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ISOLATION OF BIOACTIVE SECONDARY
METABOLITES AND PHARMACOLOGICAL STUDIES
OF VIOLA SERPENS WALL
By
RUKHSANA Ph.D
DEPARTMENT OF PHARMACY
UNIVERSITY OF PESHAWAR
2017
ISOLATION OF BIOACTIVE SECONDARY
METABOLITES AND PHARMACOLOGICAL STUDIES
OF VIOLA SERPENS WALL
Thesis submitted to the Department of Pharmacy, University of Peshawar,
Peshawar, Pakistan in partial fulfillment for the Degree of
DOCTOR OF PHILOSOPHY
IN
PHARMACEUTICAL SCIENCES
FEBRUARY, 2017
DEPARTMENT OF PHARMACY
UNIVERSITY OF PESHAWAR
APPROVAL SHEET
A Thesis presented by Rukhsana entitled “Isolation of Bioactive Secondary
Metabolites and Pharmacological Studies of Viola Serpens Wall” to the
Department of Pharmacy, University of Peshawar in partial fulfillment for the award
of the Degree of Ph.D in Pharmaceutical Sciences.
We, the undersigned have examined this thesis and do hereby approve it for the
award of Ph.D Degree.
External Examiner: _________________________________
Supervisor: ______________________________
PROF. DR. MUHAMMAD SAEED
Chairman,
Department of Pharmacy,
University of Peshawar.
Co-supervisor: ______________________________
DR. MANZOOR AHMAD
Associate Professor,
Department of Chemistry,
University of Malakand.
II DDeeddiiccaatteedd
mmyy tthhiiss hhuummbbllee eeffffoorrtt ttoo mmyy bbeelloovveedd
PPaarreennttss && FFaammiillyy
i
ACKNOWLEDGEMENT
In the name of Almighty Allah, the most merciful and beneficent, Who gave me the
courage and ability for the better understanding and completion of my PhD project. I
bow my head before Allah for His greatness, Who provided me strength and courage
to accomplish a useful and beneficial work for the benefit of mankind.
With great honor and extreme happy feelings I pay my homage and debt to my
research supervisor, Prof. Dr. Muhammad Saeed, Chairman, Department of
Pharmacy, University of Peshawar. His broad vision, advice, encouragement and co-
operation helped and guided me for the completion of my Ph.D programme and
dissertation.
I am also extremely indebted to my co-supervisor Dr. Manzoor Ahmad, Associate
Professor, Department of Chemistry, University of Malakand. His sincere help,
guidance, provision of required resources for the accomplishment of the major part of
my research work.
I would like to thank Prof. Dr. Zafar Iqbal, Meritorious professor, Tamgh-e-
Imtiaz, Department of Pharmacy, University of Peshawar and Prof. Dr. Fazal
Subhan for their support and encouragement throughout my work. I am also grateful
to all the teaching faculty of the department for their support and cooperation.
I am very thankful to Professor, Prof. Dr. Haroon Khan, Abdul Wali Khan
University Mardan, for his sincerity, guidance, co-operation and encouragement at
every stage of my PhD work. I also acknowledge the guidance and support of
Mr. Ikran Illahi, Assistant Professor and Chairman, Department of Zoology
University of Malakand.
ii
I am very grateful to Mr. Atta-ur-Rehman, Institute for Natural Product Discovery,
Universiti Teknologi, MARA Puncak Alam Selangor D.E., Malaysia for the
spectroscopic studies of the isolated compounds. I am also very thankful to the staff
of PCSIR Laboratories especially Ms. Farah Gul and Mr. Yaqoob for their co-
operation and guidance in conducting anti-inflammatory activity of V. serpens.
Thanks to Dr. Nuzhat Sultana from Khyber Medical College Peshawar and Mr.
Mohammad Shahid, PhD scholar, for their help regarding the interpretation of histo-
pathological slides. I am also very thankful to Dr. Umer Sadique Khattak,
Chairman, Department of Animal Health Sciences, Agriculture University Peshawar
and Mr. Sajjad Ali Shah, research assistant and PhD scholar Department of Animal
Health Sciences, Agriculture University Peshawar for their facilitations and guidance
in preparing the histopathological slides and taking photographs by using camera
fitted microscope in the hepatoprotective and nephroprotective activities.
I wish to pay my sincere appreciation to my lab fellows Ms. Attiqa Naz, Miss
Samreen Pervez, Miss Noor-ul-Aain, Mr. Naveed and Mr. Asif Jan for their help,
support and collective team work.
At the end I pay my regards and duly acknowledge the co-operation, guidance and
support of my parents, husband, sisters-in-law, brothers and sisters who encouraged
and enabled me to fulfill this task
.
Rukhsana
iii
TABLE OF CONTENTS
S.No. Topic Page No.
Acknowledgement i
List of Figures vii
List of Tables ix
Abbreviations xi
Abstract xiii
Chapter – 1
1. INTRODUCTION 1
1.1 General Introduction 1
1.2 Traditionally Used Medicinal Plants in Pakistan 3
1.3 Importance of Herbal Medicines in Different Traditions 4
1.3.1 Chinese Traditional Medicine 4
1.3.2 Japanese Traditional Medicine 5
1.3.3 Indian Traditional Medicine 5
1.4 Violaceae Family 5
1.5 Literature Survey of Genus Viola 6
1.6 Plant Introduction 12
1.6.1 Viola serpens Wall 12
1.6.2 Local Names 12
1.6.3 Morphology 12
1.6.4 Classification (Taxonomical position of V.serpens) 13
1.6.5 Geographical Distribution of V.serpens 13
1.6.5.1 World Wide Distribution 13
1.6.5.2 Distribution In Pakistan 14
1.7 Plant Distribution 15
1.8 Ethno medicinal Uses 16
1.9 Phytochemical Investigations 16
1.9.1 Nutritive Values 16
1.9.2 Essential and Fixed Oils 16
1.10 Isolated Compounds from Genus Viola 17
1.11 Pharmacological Studies 17
1.11.1 In vivo Biological Activities 17
1.11.1.1 Antibacterial Activity 17
1.11.1.2 Anti-fungal Activity 19
1.11.1.3 Antiprotozoal Activity 20
iv
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
v
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
vi
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
vii
LIST OF TABLES
S.No. Title Page No.
1.1 Viola species list, native to Pakistan and their worldwide distribution,
parts used chemical constituents and medicinal uses
6
1.2 List of other viola spp. of the family Violaceae, their geographical
distributions, Medicinal uses and part/s used mostly
9
1.3 List of some various other species of viola and their geographic
distributions
10
2.1 List of Drugs / Chemicals used with their Sources 36
3.1 Antimicrobial Activity of the Crude Extract along with the
Subsequent Fractions of Viola serpens
64
3.2 Antimicrobial activity of the Isolated Compounds from V. serpens 64
3.3 DPPH Scavenging Activity of Crude extract/Fractions of V.serpens
and Zones of Inhibition are Given in mm
67
3.4 Anti-oxidant Activities of Pure Isolated Compounds 1–6 from
V.serpens whole Plant
67
3.5 Larvicidal effect of the crude extract along with the subsequent
fractions of V.serpens against Aedes aegypti and Culex
quinquefasciatus species of mosquitoes
69
3.6 The Enzyme Inhibition effect of the Crude Extract and the
subsequent Fractions of V.serpens against the Enzyme Acetylcholine
Esterase
71
3.7 Acute Toxicity of the Crude Extract along with the Fractions of V.
serpens
72
3.8 Effects of the Crude Extracts/Fractions of V.serpens Wall on the
Liver Related Parameters (AST, ALT and ALP) in the Rabbits
Models
75
3.9 Effect of Crude Extract/ fractions of V.serpens Wall. on the Kidney’s
functions and Clearance in the Rabbits Models
81
3.10 The Effect of Crude Extract/Fractions of V.serpens in Acetic Acid
Induced Writhing Tests in Mice (i.p)
85
3.11 Effect of the crude/ fractions of V. serpens in formalin induced pains
for analgesia test in mice at doses of 100, 200 and 300 mg/kg, i.p
91
3.12 Anti-inflammatory effect against carrageenan and Histamine
induced paw edema in mice for V.serpens crude extract
105
viii
S.No. Title Page No.
3.13 Anti-inflammatory effect against carrageenan and Histamine
induced paw edema in mice for V.serpens n- hexane fraction
106
3.14 Anti-inflammatory effect of chloroform fraction of V.serpens
in carrageenan and Histamine induced paw edema in mice
107
3.15 Anti-inflammatory effect of Ethyl acetate fraction of V.serpens
in carrageenan and Histamine induced paw edema in mice
108
3.16 Anti-inflammatory effect against carrageenan and Histamine
induced paw edema in mice for V.serpens aqueous fraction
109
3.17 Effect of the crude extract along with the subsequent fractions of
V.serpens on xylene induced ear edema in mice
110
3.18 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-A (1)
in CDCl3
114
3.19 1H- (400 MHz.) and 13C-NMR (125 MHz) Data of Commulin-B (2)
in CDCl3
117
3.20 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-C (3)
in CDCl3
119
ix
LIST OF FIGURES
S. No. Title Page No.
1.1 Percentage of Medicinal Plants In Undeveloped and Developed
Countries
3
1.2 Illustration of Viola serpens specie of the Genus Viola 31
1.3 Flower of V. serpens species of the Genus Viola 32
1.4 Seeds of V. serpens species of the Genus Viola 32
2.1 Scheme of plant extraction and fractionation 39
2.2 Scheme representing the isolation of pure compounds using Ethyl
acetate fraction
41
3.1 % inhibition of the tested bacteria against the Crude extract/
fractions of V. serpens. Where CHCl3 represents Chloroform,
ETA represents Ethyl acetate and H2O represents the Aqueous
fraction
65
3.2 Liver photomicrographs of the rabbits treated with paracetamol,
crude extract and n-Hexane fractions of V. serpens at doses of 150
and 300 mg/kg (H&E, 100X and 400X)
77
3.2.1 Normal saline treated liver showing normal architecture of central
vein (CV), sinusoidal spaces (small arrows), hepatocytes (large
arrows) with a centrally placed nucleus and foamy cytoplasm.
(100X H&E).
77
3.2.2 Liver showing accumulation of lymphocytes (small arrows)
around the central vein (CV), fatty changes (small arrow head)
and focal area of necrosis (asterisk) with paracetamol (100X
H&E).
77
3.2.3 Liver showing regeneration, containing normal liver plates (large
arrows) along central vein (CV) with n-hexane 150 mg/kg b.w.
(H&E).
77
3.2.4 Liver showing normal appearance of central vein (CV) and plates
of hepatocytes (large arrows) with n-hexane 300 mg/kg b.w.
(100X H&E).
77
3.2.5 Liver showing hexagonal hepatocytes (large arrows) with
prominent cell borders (small arrows), nuclei (arrow heads) with
nuclear clearing and prominent nucleoli with crude extract at a
dose of 150 mg/kg b.w. (400X H&E).
77
3.2.6 Liver showing regeneration of hepatocytes (large arrows) with
congestion of sinusoids (asterisks) containing red blood cells
(small arrows) with crude extract at a dose of 300 mg/kg b.w.
(400X H&E).
77
x
S. No. Title Page No.
3.3 Photomicrogrphs of the Kidneys of Rabbits Treated with
Paracetamol and plant Extract/ Fractions at Different Doses (H&E)
83
3.3.1 Photomicrograph (100X H&E) of a section of kidney from a rabbit
treated with normal saline showing normal histological appearance
of renal cortex. The cortex contains renal corpuscles (large arrows)
embedded among proximal (arrow heads) and distal (asterisk)
convoluted tubules.
83
3.3.2 Photomicrograph (100X H&E) of a section of kidney from a rabbit
rat treated with PCM showing necrosis of cuboidal epithelial cells
(large arrows) of proximal convoluted tubules with exfoliation of
their brush border. The lumen (asterisk) of tubules contains
numerous cellular casts (small arrows).
83
3.3.3 Photomicrograph (100X H&E) of a kidney section from a rabbit
treated with n-hexane soluble fraction 150 mg/kg showing normal
histo-architecture of distal convoluted tubules with wider lumen
(asterisk) and lined by cuboidal epithelial cells (arrow heads).
Numerous loop of Henle tubules are also visible (large arrows).
83
3.3.4 Photomicrograph (100X H&E) of a section of kidney from a rabbit
treated with n-hexane soluble fraction 300 mg/kg showing normal
renal corpuscles (large arrows) with mild dilatation of proximal
(arrow heads) and distal (asterisk) convoluted tubules
83
3.3.5 Photomicrograph ((100X H&E)) of a section of kidney from a rabbit
treated with chloroform soluble fraction 150 mg/kg showing normal
renal corpuscles (large arrows), proximal (arrow heads) and distal
(asterisk) convoluted tubules
83
3.3.6 Photomicrograph (100X H&E) of a section of kidney from a rabbit
treated with ethyl acetate soluble fraction 150 mg/kg showing
normal renal corpuscles (large arrows) with mild dilatation of
proximal (arrow heads) and distal (asterisk) convoluted tubules.
83
3.3.7 Photomicrograph ((100X H&E)) of a section of kidney from a rabbit
treated with chloroform soluble fraction 300 mg/kg showing normal
renal corpuscles (large arrows) and proximal convoluted tubules
(arrow heads). The distal convoluted tubules (asterisk) exhibited
mild tubular necrosis of the cuboidal epithelial cells
84
3.3.8 Photomicrograph (100X H&E) of a section of kidney from a rabbit
rat treated with ethyl acetate soluble fraction 300 mg/kg showing
normal proximal convoluted tubules (large arrows) with numerous
loop of Henle tubules (asterisk). The interlobular blood vessels
(arrow heads) among the renal tubules exhibited mild congestion
with red blood cells.
84
xi
S. No. Title Page No.
3.3.9 Photomicrograph (100X H&E) of a section of kidney from a rat
treated with aqueous soluble fraction 300 mg/kg showing normal
renal corpuscles (large arrows). The renal tubules exhibited
dilatation (arrow heads) with exfoliation of the brush border lining
the proximal convoluted tubules into their lumen.
84
3.3.10 Photomicrograph (100X H&E) of a section of kidney from a rat
treated with aqueous soluble fraction showing mild congestion of the
renal corpuscles (large arrows) with severe dilatation of the renal
tubules (asterisk). Numerous cellular casts (arrow head) is also
visible in the lumen of renal tubules.
84
3.4 Anti-nociceptive effect of crude extract of V. serpens in Acetic acid
induced writhing test.
87
3.5 Anti-nociceptive effect of n-hexane Fraction of V. serpens in Acetic
Acid induced writhing test.
87
3.6 Anti-Nociceptive Effect of Chloroform Fraction of V. serpens in
Acetic Acid induced writhing test.
88
3.7 Antinociceptive Effect of Ethyl Acetate Fraction of V. serpens in
Acetic Acid induced writhing test.
88
3.8 Anti-Nociceptive Effect of Aqueous Fraction of V. serpens in Acetic
Acid induced writhing test.
89
3.9 Antinociceptive Effects of Formalin Induced Pain in Mice of the
Crude Extract of V.serpens
94
3.10 Antinociceptive effects of Formalin Induced Pain in Mice of the
Hexane Fraction of V.serpens
94
3.11 Antinociceptive effects of Formalin Induced Pain in Mice of the
Chloroform Fraction of V.serpens
95
3.12 Antinociceptive effects of Formalin Induced Pain in Mice of the
Ethyl Acetate Fraction of V.serpens
95
3.13 Antinociceptive effects of Formalin Induced Pain in Mice of the
Aqueous Fraction of V.serpens
96
3.14 Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on
Carrageenan Induced paw Edema
102
3.15 Anti-Inflammatory Effect (%) of the n-Hexane Fraction V. serpens
on Carrageenan Induced paw Edema
102
3.16 Anti-Inflammatory Effect (%) of the chloroform Fraction V. serpens
on Carrageenan Induced paw Edema
103
3.17 Anti-Inflammatory Effect (%) of the Ethyl Acetate Fraction V.
serpens on Carrageenan Induced paw Edema
103
xii
S. No. Title Page No.
3.18 Anti-Inflammatory Effect (%) of the Aqueous Fraction V. serpens on
Carrageenan Induced paw Edema
104
3.19 Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on
Histamine Induced paw Edema
104
3.20 Anti-Inflammatory Effect (%) of the n-Hexane Fraction V. serpens
on Histamine Induced paw Edema
105
3.21 Anti-Inflammatory Effect (%) of the chloroform Fraction V. serpens
on Histamine Induced paw Edema
105
3.22 Anti-Inflammatory Effect (%) of the Ethyl Acetate Fraction V.
serpens on Histamine Induced paw Edema
106
3.23 Anti-Inflammatory Effect (%) of the Aqueous Fraction V. serpens on
Histamine Induced paw Edema
106
3.24 Percent inhibition of Xylene induced ear edema in mice at different
doses of the crude extract and fractions of V. serpens
111
3.25 Commulin-A (1) 112
3.26 Key HMBC interaction in Compound 1 114
3.27 Commulin-B (2) 115
3.28 Key HMBC interaction in Commulin-B (2) 116
3.29 Commulin-C (3) 118
3.30 Key HMBC interaction in Commulin-C (3) 119
3.31 Structure of compound Tectochrysine (4) 120
3.32 Structure of compound Sideroxyline (5) 121
3.33 Structure of compound Cearoin (6) 122
xiii
LIST OF ABBREVIATIONS
ACh: Acetylcholine
ACh-E: Acetylcholine esterase
AIDS: Acquired Immune Deficiency Syndrome
ALP: Alkaline phosphatase
ALT: alanine aminotransferase
AST: aspartate aminotransferase
A.parasiticum: Amoebidium parasiticum
C. albicans: Candida albicans
CC: Column Chromatography
CHCl3: Chloroform
C.littoralis: Congregibacter littoralis
Co-A: Coenzyme A
COSY: Correlation Spectroscopy
COX: Cyclo-oxygenase
DPPH 2, 2-diphenyl-1-picryl hydrazyl
BHT Dibutylhydroxy toluene
DMSO: Dimethyl Sulfoxide
DNA: Deoxyribonucleic Acid
EDTA: Ethylene Diamine Tetra Acetic acid
EI-MS: Electron Ionization Mass Spectrometry
ELISA: Enzyme Linked Immunosorbent Assay
E. coli: Escherichia coli
ETA: Ethyl acetate
FAB-MS: Fast Atom Bombardment Mass Spectrum
xiv
GC: Gas Chromatography
GCMS: Gas Chromatography Mass Spectrometry
HMBC: Heteronuclear Multiple Bond Coherence
HSQC Heteronuclear single quantum coherence spectroscopy
IC50: Concentration causing 50% Inhibition
IR: Infra red
KP: Khyber Pakhtunkhwa
K. pneumonia: Klebsiella pneumonia
LD50: Median Lethal Dose
NIH: National Institute of Health
NBT nitroblue tetrazolium chloride
NMR: Nuclear Magnetic Resonance
PCM: Paracetamol
P. postuma: Hemidesmus indicus
P.aeruginosa: Pseudomonas aeruginosa
RSA Radical scavenging assay
SGOT: Serum Glutamic Oxaloacetic Transaminase
SGPT: Serum Glutamate Pyruvate Transaminase
S.aureous: Staphylococcus aureus
S. typhi: Salmonella typhi
TLC: Thin Layer Chromatography
NOESY: Nuclear Overhauser Effect Spectroscopy
UV: Ultra Violet
VLC: Vacuum Liquid Chromatography
WHO: World Health Organization
xv
µg: Micro gram
µL: Micro Litre
xvi
ABSTRACT
Viola serpens which belongs to the family Violaceae has been reported to have many
folkloric uses including use as antipyretic, laxative, emollient, expectorant, purgative,
diuretic, demulcent, diaphoretic, anti-asthmatic and anti-cancer. The current
investigation was carried out to evaluate the crude methanolic extract and various
fractions of Viola serpens for its antioxidant, antimicrobial, enzyme inhibiting
potential, larvicidal activities using using in vitro assays and for antinociceptive, anti-
inflammatory activities, hepatoprotective and nephroprotective effects using in-vivo
studies. Furthermore, bioactive secondary metabolites were also isolated and
characterized. The results showed that the crude methanolic extract and various
fractions including chloroform, ethyl acetate fraction, n-hexane fraction and aqueous
fractions possessed significant antimicrobial activities against S. typhi and E. coli, B.
subtilis, S. flexeneri and P. aerogenes.
The compounds (1-6) isolated from the plant were also tested for activity against
different spp. of Gram positive and Gram negative bacteria except the compound
commulin-C. Against S. typhi, sideroxyline showed maximum zone of inhibition (18
mm) followed by the other tested compounds, each with 17 mm zone of inhibition.
Against the P. aerogenes, tectochrysine and cearoin proved more effective followed
by sideroxylin and commulin-B. S. flexeneri proved more susceptible to the
compounds tectochrysine and cearoin (17 mm) followed by commulin-A, commulin-
B but sideroxylin was inactive against it. S. aureus was susceptible to all four
mentioned isolated compounds except commulin-B and cearoin. Commulin-B and
tectochrysine were effective against E. coli.
xvii
Viola serpens crude extract, fractions and pure compounds also showed significant
free radical scavenging activity in DPPH free radical scavenging assays.
Concentration-dependent scavenging effect is showed by the crude methanolic
extract, n-hexane and chloroform soluble fractions against DPPH with maximum
activity of 67.99%, 75.98 % and 79.00 %. In pure form the isolated compounds
commulin-A, commulin-B and commulin-C showed effective scavenging activity
with the percent values 78.05 %, 89.45% and 78.05% with IC50 value 201 ppm, 98.15
ppm and 168 ppm respectively. Larvicidal effect of the plant was tested against the A.
aegypti and C. quinquefasciatus species. The ethyl acetate soluble fraction caused
maximum percent inhibition followed by the chloroform and the crude methanolic
extract at 600 ppm with values of 89.91 %, 85.21 % and 59.67 % respectively.
Acetyl cholinesterase assay was conducted by using three different concentrations of
250, 500 and 1000 ppm. The chloroform soluble fraction caused maximum percent
inhibition followed by the ethyl acetate soluble fraction, crude extract and the aqueous
soluble fraction with 89, 70.5, 68.55 and 50.75 % at 1000 ppm and IC50 values of 149,
156, 245 and 989 ppm respectively.
The crude extract along with the subsequent fractions were found safe in the in vivo
acute toxicity screening. Viola serpens as an effective hepatoprotective and
nephroprotective plant, was proved by evaluating various blood and urine parameters
(AST, ALP, ALT, urine clearance, urea creatinine and serum creatinine) against the
Paracetamol (PCM) induced toxicity and as evident by histopathological studies.
Being a safe drug, V. serpnse proved to be significant antinociceptive and anti-
inflammatory agents by using two different protocols for analgesia (Acetic acid
induced writhing test and formalin induced nociception test) and three for anti-
xviii
inflammation (carrageenan and histamine induced paw edema and xylene induced ear
edema). The plant extract and fractions were used at three different doses (100, 200
and 300 mg/kg). The plant proved to be an effective antinociceptive and anti-
inflammatory drug following the peripheral pathway for these activities mostly in a
dose dependent manner.
The plant extract and the different fractions with the decreasing polarity produced
more attenuated antinociceptive effect in a dose dependent manner. The effect is more
significant in the crude extract followed by the n-hexane, ethyl acetate, chloroform
and aqueous soluble fractions. Peripheral pathway is followed by either due to
inhibiting/reducing the release of cyclooxygenase or lipoxygenase enzymes or may
involve the release of certain mediators.
The anti-inflammatory effect of the plant was measured by using the protocols of
carrageenan and histamine induced paw edemas and xylene induced ear edema. In
histamine induced paw edema the crude extract at doses of 200 and 300 mg/kg
showed more pronounced anti-inflammatory effects that reached the maximum in the
3rdh. The n-hexane soluble fraction at a dose of 200 mg/kg in the 3rd h of histamine
induction showed maximum anti-inflammatory effect. The chloroform and aqueous
soluble fractions showed significant effects at the doses of 200 and 300 mg/kg on the
3rd h of histamine induced edema. Different percent inhibition values were obtained in
case of each fraction. In xylene induced ear edema the crude extract and n-hexane
fraction showed a dose dependent significance (Max. values at 300 mg/kg). This was
followed by the chloroform, ethyl acetate and aqueous fraction at doses of 200 and
300 mg/kg with the maximum inhibition values of 51, 49 and 48.5 % respectively.
xix
The chloroform and ethyl acetate soluble fractions were analyzed by column
chromatography which resulted in the isolation of six pure compounds. Among the
six compounds, three were new (not reported before) and the other three were already
reported from the other sources but obtained for the first time from this plant (V.
serpens) source. Commulin-A, Commulin-B and Commulin-C were the new
compounds whereas, tectochrysine, Sideroxyline and Cearoin were the already
reported compounds. 1H-NMR, 13C-NMR, COSY, NOESY, HMBC, IR, UV, E1-MS
and HRE1-MS were the different techniques used for elucidating the structures of the
new compounds.
In conclusion, V. serpens showed significant antimicrobial, antioxidant activities,
antinociceptive, anti-inflammatory, hepatoprotective and nephroprotective activities.
