. Med. Plants. Res., 2010, 4,

161-176.

Siddharthan, S.; Yi-Zhong, C.; Harold, C.; Mei, S. Systematic evaluation of natural

phenolic antioxidants from 133 Indian medicinal plants. Food Chem. 2007,

102, 938–953.

Siddiqi, H.S; Malik H.; Rehman, N. U.; Gilani, A.H. Studies on the antihypertensive

and antidyslipidemic activities of Viola odorata leaves extract. Lipid Health

and Dis, 2012, 10(11), 6.

Singh, A. K.; Raina, V. K.; Naqvi, A. A.; Patra, N. K.; Kumar, B.; Ram, P.; Khanuja,

S. P. S. Essential oil composition and chemoarrays of menthol mint (Mentha

arvensis L. F. piperascens Malinvaud ex. Holmes) cultivar, Flavour Fragr. J.,

2005, 20, 302-305.

Souza, I.S.D.; Guimaraes, O. T.; Gotlieb, C. H.; Andrade, S.; Magalhaes, M. T.

Flavonoides from Dalbergia cearensis, Phytochem., 1975, 14(5), 1452-1453.

Stai, H.Y.; Ch, Y.F.; Wu, T.S. Anti inflammatory and analgesic activities of extract

from roots of Angelica pubescens. Planta. Medica., 1995, 61, 1-8.

Stuart M. The Encyclopedia of herbs and herbalism. Stuart. Spain: Macdonald & Co.

(Publishers) Ltd., 1989, 281.

Suyal, S., Sharma, C.M., Gairola, S., Ghildiyal, S. K., Rana, C. S., Butola, D. S.

“Phytodiversity (angiosperms and gymnosperms) in Chaurangikhal forest of

150 REFERENCES

Garhwal Himalaya, Uttarakhand, India.”.Ind. J. Sci. Technol., 2010, 3, 267–

275.

Tabba, H. D.; Chang, R.S.; Smith, K.M. Isolation, purification, and partial

characterization of prunellin, an anti-HIV component from aqueous extracts of

Prunella vulgaris. Antiviral. Res., 1989, 11(5), 263-273.

Talib, W.H. Anticancer and Antimicrobial Potential of Plant-Derived Natural

Products. Phytochemicals – Bioactivities and Impact on Health., 2011, 1, 141-

158.

Tam, J. P.; Lu, Y. A.; Yang, J. L.; Chiu, K. W. An unusual structural motif of

antimicrobial peptides containing end-to-end macro cycle and cystine-knot

disulfides. Proc. Natl. Acad. Sci. U.S.A., 1999, 96(16), 8913–8918.

Tang, J.; Wang, C.K.; Pan, X.; Yan, H.; Zeng, G.; Xu, W.; Daly N.L.; Craik D.J.;

Tan, N. Isolation and characterization of cytotoxic cyclotide from Viola

tricolor. Peptides., 2010, 31(8), 1434-40.

Teather, L.A.; Packard, M.G.; Bazan, N.G. Post-training cyclooxygenase- 2 (COX-2)

inhibition impairs memory consolidation. Learn Mem., 2002, 9(1), 41–47.

Temple, R.J.; Himmel, M.H.; Safety of newly ap-proved drugs: implications for

prescribing. JAMA. 2002, 287(17), 2273 - 2275.

Tjolsen, A.; Berge, D.G.; Hunskaar, S.; Rosland, J.H.; Hole, K. The formalin test: an

evaluation of the method. Pain., 1992, 51(1), 5-17.

Toiu, A.; Vlase, L.; Oniga, I.; Tamas, M. Quantitative analysis of some phenolic

compounds from Viola species tinctures. Farmacia J., 2008, 56, 440-445.

Valko, M.; Leibfritz, D.; Moncol, J.; Mark, T.D.; Cronin, M.; Mazur, M.; Telser, J.

Free radicals and antioxidants in normal physiological functions and human

disease. Inter. J. Biochem. Cell. Biol., 2007, 39(1), 44–84.

151 REFERENCES

Velioglu, Y.S.; Mazza, G.; Gao. L.; Oomah, B, D.; Antioxidant activity and total

phenolics in selected fruits, vegetables and grain product. J. Agr. Food. Chem.,

1998; 46(10), 4113-4117.

Verma, G.; Dua, V. K.; Agarwal, D. D.; Atul, P. K. “Anti-malarial activity

of Holarrhena anti-dysenterica and Viola canescens, plants traditionally used

against malaria in the Garhwal region of north-west Himalaya,” Malaria J.,

2011, 10, 20. http://www.malariajournal.com/content/10/1/20.