The marked pharmacological and phytochemical studies suggested further detailed
studies to confirm their folk uses and isolation of compounds that can act as potent
drug in future.
xx
Structures of the new compounds:
O
OOH
H3C
HO
OCH3
Commulin-A (1)
A
B
C
12
34
56
78
9
10
1'
2'3'
4'
5'6' O
OOH
H3C
HO
OCH3
Commulin-B (2)
HO
A
B
C
12
34
56
7
8
9
10
1'
2'3'
4'
5'6'
O
OOH
H3C
HO
OCH3
H3CO
Commulin-C (3)
A
B
C
12
34
56
78
9
10
1'
2'3'
4'
5'6'
Figure-1
O
OOH
H3C
HO
OCH3
A
B
C
12
34
56
78
9
10
1'
2'3'
4'
5'6'
H
HO
H
Figure-2: Key HMBC interactions in compound 2.
H
O
OOH
H3C
HO
OCH3
A
B
C
12
34
56
78
9
10
1'
2'3'
4'
5'6'
H
H
H
O
OOH
H3C
HO
OCH3
A
B
C
12
34
56
78
9
10
1'
2'3'
4'
5'6'
H
H3CO
Figure-2: Key HMBC interactions in compound 1.
Figure-2: Key HMBC interactions in compound 3.
xxi
Structures of the compounds isolated from V. serpens for the first time but already
reported from the other sources:
O
OOH
H3CO
9 2
34
56
78
10
Tectochrysine (4)
1'
2' 3'4'
5'6'
Figure 3.27: Structure of compound Tectochrysine (4).
O
OOH
H3C
H3CO
CH3
OH1'
2
345
6
78
9
10
2' 3'
4'
5'6'
Sideroxyline (5)
Figure 3.28: Structure of compound Sideroxyline (5).
O
OH
OCH3
OH
12
3
456
1'2'3'
4'5' 6'
AB
Cearoin (6)
Figure 3.29: Structure of compound Cearoin (6).
Chapter – 1 INTRODUCTION
1
CHAPTER – 1
1-INTRODUCTION
1.1 GENERAL INRODUCTION
The medicinal plants are enriched with ingredients that can be used in the synthesis of
drugs. Medicinal plants have been used for the cure of different ailments by the
diverse communities of the world for over thousands of years (Samuelsson, 2004). It
would be better to estimate the age of medicinal plants, being used for medication by
correlating it with the age of human civilization (Ramawat et al., 2009). The role of
medicinal plants can not be neglected in development of human cultures around the
world. World population use medicinal plants (approximatly 80-90 %) in raw and
unrefined extracts forms (Wanzala et al., 2005; Duke., 1985). The increased yearly
demand of herbal medicines globally, and particularly in the developing countries
clarifies its importance (Mahady, 2001). The consumtion of medicinal plant is going
on increasing with the passage of time (Wagner, 2009). The wide range of medicinal
plants used for the basic health care throughout the world is due to the uneconomic
and inaccessibility to the modern medical health facilities. Nature has kept treasures
of biologically active compounds in medicinal plants which facilitate the health
conditions of the huminity. The knowledge about these ethnomedicines is transferring
from one generation to another (Clark Hufford., 1993).
In most developing countries, people of the rural areas for their basic health care
mostly depend on medicine obtained from plants. This medication is comparatively
safer and inexpensive than the pharmaceutical products (Iwu et al., 1999; Idu et al.,
2007; Mann et al., 2008; Ammara et al., 2009). Isolation of active compounds from
plants is used for specific actions and characterization. In the 19th century morphine
was isolated from opium which was used particularly for the CNS related actions
Chapter – 1 INTRODUCTION
2
(Kinghorn, 2001; Samuelsson, 2004). Plants of the Himalayan region are of great
importance in herbal pharmaceutical industries. These plants are affected by various
significant climatic factors such as drought, mutagenic UV-radiation, harsh winds etc.
These factors have great influence on the active constituents of the plants and their
metabolites.
According to the 2009 botanical survey total number of plant species estimated are
250,000 to 350,000 out of which 35,000 species are used as medicinal plants for the
management of numerous ailments (Fabricant and Farnsworth, 2001). According to
the current survey, 15% of all the medicinal plants were used for phytochemical
investigations whereas, only 6% were screened for their biological activities
(Farnsworth et al., 1985; Farnsworth, 1966).
World Health Organization (WHO) reported that the use of medicinal plants is
increasing day by day. In underdeveloped countries, plants are used approximately
80% (Ernst., 2000). In developing countries the percentage in different countries is
different for example: Ethiopia (90%), Benin (80%), India (70%), Uganda (60%) and
Tanzania (60%). However, in the developed countries it is approximately 70% in
Canada, France 49%, Australia 48%, Japan 60-70%, USA 40%, and Belgium 31%.
According to a report, the budget of the world traditional medicines is about US$
60,000 million and US$ 5 trillion is supposed to be in the year 2050 (WHO, 2002). In
developed countries the use of traditional medicines has sharply expanded in the 20th
century (ESCOP, 1999; Blumenthal et al., 1998). The scientific validity of medicinal
plants has increased their importance and uses. WHO has emphasized greatly on the
scientific study of the native herbal plants remedies especially in the developing
countries (Rates, 2001). Various active ingredients have been isolated from the plants
Chapter – 1 INTRODUCTION
3
species which are particularly used for treatment of a particular disease (Qamar et al.,
2010).
Figure 1.1: Percentages of Medicinal Plants in Undeveloped & Developed Countries
1.2 TRADITIONALLY USED MEDICINAL PLANTS IN PAKISTAN
Almighty Allah has gifted our country with the treasure of medicinal plants. Both
cultivated and wild plants are the carpets of Pakistan’s land which posses great
potentialities. On the bases of photogeography, Pakistan has been divided into four
distant regions (Inrano-Turanian, Himalayan, Sindh and Indo-Pak). Pakistan has
diverse climatic zones with biodiversity found in its different parts. Species of
medicinal plants in the vast Pakistan’s flora are about 6000 (Ahmed et al., 1999). Out
of the total medically potent plants, 70% are found in the specific areas of the country
whereas; the remaining 30% are obtained from the various localities (Shinwari,
2010a). In various regions of Pakistan, from centuries the knowledge about local
medicinal plants has been practiced by about 40,000 unregistered and registered
tabibs/hakims (Saeed al., 2011). Mainly transfer of knowledge from one generation to
another occurs either verbally or through personal experiences applied and adopted
Chapter – 1 INTRODUCTION
4
for the basic health problems (Shinwari et al., 2010a; Bhardwaj and Ghakar., 2005).
In 1950’s for the basic health care conditions approximately 84 percent of the
Pakistan’s population especially of the rural areas relied on the traditional medicines
of their locality (Hocking., 1958, Ahmed et al., 1999). With the passage of time
advancements were made in the knowledge related to the medicinal plants (Balick et
al., 1996).
Research work on phytomedicines has been carried out in Pakistan in the light of
pharmacological screening based on their folkloric uses. Research is also going on
for the discovery of lead compounds from these plants by isolating active ingredients
through the application of various isolation techniques. This instrumental level
research work in the country has paved the way for finding out best economical and
safe treatments of various diseases.
1.3 IMPORTANCE OF HERBAL MEDICINES IN DIFFERENT
TRADITIONS
Herbal remidies serve as healing tools for the management of various diseases in local
areas as well as world wide. Some of the traditional roles of medicinal plants are
given below.
1.3.1 Chinese Traditional Medicines
Chinese herbal medicines are very old and most of its citizens rely on the traditional
medication. More than 50% of the Chinese of the rural areas for the basic health care,
use their traditional medicines because China has also been gifted with thousands of
medicinal plants. Out of twelve hundred medicinal plants, tabibs/hakims use five
hundred medicinal plants most commonly (Li., 2000) WTO (World Trade
Organization).
Chapter – 1 INTRODUCTION
5
1.3.2 Japanese Traditional Medicines
Japanese traditional system was derived from the Chinese system. In the 19th century.
Japanies classified their native plants in their first traditional pharmacopeia (Saito et
al., 2000).
1.3.3 Indian Traditional Medicines
Ayurveda system of traditional medicines is about 5000 years old. It was first
practiced in India for finding out solutions to the various health related problems
(Morgan., 2002).
1.4 Violaceae Family
Viola is an important genus of the family Violaceae. It is medicinal plant of great
importance on the basis of both its photochemistry and pharmacology. Violaceae is
also known as Retrosepalaceae /Alsodeiace/ Leoniaceae (Mabberley., 1987; Perveen
et al, 2009). The family includes about 23 genera which are tropical and consists of
about 930 species (Burman., 2010). Approximately 111 species were identified in
China (Wang et al., 1991) and about 17 species are distributed in different localities of
Pakistan (Qaiser et al., 1985; Marcussen et al., 2010). Eastern Asian mountains are
said to be the important taxonomical and pharmacological hubs of viola (Ballard et
al., 1999).
Chapter – 1 INTRODUCTION
6
1.5 LITERATURE SURVEY OF GENUS VIOLA
The genus is distributed through out the world in various parts of the globe. Some of
the species along with their part/s used, distribution, chemical constituents and uses
are presented in the Tables 1.1-1.3.
Table 1.1: Viola species list, native to Pakistan and their world wide distribution,
Viola parts used, chemical constituents and medicinal uses
S.No. Botanical
name of the
specie
Part/s
Used
Geographical
distribution
Chemical Constituents Uses
1 Viola
betonicifolia
Whole
plant
(Leaves,
roots,
flowers)
Pakistan,
Malaysia,
India, China,
Nepal, Sri
Lanka,Burma,
Japan,
Australia
&Taiwan
3-methoxydalbergione
(Muhammad et al 2014), 3-
Methoxy dalbergion,
Undecanoic acid, 2,4-
Dihydroxy, 5-
methoxycinnamic acid, 4-
Hydroxy coumarine, Beta-
Sitosterol, Ursolic acid,
benzoic acid, Trihydroxy
benzoic acid (Muhammad
et al., 2012a)
As astringent, antipyretic
anticancer, diaphoretic &
purgative (Shinwari et al.,
2010a). In epilepsy, nervous
disorders, for sinusitis, blood
abnormalities, skin diseases,
cough, cold, pharyngitis
(Bhatt and Negi et al., 2006)
an astringent, for cooling
effect, diuretic, laxative and
purgative (Husain et al.,
2008). For kidney diseases,
bronchitis, pneumonia and
boils (Husain et al., 2008).
Urease inhibitor (Muhammad
et al., 2014)
2 Viola biflora Whole
plant
Europe,
Central Asia,
India, China,
Korea,
Pakistan
America &
Japan
Protein [ vibi A-K,
(cycloviolacin O2, O9, varv
A, vitri A) (Burman et al.,
2010)
As antispasmodic, antiseptic,
cough, cold, diaphoretic,
laxative emetic, antipyretic,
intestinal pain, leucoderma,
Psoriasis and dermititis
(Chandra et al 2015).
3 Viola
canescens
Whole
plant (oral)
India,
Pakistan,
Nepal and
Bhutan
Alkaloid violin, methyl
salicylate, quercitrin,
glycosides and saponins
(Rana et al., 2010).
Alkaloid (emetine) malic
acid, glucoside (viola
quercitrin).
For Cold, cough, respiratory
problems, as antipyretic,
antimalarial, demulcent,
astringent, diaphoretic,
purgative, febrifuge, anti
cancerous, carminative,
demulcent, antimicrobial,
treating nervous disorders,
eczema, anti epileptic,
anticancer, anti rheumatic,
heart burn, boils and sore
throat (Hamayun et al., 2006;
Hussain et al., 2011; Rani et
al., 2013)
4 Viola cinerea Whole
plant (oral)
specially
roots and
leaves
Yemen (Kilian
et al., 2004)
Iran, Pakistan
and Oman.
Triterpenoids (Tabba et al.,
1989), cyclotide alkaloids
(Chen et al., 2005).
Flavonoids (Vukics et al.,
2008). Caffeic and
Aphrodisiac (Marwat, 2008)
Chapter – 1 INTRODUCTION
7
salicylic acid (Toiu et al.,
2008)
5 Viola
falconeri
Flower and
roots
India and
Pakistan
Triterpenoids (Tabba et al.,
1989) cyclotide alkaloids
(Chen et al., 2005).
Flavonoids (Vukics et al.,
2008). ). Caffeic and
salicylic acid (Toiu et al.,
2008).
Flower used for cold and
cough whereas, roots used
against jaundice (Saqib and
Sultan, 2005).
6 Viola
fedtschenkoan
a
Whole
plant
Central Asian
countries
including
Northern
Pakistan
Triterpenoids (Tabba et al.,
1989) cyclotide alkaloids
(Chen et al., 2005).
Flavonoids (Vukics et al.,
2008). ). Caffeic and
salicylic acid (Toiu et al.,
2008).
Not reported
7 Viola
kashmiriana
Whole
plant
India,
Pakistan,
Kashmir and
Afghanistan
Triterpenoids (Tabba et al.,
1989) cyclotide alkaloids
(Chen et al., 2005).
Flavonoids (Vukics et al.,
2008). ). Caffeic and
salicylic acid (Toiu et al.,
2008)
Applied on scores and ulcers,
heals swollen mouth and foot
disease in cattles. It also cure
bronchitis (Ishtiaq et al.,
2006)
8 Viola
kunawurensis
Whole
plant
Afghanistan,
Pakistan,
India, Nepal,
China,
Turkestan, and
Tibet.
Not reported Not reported
9 Viola
macroceras
Whole
plant
Pakistan,
Afghanistan
Not reported Not reported
10 Viola
makranica
Pakistan Not reported Anti-inflammatory and
analgesic.
11 Viola odorata Whole
plant
especially
leaves
Europe Asia
and North
Africa.
Glycoside, salicylic acid
and essential oils (Furfural
α-Terpinene, α Thujene,
para-methyl Anisole, β-
Phellandrene,
α-Pinene, Sabinene ,
Myrcene, δ-3-Carene, Z-β-
Ocimene, Benzyl alcohol,
γ-Terpinene,
Acetophenone, Z-Sabinene
hydrate , Methyl benzoate,
Linalool, Z-linalol oxide,
8-para-Menthatriene, ortho-
Menthatriene, Z-para-
menth-2-en-1-ol , 1-
Terpineol, Ethyl
benzoate,Terpinen-4 ol,
Geraniol, α- Terpineol,
Pulegone , δ-Elemene, α
Cubebene , Isoledene, α-
Copaene , β- Bournonen, β-
Cubebene , α Gurjunene ,Z-
Caryophyllene, β
Antifungal, antimicrobial, for
cold, respiratory problems
such as congestion, sore
throat and coughing. As a
laxative, sedative, analgesic,
expectorant in digestive
disorders blood cleansing,
Jaundice, and headache
(Hammami et al., 2011;
Gautam and Kuma., 2012;
Amiri et al., 2013). It is also
used for the treatment of
catarrh, chronic bronchial
asthma, upper respiratory
tract symptoms, cold,
rheumatism, oral mucosa
inflammation, hysteria,
nervous strain, antipyretic,
insomnia, headache and
diaphoretic (Fleming et al.,
1998; Zargari., 1997; Dhar et
al., 2002).
Chapter – 1 INTRODUCTION
8
Duprezianene, α-Guaiene
(Hammami et al ., 2011;
Stuart, 1989).
12 Viola
reichenbachia
na
Whole
plant
Pakistan Unknown Being an important medicinal
plant it is used in headache,
fever, cough, asthma,
constipation, bleeding piles,
skin diseases and throat
cancer. Used also as
diaphoretic and demulcent
(Kumar and Digvijay, 2014)
13 Viola
rupestris
Whole
plant
Pakistan Not reported Unknown
14 Viola serpens Whole
plant
India,
Pakistan,
Banaladash,
Ceylon, Nepal,
China and Java
(Rahman and
Choudhary.,
2012)
Flavonoids, terpenoids,
reducing sugars, amino
acids and tannins, methyl
salicylate, alkaloid voiline
gum, mucilage, glycoside,
quercitrin and saponin
(kumar et al., 2015).
As demulcent, diaphoretic
diuretic, in fever, jaundice,
asthma, piles bleeding, throat
cancer, constipation, cold,
cough, dermatitis and
headache (Kumar and
Digvijay, 2014; Kumar et al.,
2015).
15 Viola stocksii Whole
plant
India,
Pakistan, Iran,
Yemen and
Afghanistan
(Chandra et al.,
2015).
Not reported Virility in sexual masculine
power
(Marwat et al., 2008).
16 Viola tricolor Areal parts
(flowers)
Pakistan,
Europe, Asia,
America,
Australia,
Germany,
Turkey and
Spain.
Flavonoides, quercetin,
luteolin and luteolin 7-
polysaccharides, phenolic
acids, volatile oil,
Carotenoids, anthocyanins ,
cyclotides, tocopherol,
triacyl glycerolsglucoside
and Proteins (Burman et al.,
2010). Violaxanthin, vitri
peptide A, varv peptide A
(Craik et al., 1999; Molnar
et al., 2004). Monoterpenes,
sesquiterpenes, shikimic
acid derivatives and
aliphatics. Volatile
components (bisabolone
oxide) and trans-β-
farnesene
(Anca et al., 2009). Violine,
sugar resin, mucilage and
salicylic acid (Ghorbani et
al., 2012).
Dermatitis (Chevallier., 1996;
ESCOP 2009). Cystitis,
bronchitis, expectorant, anti-
inflammatory, diuretic skin
conditions, and rheumatism
(Anca et al., 2009). As anti-
epilepsy, anti-asthmatic, in
heart problems, inflammation
of lungs and heart (Ghorbani
et al., 2012).
17 Viola
turkestanica
Whole
plant
Pakistan,
India, Nepal
and Butan.
Ephidrin As nasal drops and used
mostly in veterinary (Anca et
al., 2009).
Chapter – 1 INTRODUCTION
9
Table 1.2: List of other Viola spp. of the family Violaceae, their geographical
distributions, Medicinal uses and part/s used mostly
S.No Botanical Names Geographic Distribution Medicinal uses
1 Viola arvensis
(whole plant)
Romania anti-inflammatory, expectorant, diuretic skin
conditioner, bronchitis, cystitis and rheumatism
(Anca et al., 2009)
2 Viola adunca
(whole plant
especially flowers)
Pacific Northwest Spring-flowering nectar source and as a larval
host (NRCS Plant Guide).
3 Viola Canadensis
(Roots)
North America. Roots decoction used for bladder’s pain.
(NAGPTHG, 2005).
4 Viola diffusa
(whole plant)
Japan, East Asia, South
China, & Philippines.
(Benecke et al., 1985; Zhou
et al., 2008).
Used for hepatitis treatment (Dai et al., 2015).
5 Viola hondoensis
(Whole Plant)
Korea and Japan (Hikosaka
et al., 2010)
As expectorant, anti-inflammatory, diuretic, for
bronchitis, eczema rheumatism, and skin
eruptions (Moon et al., 2004)
6 Viola japonica
(whole plant)
Korea, Eastern Asia, Taiwan,
Shikago, Japan and China
(Benecke et al., 1985).
As anti- inflammation, detoxifier, as blood cooler
and pain alleviator. Used in boils, abscesses,
ulcers, acute conjunctivitis, acute jaundice,
laryngitis, hepatitis and in various kinds of
poisonings. In chest and lungs troubles, as
expectorant and for the treatment of chronic
catarrhal accumulations. Crushed leaves applied
to the cuts, ulcers, swellings and wounds (Moon
et al., 2004).
7 Viola pedata
(whole plant)
South Kurile Islands, East
America, Siberia Japan,
Korea and Dongbei (Benecke
et al., 1985).
In headache, dysentery, colds coughs, boils, as
expectorant and for lubricating medicine
((Moerman, 1998; Native American Garden).
8 Viola pubescens
(whole plant)
Northen America Colds, cough, headache, dysentery and used for
boils. (National Audubon Society, 1979)
9 Viola vulgaris Japan, Korea, Dongbei and
Himalayan regions (Benecke
et al., 1985)
Used symptomatically in mild seborrhea skin
conditions.(Community herbal monograph)
10 Viola yedoensis
(whole plant)
Flavones, coumarins, fatty
acid, phenolic acids (Hong et
al., 2011). Polysaccharides,
flavonoides, luteolin,
quercetin, volatile oil,
phenolic acids, carotenoids,
cyclotides, anthocyanins ,
triacyl glycerols glucoside
and tocopherols (Assessment
report on Viola tricolor)
For relief of symptoms and conditioning of mild
seborrhoeic skin. (Community herbal
monograph).
It is also used as an anti-rheumatic and for
treating infections like mastitis, rhinitis and for
treating acute pyogens (Jun-Li et al., 2011).
11 Viola yazawana
(whole plant)
Japan, Korea, Dongbei and
Himalayas region. (Benecke
et al., 1985).
As an anticoagulant and antithrombotic (Kumar
et al., 2011).
12 Vi!ola hederacea
(flowers, leaves)
Australia and Melbourne Flowers eaten especially by the Victorian but the
exact use is unknown (David, 2005).
Chapter – 1 INTRODUCTION
10
Table 1.3: List of other species of Viola and their geographic distributions
S.No. Botanical Names Geographical Distribution
1 Viola alliaraefolia Mexico, North America and Japan (Benecke et al., 1985).
2 Viola bissettii Korea, Japan and Himalayas region (Benecke et al., 1985).
3 Viola banksii East Australia (Benecke et al., 1985).
4 Viola blandaeformis America and Japan (Benecke et al., 1985).
5 Viola brevistipulata Mexico, North America and Japan (Benecke et al., 1985).
6 Viola Canadensis North America (Benecke et al., 1985).
7 Viola confuse Japan and Taiwan (Benecke et al., 1985).
8 Viola diffusa East Asia, South China, Philippines and Japan (Benecke et al.,
1985; Zhou et al., 2008).
9 Viola faureana Japan (Zhou et al., 2008).
10 Viola fedtschenkoana Northern Pakistan and Central Asia (Zhou et al., 2008).
11 Viola eizanensis Korea, Japan and China (Benecke et al., 1985).
12 Viola grypoceras Japan and Korean islands (Benecke et al., 1985).
13 Viola grayii Japan (Benecke et al., 1985).
14 Viola hirtipes China and Korea (Benecke et al., 1985).
15 Viola hondoensis Korea and China (Richard et al., 1985).
16 Viola hultenii Korea, Japan, Kurile Islands and Siberia (Richard et al., 1985).
17 Viola hederacea Australia and Melbourne (Richard et al., 1985).
18 Viola iwagawai Ryukyus, Yakushima and Okinawa (Richard et al., 1985)
19 Viola japonica Japan, China, Eastern Asia, Shikago, Taiwan and Korea (Richard
et al., 1985).
20 Viola kusanoana Japan, Soviet Union and North Korea (Richard et al., 1985).
21 Viola kitamiana Australia and Malaya. (Richard et al., 1985).
22 Viola keiskei Japan, Korea, Ussuri and Manchuria (Richard et al., 1985).
23 Viola maximowicziana Japan and china (Richard et al., 1985).
24 Viola langsdorffii Alaska, Northern Eastern Asia and North America (Nathorst et
al., 1883; Richard et al., 1985).
25 Viola lactiflora Korea, Japan, North China and Manchuria (Richard et al., 1985)
26 Viola maximowicziana Japan (Richard et al., 1985)
27 Viola mandschurica Japan, China and Taiwa (Richard et al., 1985).
28 Viola nanligensis China (Jin-Zhou et al., 2008)
29 Viola nagasawai China (Jin-Zhou et al., 2008)
Chapter – 1 INTRODUCTION
11
30 Viola ovato-oblonga South Korea (Richard et al., 1985).
31 Viola obtuse Korea and Japan (Richard et al., 1985)
32 Viola orientalis Japan, Koria, China and Southern Soviet (Richard et al., 1985)
33 Viola pedata Southern Kurile Islands, Siberia, Korea, Japan and Dongbei,
Japan (Richard et al., 1985)
34 Viola palustris Europe and America (Nathorst et al., 1883).
34 Viola patrinii Canada (Richard et al., 1985).
35 Viola pubescens Headache treatment (leaves poultice), blood, cough, colds,
dysentery (infusion), boils (crushed root).
(National Audubon Society)
36 Viola phalaerocarpa Korea, Japan and China (Richard et al., 1985).
37 Viola repens Korea and Japan (Richard et al., 1985).
38 Viola rostrate Japan, North America, Asia and Georgia (Nathorst et al., 1883).
39 Viola raddeana Japan, Korea, Amur and Southern Manchuria (Richard et al.,
1985).
40 Viola rossii Japan, Korea and Himalayas region (Richard et al., 1985).
41 Viola sachalinensis Japan (Richard et al., 1985).
42 Viola shikokiana Japan, Korea, Himalyan region and Dongbei (Richard et al.,
1985).
43 Viola seiboldii Korea, Japan & china (Richard et al., 1985).
44 Viola selkirkii Afghanistan, India, Japan, North America, Sweden, Iran,
Greenland Norway, Russia, Caucasus, Siberia, Altai, Baikal,
Manchuria, Kamtschatka and British Columbiia. (Nathorst et al.,
1883).
44 Viola teshioensis Japan, china and korea (Richard et al., 1985).
45 Viola tashiroi Japan and china (Richard et al., 1985).
46 Viola tokubuchiana Japan (Richard et al., 1985).
47 Viola utchinensis Japan (Richard et al., 1985).
48 Viola verecunda Tasmania, Taiwan and New Zealand (Richard et al., 1985).
49 Viola variegata China, Japan (Richard et al., 1985).
50 Viola violacea Yakushim, Korea, Japan and Goto Islands (Richard et al., 1985).
51 Viola vaginata Korea, Japan, Himalayas region and Dongbei . (Richard et al.,
1985).
52 Viola yubariana Japan, Mexico and North America (Richard et al., 1985).
53 Viola yezoensis Japan, Hokkaido and Tokyo (Richard et al., 1985)
Chapter – 1 INTRODUCTION
12
1.6 PLANT INTRODUCTION
1.6.1 Viola serpens Wall
V. serpens Wall. is the synonym of Viola canescens Wallich ex Roxburgh (Satish et
al., 2013; Masood et al., 2014). The common names are Smooth-Leaf White Violet,
Ghatte ghans and Huikhon in different languages (Chauhan et al., 2003). Its common
English name is Himalayan White Violet because it is mostly found in the Himalayan
region (Masood et al., 2014).
1.6.2 Local Names
V. serpens is known with different names in different localities of Pakistan and India.
Its Urdu name is Banafsha (Saqib et al., 2014; Ahmad and Habib., 2014; Ahmad et
al., 2012; Barkatullah et al., 2012; Ali et al., 2011; Hamayun et al., 2006; Hamayun et
al., 2005; Shinwari et al., 2000; Naain, 1999). In KPK, it is called as Banaqsha,
Banafsha or Savar Phal (Adnan and Hoscher., 2010) in Baltistan as Skoramindoq
(Hussain et al., 2014). In lesser Himalyas it is known as bamasha, Phulnaqsha or
sweet violet. Indians call it with the name Ratmundi or Vanaksha (Rana et al., 2014),
Vanafsha (Dua et al., 2011; Suyal et al., 2010). In Himachal Pardesh, it is called
Gugluphul (Kumar et al., 2013), Banaksha and Banfasa (Rani et al., 2013). In
Uttarakhand, it is commonly called Vanfsa (Rana et al., 2010). In Nepal it is locally
called Ghatteghaans (Adhikary et al., 2011).
1.6.3 Morphology
V. serpens being a perennial herb with tufted appearance. The plant occurs both in
cluster or solitary forms. There is no clear stem (short stem) and the leaves seemed to
be originated directly from the creeping roots. Leaves are long, narrower, ovate-lance
shaped and pointed, white thin hairs may or may not be present above, 5-7 veined.
Chapter – 1 INTRODUCTION
13
Leaf-stalk and leaf blade are almost of the same length. Flowers are lilac or almost
white in color with 1-1.5 cm across. Normally the upper petals at the base bear hairs.
Stigma is beaked and having 3-lobs. Stipules are toothed and not fringed. Stem is light
green, straight, somewhat pubescent or glabrous. Fruits are in pods, which are pale
brown in color. Each pod contains many tiny blackish seeds. The plant odor is very
good and attractive.
1.6.4 Classification (Taxonomical position of V. serpens)
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Sub class: Dilleniidae
Super order: Dillenianae
Order Malpighiales
Family: Violaceae
Genus: Viola
Species: Serpens/ Pilosa.
Herbarium: Submitted in the Department of Botany, University of
Peshawar, Peshawar.
Voucher No: Bot. 20158 (PUP).
1.6.5 Geographical Distribution of V.serpens
1.6.5.1 World Wide Distribution
The world wide geographic distribution of V. serpens is in Pakistan, China, India, Sir
Lanka, Nepal, Java, Philippines, Thialand and Nagaland.
Chapter – 1 INTRODUCTION
14
1.6.5.2 Distribution in Pakistan
In Pakistan V. serpens is distributed widely in Swat, Shangla, Buner, Chitral, Hazara,
Kaghan valley and Shogram (Barkatullah et al., 2012; Hamayun et al., 2006; Shinwari
et al., 2000). It is also found in Rawalpindi, Kotli Sattian, Azad Jammu Kashmir and
NeelumValley (Ahmal et al., 2012; Ahmad and Habib., 2014; Saqib et al., 2014).
Tropical and temperate zones are the hubs of this plant which is restricted only to the
hilly areas (Singh et al., 2005). In Pakistan, V. serpens is also found in the localities of
Shawal (NorthWaziristan), Parachinar (Kurrum Agency), Swat, Dara Adam Khel,
Teera (Orakzai Agency), Bajour, Razmak, Miran Shah (South Waziristan), Fizagut
and Kaalam (Shinwari et al., 2010a). In the temperate Himalayas Mountains the plant
exsists at about 2000 m of elevation (Kumar et al., 2013).
Chapter – 1 INTRODUCTION
15
1.7 PLANT DESCRIPTION
They grow as perennial herbs, shrubs, and trees or treelets (Hekking., 1984,
Munzinger et al., 2003). Viola is the Violaceae largest genus, distributed greatly in the
northern hemisphere (Marcussen et al., 2012). Rhizomes of viola are somewhat
elongated but mostly short. Usually maximum height of the plant is about 4 inches or
they lack the short stem. Similarly the branched stem arises from the hairy rhizomes if
present (Muhammad et al., 2012), which ends with winged or non-winged leaves
which may be ovate-triangular stalked, cordate, crenate or serrate. Flowers are
zygomorphic, different colors in different species like pale, blue, white and violet.
Most of the species have violet color flowers due to which the family is also known as
violet family. Each flower arises singly from the axil of the leaves with a along stalk
comprises of unequal sized five petals. A spur is formed by the lower ones containing
nectar for attracting insects for pollination. (Clark and Trelawny., 1998). The number
of calyx is five which surround the petals of the flower. Three united carpels form a
compound pistil. Stamens number is also five, short filaments with coherent anthers
and form ring around the gynoecium. Fruits are inferior (hypergynous) to ovules.
Ovary is surrounded by the anthers which are connate, filaments are broad separated
and short, the lateral two ends resulting in the corolla spur formation. Stigma is lobed,
usually beaked or straight. The shape of the seeds pod (ovary) differs in different
species which may be pointed or triangular. Seeds are smooth, shiny with caruncle
(fleshy outgrowth) important for dispersion automatically by wind when the seed pod
bursts (Qaiser et al., 1985; Reznick and Voss, 2012; Clark and Trelawny., 1998).
Such flowers are known as cleistogamous flowers. The floral formula of the flower is
K5 Co5 S5 P (3). Ovary is sessile with curved style at base.
Chapter – 1 INTRODUCTION
16
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,
Chapter – 1 INTRODUCTION
17
monoterpenes, sesqueterpenes and shikimic acid derivatives. The total reported
essential oils are 35 in number (Anca et al., 2009).
1.10 ISOLATED COMPOUNDS FROM GENUS VIOLA
The literature survey describes isolation of important pharmacologically active
compounds from various species of the genus. These isolated compounds belong to
various naturally occurring classes such as cyclotide alkaloids (Simonsen et al., 2005),
salicylic acid (Toiu et al., 2008), flavoniods (Vukics et al., 2008a; Vukics et al.,
2008b), derivatives of caffeinec acid (Toiu et al., 2008) and triterpenoids (Tabba et
al., 1989). Some of the secondary metabolites from various species of the genus are
presented in the Table 1.1.
1.11 PHARMACOLOGICAL STUDIES
Various pharmacological studies including in vivo and in vitro screening have been
carried out by different researchers in different eras.
1.11.1 In vivo Biological Activities
1.11.1.1 Antibacterial Activity
Crude aqueous methanolic extract of V. betonicifolia whole plant with the subsequent
fractions were applied to the antibacterial activity. The in vitro anti-bacteria bioassay
was conducted against Bacillus cereus, Escherichia coli, Staphylococcus aureus,
Enterobacter aerogenes, Proteus mirabilis, Salmonella typhi and Enterococcus fecalis
in which remarkable activity against E. coli and Salmonella typhi was observed
(Naveed et al., 2013b). V. odorata showed pronounced effect against the tested
microorganisms (Hassan and Naeem., 2014). The methanol soluble extract and its
subsequent fractions, the aqueous, acetone and petroleum ether soluble extracts
showed antimicrobial activity. The methanolic extract showed maximum inhibition
Chapter – 1 INTRODUCTION
18
against Haemophilus influenzae (24 mm) and Streptococcus pneumoniae (19 mm).
Whereas, lowest inhibition observed against Pseudomonas aeruginosa (13 mm) was
caused by aqueous, acetone and petroleum ether in the descending order (Kinghorn et
al., 2007). V. odorata possessed about 5.2 % triterpene, saponins, ursolic acid in the
form of glycone and galacturonic acid or galactose (Rastogi., 1984). Some toxic
metabolites may be responsible for the significant antimicrobial activity. V. odorata
aqueous fraction (flowers) revealed effective antibacterial activities against S. aureus
E. coli and B. subtilis (Khatibi et al., 1989). The aerial part at a concentration 3, 2 and
1 mg/kg was effective against B. subtilis, S. aureus and E. coli (Ramezani et al.,
2012). Cyclotides (Cyclotide cycloviolacin O2) are peptides which are rich in small
disulfide, obtained from the dried areal plant of V. odorata. Oils of V. odorata
methanolic extract are also effective against microbes (Hammami et al., 2011). It
efficiently inhibits the growth of E. coli, K. pneumonia and P. aeruginosa but does
not show any effect against S. aureus (Pranting et al., 2010; Ashfaque Khan et al.,
2011). The range of MICs showed by the standard antibiotic erythromycin was 3.12
to 12.5 mg/ml whereas, MICs presented by V. odorata fractions against S.
pneumoniae, H. influenzae and S. pyogenes were similar to the standard (6.25 mg/ml).
Better MIC showed by the methanolic extract against S. aureus (3.12 mg/ml) and
least MIC (12.5 mg/ml) was recorded against P. aeruginosa. MICs of ethyl acetate
fraction for E. coli and K. pneumoniae were 10.l g/L and 5.5 lg/L respectively
(Gautam and Kumar., 2012). The use of V. odorata for treating respiratory infections
provides a rationale for future study (Khan et al., 2011; Salve et al., 2014).
Similarly, ethanolic extract of leaves of V. serpens Wall. was used (in vitro) against
bacterial diseases. The selected species of microbes were S. typhi, E. coli, S. aureus
and K. pneumonia. The result was that maximum antibacterial activity was shown by
Chapter – 1 INTRODUCTION
19
the ethanolic extract of V. serpens. The zone of inhibition values of the plant extract
and different antibiotics were compared which proved to be more effective against the
microbes (Kumar et al., 2015). The isolated cyclotide (vhl-1) from the V. hederaceae
was also tested for the same activity against E.coli and S. aureus but the effect was
negative and showed no inhibition (Chen et al., 2005).
1.11.1.2 Anti-fungal Activity
The hydromethanolic extract/fractions of V. betonicifolia were subjected to antifungal
activity. In vitro antifungal bioassay of the plant was performed against Aspergillus
niger, Aspergillus parasiticus, Candida albicans, Juncus effuses, Saccharomyces
cerevisiae, Trichophyton rubrum and Fusarium solani. The tested samples including
crude aqueous methanolic extract, ethyl acetate and chloroform fractions showed to
be effective against the selected fungi except C. Albicans. V. betonicifolia plant is
used effectively as an antifungal source (Muhammad et al., 2013). Various fractions
of V. canescens with different solvents (acetone, ethanol, petroleum ether & water)
were tested for the antifungal activity. Ethanol, petroleum ether & water showed
intermediate antifungal activity whereas, a dose of 1000 mg/ml acetone extract
showed maximum inhibited zone. The extracts of ethanol as well as the petroleum
ether also showed highest MIC (Rawal et al., 2015). The antifungal activity of V.
odorata crude extract and its essential oil were also investigated against Botrytis
cinerea. Against the tested pathogenic fungi the applied oils obtained from the plant
also showed strong antifungal effect (Kumar et al., 2011). The whole plant of the
crude extract of V. tricolor was tested against C. albicans which showed mild
antifungal activity against fungal diseases/infections (Banaszezak et al., 2005). The
isolated cyclotide (vhl-1) from the V. hederaceae was also tested for the antifungal
Chapter – 1 INTRODUCTION
20
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
Chapter – 1 INTRODUCTION
21
showed outstanding antispasmodic activity against the strain of P. falciparum having
sensitivity against chloroquine (Lee et al., 2009; Moon et al., 2007).
1.11.1.7 Anti-malarial Activity
Petroleum ether fraction of V. canescens collected from Northwestern Himalaya
proved as an anti-malarial. The extract showed its inhibitory action in comparison
with the standard drug against the causative agent of malaria (P. falciparum) (Verma
et al., 2011).
1.11.1.8 Anthelmintic activity
V. betonicifolia was tested for nematocidal activity. Anthelmintic activity of the plant
extract along with the subsequent fractions was tested against worms of various
species which resulted in significant vermicidal activity. Ethyl acetate and chloroform
fractions proved to be more effective showing 42 and 58 min. death time against P.
posthuma, respectively. The same fractions mortality rates after 48 h against C.
littoralis were 66 and 62% respectively. Whereas, against H. indicus ethyl acetate and
chloroform fractions showed 49 and 57% mortality rates respectively. On the bases of
these results V. betonicifolia being a natural source can be significantly used for
anthelmintic activity (Naveed et al., 2012).
Cyclotide shows anthelmintic activity. Various cyclotides isolated from V. odorata
were cycloviolacin O14, cycloviolacin O2, cycloviolacin O13, cycloviolacin O15,
cycloviolacin O8, cycloviolacin O16 and cycloviolacin O3. In nematode larval
development assays these cyclotide showed about 18-fold higher potency as
compared to the kalata B1 prototypic cyclotide. Cycloviolacin O14 and cycloviolacin
O2 are more potent than kalata. Cycloviolacin O2 residues, lysine and glutamic acid
residues are the most effective anthelmintic cyclotide. The anthalmentic activity of
Chapter – 1 INTRODUCTION
22
cyclotide e.g methylation results in six times decrease in its activity. Acetylation
masks the positive charge effectively and the anthalmentic activity is decreased by18-
fold. It is concluded that V. odorata is a significant vermicidal or anthelmintic in
nature (Wang et al., 2008; Muhammad et al., 2012).
1.11.1.9 Antioxidant Activity
The crude ethanolic extract of V. serpens was investigated for the antioxidant activity.
The presence of certain phytochemicals in the ethanolic extract of the V. serpens
showed that it is an effective antioxidant plant. Ascorbic acid a non-enzymatic
antioxidant and the antioxidants enzymes such as catalase, ascorbate oxidase
peroxidase are present in V. serpens (Kumar et al., 2011). DPPH, standard scavenger
was used for the evaluation of antioxidant potential of Viola tricolor (Vukics et al.,
2008; Nikolova et al., 2011). Viola odorata also proved a strong antioxidant natural
source. It was verified by using DPPH reducing power assay, hydrogen peroxide and
ferric thiocynate scavenging protocols. Phytochemical investigation of the flavonoids
and the total phenolic compounds measurement in the plant extracts scientifically
strengthen it as strong antioxidant source. (Ebrahimzadeh et al., 2010)
1.11.1.10 Anti-Tubercular Activity
Subsequent fractions of the crude ethanolic extract of V. odorata were investigated for
the anti-tubercular activity. T.B causing bacterial stains, M. tuberculosis H37Rv and
MDRTB were used in the study. Among various fractions the dichloromethane and n-
hexane showed significant activity against both the strains. Isolated compounds
salicin and phenyl alanine ethyl ester from V. odorata were tested purely against the
tested strains. It was concluded that V. odorata is a rich source of important chemical
constituent which are effectively used against the M. tuberculosis H37Rv and M.
Chapter – 1 INTRODUCTION
23
avium strains. Thus this plant is a lead potential anti-TB drug (Hassan and Naeem.,
2014).
1.11.1.11 Treatment of Jaundice
Leaves extract of V. serpens was tested for the antibacterial activity against the
jaundice causing bacteria. Bacterial strains were isolated from patients suffering from
jaundice and treated against the crude methanolic extract which showed pronounced
effect against the particular strains. Crude methanolic extract was applied which
showed pronounced effect against jaundice. The scientific proof increases the plant
importance for its use as remedies in jaundice along with many other infectious
diseases (Kumar et al., 2015).
1.11.1.12 Urease Inhibitory Activity
3-methoxy dalbergion is an isolated compound from V. betonicifolia (Naveed et al.,
2014). The mechanistic study on this natural compound as a urease inhibitor was
carried out by linking docking studies along with the enzyme kinetics. Findings of the
study are that the new naturally isolated compound (3-Methoxydalbergione) proved to
be helpful in the urease linked diseases. Urease is a harmful compound which is
directly involved in infectious stones formation, encrustation pyelonephritis,
urolithiasis, ammonia, hepatic coma, hepatic encephalopathy and urinary catheter.
Moreover, it is the main cause of peptic and gastritis ulcers induced by Helicobacter
pylori (Naveed et al., 2014).
1.11.1.13 Anti-HIV Effect
From various species of viola more than hundred macrocyclic compounds have been
isolated, having three disulphide bonds known as cyclotides. Important five cyclotides
have been isolated from V. yedoesis, having significant effects against HIV activities.
Chapter – 1 INTRODUCTION
24
Among these cycloviolacin Y5 is one of the most important cyclotide tested
effectively in anti-HIV in vitro XIT-based assay (Wang et al., 2008).
1.11.1.14 Insecticidal Activity
Cyclotides are plants peptides having cyclic structures, which possess activities like
insecticidal, anthalmentic and anti-HIV. More than 100 cyclotid have been isolated
from viola family. These compounds are more effective insecticidal (Wang et al.,
2009).
1.11.2 In vitro Biological Activities
1.11.2.1 Acute Toxicity
V. odorata was tested for the acute toxicity in in vivo animal’s model (rats). V.
odorata was scientifically proved as a safe drug even at higher dose (2000 mg/kg)
which can be used safely for clinical purpose (Vishala et al., 2009). In 24h assessment
time duration V. betonicifolia methanolic extract also proved safe at high dose (2000
mg/kg i.p.) and one of its isolated compound [4-hydrose of oxyl coumarin (4HC)] was
also showed to be a safe drug at high doses. Both the plant in the crude form or the
isolated compound can be safely used for clinical purpose (Naveed et al., 2012;
Naveed et al., 2013).
1.11.2.2 Antinociceptive Activity
Antinociceptive activity of V. canescens aqueous ethanolic extract showed significant
effect (Barkatullah et al., 2012). Crude aqueous methanolic extract and the fraction of
n-hexane of V. betonicifolia Sm. were tested for the same activity at three different
doses. Both the methanolic extract and the fraction showed strong dose-dependent
analgesic effect against the acetic acid induced writhing test model (Naveed et al.,
2012, Naveed et al., 2012). At a dose of 400 mg/kg body weight V. odorata proved an
Chapter – 1 INTRODUCTION
25
outstanding anti-nociceptive drug (acetic acid and tail immersion tests) (Antil et al.,
2011). It also proved to be a dose dependent effective analgesic natural source
(Barkatullah et al., 2012). The flowers of V. tricolor were investigated for the anti-
nociceptive effect of a gel containing extract. V. tricolor flowers were investigated for
thermal burn by UVB irradiation and for gel stability performance study which
provided scientific proof to the plant as a natural source of analgesia in the ultraviolet
induced burn, at a temperature below 25 ºC (Piana et al., 2013).
1.11.2.3 Anti-Inflammatory Activity
Clear anti-inflammatory observations were obtained by testing the plant of V.
betonicifolia (n-hexane fraction) in BALB/c mice, following two different protocols
(carrageenan and histamine-induced protocols). It was found that the aqueous
methanolic extract at different test doses showed significant anti-inflammatory
activity. This justifies its use as pain killer in traditional medicine (Naveed et al.,
2012). V. odorata (whole plant) also proved significant anti-inflammatory plant
(Chatterjee et al., 1991; Kaul., 1997). The flowers of V. tricolor were investigated for
the anti-inflammatory effect of a gel containing extract and induced thermal burn by
UVB irradiations for gel stability performance study. Conclusion of the study was that
V. tricolor showed significant anti-inflammatory effect (Piana et al., 2013).
1.11.2.4 Antipyretic Activity
Viola betonifolia crude aqueous methanolic extract/n-hexane fraction were both
analysed agaist the induced pyrexia. The in vivo study conclusion was that both of
these (crude extract and the n-hexane fraction) showed excellent antipyretic activity.
This study provides preciseness and solid reasons to the plant that shows antipyretic
activity (Naveed et al., 2012). In the induced pyrexia, V. betonicifolia at 300 mg/kg
Chapter – 1 INTRODUCTION
26
body weight showed dose dependent (78.23%) antipyretic effect (Naveed et al.,
2012). V. odorata also is used effectively for antipyretic activity (Chatterjee et al.,
1991; Kaul., 1997).
1.11.2.5 Gastrointestinal Motility
V. canescens was tested for the gastrointestinal motility. The test was performed in
the in vivo model in mice. The findings of the study was that the plant purgative
activity. This investigation verifies its traditional use for this purpose (Vishala et al.,
2009).
1.11.2.6 Laxative Effect
The n-butanol and aqueous fractions of V. odorata in 200 and 400 mg/kg doses
proved better laxative effect in the animal model (rats) in in-vivo protocol (Vishala et
al., 2009). Traditionally V. canescens is used as an effective laxative in traditional
medicines in the crude form (Vishala et al., 2009). Both the screening procedures
were carried out either in vitro or in vivo, of the whole plant of V. betonicifolia crude
methanolic extract at different doses. Partially atropine-sensitive prokinetic in both
low and high doses 50 and 100 mg/kg respectively were used. Laxative effect in the
doses 30 and 100 mg/kg also showed significant effects. Isolated rabbit guinea-pig
ileum and jejunum showed dose-dependent contractions at 0.03-5 mg/mL and 0.01-
0.3 mg/mL respectively. More effective spasmodic effect of the crude methanolic
extract was observed in guinea-pig ileum as compared to the preparation of rabbit
jejunum (Naveed et al., 2013).
Chapter – 1 INTRODUCTION
27
1.11.2.7 Hepatoprotective Activity
V. odorata is traditionally used against liver diseases. Scientific background was
given to the folk uses by paracetamol induced hepatotoxicity protocol. Plant’s
aqueous methanolic extract was tested at two different doses (250 and 500 mg/kg) in
mice (in vivo). The results showed that the increased levels of AST, ALT, ALP (liver
enzymes) and the total bilirubin were significantly reduced to the normal levels.
Regeneration from hepatocellular necrosis and inflammation of the plant extract
treated groups with pure paracetamol was clear from the histopathological slides
(Qadir et al., 2014).
1.11.2.8 Diuretic Activity
The significant diuretic activity of the aqueous extract V. odorata’s aerial parts was
observed by adjusting two different oral doses, using in vivo animals model (Vishala
et al., 2009).
1.11.2.9 Anxiolytic Activity
The crude aqueous methanolic extract of V. betonicifolia was used for the in vivo
animal model. Results of the study were the dose dependent significant anxiolytic
activity of the plant extract. The test used for the activity was staircase test (Naveed et
al., 2013). The whole plant of Viola betonicifolia and its n-hexane fraction were tested
for sedation and different nervous disorders like muscle relaxant, antidepressant,
anxiolytic to assure the folk use. Various tests protocols were adopted for this purpose
i.e staircase test (anxiolytic activity) rota rod, traction test, chimney test and inclined
plane (muscle relaxant). Different doses of the crude and the n-hexane fractions gave
different results when administered to the test mice (i.p.). The fraction of n-hexane
was also monitored by using the forced swimming test for the effect of sleep-
Chapter – 1 INTRODUCTION
28
induction activity and staircase test for the anxiolytic action. The fraction of n-hexane
as well the crude methanolic extract showed a noteworthy sleep inducing effect and
reduced locomotive activity. Sleep duration was increased in a dose-dependent mode.
The outcomes of the tests were that the n-hexane fraction showed sedative, muscle
relaxant, anxiolytic and for the assessment of various nervous disorders (Naveed et
al., 2013).
1.11.2.10 Muscle Relaxant
V. betonicifolia crude aqueous methanolic extract (100, 200 and 300 mg/kg) and its
isolate, 4-hydroxyl (20 and 30 mg/kg) showed significant muscle relaxant effects.
Thus crude aqueous methanolic extract had strong muscle relaxant and sedative-
hypnotic activities. Similarly, significant muscle relaxant activity was showed by 4-
hydroxyl coumarin. This gives a scientific proof to the folkloric uses of the plant as a
muscle relaxant (Naveed et al., 2013).
1.11.2.11 Sedative-Hypnotic Effect
In sedative-hypnotic activity the latency time was remarkably reduced and the
sleeping time was increased by the crude aqueous methanolic extract of V.
betonicifolia (Naveed et al., 2013). V. odorata was investigated for its sedative and
hypnotic effect in animal models (rats), adjusting different test doses. Dose dependent
sedation of the crude methanolic extract was proved from the study designed (Monadi
et al., 2013).