Vishala, A.; Parveena, K.; Poojab, S.; Kannappanc, N.; Kumar, S. Diuretic, Laxative

and Toxicity Studies of Viola odorata aerial parts. Pharmacol. Online., 2009,

1, 739-748.

Vukics V.; Toth, B.H.; Ringer, T.; Ludanyi, K.; Kery, A.; Bonn, G.K.; Guttman, A. .

Quantitative and qualitative investigation of the main flavonoids in heartsease

(Viola tricolor L.). J. Chromatogr. Sci., 2008, 46(2), 97-101.

Vukics, V.; Kery, A.; Bonn, G.K.; Guttman, A. Major flavonoid components of

heartsease (Viola tricolor L.) and their antioxidant activities. Anal. Bioanal.

Chem. 2008, 390(7), 1971-1925.

Vukics, V.; Kery, A.; Guttman, A. Analysis of polar antioxidants in heartsease (Viola

tricolor L.) and garden pansy (Viola wittrockiana Gams.). J. Chromatogr. Sci.,

2008, 46(9), 823-827.

Wang. C.K.; Colgrave, M.L.; Gustafson, K.R; Ireland, D.C. Goransson, U.; Craik,

D.J. Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis, J.

Nat. Prod., 2008, 71, 47-52.

Wanzala, W.; Hassanali, A.; Mathias, E.; Baumann, M.P.O.; Kyule, N.M.; Zessin,

K.H. “Ethnoveterinary medicine: a critical review of its evolution, perception,

152 REFERENCES

understanding and the way forward.” Livest. Res. Rur. Devel., 2005, 17, 11, 1-

29.

WHO Guidelines for Assessing Quality of Herbal Medicines with Reference to

Contaminants and Residues. Geneva: WHO.2002.

Winter, C.A.; Poster, C.C. Effect of alteration in side chain up on anti inflammatory

and liver glycogen activities in hydrocartisone ester. J. Am. Pharma. Assoc.

Am. Pharm. Assoc, 1957, 46, 515-519.

Wittwer, F.M.; Bohmwald, L.H. Manuel de patologia clinica veterinaria. Valdivia

Chile. 1986, 53-93.

Xiao-Jia, H.U.; Hui-Zi J.I.N.; Wen-Zheng, X.U.; Ming, C.; Xiao-Hua, L.; Wei, Z.;

Juan, S.; Chuan, Z.; Wei-Dong, Z. Anti-inflammatory and analgesic activities

of edgeworthia chrysantha and its effective chemical constituents. Biol.

Pharm. Bull., 2008, 31(9), 1761-1765.

Yerra. R.; Senthil, G.P.; Gupta, M.; Mazumder, U.k. Studies on in vitro antioxidant

activities of methanol extract of Mucuna pruriens (Fabaceae) seeds. Eur. Bull.

Drug. Res. 2005, 13, 31-39.

Young, J.M.; Spires, D.A.; Bedord, C.J.; Wagner, B.; Ballaron, S.J.; Deyoung, L.M.

The mouse ear inflammatory response to topical arachidonic acid. J. Investig.

Dermatol., 1984, 82, 367-371.

Zaninir J. C..; Medeiros Y. S..; Cruz A. B.; Yunes R. R. A.; Calixto, J. B. Action of

compounds from Mandevilla velutina on croton oil-induced ear oedema in

mice. A comparative study with steroidal and non-steroidal anti-inflammatory

drugs. Phytother. Res. 1992, 6, 1-5.

Zargari A. Medicinal Plants. Tehran University Publication, Tehran., 1997, 4(6)., 103.

153 REFERENCES

Zeashana, H.; Amresha, G.; Raoa, C.V.; Singhb S. Antinociceptive activity of

Amaranthus spinosus in experimental animals. J. Ethnopharmacol., 2009,

122(3), 492–496.

Zhon, Y.C.; Zheng, R.L. Phenolic compounds and an analog as superoxide anion

scavengers and antioxidants. Biochem. Pharmacol. 1991, 42, 1177-1179.

Zhou, J.S.; Gong, Q.; Xing, F.W. Viola nanlingensis (Violaceae), a new specie from

Guangdong, Southern China. Ann. Bot. Fennici., 2008, 45, 233-236.

Zhou, M.; Wang, H.; Suolangjiba, K.J.; Yu, B. Antinociceptive and anti-inflammatory

activities of Aquilaria sinensis (Lour.) Gilg. Leaves extract. J.

Ethnopharmacol., 2008. 117, 345-350.

154