1.11.2.12 Anesthetic Effect
V. odorata was investigated for its pre-anesthetic effects. The results reveal the dose
dependency of the sedative effect in the plant extract. The extract at three different
Chapter – 1 INTRODUCTION
29
doses (100, 200 and 400 mg/kg) were used in which only 400 mg/kg increased the
sedation effect (Monadi et al., 2013).
1.11.2.13 Uterotonic Effect
The presence of cyclotides in various species of violaceae family is also responsible
for the uerotonic activity. For example about 30 cyclotide have been isolated from V.
odorata and five important have been isolated from V. yedoesis (Schöpke et al.,
1993).
1.11.2.14 Anti-neurotensive
Naturally equipped plants with cyclotide are also rich sources of anti-neurotensive
effect. Most of Viola species are gifted with cyclotide so they are significantly used
for anti-neurotensive activity (Schöpke et al., 1993; Tam et al., 1999).
1.11.2.15 Anti-cancer Activity
V. odorata acetone extract possess chemo-preventive effect in the in vivo animal
model (Perwaiz and Sultana., 1998). Cycloviolacin O2 isolated from V. odorata,
showed cytotoxic effect against the ten different lines of the cancer cell which include
myeloma, leukemia, lymphoma & renal adenocarcinoma and small-cell lung cancer.
V. odorata proved as a more significant antitumor drug (Melo et al., 2011; Talib.,
2011). A cyclotide, Cycloviolacin O2, an isolate of V. odorata showed outstanding
antitumor activity. The cyclotides also showed positive results in the management of
breast cancer by using doxorubicin (presence/absence) by assay of cell proliferation
for the establishment of chemosensitization abilities (Gerlach et al., 2010). Cyclotides
of V. tricolor are toxic in nature (Tang et al., 2010). The study provides a legal
scientific background to the plant for its folkloric uses in treating throat, tongue breast
and lung cancers (Lindholm et al., 2002; Salve et al., 2014).
Chapter – 1 INTRODUCTION
30
1.11.2.16 Anti-hypertensive Effect
The leaves extract in crude form of V. odorata possess anti-hypertensive action by
using both the in vivo and in vitro protocols. This resulted in lowering of the mean
arterial blood pressure in vivo by using rats in the animal models. Guinea-pig isolated
atria were used in the in vitro antihypertensive protocol. This resulted in spontaneous
atria contraction by force and rate inhibition (Hasan et al., 2012). V. mandshurica
specie of Violaceae family also plays a vital role in antihypertensive activity.
Angiotensin-converting enzyme inhibitors (ACE) are responsible for blood vessels
contraction. The absorbance of V. mandshurica extract at 228 nm was maximum. For
ACE inhibition, captopril, a standard reagent was used. The roots extract showed
more effectiveness than petioles and leaves for this activity (Huh et al., 2015).
1.11.2.17 Anti-dyslipidemic Effect
V. odorata Linn. showed dyslipidemic effect when used in the in vivo and in vitro
assays. Tyloxapol-induced dyslipidemia protocol was followed. Significant reduction
in the total cholesterol lever in the dyslipidimic induced rats was caused by the plant
extract. So the conclusion was that the vasodilatation action of the plant extract was
mediated through various pathways such as its release from intracellular stores, Ca++
influx inhibition through Ca++ channels membrane, and NO mediated pathways,
resulted in the fall in blood pressure. Thus V. odorata is significantly used for
antidyslipidemic activity (Hasan et al., 2012).
1.11.2.18 Expectorant and Anti-tussive Effect
V. odorata different parts i.e roots, leaves and flowers were used for the expectorant
and anti-tussive activities. The active constituents responsible for the said activities
are alkaloids, salicylic acid, saponins, volatile oil and methyl ester, which give
Chapter – 1 INTRODUCTION
31
scientific approval to the plant’s folk uses for this activity (Gairola et al., 2010;
Sellappan., 2015).
Figure 1.2: Illustration of V.serpens specie of the genus Viola
Chapter – 1 INTRODUCTION
32
Figure 1.3: Flower of V. serpens specie of the genus Viola
Figure 1.4: Seeds of V. serpens specie of the genus Viola
Chapter – 1 INTRODUCTION
33
1.12 AIMS AND OBJECTIVES
To isolate medicinally important bioactive secondary metabolites from Viola
serpens.
To elucidate the structure of isolated compound(s) using various spectroscopic
techniques.
To evaluate the pharmacological activities (antioxidant, analgesic, acute
toxicity, anti-inflammatory, nephroprotective, hepatoprotective, enzyme
inhibition and larvicidal activity) of the crude extracts and fractions
.
Chapter – 2 EXPERIMENTAL
34
CHAPTER – 2
2. EXPERIMENTAL
2.1 GENERAL EXPERIMENTAL CONDITION
Chemical studies along with the different biological activities were carried out in the
Departments of Pharmacy and Chemistry, University of Malakand, Chakdara.
However, a part of the biological activities was performed in PCSIR laboratories,
Peshawar and Department of Animals Health Sciences, Agriculture University
Peshawar. Spectroscopic studies were performed in Atta-U-Rehman Institute for
Natural Product Discovery, Universiti Teknologi MARA Puncak Alam Selangor D.E.
Malaysia.
2.2 SPECTROSCOPIC TECHNIQUE
The isolated compounds were characterized by means of various spectroscopic
techniques including UV, IR, 1H and 13C-NMR, NOESY, COSY, HSQC
(Heteronuclear Single-Quantum Correlation) and HMBC. Fully automated, Hitachi
Spectrophotometer (model U-3200) was used for the UV spectra determination.
Spectrometer, model JASCO 302-A was used for the IR spectra on potassium
bromide (KBr) discs. The compounds low-resolution electrons impact spectra were
determined by means of mass spectrophotometer (model MAT311A) linked with
PDP11/34 system of computer. The 1H-NMR spectra of the compounds were
determined by using Nuclear magnetic resonance spectrometer by Bruker AM-300,
AM-400 and AMX-500. Internal reference TMS was used for the spectra at 300, 400,
or 500 MHz. Distortion less Enhancement by Polarization Transfer (DEPT)
experiments for the moieties CH, CH2 and CH3 at 90o and 135o were performed.
2.3 PHYSICAL CONSTANTS
Chapter – 2 EXPERIMENTAL
35
The melting point instrument known as Gallenkamp electrothermal melting point
apparatus of the Model 5A-6797 (England) was used for the determination of the
melting points of the isolated compounds. Optical rotation of the compounds was also
recorded by using a digital polarimeter (JASCO DIP- 140).
2.4 COLUMN CHROMATOGRAPHY (CC).
In column chromatography (CC) silica gel was used. Isolation and purification, of the
compounds required silica gel 60 (Merck) on mesh size 230-270. The organic
solvents (n-hexane, chloroform, ethyl acetate and methanol) were used in column
chromatography as mobile phases
2.5 THIN LAYER CHROMATOGRAPHY (TLC)
Samples purification was insured by using thin layer chromatography technique
(TLC). F254 aluminum sheets, pre-coated Kiesel gel 60 (Merck) were used for TLC.
2.6 DRUGS AND REAGENTS
In various experiments different drugs and commercial grade chemicals were used.
Details of which are given in the Table 2.1. Different doses of crude methanolic
extract and the fractions were prepared in normal saline and distilled water for various
biological activities. These solvents were also used as negative control. Commercial
grade organic solvents (n-hexane, ethyl acetate, chloroform, n-butanol, and methanol)
were selected and used.
Chapter – 2 EXPERIMENTAL
36
Table 2.1: List of Drugs/Chemicals Drugs/ Chemicals Source
Acetic acid
Acetyl choline esterase
Acetyl thiocholine iodide
Sigma Chemical Co, St Louis, MO, USA
Acid alcohol
Amino alcohol
Merck Co (Darmstadt’s Germany)
Aspirin Reckitt Benckiser Pakistan
Ascorbic acid China fooding Ltd. China.
Acetyl choline esterase
Acetyl thiocholine iodide
Sigma Chemical Co, USA
Brewer’s yeast Vahine Professional, France
Bismith nitrate Sigma Chemical Co,
St Louis, MO, USA
Chloroform Merck Co (Darmstadt Germany
Carrageenan Sigma Chemical Co, St Louis, MO, USA
Ceric sulphate Merck, Darmstadt, Germany
Diclofenac sodium Sigma Chemical Co, St Louis, MO, USA
Dragendorff's reagent Searle pharmaceuticals Pakistan Limited
DPPH Merck Millipore Corporation, Germany
5,5 dithio-bis-nitro benzoic acid Sigma Chemical Co,
St Louis, MO, USA
Ethyl acetate
Eosin
Ethyl alcohol Merck Co Darmstadt Ltd., Germany
Formalin Sigma Chemical Co,
St Louis, MO, USA
Folin & Ciocalteu’s Merck, Millipore Corporation, Germany
Galanthamine
Hematoxyline Merck, Darmstadt, Germany
H2SO4 Sigma Chemical Co,
St Louis, MO, USA
Imipenem Sigma Chemical Co,
St Louis, MO, USA
Ibuprofen Munawar pharma (pvt) Ltd. Pakistan.
Methanol
n-Butanol
n-Hexane
n-propylgallate
Merck Co (Darmstadt Germany)
Normal saline Santa Cruz Biotech, USA
Nutrient agar Sigma Chemical Co,
St Louis, MO, USA
Paracetamol Alfa Aesar - A Johnson Matthey Company
Paraffin China Shengtong petrochemical co. Ltd. China.
Phosphate buffer Santa Cruz Biotech, USA
Potassium Bromide
Potassium Iodide
Sigma Chemical Co,
St Louis, MO, USA
Riboflavin Alibaba Ltd. Pakistan.
Silica gel Sigma Chemical Co, St Louis, MO, USA
Silymarin Hisunny Chemicals, China
Sod. Carbonate
Sod. Pentobarbital Merck, Darmstadt, Germany
Sulfuric acid Sigma Chemical Co,
St Louis, MO, USA
Tramadol Searle products Ltd. Pakistan
Xylene Sigma Chemical Co,
St Louis, MO, USA
Chapter – 2 EXPERIMENTAL
37
2.7 PLANT MATERIALS
The plant collection was done from District Shangla, (Village, Puran) Khyber
Pakhtunkhwa, Pakistan in the month of April, 2011. In order not to affect the flora of
the area, collection was done with the permission of Swat forest officer. Plant
specimen was identified by Professor Dr. Mohammad Ibrar, (Taxonomist)
Department of Botany, University of Peshawar. The plant specimen was deposited
with voucher number Bot. 20158 (PUP) kept in the herbarium of the same
Department. The whole plant (13 kg) was collected and shade dried at ambient
temperature.
2.7.1 Extraction and Fractionation
The plant was shade dried powdered (10 kg) and soaked in 90% organic solvent
methanol (25 L) for 10 days at a temperature of 25-30oC. The soaked plant was
vigorously stirred twice daily (morning and evening). After each three days and
finally after four days, colorless thin cloth was used to filter the aqueous-methanol
soluble residues which was filtered finally by Whatmann filter paper No. 1. After
each filtration the residue was soaked in the said solvents till the day 10. Through
rotary evaporator (Model: R-210, Buchi, Switzerland) the filtrate was dried by using a
heating bath (B-491) at 40-45oC fitted along with a re-circulating chiller (NESLAB
instruments). The total of 1.57 kg crude methanolic extract was obtained. 30 g of the
crude extract was separated for various biological activities whereas, the remaining
extract was fractionated by using a separating funnel of capacity 5 L. The crude
extract (1.37 kg) was dissolved in distilled water (1 L) and transferred into a
separating funnel along with 1.5 L n-hexane and shaken vigorously. Separating funnel
was kept on stand till the appearance of immiscible layers in which n-hexane
accumulated as an upper layer.
Chapter – 2 EXPERIMENTAL
38
The procedure was repeated three times. The n-hexane soluble layer collected was
concentrated at low temperature (40-45oC) under reduced pressure. The n-hexane
fraction collected was 706 g. Chloroform was then added to the layer separated from
n-hexane layer in the separating funnel, vigorously shaken and kept for separating the
mixture into layers. As chloroform is a denser solvent so its fraction is collected as a
lower layer. The mixture (upper layer) was conducted for the acquisition of further
fractions which were carried out three times. The fraction of chloroform was collected
and concentrated under vacuum resulting into a semi solid mass of chloroform soluble
fraction (17 g). The same procedure was followed for the fractions of ethyl acetate
and n-butanol thus ethyl acetate (22.7 g), n-butanol (35 g) were finally obtained as
solid masses. The finally left fraction, after recovering the above mentioned soluble
fraction was concentrated and recovered as an aqueous soluble fraction (45 g). The
crude methanolic extract was subjected along with its five subsequent fractions for
isolation and different phytochemical and pharmacological activities.
Chapter – 2 EXPERIMENTAL
39
This whole process is presented in Figure 2.1.
Figure 2.1: Scheme of plant extraction and fractionation
Chapter – 2 EXPERIMENTAL
40
2.7.2 Isolation and Purification
The ethyl acetate (65 g) soluble fraction was subjected to fractions by means of VLC
over silica gel (1300 g). The elution was started from n-hexane, followed by
increasing polarity of n-hexane-chloroform gradients. Finally the column exhausted
by gradual increased in polarity of the mobile phase with methanol-chloroform (20%)
gradient that afforded five sub-fractions (FMC1-FMC5). Sub fraction FMC3 (100 mg),
was used in column chromatography on silica gel repeatedly and eluting with ethyl
acetate-n-hexane (20%). Finally, the compounds were separated through preparative
TLC, and checked for purity using TLC, yielded commulin-A (1), commulin-B (2)
and tectochrysine (4). Similarly, further purification of the sub-fraction FMC-5 was
done by using column chromatography on silica gel repeatedly by treatment with
ethyl acetate-n-hexane (30%) resulted in the isolation of pure compounds, Commulin-
C (3), sideroxylin (5) and (cearoin) (6).
Chapter – 2 EXPERIMENTAL
41
.
using Silica gel (1300 g),
Et-Acetate fraction
VS (65 g)
RFCC, 3:7 Et.acetate:hexaneRepeated Flash Column Chromatography (RFCC), 2:8, Et-acetate:Hex
commulin-B
tectochrysine
FMC1 FMC2 FMC3
commulin-A
FMC4 FMC5
Commulin-C sideroxylin
cearoin
Figure 2.2: Scheme representing the isolation of pure compounds using Ethyl acetate
fraction.
Chapter – 2 EXPERIMENTAL
42
2.8 EXPERIMENTAL DATA OF NEW COMPOUNDS FROM VIOLA
SERPENS
2.8.1 Commulin-A (1)
Physical State: Yellowish amorphous powder
Yield: 17.5 mg
Melting Point: 138-140oC
Solvent system used: Ethyl acetate and n-hexane (2:8)
Solubility: At room temperature soluble in methanol
[α] 30D : + 375.0o (c = 0.8, CHCl3)
UV activity: UV visible on TLC
IR max cm-1: 3450 (OH), 2968 1591, and 1463 cm-1 (aromatic CH), 1719
(saturated ketone), 1250-1383 (C - C)
EI-MS m/z: 298.084 [M]+ (C17H14O5, calcd.298.2940)
EI-MS m/z (Peak %): 298 (80), 267 (65), 250 (35), 235 (20), 218 (10), 141(6).
1H & 13C-NMR (300 & 100 MHz CDCl3): Details mentioned in
the Table 3.18.
2.8.2 Commulin- B (2)
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
[α] 30D : -166.7o (c = 1.2, CHCl3)
UV activity: UV visible on TLC
IR max cm-1: 3422 (OH), 2968 (aromatic CH), 1719 (saturated ketone), 1601
(aryl), 1659 (C = C), 1250-1383 (C - C)
EI-MS m/z: 314.068 [M]+ (C17H14O6, calcd. 314.2930)
EI-MS: m/z (Peak %): 314.068 (79), 283.06 (55), 266.06 (30), 251.03 (19),
234.03(11), 17.00 (8), 76.03 (5)
1H & 13C-NMR (300 & 100 MHz CDCl3): Details mentioned in the
Table 3.19
Chapter – 2 EXPERIMENTAL
43
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
[α]30D: + 13.0o (c = 1.5, CHCl3)
UV activity: UV visible on TLC
IR max cm-1: 3560 (OH), 2968 (aromatic CH), 1717 (saturated ketone), 1628
(aryl)
FAB-MS m/z: 328.081 [M]+ (C18H16O6, calcd 328.32)
EI-MS: m/z (Peak %): 328.081 (89), 297.08 (64), 280.07 (35), 265.05 (21), 248.04
(17), 218.04 (9), 77.04 (5).
1H & 13C-NMR (300 & 100 MHz CDCl3): Details mentioned in the
Table 3.20
2.9 EXPERIMENTAL DATA OF KNOWN COMPOUNDS FROM VIOLA
SERPENS
2.9.1 5-Hydroxy-7-methoxy flavone (tectochrysine) (4)
Physical State: Colorless Solid
Yield: 13.1 mg
Melting Point: 162-168 oC
Solvent system used: Ethyl acetate and n-Hexane (3:7)
Solubility: At room temperature soluble in methanol
IR max cm-1: 3400, 3000, 1649, 1475, and 1460 cm-1
HR-EIMS m/z: 268.2013 [M]+ (calcd. for C16H12O4, 268.2011)
1H-NMR (500 MHz, CDCl3): δ 7.87 (1H, m, H-2′), 7.52 (1H, m, H-6′ & H-3′,), 6.65
(1H, s, H-3), 6.48 (1H, J = 2.2, H-6) δ 6.39 (1H, d, J = 2.2 Hz,
H-8), 3.94 (3H, s, OCH3)
Chapter – 2 EXPERIMENTAL
44
2.9.2 4́, 5-Dihydroxy-7-methoxy-6, 8-dimethylflavone (Sideroxylin) (5)
Physical State: Yellow needles
Yield: 8.4 mg
Melting Point: 565.5°C
Solvent system used Ethyl acetate and n-Hexane (3:7)
Solubility: At room temperature soluble in methanol
IR max cm-1: 3500 and 1655 cm-1 (hydroxyl and ketonic)
HR-EIMS m/z: 312.0125 (calcd. for C18H16O5, 312.0123)
1H-NMR (500 MHz, CDCl3): δ 13.07 (1H, s, OH-5), 7.97 (1H, d, J = 8.8 Hz, H-2 &
H-6′), 6.87 (1H, s, H-3), 3.94 (3H, s, OCH3), 2.32 (3H, s, -CH3
ring A), 2.08 (3H, s, -CH3 ring A)
2.9.3 5-Dihydroxy-4-methoxybenzophenone (Cearoin) (6)
Physical state: Yellow amorphous powder
Yield: 12.3 mg
Melting Point: 182-189 C
Solvent system used: Ethyl acetate and n-Hexane (3:7)
Solubility: At room temperature soluble in methanol
IR max cm-1: 3448 (OH), 1743 (C = O) cm-1
HR-EIMS m/z: 244.1322 [M+1]+ (calcd. for C14H12O4, 244.1334)
1H-NMR (500 MHz, CDCl3): δ 11.95 (1H, s, OH-2), 8.89 (1H, s, OH-5), 7.61, 7.54
(m, aromatic protons, ring B), 6.88 (1H, s, H-6), 6.59 (1H, s, H-
3), 3.48 (3H, s, OCH3
.
Chapter – 2 EXPERIMENTAL
45
2.10 IN-VIVO BIOLOGICAL ACTIVITIES
Different in-vivo biological activities of the crude methanolic extract/fractions (whole
plant) were performed by using different protocols.
2.10.1 Experimental Animals
BALB/C mice of both sexes, male and female were employed for the bio-assays. The
mice were bought from NIH (National Institution of Health) Islamabad, bred in the
animal house, Department of Pharmacy, University of Malakand. The animals were
kept at standard laboratory formula of 25oC temperature and at 12/12 h light/dark
cycle with free access to the food and water. Rules of the ethical committee were
followed before and after the experiments. Food and health, guidelines of the mice
were adjusted throughout the experiments according to the rules provided by the
institute of laboratory animal resources, Commission on life sciences, National
Research Council.
2.10.2 Acute Toxicity
The study was performed on crude methanolic extract (whole plant) at various doses
ranging from 1-2 g/kg body weight. The animals (mice) were uniformly grouped into
three, each of which contained six mice. The negative control group was treated with
distilled water (10 ml/kg dose) and the two remaining groups were given doses of the
crude methanolic extract (1mg/kg and 2mg/kg body weight). After the test doses
administration, 24 h observations of the animals were done. Observations of the first 4
h of the animals were for the effect of acute toxicity. Death/s, if occurred is identified
after 24 h (Araujo et al., 2014).
Chapter – 2 EXPERIMENTAL
46
2.10.3 Analgesic Activity
Two different protocols were used to determine the mechanism(s) of the anti-
nociceptive effect of the plant V. serpens crude methanolic extract/ fractions.
2.10.3.1 Acetic Acid Induced Writhing
Crude methanolic extract and fractions of the whole plant were screened for analgesic
activity. BALB/C mice of both the sexes with body weights ranged from 18-22 gms
body weights. The animals were categorized into fourteen different groups (n=6).
Group I (negative control) and II (positive control) were treated with a dose of 10
ml/kg normal saline and 10 mg/kg Diclofenac sodium respectively. The supplied food
was withdrawn 2 h before starting the activity (Koster et al., 1959; Adzu et al., 2001;
Khan et al., 2010). Different fractions (n-hexane, Chloroform, Ethyl acetate, and
aqueous) along with the crude methanolic extract were administered to the remaining
groups, III to XIV in three different doses i.e. 100, 200 and 300 mg/kg body weight.
After 30 min of the previous treatments all the groups were administered equally with
1% acetic acid (i.p). After 5 min of acetic acid injection counting of the abdominal
writhes (constrictions) for 10 min duration was done (Collier et al., 1968). The
percentage of analgesic activity was calculated according to the designed formula
expressed below.
% Analgesic effect = 100 – No of Writhes in the Test Animals x 100
No of Writhes in Control Animal
2.10.3.2 Formalin Test
The formalin test was conducted according to the method of the previous study (Liu et
al., 2007). BALB/C mice of both the sexes (male and female) were selected in the
range of 18-22 g body weights. The animals were categorized into seven groups each
having six animals (n=6). Pains were induced in animals by injecting in the right hind
Chapter – 2 EXPERIMENTAL
47
paw 0.05ml of 2.5% formalin (40% formaldehyde i.p). The Group I (control) and the
Group II (standard) received normal saline and standard drug (Diclofenac sodium) in
the dose of 10 mg/kg (i.p) respectively. Different fractions (n-hexane, chloroform,
ethyl acetate, and aqueous) along with the crude methanolic extract were administered
to the reset of the groups, III –XIV at doses of 100, 200 and 300 mg/kg (body weight
p.o) 60 min prior to the formalin injections. The pains indicators were the time spent
with responses of licking and biting of the injected paw. Measurement of responses
were done in two phases after injecting the formalin doses i.e for first 5 min (early
phase) and then the next 20 - 30 min (late phase).
2.10.4 Anti-inflammatory Activity
Crude methanolic extract along with different fractions were screened for anti-
inflammatory effect. The action of crude extract of the plant and its different fractions
were determined using three different protocols in order to make clear the mechanism
involved behind anti- inflammatory activity of the plant.
2.10.4.1 Carrageenan Induced Paw Edema
Crude methanolic extract along with its different fractions were incorporated for
judgment of anti-inflammatory effect in the plant. BALB/C mice of both sexes of
body weight 25-30 g were selected and divided into fourteen groups. Each group
included 6 mice (n=6). Group I and II were used as negative and positive control
respectively. Group I animals were treated with normal saline (10 ml/kg body weight)
whereas, the group II animals were treated with diclofenac sodium at a dose of 10
mg/kg body weight. The rest of the groups (III-XIV) were treated with the crude
methanolic extract and its various fractions (n-hexane, chloroform, ethyl acetate, and
aqueous), at different doses of 100, 200 and 300 mg/kg (body weight) respectively.
Chapter – 2 EXPERIMENTAL
48
Each mouse after 30 min of treatment of the test samples dose, was treated with sub-
planter injection in right hind paw with 1% carrageenan. Anti-inflammatory effect
was measured with the help of plethysmometer (LE 7500 plan lab S.L) for 5 h i.e. at
0, 1st , 2nd , 3rd , 4th and 5th h (Collier et al., 1968). The below mentioned formula was
used for calculating the percent inhibition for edema.
% Inhibition = A–B / B x100
Where A and B represents edema volume of negative control, paw edema of tested
groups respectively.
2.10.4.2 Histamine Induced Paw Edema
The trial was adopted according to authentic protocol (Amann et al., 1995). The test
sample indomethacin and distilled water at doses of 10 mg/kg and 10 ml/kg
respectively were administered orally. Histamine (0.1 ml) was administered as sub-
plantar injection to right hand paw tissues after one hour to the test samples treatment.
After the histamine injection the paw thickness measurement was done for 3 h at a
regular interval of 30 min each. The % inhibition was calculated by using the formula.
Inhibition (%) = 100 x Value of Control Group – Value of the Test Sample
Value of Control Group
2.10.4.3 Xylene Induced Ear Edema
The xylene induced ear edema test was conducted by following the authentic
protocols (Dai et al., 1995; Amin et al., 2012). The positive- control group of mice
(BALB/C of both sex 25-30g body weights) was administered orally with Ibuprofen
(100 mg/kg). Plant crude extract and the fractions at doses of 100, 200 and 300 mg/kg
were used (p.o). The test animal after an hour, received 20 μl (0.02 mL) of xylene on
the right ear lobe at both posterior and anterior surfaces. The lobe of the left ear was
measured as control ones. Cervical dislocations of the treated mice were done after
Chapter – 2 EXPERIMENTAL
49
one hour of the xylene injection. Using a cork borer circular ear section of 3 mm
diameter were taken from each ear of the killed mice and weighted. The ear edema
was calculated by taking out the percentage by comparing the weights of untreated
left ear with the treated one.
Inhibition (%) = 100 x Value of Controlled Group – Value of the Tested Sample
Value of Controlled Group
2.10.5 Larvicidal Bioassay
Crude extract of the whole plant along with its various fractions were tested for
larvicidal bioassay (Ikram et al., 2012). Larvae were collected from ponds water in a
plastic jar and kept in the laboratory conditions. The crude extract and its subsequent
fractions (n-hexane, chloroform, ethyl acetate, and aqueous) were subjected to 10,000
ppm stock solutions in distilled water. From the stock solution each 100 ml of 2000,
1500, 1000, 500, 100 and 50 ppm dilutions in 500 ml plastic jars were prepared
separately. Each jar was accordingly labeled and fed with 25 active larvae. Wide
mouthed glass dropper was used to transfer the calculated number of larvae to the
labeled plastic jar. Each jar, including the controlled one was also provided with a diet
of finely ground dog biscuits and brewer’s yeast in 2:3 ratios. The adjusted laboratory
conditions were 30 ± 2°C temperature and 70-75 relative humidity. After 24 h of
exposure, dead larvae numbers were counted. Cervical or siphon region was used for
the identification of dead larvae, as they fail to move after being prodded. The
experiment was repeated five times. Number of dead and live larvae (dead and live if
present) was identified and the species were confirmed.
Formula given below was used for Percentage mortality determination:
% Mortality = 100 – Number of Living Larvae in Test Sample x 100
Number of Living Larvae in Control
Chapter – 2 EXPERIMENTAL
50
2.10.6 Nephroprotective and Hepatoprotective Activities
2.10.6.1 Animals Used
Sixty domestic local mature rabbits (Oryctolaguscuniculus) of both sexes were
purchased from local market. They were kept in a well ventilated and wide chambered
animal house at the University of Malakand, KP, Pakistan. The rabbits were fed on
chaw pellets along with fresh green vegetables, grasses and open access to fresh water
at libitum. Acclimatization of the animals was done for at least two weeks before
commencement of experiment.
2.10.6.2 Animals Grouping and Dosing
The rabbits were grouped into fifteen groups for eight days protocol (Ikram et al.,
2012; Gulati et al., 2012). Four rabbits were kept in each group. Two doses low (150
mg/kg) and high (300 mg/kg) were tested for each extract and fraction. Each group
was tagged separately for the purpose of identification. Group 1, administered with
normal saline, served as normal control, group 2 was treated with paracetamol only
(controlled group); group 3 served as standard control which was treated with
paracetamol from the first day followed by silymarin, a well-known standard
hepatoprotective drug. Groups 4, 5 received paracetamol followed by crude methnolic
extract at doses of 150 and 300 mg/kg body weight. The groups 6, 7 received
paracetamol followed by n-hexane fraction at doses of 150 and 300 mg/kg body
weight, groups 8, 9 received paracetamol followed by the fraction of ethyl acetate
(150 and 300 mg/kg). Group 10, 11 received paracetamol followed by the fraction of
chloroform at doses of 150 and 300 mg/kg body weight whereas, groups 12,13
received paracetamol followed by the fraction of n-butanol at doses of 150 and 300
mg/kg body weight. The groups 14 and 15 were treated with paracetamol followed by
Chapter – 2 EXPERIMENTAL
51
aqueous fraction with same doses. The doses’ details were: paracetamol 1 g /kg body
weight (Sasidharan, 2012), silymarin 50 mg/kg body weight (Bak et al., 2012).
2.10.6.3 Chemicals used
ALT, AST, ALP and the serum levels were estimated by using commercially
available kits (purchased from AMP Diagnostics, Austria) on a UV visible light
spectrophotometer (Agilent 8453) and silymarin.
2.10.6.4 Histopathology
Examination of the dissected rabbit’s tissue from kidney and liver were collected and
stored in 10 % formalin solution. Standard protocol was adopted for processing the
samples (Bancroft and Gamble (2007).
Procedure for Histopathology
One centimeter of the kidney and liver were cut for tissue processing. After washing
with running tap water the samples were threaded and placed in water. The tissues
were washed in such a manner that they could not be damaged and washing was
continued overnight. Automatic processor of tissues (Tissue-Tek® Sakura, Japan) was
used for placing the tissues in ascending grade of alcohol for dehydration with
decreasing time period. Alcohols of various grades were used for tissues to be placed
for definite time as follows;
Dehydration
30% alcohol 3-4 hrs
50% alcohol 2 hrs
70% alcohol 2hrs
80% alcohol 1.5 hrs
95% alcohol 1.5 hrs
Absolute alcohol I 1 hr
Absolute alcohol II 1 hr
Chapter – 2 EXPERIMENTAL
52
Clearing
Alcohol with Xylene 45 min
Xylene I 30 min
Xylene II 15 min
Impregnation
Paraffin, melted at 72ºC was used for tissue samples impregnation.
Paraffin I 2 hrs
Paraffin II 2hrs
Embedding
Blocks were made after tissues processing. Automatic tissue embedding assembly
(Tissue-Tek® TEC™ Sakura) was used for tissue blocks preparation. Tissues placed in
plastic cassettes were poured with molten paraffin for the preparation of blocks. Cold
plate of tissue blocks were shifted and allowed to dry.
Sectioning
By Microtome (Accu-Cut® SRM™ 200 Sakura) tissue blocks were sectioned, with
about 4-5 µm thickness. The folds were removed at 56oC by placing the obtained
sections in water bath (Sakura) which floated over the water surface. On slides
albumin was applied for proper cleaning and sticking of the sections to slides.
Sections were mounted over the slides and dried by keeping in oven (Daihan Lab
Tech Co., ltd) for 3-4 h.
Staining
Slides after final drying were placed for staining. Hematoxylin and Eosin (H & E)
staining of slides sections and automatic slide stainer (Tissue-Tek® DRS™ 2000
Sakura, Japan) was used. Standard staining protocol was followed;
Chapter – 2 EXPERIMENTAL
53
Removal of Paraffin
Reagents Time period
Xylene 3 min
Xylene 3 min
Xylene 3 min
Removal of Xylene with Alcohol
Ethyl alcohol 100% 1 min
Ethyl alcohol 100% 1.30 min
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
Chapter – 2 EXPERIMENTAL
54
Clearing
Xylene 1.30 min
Xylene 1 min
Xylene 1.30 min
Mounting of Cover Slip
Slides were cleaned properly after completion of staining process. DPX (Scharlau)
pouring and covering was done with great caution so as to avoid formation of bubbles
and form clear and neat slides.
2.10.6.5 Hematological and Serological profile of infected Rabbits
Blood samples were collected from rabbit in clean EDTA tubes. Serology required the
collection of 3 mL blood in tubes and allowed to clot. By centrifugation the blood for
10 min at 3000 rpm serum was collected in 1 mL Eppendorf tubes and was kept at
4°C until further use.
Serology
3 mL blood samples were collected in clean tubes, centrifuged for 10 min at 3000
rpm, 1mL Eppendorf tubes were used for serum separation. Serum glutamic pyruvate
transferase (SGPT), total serum proteins, albumin and globulin were measured by
using Biochemistry analyzer (PS-520 SHENZHEN PROCAN ELECTRONICS,
CHINA).
Serum Glutamic Pyruvate Transferase (SGPT)
Reagents R1 and R2 are two reagents in kit (Reactivos, GPL Barcelona, Spain) for
estimation of serum glutamic pyruvate transferase (SGPT). As per manufacturer
instruction solution was prepared by mixing 4 volumes of R1 and 1 volume of R2.
Chapter – 2 EXPERIMENTAL
55
1mL of solution was then mixed with 100 µL of serum sample, incubated at 37 ºC for
1 min. In Automatic Biochemistry analyzer SGPT activity sample was loaded.
Total Serum Protein (TSP)
Estimation of total serum protein was done by using Kit (Reactivos, GPL Barcelona,
Spain) which contains a calibrator and reagent (R). Reading was obtained by mixing a
reagent (R) 1 mol with 25 µL of calibrator. Reagent (R) (1mL) was then mixed with
25 µL of serum sample and incubated at 37 ºC for 5 min. The sample was loaded in
an Automatic Biochemistry analyzer and result was recorded.
Serum Albumin
Estimation of serum albumin was used by using kit (Reactive, GPL Barcelona, Spain)
containing reagent (R) and a calibrator. Reading was obtained by using reagent (R) 1
mol mixed with 5 µL of calibrator. By incubation at 37 ºC for 5 min, 1mL of reagent
(R) was mixed with 5 µL of serum sample. The sample was loaded in an Automatic
Biochemistry analyzer and the result was recorded.
Serum isolation and assessment of some liver related serum enzymes and
kidney parameters
This is an 8 day protocol. On the 9th day the animals were dissected. Before twelve
hours from dissection, food was withdrawn and then anesthetized by chloroform
inhalation. Directly after dissection, blood was directly drawn with 21 Gauge needle
in 3 mL syringes from the heart chambers by cardiac puncture (Illahi et al., 2012).
The blood samples for haematological analysis were collected into EDTA (Ethylene
Diamine Tetra-acetic Acid-coated) tubes (K2-EDTA) with coagulant and kept at room
temperature for 1 hour. By centrifugation the blood for 5 min at 3000 rpm serum was
harvested and collected in Eppendorf (5702R, Germany) tubes and kept at –20˚C until
Chapter – 2 EXPERIMENTAL
56
analyzed. The analyzed biochemical markers for hepatotoxicity were aspartate amino
transferase (AST), enzymatic activities of serum alanine amino transferase (ALT) and
alkaline phosphatase (ALP). Serum urea and creatinine tests were done for
nephrotoxicity. They were conducted as:
Determination of Glomerular Filtration Rate
The urea and creatinine clearance tests were used to estimate the glomerular filtration
rate.
Urea Clearance
The following formula was used for the urea clearance test:
GFR = [Urine urea x Urine volume]/Serum urea.
Creatinine clearance
The following formula was used for the creatinine clearance test:
GFR = [Serum creatinine x Urine volume]/Serum creatinine.
2.10.6.6 Statistical Analysis
One way ANOVA, Tukey Test of Post Hoc was applied on means of the data and
analyzed by using computer software SPSS 16.0.
2.10.6.7 Collection and analysis of urine
On the 9th day all the animals for collection of urine samples were kept in individual
cages. 24 h urine samples were collected. During this period the animals had free
access to drinking water. In graduated cylinder 24 h total urine volume in mL of each
rabbit was measured. The sample was analyzed for urinary creatinine and urinary urea
after storage at 4oC for one day. The parameters were estimated through COBAS
chemistry automation using Roche Diagnostic kits.
Chapter – 2 EXPERIMENTAL
57
2.11 IN VITRO BIOLOGICAL ACTIVITIES
The crude aqueous methanolic extract along with the various fractions were subjected
for various in vitro biological activities of the whole parts of the V. serpens.
2.11.1 Anti-oxidant Activity
2.11.1.1 Superoxide Anion Radical Scavenging Assay
The activity was based on a system of riboflavin- light-NBT (Beauchamp and
Fridovich, 1971). The reaction mixture contains 0.5 mol of phosphate buffer (50 mM,
pH 7.6), 0.25 mol PMS (20 mM), 0.3 mol riboflavin (50 mM) and 0.1 mol NBT (0.5
mM), before1 mol sample solution addition. Start of reaction was taken by using
fluorescent lamp for illuminating the reaction mixture with different concentrations of
the methanolic extract.
Keeping ascorbic acid as a standard at 560 nm wavelength, the absorbance was
measured by incubating for a period of 20 min.
The following formula was used for calculating the generated superoxide anions.
Hydrogen Peroxide Scavenging Activity = (1-Absorbance of the Sample) x 100
Absorbance of Control
2.11.1.2 DPPH Radical Scavenging Activity
The antioxidant activity ( in vitro) of the crude methanolic extract and its fraction i.e
Ethyl acetate, chloroform, butanol and aqueous fractions were evaluated by using
DPPH (2, 2-diphenyl-1-picryl-hydrazyl) scavenging assay established procedures
(Bursal and Gulcin, 2011). In methanol 500 ppm of each extract was prepared from
25 mol stock solution. From each extract in separate test tubes the stock solution of a
5 mol solution each of 0, 20, 40, 60, 80 and 100 ppm was prepared. Triplicate of each
concentration was taken. Ascorbic acid was taken as a standard chemical. The same
procedure was repeated thrice. To each test tube DPPH of 1 mol was added to each
Chapter – 2 EXPERIMENTAL
58
test tube. A control was set by the addition of 1 mol of DPPH to 5 mol of methanol in
a test tube. For 30 min in dark and at room temperature, the test tubes were incubated
and then the absorbance of each extract and fractions, standard and control was
measured at 517 nm by using UV spectrophotometer (1700 Shimadzu Japan). There is
inverse proportionality between the scavenging effect of the free radical and the
absorbance of the reaction mixture. DPPH free radical percent scavenging effect was
expressed as the antioxidant activity of the extracts and the standard. The formula
used was as follow:
Percent Radical Scavenging Activity = (Ac – As / Ac) × 100
Where; Ac is the absorbance of control As is the absorbance of sample.
Chemicals Used
Diphyneyl-1-picryl-hydrazyl (DPPH), ascorbic acid, Folin-Ciocalteu, sodium
carbonate (Na2CO3) and sodium pentabarbital were purchased from Sigma Co.
(USA). Methanol, n-Hexane, ethyl acetate, chloroform and n-butanol used for plant
extraction were of analytical grade and were purchased from Merck Co. (Darmstadt,
Germany).
2.11.2 Antibacterial Assay
All the isolated compounds were screened against the various strains of bacteria
including: Bacillus subtilis Escherichia coli, Pseudomonas aeruginosa, Salmonella
typhi, Staphylococcus aureus and Shigella flexneri. Antibacterial screening was done
by dissolving 3 mg of sample in 3 mol DMSO. Nutrient agar media in the molten
state (45 ml) was poured into sterilized Petri dishes and let for solidifying. Soft agar
(sterile) having 100 mol of test-organisms culture were poured in the wells bored at
certain distance through the 6-mm metallic sterile borer. Then each well was poured
Chapter – 2 EXPERIMENTAL
59
with 100 L of the test sample and incubated at a temperature of 37ºC for 24 h. The
measurements of zones of inhibition give the test results. The broad spectrum
antibiotic, Imipenem used as a positive control drug. DMSO was used as a negative
control drug (Imran et al., 2014; Boyanova et al., 2005).
2.12 ENZYME INHIBITION
2.12.1 Chemicals Required for Anticholine Esterase
Phosphate buffer (pH 8), acetyl choline esterase, acetyl thiocholine iodide, 5, 5 dithio-
bis-nitro benzoic acid (DTNB), galanthamine (standard drug).
2.12.2 Acetylcholinesterase Inhibition
Acetylcholinesterase enzymatic activity was measured using an authentic protocol
(Ferreira A et al, 2006) 98 lL (50 mM) Tris–HCl buffer pH 8, 30/L sample and 7.5 lL
acetylcholinesterase solutions containing 0.26 U ml _1 were mixed well in a plate of
ELISA and incubate for 15 min. Subsequently, 22.5 /L of AchI 0.023 mg ml_1 and
142 lL of (3 mM) DTNB were added. At equilibrium point of the reaction the
absorbance at 405 nm was noted. Water was used as a control in the reaction in place
of the extract/ fractions. The 100% activity was obtained from the value of
absorbance. The % Inhibition was calculated using the formula mentioned below:
Inhibition (%) = 100 x Value of Controlled Group – Value of the tested Sample
Value of Controlled Group
Tests were conducted three times and Tris–HCl was used as a blank with buffer as a
substitute for the enzyme solution. The percentage of inhibition was plotted against
the concentration of the extract solution in order to obtain the value of 50% inhibition
(IC50).
Chapter – 3 RESULTS & DISCUSSION
61
CHAPTER – 3
3. RESULTS & DISCUSSION
3.1 BIOLOGICAL ACTIVITIES
3.1.1 In-vitro Biological Activities
3.1.1.1 Antimicrobial Activity
Antibacterial Activity of the Crude Extract and Subsequent Solvent Fractions of
V. serpens
The antibacterial effect of crude extract/fractions of V. serpens is presented in Table
3.1 and 3.2. The crude extract, chloroform and ethyl acetate soluble fractions of the
plant showed significant effects against all the tested bacteria whereas, the n-hexane
and aqueous soluble fractions did not show significant effects. The imipenem, a broad
spectrum antibiotic was used as a standard drug which showed marked effect against
all the tested bacteria. Maximum zones of inhibition were shown by the soluble
fraction of chloroform followed by ethyl acetate and the crude extract. Chloroform
soluble fraction showed maximum zone of inhibition (16 mm) against S. typhi
followed by ethyl acetate soluble fraction (13 mm), crude extract (10 mm) and the
aqueous soluble fraction (5 mm). The maximum activity against E. coli was observed
for the chloroform soluble fraction followed by ethyl acetate with 18 and 14 mm
zones of inhibition respectively. B. subtilis was most susceptible to the chloroform
soluble fraction having a value of 16 mm as its zone of inhibition. Both the
chloroform and the aqueous soluble fractions showed sensitivity against S. flexeneri
with the zone of inhibition of 8 mm. The n-hexane soluble fraction did not show any
effect against S. aureus. The aqueous soluble fraction followed by the crude extract
showed antibacterial activity against P. aeruginosa with the values of 6 and 5 mm
respectively as zones of inhibition. The results clearly demonstrate that the crude
Chapter – 3 RESULTS & DISCUSSION
62
extract of the plant as well as various fractions possessed significant antibacterial
effect.
Similarly, the antibacterial effect of the isolated compounds commulin-A (1),
commulin-B (2), tectochrysine (4), sideroxylin (5) and 2, 5-dihydroxy-4-
methoxybenzophenone (6) (cearoin)} is depicted in Table 3.2. All of the isolated
compounds except compound 3 (due to insufficient quantity) were screened against
Gram positive and Gram negative strains of bacteria (S. aureus, B. subtilis, E. coli, P.
aeruginosa, S. flexneri and S. typhi). The results showed that all the pure compounds
exhibited significant antibacterial effects against most of the selected bacteria when
compared with the standard broad spectrum antibiotic, imipenem. The maximum zone
of inhibition (18 mm) was shown against S. typhi, by sideroxylin followed by
commulin-B, tectochrysine and cearoin having the same zones of inhibition (17 mm).
When studied against P. aeruginosa, maximum inhibition was caused by the
compounds, tectochrysine (17 mm) and cearoin (17 mm) followed by the compounds
sideroxylin (16 mm) and then commulin-B (13 mm). Similarly, when tested against S.
flexeneri, the maximum zone of inhibition was induced by tectochrysine (15 mm) and
cearoin (15 mm) followed by commulin-A, commulin-B (12 and 10 mm
respectively). Whereas, sideroxylin was found inactive against S. flexeneri.
Commulin-A, tectochrysine and cearoin possessed antimicrobial effect against S.
aureus (24 mm, 15 mm and 15 mm respectively) while commulin-B and cearoin were
found inactive. Compounds commulin-B and tectochrysine showed no significant
inhibitory effects against E. coli whereas, rest of the compounds were totally inert
against this particular bacterium.
Chapter – 3 RESULTS & DISCUSSION
63
The antibacterial activity of V. serpens against various bacteria including both Gram
positive and Gram negative showed that extract/fractions as well as the isolated
compounds possessed marked activity. Currently various microbes/pathogens have
become more resistant and have developed certain mechanisms for their protection.
For instance, causing mutation in their own genes or acquire genes from other bacteria
to resist the anti-metabolic actions of the antimicrobial drugs. E. coli, Shigella,
Salmonella, S. aureus and P. aeruginosa causing various bacterial infections
(Goosney, et al., 1999; Daniel et al., 2012; Bhattacharya, et al., 2012; Reygaert,
2013). Most of the antibiotics in clinical practice are resistant to most of the tested
pathogens so there is need for more effective antimicrobial drugs.
As the plant extract/fractions and its isolated compounds showed marked activity
against most of the tested pathogens, therefore, it can be assumed that the
extract/fractions as well as its isolated compounds could be useful natural alternatives
against the infections caused by these resistant bacteria. In this connection, further
detail studies are required to ascertain its therapeutic potential, safety and clinical
uses.
Chapter – 3 RESULTS & DISCUSSION
64
Table 3.1: Antimicrobial Activity of the Crude Extract along with the
Subsequent Fractions of Viola serpens.
Zone of Inhibition (mm)
Group Salmonella
typhi
Escherichia
coli
Bacillus
subtilis
Shigella
flexneri
Staphylococcus
aureus
Pseudomonas
aeruginosa
Imipenem 33 30 33 27 33 24
Crude
Extract
10 6 6 8 3 5
n-Hexane - 2 - 2 - -
Chloroform 21 18 16 5 2 -
Ethyl
acetate
13 14 7 8 3 -
Aqueous 5 5 - 5 2 6
Data are presented as mean ± SEM of three independent assays.
Table 3.2 Antimicrobial activity of the Isolated Compounds from V. serpens
Compound Zone of Inhibition (mm)
Staphylococcus
aureus
Bacillus
subtilis
Shigella
flexneri
Escherichia
coli
Pseudomonas
aeruginosa
Salmonella
typhi
Imipenem 33 33 27 30 24 25
1 (Commuline
A)
24 15 12 _ _ 15
2 (Commuline
B)
_ 15 10 10 13 17
4
(Tectochrysine)
15 16 15 11 17 17
5
(Sideroxyline)
15 12 _ _ 16 18
6
(Cearoin)
_ 15 15 _ 17 17
Data are presented as mean ± SEM of three independent assays.
Chapter – 3 RESULTS & DISCUSSION
65
Extract Hexane CHCl3 ETA H2O Imipenem
0
10
20
30
40 P.aerogenes
% i
nh
ib
itio
n
Extract Hexane CHCl3 ETA H2O imipenem
0
10
20
30
40S.flexeneri
% i
nh
ib
itio
n
Extract Hexane CHCl3 ETA H2O Imipenem
0
10
20
30
40S.typhi
% i
nh
ib
itio
n
Figures 3.1: % inhibition of the tested bacteria against the Crude extract/
fractions of V. serpens. Where CHCl3 represents Chloroform, ETA
represents Ethyl acetate and H2O represents the Aqueous fraction.
Extract Hexane CHCl3 ETA H2O Imipenem
0
10
20
30
40B. subtilis
% i
nh
ib
itio
n
Hexane CHCl H 2 O Imipenem
0
10
20
30
40 E. coli
% i
nh
ibit
ion
inh
ibit
ion
Extract ETA 3
Extract Hexane CHCl ETA H 2 O Imipenem
0
10
20
30
40 S.aureous
% I
nh
ibit
ion
Inh
ibit
ion
3
Chapter – 3 RESULTS & DISCUSSION
66
3.1.2 Effect of Crude extract/Fractions of V. serpens in DPPH free Radical
Scavenging Assay
The free radical scavenging effect of crude/fractions of V. serpens at various
concentrations is shown in Table 3.3. The crude extract caused concentration
dependent scavenging effect against DDPH with maximum activity of 67.99% at 500
ppm and IC50 value 182 ppm. Upon fractionation, considerable change in effect was
observed. Only the n-hexane and chloroform soluble fractions were significant with
the dose dependent scavenging activity of 75.98 and 79.00 % at 500 ppm having IC50
164 ppm and 144 ppm respectively.
The compound (1-6) isolated from the plant were also investigated for the radical
scavenging activity at different concentrations. The compounds antioxidant activity is
presented in the Table 3.4. Maximum radical scavenging activity was showed by
commulin-C (78.05 %) with an IC50 value 168 ppm followed by commulin-B (89.45
%) and IC50 value was 98.15 ppm. It was then followed by the tested pure compound
commulin-A having 78.05 % as the values of its radical scavenging activity with IC50
201 ppm.
The DPPH free radical scavenging assay is a simple, economical and most commonly
used for the assessment of test articles (Marinova and Blatchvarov, 2011; Anwar et
al., 2009). The scavenging effect is either by the loss of proton, radical’s dismutation
and formation of chelate by donating hydrogen, resulting in the stable phenoxyl
radicals (Das and Pereira, 1990; De Gaulejac et al., 1989; Hatano et al., 1989; Nahak
and Sahu, 2010; Illahi et al., 2013). Free radical generation or oxidative stress has
been observed in various diseases such as inflammation, coronary heart, diabetes,
aging and various types of cancer (Valko et al., 2007; Maaz et al., 2010). Abundant
Chapter – 3 RESULTS & DISCUSSION
67
existence of phenols and polyphenols in the plant species are the signs of antioxidant
property which works through various mechanisms. The presence of hydroxyl groups
is responsible for the chemical structure of phenolic compounds for free radical
scavenging activity (Anu et al., 2011; Siddharthan et al., 2007). As V. serpens
contained various phenolic compounds (Anu et al., 2011) was responsible for the
antioxidant activity of the plant. Moreover, the isolated flavonoids 1-6 from the ethyl
acetate soluble fraction of the plant also showed marked scavenging effect against
DPPH. The study provides a strong scientific background to use the plant in diseases
related to oxidative stress.
Chapter – 3 RESULTS & DISCUSSION
68
Table 3.3: DPPH Scavenging Activity of Crude extract/Fractions of V.serpens
and Zones of Inhibition are Given in mm.
Concentrations
(ppm)
Crude
extract
n-Hexane Chloroform Ethyl
acetate
Aqueous BHT
20 0.62±1.45 3.65±1.33 3.99±0.13 1.66±0.00 0.93±0.02 8.51±1.01
40 6.63±1.54 7.63±0.21 6.04±0.08 1.99±0.02 3.95±0.11 20.58±2.00
60 10.43±1.11 11.79±1.03 9.08±0.16 10.41±0.22 10.68±0.26 59.52±3.10
80 27.01±1.09 29.13±1.11 33.21±1.45 19.77±0.21 14.80±0.23 66.64±2.45
250 66.31±0.21 69.75±1.54 74.04±3.21 25.90±0.20 15.06±1.32 76.45±3.02
500 67.99±2.14 75.98±2.43 79.00±2.56 37.52±0.31 26.76±2.13 87.43±2.63
IC50 182±3.70 164±4.21 144.0±2.56 - - 54±2.15
Values are mean ± SEM of three independent readings. Control= Methanol, Standard
= BHT (Dibutylhydroxytoluene).
Table 3.4 Anti-oxidant Effects of the Isolated Compounds 1–6 from V.
serpens whole Plant
Compounds % RSA IC50 μM
1 (Commuline-A) 78.05 201±2.01
2 (Commuline-B) 89.45 98.15±1.26
3 (Commuline-C) 82.12 168±3.19
4 (Tectochrysin) NA NA
5 (Sideroxyline) NA NA
6 (Cearion) NA NA
n-Propyl gallate (standard) 90.13 106±1.45
Data is presented as mean ±SEM of three independent assays.
Chapter – 3 RESULTS & DISCUSSION
69
3.1.3 Effect of Crude Extract/ Fractions of V.serpens in Larvicidal Effect
The crude methanolic extract and fractions of V. serpens were investigated for
larvicidal activity against Aedes aegypti and Culex quinquefasciatus species of
mosquito (Tables 3.5). The crude extract showed dose dependent activity against A.
aegypti. The maximum effect (59.67 %) was showed by crude extract against the
larvae of A. aegypti at 600 ppm. Upon fractionation, changes in the overall activity
were observed. The maximum percent inhibition was caused by the ethyl acetate
(89.91%) fraction followed by the chloroform (85.21%) at the concentration of 600
ppm. The n-hexane and aqueous fractions showed insignificant effect against the
larvae A. aegypti at any concentration.
Similarly, the crude extract and various fractions of V. serpens were investigated
against larva of Culex quinquefasciatus at different dilutions (Table 3.5). The crude
methanolic extract was effective at a concentration of 600 ppm with percent mortality
51 and IC50 value 539 ppm. The mere significant effect was observed in the
chloroform fraction with maximum percent mortality at a concentration of 600 ppm
(53 %) with the IC50 value 500 ppm followed by the fraction of ethyl acetate. The
ethyl acetate fraction with LC50 value 510 ppm also showed more significant effect
(52.43%) at a concentration of 600 ppm. Rest of the fractions (n-hexane and aqueous)
showed no significant effects against C. quinquefasciatus.
Throughout, the world, insects born diseases are mainly responsible for serious
diseases which may lead to mortality (Pavela, 2009).Various serious diseases (Dengue
fever, malaria, Japanies encephalitis, yellow fever, angioedema and filariasis) are
mainly caused by mosquitoes which annually causes millions of deaths (Peng et al.,
1999). Various insecticidals available in the market with the names; Methoprene,
Chapter – 3 RESULTS & DISCUSSION
70
Temephos, Arosurf MSF, Agnique MMF (monomolecular films) Bonide, BVA2, and
Golden Bear-1111 (GB-1111) (oils) but all of these have some side effects either
direct on the human life/ aquatic animals/ environment (Larvicides for Mosquito
Control, United State Environmental protection agency 2000, 735-F-00-002). So there
was need for a safer natural larvicidal with no side effects. From the results it is clear
that the crude extract and the subsequent fractions of V. serpens showed significant
larvicidal activity against both the species of mosquitoes (A. aegypti and C.
quinquefasciatus). Phytochemicals like alkaloids, flavonoids, saponins, tannins and
steroids contribute mostly to the larvicidal activity (Pedro et al., 2014). V. serpens has
also been provided by nature with these phytochemical which may be responsible for
its strong larvicidal activity (Pratik et al., 2011). Further detailed studies in this
connection, are required for assuring the therapeutic potential, safety, economical
source and clinical uses of V. serpens.
Table 3.5: Larvicidal effect of the crude extract along with the subsequent
fractions of V. serpens against Aedes aegypti and Culex
quinquefasciatus specie of mosquitoes.
Extract/Fractions Aedes aegypti LC50
ppm Percent Mortility
10 ppm 100 ppm 200 ppm 400 ppm 600 ppm
Crude extract 33.13±1.21 37.88 47.69 55.19 59.67 325 ppm
n-Hexane 12.0±1.40 14.13 15.09 16.20 16.90 -
Chloroform 49.11±1.21 57.21 62.32 77.31 85.21 59 ppm
Ethyl acetate 43.21±1.03 59.19 64.32 72.34 89.91 88 ppm
n-Butanol 6.91±1.33 10.31 13.37 25.25 29.44 -
Aqueous 7.21±1.43 7.89 8.32 8.84 9.12 -
Culex quinquefasciatus
Percent Mortility
Crude extract 34.24 39.33 43.12 47.67 51 539
n-Hexane 10.10 12.70 14.56 16.89 19 -
Chloroform 22.61 31.05 44.51 49.15 53 500
Ethyl acetate 20.16 30.98 34.88 46.18 52.43 510
Aqueous 13.10 16.14 18.36 20.05 22 -
Data is presented as mean ±SEM of three independent assays.
Chapter – 3 RESULTS & DISCUSSION
71
3.1.4 Effect of Crude Extract/ Fractions of V.serpens in Acetyl Cholinesterase
Assay
The effects of crude extract/fractions of V. serpens against acetylcholine esterase are
presented in the Table 3.6. The effect was observed at three different concentrations
(250, 500 and 1000 ppm). The crude extract showed concentration dependent
inhibition on acetylcholine esterase enzyme with maximum activity (68.55%) at 1000
ppm and IC50 value of 245 ppm. When crude extract was fractionated, considerable
changes in inhibitory profile was noted. Among the fractions, chloroform was the
most effective and caused maximum inhibition of 89% at 1000 ppm and IC50 value of
149 ppm. It was followed by ethyl acetate with maximum activity (70.5%) at a
concentration of 1000 ppm IC50 value of 156 ppm. The aqueous fraction also induced
significant inhibition (50.75 %) at a concentration of 1000 ppm with IC50 value 989
ppm. However, the n-hexane fraction was unable to produce significant effect at
tested concentrations.
Acetylcholinesterase being a secretary protein and important cholinergic synaptic
element hydrolyzes and releases acetylcholine from the nerves’ terminals
(presynaptic) (Brufani et al., 1986). Acetylcholine plays a key role in cognitive
functions such as learning and memorization (Rusted et al., 2000). The inhibitory
effect of acetyl cholinesterase plays a vital role in the management of neurological
disorders like Alzheimer’s disease (Rhee et al., 2001), Parkinson’s disease, senile
dementia, myasthenia gravis and ataxia (Repchinsky, 2004; Rahman and Choudhary,
2001). Acetyl cholinesterase inhibitor available in market now a days are donepezil,
tacrin and rivastigmin etc with side effects even for mild type of Alzheimer’s disease
(Schneider, 2001). So there is need for safe and effective drug. Medicinal plants with
Chapter – 3 RESULTS & DISCUSSION
72
different geographical conditions are sources of acetyl cholinesterase inhibitors
(Mukherjeea et al., 2007).
As the crude extract/fractions of V. serpens demonstrated marked inhibitory effect
against the acetyl cholinesterase in vitro therefore, it can be assumed that the plant
could be an effective natural source of acetyl cholinesterase inhibitors. Moreover,
quercetin has been reported from the plant which exhibited marked cholinergic
activity (Park et al., 1996; Jung and Park, 2007). Thus, it could be partially
responsible for the current action of the plant.
Table 3.6: Enzyme Inhibition effect of the Crude Extract and the subsequent
Fractions of V. serpens against the Enzyme Acetylcholine Esterase
Compounds Concentrations (ppm) Activity % IC50 (ppm)
Crude Extract 250 50.51±2.14
245
500 57±2.52
1000 68.55±3.10
n-hexane 250 47.25±1.41
189
500 34±1.35
1000 47.25±1.81
Chloroform 250 68.75±2.46
149
500 82.5±3.71
1000 89±3.90
Ethyl acetate 250 67.5±2.31
156
500 65±1.63
1000 70.5±2.70
Aqueous 250 13.75±0.15
989
500 45±1.67
1000 50.75± 2.21
Chapter – 3 RESULTS & DISCUSSION
73
3.2 IN-VIVO BIOLOGICAL ACTIVITIES
3.2.1 Acute Toxicity
V. serpens Wall. crude extract along with the subsequent fractions (n- hexane,
chloroform, ethyl acetate, n-butanol and aqueous) tested for acute toxicity at different
doses (1000, 1500 and 2000 mg/kg, i.p.) proved a safe herbal medicine. The mice
were safe and behaved normal when observed in the first 4 h and no death occurred
after 24 h. Assessment bioassay time period is represented in the Table 3.7.
Table 3.7: Acute Toxicity of the Crude Extract along with the Fractions of V.
serpens
Extract/Fraction Doses (mg/kg) Gross effect after 4h Mortality rate
after 24 h
Crude extract 1000
1500
2000
-
-
-
-
-
-
n-Hexane fraction
1000
1500
2000
-
-
-
-
-
-
Chloroform fraction 1000
1500
2000
-
-
-
-
-
-
Ethyl acetate fraction 1000
1500
2000
-
-
-
-
-
-
Aqueous fraction 1000
1500
2000
-
-
-
-
-
-
3.2.2 Hepatoprtotective and Nephroprotective Effects of Crude Extract/
Fractions of V. serpens
3.2.2.1 Hepatoprotective Effect
The hepatoprotective effects of the crude extract and subsequent fractions of V.
serpens are given in the Table 3.8 and Figures 3.2. Different blood parameters (ALT,
AST and ALP) along with the histopathological slides of the kidneys were selected.
The results showed that the ALT values noted in the groups of rabbits treated with
Chapter – 3 RESULTS & DISCUSSION
74
paracetamol alone showed a significant increase (six folds) than the values noted in
the normal saline treated animals. Silymarin, a standard hepatoprotective drug has
reduced the ALT value by two folds than the paracetamol value. Crude extract along
with all the fractions caused a greater reduction in the value as compared with the
paracetamol value. Chloroform soluble fraction at a dose of 150 mg/kg and ethyl
acetate soluble fraction at a dose of 300 mg/kg showed pronounced effects. Whereas,
the high doses of the crude extract and n-hexane soluble fraction showed no
significant effects. There was marked reduction in the AST values of the crude extract
along with all the fractions at both the low and high doses in comparison with the
paracetamol values. However, chloroform at low (150 mg/kg) and high (300 mg/kg)
doses, ethyl acetate and n-butanol at high doses (300 mg/kg) showed less significant
effect of AST values. Rest of the fractions in both the doses showed similar values to
the standard drug silymarin.
Similarly, there was a marked reduction in the ALP values of all the fractions along
with the crude extract at the doses of 150 and 300 mg/kg in comparison with the
paracetamol value. The ALP results showed to be even more pronounced than the
silymarin. The values are closer to the values of normal saline and silymarin. The
arrangement is given in the decreasing order of their effectiveness i.e silymarin >
aqueous fraction > n-butanol > chloroform > crude extract > ethyl acetate > n-hexane.
Effects of Histopathological Analysis
Histological sections of the liver of the rabbits treated with saline solution showed
normal tissue architecture with a centrally placed nucleus and foamy cytoplasm of
hepatocytes (Figures 3.2.1). No vascular disturbance was noted in the arterial and
Chapter – 3 RESULTS & DISCUSSION
75
venous system. The sinusoidal spaces were neither enlarged nor reduced but of
normal sizes.
The hepatocytes of the rabbits treated with paracetamol alone showed cellular
swelling and vacuolation (Figure 3.2.2). The rounded and sharply demarked
boundaries of the vacuoles were suggesting fatty changes. The sinusoidal spaces were
significantly decreased due to increased cell sizes. No vascular changes such as
congestion or hemorrhages were noted.
The crude extract of the plant showed a significant reduction in the paracetamol
induced damage to hepatocytes (Figure 3.2.3). Amelioration in the toxic effects of
paracetamol on the hepatocytes was noted in rabbit which were given crude extract at
both low (150 mg/kg) and high (300 mg/kg) doses (Figure 3.2.4). The protective
effects were more pronounced at a higher dose.
The rabbits treated with n-hexane extract of the plant showed a protective effect
against paracetamol mediated damage to hepatocytes. However, the higher doses of
plant extract exhibited minimal protection as noted in the lower dosed group.
Likewise, plant material extracted with chloroform, ethyl acetate and n-butanol
showed a lesser decrease in liver lesions at higher doses than the lower doses.
However, liver histology indicated a significant improvement in the group of rabbits
given aqueous plant extract at a higher dose than the lower dose (150 mg/kg).
Chapter – 3 RESULTS & DISCUSSION
76
Table 3.8: Effects of the Crude Extracts/Fractions of V. serpens Wall on the
Liver Related Parameters (AST, ALT and ALP) in the Rabbits
Models
*P<0.05, **P<0.01 ***P<0.001 when compared with PCM treated group % change =
Extract Treatment Value - Paracetamol Toxic Value/Test Sample Value X 100.
Groups Dose
mg/kg
Liver-related parameters with % change values
ALT AST ALP
Normal saline 1 mL/kg 20 ± 4.6 29.8±6.0 30.3 ± 4.3
Paracetamol Control 1000 129 ± 5.3 75.3 ± 18.8 185 ± 7.8
Standard Silymarin 50 65 ± 2.8*** 40 ± 6.9*** 81 ± 7.2
Crude extract 150 76 ± 9*** 65 ± 18.3*** 71.8 ± 10.4***
300 103 ± 1.8 47 ± 8.25*** 66.3 ± 3.1***
n-hexane 150 53 ±7.53*** 44 ± 8.3*** 89 ± 11.7***
300 96 ± 6.3 45 ± 2.6*** 80.3 ± 9.5***
Chloroform 150 27 ± 7.9*** 67.3 ± 4.9** 50 ± 8.6***
300 68 ± 8.2*** 82 ±15.4** 70 ± 9.5***
Ethyl Acetate 150 66 ± 1.9*** 66 ± 4.39*** 85 ± 16.3***
300 47 ± 4.039*** 83 ± 2.5 * 62 ± 6.7***
n-Butanol 150 68 ±14.3*** 67.3 ± 1.5** 44.3 ± 4.5***
300 73 ± 3.4*** 65 ± 1.96*** 45 ± 33.3***
Aqueous 150 75 ± 6.79*** 48 ± 1.9*** 39.5 ± 2.4***
300 62 ± 2.2*** 45.5 ± 5.3*** 34 ± 3.1***
Chapter – 3 RESULTS & DISCUSSION
77
Figure 3.2.1: Normal saline treated liver showing normal architecture of central vein (CV), sinusoidal spaces (small
arrows), hepatocytes (large arrows) with a centrally placed
nucleus and foamy cytoplasm. (100X H&E).
Figure 3.2.2: Liver showing accumulation of lymphocytes (small arrows) around the central vein (CV), fatty changes (small arrow
head) and focal area of necrosis (asterisk) with paracetamol
(100X H&E).
Figure 3.2.3: Liver showing regeneration, containing normal
liver plates (large arrows) along central vein (CV) with n-hexane
150 mg/kg b.w. (H&E).
Figure 3.2.4: Liver showing normal appearance of central vein
(CV) and plates of hepatocytes (large arrows) with n-hexane 300
mg/kg b.w. (100X H&E).
Figure 3.2.5: Liver showing hexagonal hepatocytes (large
arrows) with prominent cell borders (small arrows), nuclei (arrow
heads) with nuclear clearing and prominent nucleoli with crude
extract at a dose of 150 mg/kg b.w. (400X H&E).
Figure 3.2.6: Liver showing regeneration of hepatocytes (large
arrows) with congestion of sinusoids (asterisks) containing red
blood cells (small arrows) with crude extract at a dose of 300
mg/kg b.w. (400X H&E).
Figures 3.2.1-3.2.2 Liver photomicrographs of the rabbits treated with
paracetamol, crude extract and n-Hexane fraction of V.serpens at
doses of 150 and 300 mg/kg (H&E, 100X and 400X).
Chapter – 3 RESULTS & DISCUSSION
78
3.2.2.2 Nephroprotective Effect of V. serpens Crude Extract and its Subsequent
Fraction
The nephroprotective effects of the crude extract/fractions of V. serpens from the
blood biomarkers and histopahtological slides are summarized in the Table 3.9 and
Figures 3.3 respectively.
In kidney related blood parameters, blood urea of the crude extract and some of the
fractions are insignificant in comparison with the paracetamol values. Whereas,
aqueous fraction, in both the low and high doses (150 and 300 mg/kg), crude extract
and chloroform in low doses (150 mg/kg), n-hexane, ethyl acetate and n-butanol in
high doses (300 mg/kg) are comparatively more effective than the paracetamol values
and closer to the values of normal saline.
Serum creatinine values of all the fractions along with the aqueous methanolic extract
are significant in comparison with the paracetamol and normal saline values.
Creatinine clearance value has been reduced to the low level than the normal value by
paracetamol dose at 1mg/kg body weight for 8 days. No significant effects except
chloroform soluble and aqueous soluble fractions at high doses (300 mg/kg) were
obtained. Aqueous soluble fraction at low dose (150 mg/kg) is also comparatively
significant. The creatinine clearance values of all the fractions along with aqueous
methanolic extracts at both (low and high) doses are closer to the values of normal
saline (nephrprotective).
The histological section of the kidneys of rabbit treated with saline solution showed
normal tissue structure with normally placed glomeruli and tubules. The size of
glomerular cells and urinary spaces were normal. The tubular epithelial cells were
normal in size and adhered to basement membranes. No vascular disturbance was
observed (Figure 3.3.1-3.3.2).
Chapter – 3 RESULTS & DISCUSSION
79
Effects of Histopathological Analysis
The histological sections of the kidneys of the rabbits treated with paracetamol alone
showed a wide spread signs of toxicities. The most obvious ones were degeneration
changes in tubules, where the tubular epithelial cells were swollen (most probably
hydropic change) with some clear fatty changes. The sloughing of tubular epithelial
cells from the basement membrane and accumulation in the tubular lumen was
another prominent lesion in the tubular cells. The glomeruli showed shrinkages and
increase urinary spaces. No histological observable difference was noted in the
sections.
The protective role of the plant materials extracted with methanol and chloroform
were obvious from kidneys histology. The groups of rabbit given n-hexane, ethyl
acetate and n-butanol factions showed inverse dose dependent relationship in the
kidneys histology. The improvements in the lesions were lesser in groups given
higher dose of plant extract as compared to lower dosed group. However, the aqueous
fraction showed a dose dependent response.
Exposure of liver and kidneys to the drugs itself or its active metabolites results either
into direct toxicity or may get a chance of immunological reaction (Maaz et al., 2010).
Toxic metabolites are the results of about 62% of withdrawn drugs administration.
Paracetamol is a commonly used analgesic and antipyretic drug, results in acute
centrilobular necrosis and centrizonal heamorrhagic (Boyd and Bereckzky, 1966;
Clark et al., 1973). 90-95% PCM metabolism occurs through the liver and excreted
through kidneys (Temple and Himmel, 2002; Edwige et al., 2012). In body various
reactive radicals like hydroxyl radicals, hydrogen peroxide, superoxide anions, nitric
oxide, nascent oxygen and lipid oxides generation occur due to certain internal and
external factors resulting in disorders like hepatic ailment and kidneys disorders
Chapter – 3 RESULTS & DISCUSSION
80
(Beris et al., 1991; Malila et al., 2002; Yerra et al., 2005). In therapeutic doses of
PCM, only 5% of the drug was converted to N-acetyl-p-benzoquineimine (NAPQI), a
highly reactive cytochrome P450 mediated intermediate metabolite (Raucy et al.,
1989).Whereas, in toxic doses it is mostly oxidized by cytochrome P-450 enzymes to
highly reactive NAPQI (Kassem et al., 2013). Decreased glutathione store or
metabolites NAPQI covalently bond to vital proteins, hepatocyte membrane’s lipid
bilayer and raise the lipid peroxidation (McConnachie et al., 2007) responsible for
mediating liver and kidneys toxicity. Biochemical parameters (AST, ALT and ALP)
with increased levels better reflect the liver injury (Benjamin, 1978; Wittwer et al.,
1986; Edwards and Bouchier, 1991).
In the present study, the liver biomarkers, ALT, AST and ALP values were
significantly reduced and comparable to silymarin treated group in comparison with
the values of purely paracetamol intoxicated groups. This suggests the protection,
regeneration, and restoration of the cellular permeability of the plant extract and
fractions in the paracetamol intoxicated rabbit models. The mechanisms involved
behind this may be the free radical scavenging effect by intercepting the radicals
involved in paracetamol metabolism (microsomal enzymes). Antioxidants are agents
that can neutralize deleterious effects of free radicals. Exogenous support is taken for
keeping a balance between oxidants and antioxidants. Plants with antioxidant
properties are becoming more and more popular all over the world (Jayaprakash et al.,
2001). There is a strong relationship between the phenols and antioxidant activity
(Velioglu et al., 1998; Kahkonen et al., 1999; Javanmardi et al., 2003). The
antioxidant constituents and the phenolic compounds showed the potential to prevent
oxidative degradation of cellular components (Zhon and Zheng, 1991; Kahkonen et
al., 1999).
Chapter – 3 RESULTS & DISCUSSION
81
Phytochemically analysis showed that V. serpens contained antioxidant constituents
such as ascorbic acid, ascorbate oxidase, peroxidase and catalase (Vukics et al., 2009)
along with the phenolic contents which could be the reason behind its
hepatoprotective and nephroprotective effects against the paracetamol induced
hepatotoxicity and nephrotoxicity. Additionally, there was a linear positive correlation
between the total phenolic contents and antioxidant capacities of V. serpens
(Siddharthan et al., 2007). Moreover, one of the mechanisms in the hepatoprotection
and nephroprotection may be due to the phytochemicals presence like flavonoids,
glycosides, alkaloids, coumarins and tannins present in V. serpens plant (Pratik et al.,
2011). The scientific reports also indicated the hepatoprotective and nephroprotective
role of certain flavonoids, triterpenoids and steroids in toxicity (Garba et al., 2009).
Purely paracetamol treated rabbit groups histopathology showed cellular swelling and
vacuolation of the hepatocytes. Fatty changes with swollen vacuoles and decreased
sinusoidal spaces due to increased cell sizes have also been indicated. The histological
slides of crude extract of the plant both at low and high doses showed significant
recovery of the paracetamol-induced toxicity. The mentioned biochemical
constituents in the extract showed the presence and recovery of the toxified
hepatocytes which is dose dependent. The histopathology of rabbits treated with the
plant fractions showed protective effects. The effectiveness was more at low doses
than high doses whereas, the case was reversed in n-butanol.
The histological sections of the kidneys of the rabbits treated with paracetamol alone
showed a wide spread signs of toxicities like degeneration of the tubular epithelial
cells, swelling and fatty changes, shrunk glomeruli and increased urinary spaces. It is
clear from the histological slides of the groups treated with methanol (crude extract)
and chloroform along with the toxic paracetamol doses that the presence of certain
Chapter – 3 RESULTS & DISCUSSION
82
biochemical constituents in the plant extract/fraction, secure the kidneys against the
toxicity. The rabbit groups of n-hexane, ethyl acetate and n-butanol factions showed
inverse dose related relationship in the kidneys histopathology.
It is concluded from the present study that the crude extracts and different fractions of
V. serpens Wall. possess strong hepatoprotective and nephroprotective activities and
thus provided a scientific rationale for the uses of the plant in the treatment of liver
and kidney toxicities. In this regard, a further detailed study regarding the
phytochemistry and pharmacology is required to ascertain its chemical background.
Table 3.9: Effect of the Crude Extract/ Fractions of V. serpens Wall. on the
Kidney’s Function and Clearance in the Rabbits Models
Groups Dose
mg/kg
Kidney Related Parameters with % Change Values
Blood urea Serum Creatinine Creatinine
Clearance
Saline 1mL/kg 12.0 ± 2.6 0.3 ± 0.12 4.7 ± 2.8
PCM Control 1000 24.3 ± 2.3 1.5 ± 0.29 0.36 ± 1.3
Crude extract 150 15.3*** ± 1.3 0.6 ± 0.04*** 1.5** ± 0.29
300 21*± 2.5 0.5 ± 0.00 *** 1.35**± 0.26
n-hexane 150 25 ± 2.6 0.05 ± 0.03*** 1.1** ± 0.21
300 19.8**± 3 0.52 ± 0.02*** 1.36 ± 0.27
Chloroform 150 18.5**± 1.5 0.5 ± 0.11*** 2.0**± 0.6
300 22.3*± 2.3 0.4 ± 0.03*** 4.0 ± 0.9
Ethyl Acetate 150 24 ± 3.1 0.6 ± 0.12*** 0.84 ***± 0.18
300 19.8* ± 4.5 0.6 ± 0.06*** 0.93*** ± 0.24
n-Butanol 150 23.3 ± 3.1 0.62 ± 0.04*** 1.26**± 0.12
300 18.8**± 3.3 0.6 ± 0.04*** 1.5**± 0.4
Aqueous 150 17.7***± 2.0 0.7 ± 0.08*** 2.5* ± 0.59
300 14.3***± 2.0 0.5 ± 0.08*** 4.7 ± 1.0
*P<0.05, **P<0.01 ***P<0.001 when compared with PCM treated group
% change = Extract Treatment Value ˗ PCM Toxic Value/Test Sample Value X100
Chapter – 3 RESULTS & DISCUSSION
83
Figure 3.3.1: Photomicrograph (100X H&E) of a section of kidney
from a rabbit treated with normal saline showing normal
histological appearance of renal cortex. The cortex contains renal
corpuscles (large arrows) embedded among proximal (arrow
heads) and distal (asterisk) convoluted tubules.
Figure 3.3.2: Photomicrograph (100X H&E) of a section of
kidney from a rabbit treated with PCM showing necrosis of
cuboidal epithelial cells (large arrows) of proximal convoluted
tubules with exfoliation of their brush border. The lumen
(asterisk) of tubules contains numerous cellular casts (small
arrows).
Figure 3.3.3: Photomicrograph (100X H&E) of a kidney section
from a rabbit treated with n-hexane soluble fraction 150 mg/kg
showing normal histo-architecture of distal convoluted tubules
with wider lumen (asterisk) and lined by cuboidal epithelial cells
(arrow heads). Numerous loop of Henle tubules are also visible
(large arrows).
Figure 3.3.4: Photomicrograph (100X H&E) of a section of
kidney from a rabbit treated with n-hexane soluble fraction 300
mg/kg showing normal renal corpuscles (large arrows) with
mild dilatation of proximal (arrow heads) and distal (asterisk)
convoluted tubules.
Figure 3.3.5: Photomicrograph ((100X H&E)) of a section of
kidney from a rabbit treated with chloroform soluble fraction 150
mg/kg showing normal renal corpuscles (large arrows), proximal
(arrow heads) and distal (asterisk) convoluted tubules.
Figure 3.3.6:Photomicrograph (100X H&E) of a section of
kidney from a rabbit treated with ethyl acetate soluble fraction
150 mg/kg showing normal renal corpuscles (large arrows)
with mild dilatation of proximal (arrow heads) and distal
(asterisk) convoluted tubules.
Chapter – 3 RESULTS & DISCUSSION
84
Figure 3.3.7: Photomicrograph ((100X H&E)) of a section of
kidney from a rabbit treated with chloroform soluble fraction 300
mg/kg showing normal renal corpuscles (large arrows) and
proximal convoluted tubules (arrow heads). The distal convoluted
tubules (asterisk) exhibited mild tubular necrosis of the cuboidal
epithelial cells.
Figure 3.3.8: Photomicrograph (100X H&E) of a section of
kidney from a rabbit rat treated with ethyl acetate soluble
fraction 300 mg/kg showing normal proximal convoluted
tubules (large arrows) with numerous loop of Henle tubules
(asterisk). The interlobular blood vessels (arrow heads) among
the renal tubules exhibited mild congestion with red blood
cells.
Figure 3.3.9: Photomicrograph (100X H&E) of a section of kidney
from a rat treated with aqueous soluble fraction 300 mg/kg
showing normal renal corpuscles (large arrows). The renal tubules
exhibited dilatation (arrow heads) with exfoliation of the brush
border lining the proximal convoluted tubules into their lumen.
Figure 3.3.10: Photomicrograph (100X H&E) of a section of
kidney from a rat treated with aqueous soluble fraction
showing mild congestion of the renal corpuscles (large arrows)
with severe dilatation of the renal tubules (asterisk). Numerous
cellular casts (arrow head) is also visible in the lumen of renal
tubules.
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
85
3.2.2.3 Antinociceptive Activity
Effect of Crude Extract/ Fractions of V. serpens Wall. in Acetic Acid Induced
Writhing Test
The results of crude extract/fraction of V. serpens in acetic acid induced writhing test
at various doses (100, 200 and 300 mg/kg i.p.) are shown in Table 3.10. The standard
drug diclofenac was used for comparison. The crude extract caused significant
attenuation of writhes induced by the injection of acetic acid in a dose dependent
manner with a more significant value of 19.77 with the inhibition of 70.05% reduction
in pain at 300 mg/kg i.p represented in the Figure 3.4. The crude extract upon
fractionation provoked different effects. The maximum effect is produced by the
soluble fraction of n-hexane in a dose dependent manner followed by the soluble
fraction of ethyl acetate. At a dose of 300 mg/kg maximum reduction in the numbers
of writhes were noted (21) with a percent reduction value of 68.8% represented in the
Figure 3.5. The chloroform soluble fraction also incorporated a significant effect in a
dose dependent manner with a significant value of 31.50 at a maximum dose of 300
mg/kg and at a percent reduction value of 50.37 % represented in the Figure 3.6. The
soluble fraction of ethyl acetate also produced a dose dependent analgesic effect with
the more significant value 34.75 at a dose of 300 mg/kg body weight with a percent
reduction value 50.37 % showed in the Figure 3.7. Whereas, the aqueous soluble
fraction does not produce significant antinociceptive effect at any test dose
represented in the Figure 3.8.
Chapter – 3 RESULTS & DISCUSSION
86
Table 3.10: The Effect of Crude Extract/Fractions of V. serpens in Acetic Acid
Induced Writhing Test in Mice (i.p)
Drugs Dose mg/kg No. of writhing (10min) % Protection
Saline 10 ml/kg 66±2.90
Crude 100 37.55±2.70* 43
200 25.80±2.90** 60.9
300 19.77±2.00** 70.05
n- hexane 100 39.40±2.50* 40.30
200 27.90±2.56** 59.09
300 21.50±1.9** 68.18
Chloroform 100 49.90±2.90 24.4
200 38.70±2.80* 42.42
300 31.50±1.70* 52.27
Ethyl acetate 100 45.33±2.10 31.31
200 37.12±2.75* 43.76
300 34.75±2.45* 50.37
Aqueous 100 55.50±2.90 15.9
200 50.12±3.10 24.1
300 45.27±2.90 31.8
Diclofenac 10 11.15±1.5 83.1
Values are reported as mean ±SEM for group of six mice. ANOVA followed by
Dunnett tests were used for data analysis. Significant and satisfactory values are
represented by asterisks from the control. *P<0.05 or **P<0.01
Chapter – 3 RESULTS & DISCUSSION
87
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
1 0 0
**
**
***
*
% P
ro
te
ctio
n
C r u d e e x tr a c t
Figures 3.4: Antinociceptive Effect of Extract of V. serpens in Acetic acid
Induced Writhing Test. Significant and satisfactory values are represented by
asterisks from the control. *P<0.05 or **P<0.01
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
1 0 0
**
*
**
*
**
*
% P
ro
te
ctio
n
H e x a n e
Figures 3.5: Antinociceptive Effect of n-hexane Soluble Fraction of V. serpens
in Acetic Acid Induced Writhing Test. Significant and satisfactory values are
represented by asterisks from the control. *P<0.05 or **P<0.01
Chapter – 3 RESULTS & DISCUSSION
88
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
1 0 0 **
*
*
*
% P
ro
te
ctio
n
C h lo r o f o r m
Figures 3.6: Anti-Nociceptive Effect of Chloroform Soluble Fraction of V.
serpens in Acetic Acid Induced Writhing test. Significant and satisfactory values
are represented by asterisks from the control. *P<0.05 or **P<0.01
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
1 0 0
*
**
*
% P
ro
te
ctio
n
E t h y l a c e ta t e
Figures 3.7: Antinociceptive Effect of Ethyl Acetate Soluble Fraction of V.
serpens in Acetic Acid Induced Writhing Test. Significant and satisfactory values
are represented by asterisks from the control. *P<0.05 or **P<0.01
Chapter – 3 RESULTS & DISCUSSION
89
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
1 0 0
*
**
***
% P
ro
te
ctio
n
A q u e o u s
Figures 3.8: Antinociceptive Effect of Aqueous Soluble Fraction of V. serpens in
Acetic Acid Induced Writhing Test. Significant and satisfactory values are
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
90
The maximum percent inhibition of the fraction was 26.5% and 37.14% respectively
in the dose dependent manner. The Ethyl acetate fraction also showed significant
effect with 25.0 and 38.57% inhibitions in the early and late phases respectively. The
lowest antinociceptive effect among all the fractions of the plant extract was noted in
the aqueous fraction with the percent inhibition values of 23% and 25% in the early
and late phases respectively.
Acetic acid induced abdominal constriction/writhes is an assay used for the
measurement/determination of antinociceptive activity by the peripheral mechanism
(Du et al., 2007; Duarte et al., 1988). Writhes are define as the stereotypical response
of the mice/rats (serous membrane) resulting with the intraperitoneal administration of
the irritating agent in which the coordination of the motor activity is disturbed along
with the movement of the body and muscles (Zeashana et al., 2009) stressful
constriction of the abdominal cavity occur. As a result of acetic acid (pain inducer)
administration, release of pain mediators/ endogenous substance causes the increased
production of lipooxygenase as well as prostaglandin (PGE2 and PGE2α) in the
peritoneal fluid (Deraedt et al., 1980; Khan et al., 2010; Mbiantcha et al., 2011).
Capillary permeability is increased which ultimately causes the stimulation of
inflammatory pain through the peritoneal receptors (Collier et al., 1968; Choi 2007).
The reduction in the number of writhes indicates the antinociceptive activity. The
plant extract and its different fractions produced more attenuated antinociceptive
effect in dose dependent manners. The significance of the activity decreased with the
increased polarity of the solvents. Means that the crude extract showed more
attenuated analgesic effect whereas, the effect decreases as we go on increasing the
polarity i.e in n-hexane, ethyl acetate, chloroform and aqueous soluble fractions. In
the three test doses, 300 mg/kg (i.p) was more effective in all the fractions/crude
Chapter – 3 RESULTS & DISCUSSION
91
extract of the plant as compared with the low doses (100 & 200 mg/kg, i.p). The
peripheral pathway followed by the plant for this activity may be due to the
inhibition/hindrance of the local peritoneal receptors that may cause the
inhibition/reduction in the release of cyclooxygenase or lipoxgenase enzymes. The
release of certain mediators may also be involved in the analgesic activity of the plant.
Due to the pronounced antinociceptive effect, the plant may be effectively
recommended for clinical purpose.
In general, the acetic acid induced writhing test is nonspecific and therefore,
mechanistic approach was performed by using formalin induced paw licking test.
Formalin induced paw licking and flicking protocol is a suitable method for the
qualitative measurement of centrally acting analgesia (Dubuisson & Dennis 1977;
Tjolsen et al., 1992). Formalin induced nociception being a biphasic analgesic
behavioral protocol with the involvement of two clearly different stimuli. The
chemical released in the early phase (neurogenic phase) are the bradykinin and
substance P. The late inflammatory phase, involve the release of prostaglandins,
histamine, serotonin and bradykinin (Tjolsen et al., 1992). The plant extract/fractions
of V. serpens showed more pronounced antinociceptive effect in late phase as
compared to the early phase. The effect decreased with the increasing polarity of the
solvent in the subsequent manner. Means more significant effects were shown by the
n-hexane fraction followed by the chloroform, ethyl acetate and aqueous fraction in a
dose dependent manner. The effect of the plant being more significant in the second
phase indicates its similarity with the non-steroidal anti-inflammatory drug like
indomethacin and aspirin (Santos et al., 1994; Choi et al., 2001). Centrally acting dugs
like narcotic analgesics show effectiveness in both the early and late phases (Stai et
al., 1995; Santos et al., 1994).
Chapter – 3 RESULTS & DISCUSSION
92
The main constituents in the plant of V. serpens are alkaloids, saponins, tannins and
flavonoids (some also isolated in the present study). These compounds may be
responsible for the inhibition/decrease in mediators release like prostaglandins,
histamine, serotonin or bradykinin, responsible for the pain suppression in the late
phase of formalin induced pain. (Naveed et al., 2012; Naveed et al., 2012). The
outcome of the study is that the anti-nociceptive property of the plant was mediated
through the peripheral mechanism; augmented by interference of centrally acting pain
mediators. Thus, this study provided a scientific rationale for the traditional use of the
plant in different animal protocols.
Chapter – 3 RESULTS & DISCUSSION
93
Table 3.11: Effect of the Crude Extract/ Fractions of V. serpens in Formalin
Induced Pains for Analgesia Test in Mice at Doses of 100, 200 and
300 mg/kg, i.p
Drugs Dose mg/kg Early phase
(0-5min)
Late phase
(15-30 min)
Saline 10 ml/kg 68±1.79 93±2.30
Crude 100 59±2.95 50±2.00*
200 51±2.20* 42±2.25**
300 44±2.90** 33±1.70***
n-hexane
100 60±1.36 50±2.19
200 53±1.79* 41±2.25*
300 42±2.24** 29±1.35**
Chloroform
100 62±2.45 51±2.50*
200 56±2.50 38±2.35**
300 50±2.70* 29±2.50***
Ethyl acetate
100 60±2.36 44±2.50*
200 56±2.50 36±2.35**
300 51±2.10* 28±2.50***
Aqueous
100 62±3.10 53±2.50
200 57±2.90 51±2.35*
300 52±2.90* 46±2.50*
Tramadol 30 39±1.34 20±1.10**
Values are reported as mean ±SEM for group of six mice. ANOVA followed by
Dunnett tests were used for data analysis. Significant and satisfactory values are
represented by asterisks from the control. *P<0.05 or **P<0.01.
Chapter – 3 RESULTS & DISCUSSION
94
100 200 300 Tramadol
0
20
40
60
80Early PhaseLate PhaseCrude extract
% P
ro
tecti
on
Figure 3.9: Antinociceptive Effects of Formalin Induced Pain in Mice of the
Crude Extract of V.serpens
100 200 300 Tramadol
0
20
40
60
80 Early Phase
Late Phasen-Hexane
% P
ro
tecti
on
Figure 3.10: Antinociceptive Effects of Formalin Induced Pain in Mice of the n-
Hexane Soluble Fraction of V.serpens.
Chapter – 3 RESULTS & DISCUSSION
95
100 200 300 Tramadol0
10
20
30
40
50Early PhaseLate Phase
Chloroform%
Pro
tecti
on
Figure 3.11: Antinociceptive Effects of Formalin Induced Pain in Mice of the
Chloroform Soluble Fraction of V.serpens.
100 200 300 Tramadol0
20
40
60
80Early PhaseLate Phase Ethyl acetate
% P
ro
tecti
on
Figure 3.12: Antinociceptive Effects of Formalin Induced Pain in Mice of the
Ethyl Acetate Soluble Fraction of V.serpens
Chapter – 3 RESULTS & DISCUSSION
96
100 200 300 Tramadol0
20
40
60
80 Early PhaseLate Phase
Aqueous
% P
ro
tecti
on
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
97
dose of 100 to 300 mg/kg i.p in the 2nd and 3rdh. Whereas, the effectiveness of the
carageenan induced anti-inflammatory effect remained till the 5th hour of injection.
The % protection is represented in the Figure 3.15. The chloroform and aqueous
soluble fractions showed significant effect only at a dose of 200 and 300 mg/kg in the
3rd h of induced inflammation and the significant % protections represented in the
Figure 3.16 and 3.18 respectively. Ethyl acetate soluble fraction showed anti-
inflammatory activity at a dose of 300 mg/kg in the 2nd h. Moreover, in the 3rd h both
the doses, 200 and 300 mg/kg were significant and the percent protection values
represented on the Figure 3.17.
Effect of Crude Extract/Fractions of V. serpens on Paw Edema Induced by
Histamine
The effect of the crude extract/fractions of V. serpens in histamine induced paw
edema at various doses (100, 200 and 300 mg/kg i.p) in various durations (1-5 h) is
presented in the Tables 3.12-3.16. The crude extract at doses of 200 and 300 mg/kg
showed more pronounced anti-inflammatory effects in the 2nd to the 5th hours of the
histamine induced edema. The significance level reached the maximum in the 3rd hour
and then decreased slowly till the 5th hour. The crude extract was then subjected to
various fractions, exhibiting different inhibitory effects showed in the Figure 3.19. In
fractions maximum anti-inflammatory effect against the histamine induced paw
edema was produced by the n-hexane soluble fraction at a dose of 200 mg/kg in the
3rd h with percent inhibition 46.70% represented in the Figure 3.20. The significant
anti-inflammatory effect started from the 2nd h and lasted till the 5th h of the edema
induction. On the other hand chloroform and aqueous soluble fractions showed
significant effects at the doses of 200 and 300 mg/kg on the 3rd h of histamine induced
edema with percent inhibition values of 31.48 and 34.60 % represented in the Figures
Chapter – 3 RESULTS & DISCUSSION
98
3.21 and 3.23 respectively. Whereas, the ethyl acetate soluble fraction was non-
significant in the three test doses at all the 5 mentioned test hours with the %
inhibition representation in the Figure 3.22.
Effect of Crude Extract/Fractions of V. serpens on Ear Edema Induced by
Xylene
Results of anti-inflammatory effect of V. serpens on xylene induced ear edema are
presented in the Table 3.17 and the percent inhibition in the Figures 3.24. The crude
extract/subsequent fractions of V. serpens were subjected for the anti-inflammatory
effect by using xylene induced ear edema protocol. Three test doses were selected
(100, 200 and 300 mg/kg Oral administration) for the anti-inflammatory effective
results determination. The crude extract showed maximum inhibitory effect (57.6 %)
at a dose of 300 mg/kg. The effect of the crude extract was significant in a dose
dependent manner. Upon treatment with different solvents the fractions obtained
showed different anti-inflammatory effects. The most effective and significant
fraction considered was the n-hexane which also showed significance in a dose
dependent manner with the maximum percent inhibition value of 55 % at 300 mg/kg.
This was followed by the chloroform and ethyl acetate soluble fractions and then by
the aqueous soluble fraction whose considerable effects were shown at doses of 200
and 300 mg/kg with the maximum inhibition values of 51, 49 and 48.5 %
respectively.
Inflammation being a complex process has direct association with pain which may
involve increase in: vascular permeability, cells migration (mononuclear and
granulocytes) and proliferation of granulomatous tissue. Anti inflammatory
compounds act through different mechanisms, either by blocking the pro-
inflammatory mediators (directly via enzyme like COX-2 inhibition) or enzyme
Chapter – 3 RESULTS & DISCUSSION
99
expression is decreased such as anti-inflammatory steroidal compounds or substrate
levels are decreased like reduction in the release of arachidonic acid. Immuno-
stimulation is also one of the mechanism i.e phagocytosis activation as well as
maturation of myeloid cells which ultimately response to the challenge of allergen
(Safaihy and Sailer, 1997). The plant extract/fractions of V. serpens demonstrated its
effectiveness against the induced inflammation protocols in carrageenan and
histamine paw edema and xylene ear edema.
Carrageenan being a choice of phlogistic agent is used for anti-inflammatory drugs
testing and having an extensive measurement of reproducibility (Winter, 1957). It is a
biphasic model with the early phase including 1–2 h, mediated mostly by the release
of serotonin, histamine and prostaglandins increased level. The late phase includes the
release of prostaglandin whereas, kinine releases in between the two phases (Antonio
and Souza, 1998; Zhou et al., 2008). The enzyme cyclooxygenase (COX) catalyses
the biosynthesis of prostaglandin metabolites (arachidonic acid) in the early phase
(Teather et al., 2002). COX-1 (constitutive form of COX) is involved in cellular
function (Herschman, 1996).COX-2 (inducible isoform) increases response to various
tissue inflammatory stimuli (Teather et al., 2002). COX-3 is determined in the heart
tissue and brain cortex (Chandrasekharan et al., 2002).
In carrageenan induced paw edema protocol the crude extract and n-hexane fractions
were effective against the inflammation challenge from the 2nd till the 5th h at 200 and
300 mg/kg. whereas the chloroform, aqueous and ethyl acetate fractions also showed
significant effects and reduced paw edema in the 3rd h at 300 mg/kg i.p. The crude
extract and the n-hexane fraction of V. serpens are effective in both the phases
whereas, rest of the fractions showed significant effects only in the late phase.
Chapter – 3 RESULTS & DISCUSSION
100
Histamine, a fundamental amine and mediator associated with inflammation and
allergic reactions, causes both increase in the vascular permeability and vasodilatation
(Rang et al., 2001; Linardi et al., 2002; Cuman et al., 2001). The lipoxygenase and
cyclooxygenase pathways are followed by the arachidonic acid metabolites.
Prostaglandin (PG) and prostaglandin E2 (PGE2) are mainly involved in the cause or
enhancement of the signs of cardinal inflammation. These enzymes of the arachidonic
acid provoke the inflammatory response (Young et al., 1984). The results of the
present study revealed that the two doses (200 and 300 mg/kg i.p) of V. serpens in the
crude as well as in the subsequent fractions suppressed the histamine induced edema
effectively which may be due to the presence of such compounds capable of resisting/
inhibiting the release of histamine, prostaglandins or mediators of the mast cells
(histamine, PG and 5-HT) (Rao et al., 2005).
The xylene-induced ear edema in mouse is a testing and investigating procedure for
acute anti-inflammatory activity response, resulting in severe vasodilatation and skin
edema (ear) (Atta and Alkofahi, 1998; Kim et al., 2007; Xiao-Jia et al., 2008). Xylene
tropical application on ear leads to an immediate mouse ear irritation resulting in the
fluid accumulation (edema formation) and acute response of inflammation (Okoli et
al., 2006). Anti-inflammatory steroidal and non-steroidal antiphlogistic agents are
evaluation by this method especially the ones inhibiting phospholipase A2 (Zaninir et
al., 1992). The results obtained from the study showed that the ear edema of the crude
extract as well as in the fractions subsided in a dose dependent manner (crude extract
and n-hexane). Whereas, in the other fractions significant effects were found only at
high doses (300 mg/kg). Thus, the effectiveness of V. serpens in the model suggests
that the plant extract and its fractions possibly act by inhibiting the enzyme
phospholipase A2 (PLA2) (Atta and Alkofahi, et al., 1998).
Chapter – 3 RESULTS & DISCUSSION
101
Phytochemically, different groups of compounds are reported to be present in Viola
species including triterpenoids, cyclotide, alkaloids and flavonoids. (Naveed et al.,
2012; Naveed et al., 2012). Triterpenoids are one of the important contributors of anti-
inflammatory activity (Safaihy and Sailer, 1997; Andrikopoulos et al., 2003). Along
with this the presence of inflammation sites in high concentration oxidant and free
radicals also contributes to the anti-inflammatory process and play an important role
in avoiding the process of inflammation (Salvemini et al., 1996). V. serpens also
contains various phenolic compounds (Anu et al., 2011) and possesses antioxidant
activity along with the triterpenes (anti-inflammatory compounds) which may be the
major contributors for its anti-inflammatory activity. In the present study the isolated
flavonoids 1-6 from the chloroform fraction of the plant also showed marked
scavenging effect against DPPH so this may also give a solid scientific background to
the plant as a strong anti-inflammatory agent. Moreover, further work in future is
required to be focused on this plant as an anti-inflammatory agent to make its use
more authentic and more common with the scientific knowledge.
Chapter – 3 RESULTS & DISCUSSION
102
1h 2h 3h 4h 5h0
20
40
60
80
100 100/kgmg 200mg/kg 300mg/kg Diclofenac
*
*
*
**
**
*** **
***
*
*
**
*
*
*
***
% i
nh
ibit
ion
Figure 3.14: Anti-inflammatory Effect (%) of the Crude Extract of V. serpens on
Carrageenan Induced Paw Edema
1h 2h 3h 4h 5h0
20
40
60
80
100 100mg/kg 200mg/kg 300mg/kg Diclofenac
*
**
*
**
**
* **
*
**
*
***
**
*
* *
**
% I
nh
ibit
ion
Figure 3.15: Anti-inflammatory Effect (%) of the n-Hexane Soluble Fraction of
V. serpens on Carrageenan Induced Paw Edema
Chapter – 3 RESULTS & DISCUSSION
103
1h 2h 3h 4h 5h0
20
40
60
80
100100mg/kg 200mg/kg 300mg/kg Diclofenac
*
**
*
**
* **
*
**
*
*
% I
nh
ibit
ion
Figure 3.16: Anti-Inflammatory Effect (%) of the Chloroform Soluble Fraction of V.
serpens on Carrageenan Induced Paw Edema
1h 2h 3h 4h 5h0
20
40
60
80
100
100mg/kg 200/kgmg 300mg/kg Diclofanec
**
**
*
**
**
*
**
*
**
% I
nh
ibit
ion
Figure 3.17: Anti-Inflammatory Effect (%) of the Ethyl Acetate Soluble
Fraction of V. serpens on Carrageenan Induced Paw Edema
Chapter – 3 RESULTS & DISCUSSION
104
1h 2h 3h 4h 5h0
20
40
60
80
100
100mg/kg 200mg/kg 300mg/kg Diclofenac
*
**
**
*
**
**
*
**
*
*
% I
nh
ibit
ion
Figure 3.18: Anti-Inflammatory Effect (%) of the Aqueous Soluble Fraction of
V. serpens on Carrageenan Induced Paw Edema
1h 2h 3h 4h 5h0
20
40
60
80
100 100mg/kg 200mg/kg 300mg/kg Diclofenac
*
**
**
**
* *
**
*
**
*
***
**
*
**
**
*
**
*
% I
nh
ibit
ion
Figure 3.19: Anti-Inflammatory Effect (%) of the Crude Extract of V. serpens on
Histamine Induced Paw Edema
Chapter – 3 RESULTS & DISCUSSION
105
1h 2h 3h 4h 5h0
20
40
60
80
100 100mg/kg 200mg/kg 300mg/kg Diclofenac
**
*
**
**
* *
**
**
**
*
* ** *
**
*
% I
nh
ibit
ion
Figure 3.20: Anti-Inflammatory Effect (%) of the n-Hexane Soluble Fraction of V.
serpens on Histamine Induced Paw Edema
1h 2h 3h 4h 5h0
20
40
60
80
100
100mg/kg 200mg/kg 300mg/kg Diclofenac
*
**
**
* **
*
**
*
**
*
**
% I
nh
ibit
ion
Figure 3.21: Anti-Inflammatory Effect (%) of the Chloroform Soluble Fraction
of V. serpens on Histamine Induced Paw Edema
Chapter – 3 RESULTS & DISCUSSION
106
1h 2h 3h 4h 5h0
20
40
60
80
100 100mg/kg 200mg/kg 300mg/kg Diclofenac
*
**
*** ******
**%
In
hib
itio
n
Figure 3.22: Anti-Inflammatory Effect (%) of the Ethyl Acetate Soluble Fraction of V. serpens on Histamine Induced Paw Edema
1h 2h 3h 4h 5h0
20
40
60
80
100100 mg/kg 200mg/kg 300mg/kg Diclofenac
***
**
**
**
***
***
*% I
nh
ibit
ion
Figure 3.23: Anti-Inflammatory Effect (%) of the Aqueous Soluble Fraction of V.
serpens on Histamine Induced Paw Edema
Chapter – 3 RESULTS & DISCUSSION
107
Table 3.12: Anti-Inflammatory Effect of Crude Extract of V. serpens Against Carrageenan and Histamine Induced Paw Edema
in Mice
Treatment Dose
mg/kg
Normal Paw Size
(NPS)
0h 1h 2h 3h 4h 5h
Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2092 ± 0.14
Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12
Anti-inflammatory effect against carrageenan induced paw edema
Crude extract
100 0.0915 ± 0.10 0.2134 ± 0.10 0.2018 ± 0.15 0.1803 ± 0.10 0.1408* ± 0.10 0.1572 ± 0.15 0.1590 ± 0.20
200 0.0965 ± 0.13 0.2050 ± 0.11 0.1700* ± 0.19 0.1495* ± 0.21 0.0901** ± 0.17 0.1130* ± 0.19 0.1230* ± 0.13
300 0.0970 ± 0.11 0.2178 ± 0.05 0.1670* ± 0.18 0.1233* ± 0.22 0.0702** ± 0.18 0.0830** ± 0.25 0.1003** ± 0.19
Anti-inflammatory effect against histamine induced paw edema
Crude extract
100 0.0970±0.05 0.2001±0.05 0.2005±0.19 0.1966±0.25 0.1850±0.25 0.1960±0.05 0.1990±0.35
200 0.0955 ± 0.10 0.2055 ± 0.20 0.1614* ± 0.15 0.1510* ± 0.20 0.0990** ± 0.23 0.1083* ± 0.25 0.11087* ± 0.10
300 0.0962 ± 0.15 0.2087 ± 0.30 0.1180* ± 0.55 0.1150* ± 0.10 0.0803** ± 0.20 0.0921** ± 0.30 0.1231** ± 0.30
Values are reported as mean ±SEM for group of six mice each for carrageenan and Histamine by applying ANOVA followed by Dunnett
tests for data analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01.
Chapter – 3 RESULTS & DISCUSSION
108
Table 3.13: Anti-Inflammatory Effect Against Carrageenan and Histamine Induced Paw Edema in Mice for V. serpens n- Hexane
Soluble Fraction
Treatment Dose
mg/kg
Normal Paw Size
(NPV)
0h 1h 2h 3h 4h 5h
Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2092 ± 0.14
Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12
Anti-inflammatory effect against carrageenan induced paw edema
n-Hexane 100 0.0910 ± 0.20 0.2130 ± 0.10 0.2015 ± 0.05 0.1900 ± 0.20 0.1485* ± 0.20 0.1575 ± 0.10 0.1670 ± 0.15
200 0.0970 ± 0.15 0.2053 ± 0.10 0.1715* ± 0.20 0.1500* ± 0.24 0.0985** ± 0.19 0.1135* ± 0.20 0.1232* ±0.15
300 0.0970 ± 0.10 0.2180 ± 0.02 0.1672* ± 0.25 0.1235* ± 0.20 0.0785** ± 0.20 0.0930** ± 0.25 0.1003**±0.20
Anti-inflammatory effect against histamine induced paw edema
n-Hexane 100 0.0982±0.03 0.2091±0.15 0.2100±0.18 0.1963±0.19 0.1547±0.25 0.1700±0.12 0.1960±0.27
200 0.0895 ± 0.11 0.2119 ± 0.22 0.2021* ± 0.16 0.1506* ± 0.22 0.1052** ± 0.24 0.1285* ± 0.25 0.1330*±0.12
300 0.0901 ± 0.14 0.2067 ± 0.24 0.178`5* ± 0.25 0.1612* ± 0.11 0.1078** ± 0.15 0.1338** ± 0.30 0.1421**±0.26
Values are reported as mean ±SEM for group of six mice each for carrageenan and Histamine by applying ANOVA followed by Dunnett tests for data
analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01.
Chapter – 3 RESULTS & DISCUSSION
109
Table 3.14: Anti-Inflammatory Effect of Chloroform Soluble Fraction of V. serpens in Carrageenan and Histamine Induced
Paw Edema in Mice
Treatment Dose mg/kg Normal Paw Size
(NPS)
0h 1h 2h 3h 4h 5h
Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2090 ± 0.14
Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12
Anti-inflammatory effect against carrageenan induced paw edema
Chloroform 100 0.0917 ± 0.11 0.2135 ± 0.15 0.2020 ± 0.13 0.1976 ± 0.17 0.1610* ± 0.11 0.1669 ± 0.15 0.1699 ± 0.15
200 0.0972 ± 0.10 0.2001 ± 0.20 0.1811* ± 0.21 0.1702* ± 0.15 0.1433** ± 0.12 0.1692* ± 0.19 0.1734* ± 0.17
300 0.0999 ± 0.21 0.1788 ± 0.23 0.1627* ± 0.14 0.1601* ± 0.20 0.1386** ± 0.19 0.1630** ± 0.19 0.1688** ± 0.21
Anti-inflammatory effect against histamine induced paw edema
Chloroform 100 0.0964± 0.05 0.2112±0.05 0.2120±0.19 0.1989±0.25 0.16702±0.25 0.1761±0.05 0.1805±0.35
200 0.0955 ± 0.10 0.2050 ± 0.20 0.2003* ± 0.15 0.1770* ± 0.20 0.1483** ± 0.23 0.1709* ± 0.25 0.1790* ± 0.10
300 0.0962 ± 0.15 0.2085 ± 0.30 0.1831* ± 0.55 0.1697* ± 0.10 0.1432** ± 0.20 0.1670** ± 0.30 0.1699** ± 0.30
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.
Chapter – 3 RESULTS & DISCUSSION
110
Table 3.15: Anti-Inflammatory Effect of Ethyl Acetate Soluble Fraction of V. serpens in Carrageenan and Histamine Induced
Paw Edema in Mice
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
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
Ethyl Acetate
100 0.0888 ± 0.21 0.2100 ± 0.19 0.2029 ± 0.11 0.1991 ± 0.12 0.1665* ± 0.20 0.1865 ± 0.11 0.2011 ± 0.19
200 0.0972 ± 0.13 0.2021 ± 0.19 0.1811* ± 0.22 0.1732* ± 0.20 0.1500** ± 0.16 0.1692* ± 0.13 0.1734* ± 0.12
300 0.0892 ± 0.18 0.1868 ± 0.19 0.1699* ± 0.22 0.1598* ± 0.23 0.1416** ± 0.16 0.1689** ± 0.14 0.1723** ± 0.17
Anti-inflammatory effect against histamine induced paw edema
Ethyl Acetate
100 0.0932 ± 0.02 0.2109 ± 0.10 0.2021 ± 0.20 0.1980 ± 0.22 0.1773±0.10 0.1859 ± 0.12 0.1970 ± 0.33
200 0.09032 ± 0.13 0.2067 ± 0.18 0.1810* ± 0.22 0.1751* ± 0.25 0.1642 ± 0.18 0.1752 ± 0.22 0.1820 ± 0.09
Chapter – 3 RESULTS & DISCUSSION
111
Table 3.16: Anti-Inflammatory Effect of Aqueous Soluble Fraction of V. serpens against Carrageenan and Histamine Induced
Paw Edema in Mice
Treatment Dose mg/kg NPS 0h 1h 2h 3h 4h 5h
Saline 10ml 0.0950 ± 0.10 0.2150 ± 0.20 0.2160 ± 0.15 0.2160 ± 0.10 0.2090 ± 0.20 0.2070 ± 0.10 0.2090 ± 0.14
Diclofenac 10mg 0.0910 ± 0.25 0.2130 ± 0.15 0.1475* ± 0.05 0.0970** ± 0.05 0.0486** ± 0.07 0.0610** ± 0.05 0.0811** ± 0.12
Anti-inflammatory effect against carrageenan induced paw edema
Aqueous 100 0.0864 ± 0.03 0.2092 ± 0.19 0.2101 ± 0.20 0.1833 ± 0.13 0.1571* ± 0.22 0.1782 ± 0.10 0.1968 ± 0.11
200 0.0921 ± 0.12 0.2090 ± 0.21 0.2152* ± 0.12 0.1705* ± 0.24 0.1423** ± 0.14 0.1680* ± 023 0.1902* ± 0.16
300 0.0906 ± 0.15 0.2099 ± 0.17 0.2012* ± 0.19 0.1643* ± 0.16 0.1230** ± 0.13 0.1598** ± 0.15 0.1860** ± 0.14
Anti-inflammatory effect against histamine induced paw edema
Aqueous
100 0.0961± 0.02 0.2009 ± 0.02 0.2013 ± 0.19 0.1993 ± 0.15 0.1603 ± 0.25 0.1803 ± 0.05 0.1995±0.30
200 0.0894 ± 0.13 0.2003 ± 0.23 0.1959 ± 0.15 0.1823± 0.21 0.1530 ± 0.23 0.1721 ± 0.21 0.1920 ± 0.20
300 0.0905 ± 0.13 0.2011 ± 0.32 0.1901 ± 0.55 0.1603 ± 0.14 0.1413 ± 0.20 0.1550 ± 0.24 0.1828 ± 0.28
Values are reported as mean ±SEM for group of six mice each for carrageenan and Histamine by applying ANOVA followed by Dunnett tests for
data analysis. Significant and satisfactory values are represented by asterisks from the control. *P<0.05 or **P<0.01
Chapter – 3 RESULTS & DISCUSSION
110
Table 3.17: Effect of the Crude Extract/ Subsequent Fractions of V. serpens on
Xylene Induced Ear Edema in Mice
Group Doses (mg/kg) Ear weight (mg) Inhibition (%)
Control 10ml/kg 185
Ibuprofen 100 57.32±3.51 69.0
Crude extract
100 129.5±2.34 30.0
200 101.9±1.91 45.4
300 78.3±2.09 57.7
n-Hexane
100 135±2.56 27.0
200 105±4.01 43.0
300 83±2.11 55.0
Chloroform
100 137±4.12 25.9
200 112±2.78 39.5
300 90.5±3.11 51.0
Ethyl acetate
100 133±2.34 28.0
200 117±1.77 36.8
300 93.2±2.05 49.6
Aqueous
100 132±3.00 28.6
200 107±2.13 42.0
300 95.3±2.34 48.5
Chapter – 3 RESULTS & DISCUSSION
111
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
**
**
*
*
**
% P
ro
te
ctio
n
C ru d e e x tr a c t
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
**
*
**
*
% P
ro
te
ctio
n
C h lo r o f o r m
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
*
**
*
**
**
*
% P
ro
te
ctio
n
H e x a n e
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
*
**
*
**
% P
ro
te
ctio
n
E th y l a c e ta te
1 0 0 2 0 0 3 0 0 D ic lo
0
2 0
4 0
6 0
8 0
*
**
**
*
% P
ro
te
ctio
n
A q u e o u s
Figures 3.24: Percent Inhibition of Xylene Induced Ear Edema in Mice at Different
Doses of the Crude Extract and Fractions of V. serpens
Chapter – 3 RESULTS & DISCUSSION
112
3.3 ISOLATED COMPOUNDS
3.3.1 New Compound from Viola serpens
3.3.1.1 Commulin-A (1)
Commulin-A (1) was isolated from the methanolic extract of V. serpens as yellowish
amorphous powder (see section 2.7.2). The compound 1 was assigned molecular formula
C17H14O5 on the bases of ion peak at m/z 299 [M]+ in EI-MS and NMR spectral data. The
UV maxima at 340 and 264 nm indicated a flavonoid skeleton in compound 1 (Chauhan
et al., 1977). The IR spectrum revealed the presence of hydroxyl (3450 cm-1), conjugated
ester (1695 cm-1), conjugated carbonyl (1719 cm-1), and aromatic functionalities (2968,
1591, and 1463 cm-1).
Figure 3.25: Commulin-A (1)
The 1H-NMR spectrum of commulin-A (1) showed the presence of a methoxy, a methyl,
a methine and aromatic protons. In the downfield region of the spectrum a sharp singlet at
δ6.66was assigned to H-3methine proton. A broad singlet of one proton integration at δ
13.16 was assigned to the hydrogen-bonded hydroxyl group present at C-5. Similarly a
sharp signal of three proton integration was observed at δ 4.04 due to the presence of
Chapter – 3 RESULTS & DISCUSSION
113
methoxyl group in compound (1). The 1H-NMR spectrum also displayed multiplet in the
range of 7.04-8.01, were due aromatic protons
The 13C-NMR spectrum (Broad band decoupling (BB), Distortionless Enhancement
Polarization Transfer (DEPT)) (Table 3.18) showed seventeen signals, including one
methyl, one methoxy, six methines, and nine quaternary carbons. The downfield region
signals at 181.1 were assigned to the ketonic carbons of ring C. Similarly, signals at
158.5, 154.3, 153.4 and 147.7 were due to the C-2, C-5, C-7 and C-10 of the ring A
respectively. In the upfield region signals at 29.7 and 60.1 were assigned to methyl and
methoxyl group of the molecule.
Further structural assignments were made by using 2D-NMR experiments. In the HMBC
spectrum, the C-3 methine proton (δ 6.66) showed correlations with C-2 (δ 158.5), C-4 (δ
181.1) and C-9 (δ 111.3).The C-2′ methine proton showed correlations with C-1′ (δ
112.4) and C-3′ (δ 126.7). Similarly correlation of the C-4′ methine proton (δ 7.42) with
the C-3′ (δ 126.7), C-5′ (δ 126.5) and C-2′ (δ 122.3) were established from the same
HMBC spectrum. Moreover C-6′ (δ 7.62) methine proton exhibited interactions with C-1′
(δ 112.4) and C-5′ (δ 126.5). Also, C-6 (δ 1.57) methyl protons showed HMBC
interactions with C-5 (δ 154.3), C-7 (δ 153.4) and C-6 (δ 111.3). Similarly C-5 (δ 13.16)
hydroxyl proton showed HMBC interactions with C-4 (δ 181.1), C-9 (δ 111.3), and C-6
(δ 111.3). Thus on the basis all the spectroscopic data the structure of compound 1 was
deduced as flavone and having 8-methoxy, 6-methyl, 5, 7-dihydroxyflavone (Figure-3.21)
Chapter – 3 RESULTS & DISCUSSION
114
Figure 3.26: Key HMBC interaction in Compound 1
Table 3.18: 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-A (1) in
CDCl3
C.No. 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC
Correlations
2 - 158.5 C
3 6.66, s 94.2 CH 2, 4, 9
4 - 181.1 C
5 - 154.3 C
6 - 111.3 C
7 - 153.4 C
8 - 125.4 C
9 - 111.3 C
10 - 147.7 C
1´ - 112.4 C
2´ 8.01, d, 7.32 122.3 CH 1´, 2´, 3´, 4´
3´ 7.04, t, 7.38 126.7 CH
4´ 7.42, t, 6.98 128.2 CH
5´ 7.04, t, 7.38 126.7 CH
6´ 8.01, d, 7.32 122.3 CH 1´, 6´, 5´
CH3 1.57, s 29.7 ) CH3 5, 6, 7,
OCH3 4.04, s 60.1 CH3 10, 8, 7
3.3.1.2 Commulin-B (2)
Commulin-B (2) was isolated from the methanolic extract of V. serpens as yellow
amorphous powder (see section 2.7.2). The compound 2 had molecular formula
C17H14O6, as established on the basis of ion peak at m/z 314 [M+]+ in HREI-MS and
Chapter – 3 RESULTS & DISCUSSION
115
NMR spectral data .The HREI-MS exhibited an ion at m/z298 [M -17] + which resulted
from the loss of a hydroxyl group from the molecule. The HREI-MS also showed
fragments at m/z196, and 118 resulting from the retro Diels-Alders cleavage of the ring C
of compound 2 (Grisebach and Grambow, 1968). Similarly these ions confirmed the ring
A was substituted by two hydroxyl groups, one methyl and one methoxy group, while
ring B with one hydroxyl group. The IR spectrum of 2 included hydroxy (3422 cm-1),
conjugated ketone (1719 cm-1), and aromatic absorption (1601, 1500, 2968 cm-1).
Figure 3.27: Commulin-B (2)
The 1H-NMR spectral data of Commulin-B (2) showed the distinct resemblance with that
of compound 1 except for the absence of C-2′ methine proton in 1H-NMR and the
presence of additional quaternary signals at δ 154.3 in 13C-NMR spectrum (Table-3.19) of
compound 2, indicating the presence of a hydroxyl group at C-2′ in Commulin-B.
In the downfield region of the 1H-NMR spectrum, a sharp singlet at δ 6.54 was assigned
to H-3 methine proton. The 1H-NMR spectrum also displayed a singlet of three protons
integration at 4.04 due to the methoxyl group at C-8 position of ring A. Another singlet
of three proton integration at 1.29 was assigned to the methyl group of the same ring A.
A multiplet of four protons integration in the range of 7.07-7.9 was assigned to aromatic
methine protons of ring of ring B.
Chapter – 3 RESULTS & DISCUSSION
116
The 13C-NMR spectrum (Broad band decoupling (BB), Distortionless Enhancement
Polarization Transfer (DEPT) (Table 3.19) showed seventeen signals, including one
methyl, one methoxy, five methines, and ten quaternary carbons. The downfield region
signals at 179.8 were assigned to the ketonic carbons of ring C. Similarly, signals at
160.9, 154.3 and153.4 were due to the C-2, C-5, and C-7 of the ring A respectively. In the
upfield region signals at 29.4 and 60.9 were assigned to methyl and methoxyl group of
the molecule
In the HMBC spectrum, the C-3 methine proton (δ 6.54) showed correlations with C-2 (δ
160.9), C-4 (δ 179.8) and C-9 (δ 102.4). Similarly correlation of the C-4′ methine proton
(δ7.34) with the C-3′ (δ 125.5), C-5′ (δ 126.9) and C-2′ (154.3) were established from the
same HMBC spectrum. Moreover C-6′ (δ7.16) methine proton exhibited interactions with
C-1′ (δ 122.4) and C-5′ (δ 126.9). Thus structure of compound 2 was deduced as flavone
and having 8-methoxy, 6-methyl, 5, 7, 2′-trihydroxyflavone.
Figure 3.28: Key HMBC interaction in Commulin-B (2)
Chapter – 3 RESULTS & DISCUSSION
117
Table 3.19 1H- (400 MHz.) and 13C-NMR (125 MHz) Data of Commulin-B (2) in
CDCl3
C.No. 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC
Correlations
2 - 160.9 C
3 6.54, s 96.6 CH 2, 4, 9
4 - 179.8 C
5 - 154.3 C
6 - 110.8 C
7 - 153.4 C
8 - 123.2 C
9 - 102.4 C
10 - 147.7 C
1´ - 122.4 C
2´ - 154.3 C
3´ 7.07, d, 7.9 125.5 CH
4´ 7.34, t, 7.2 121.7 CH 2´, 3´, 5´
5´ 7.14, t, 7.1 126.9 CH
6´ 7.16, d, 6.8 111.3 CH 1´, 5´
CH3 1.29, s 29.4 CH3
OCH3 4.04 60.9 CH3
3.3.1.3 Commulin-C (3)
Commulin-C (3) was also isolated from the methanolic extract of V.serpens as an
amorphous powder. The compound 3 was assigned the formula C18H16O6 on the basis of
an ion peak at m/z 329 [M]+in FAB-MS and based on NMR spectral data. The FAB-MS
of Commulin-C (3) exhibited ion peak at m/z 297 which resulted from the loss of
methoxyl group from M+. The IR spectrum of compound 3 showed absorption bands
3560 (OH), 2968, 1610, 1515 (aromatic), 1717 (conjugated ketone).
Chapter – 3 RESULTS & DISCUSSION
118
Figure 3.29: Commulin-C (3)
The 1H-NMR spectral data of compound 3 indicated its resemblance with the compound
2 except for the presence of a methoxy group instead of hydroxyl group at C-2` in
compound 3. In the downfield region of the 1H-NMR spectrum, a sharp singlet at δ 6.54
was assigned to H-3 methine proton. The 1H-NMR spectrum also displayed two singlets
of three protons integration at 3.96 and 3.89 were due to the methoxyl groups of the
compound.
The 13C-NMR spectrum (BB, DEPT) (Table 3.20) showed eighteen signals, including one
methyl, two methoxy, five methines and ten quaternary carbons. In HMBC spectrum
(Figure 3.26), the C-3 methine proton (δ 6.54) showed correlations with C-2 (δ 161.5), C-
1′ (δ 123.2), C-4 (δ182.2), and C-9 (δ 102.4). The presence of the second methoxy group
at C-2′ (δ 158.1) was further confirmed by the HMBC correlations of methoxy protons (δ
3.89) with C-2′ (δ 158.1), C-1′ (δ 123.2), and C-3′ (δ 119.7).Thus on the basis of 1D and
2D NMR specral data the structure of compound 3 was deduced as flavone and having 2′,
8-dimethoxy, 6-methyl, 5, 7-dihydroxyflavone.
Chapter – 3 RESULTS & DISCUSSION
119
Figure 3.30: Key HMBC interaction in Commulin-C (3)
Table 3.20: 1H- (400 MHz.) and 13C-NMR (100 MHz) Data of Commulin-C (3) in
CDCl3
C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC
Correlations
2 - 161.5 C
3 6.54, s 96.6 CH 2, 4, 9
4 - 182.2 C
5 - 155.9 C
6 - 110.8 C
7 - 155.9 C
8 - 123.2 C
9 - 102.4 C
10 - 149.8 C
1´ - 123.2 C
2´ - 158.1 C
3´ 7.03, d, 8.7 119.7 CH
4´ 7.45, t, 7.4 129.8 CH
5´ 7.09, t, 7.2 121.3 CH 5´, 6´, 4´
6´ 7.65, d, 7.9 130.8 CH 1´, 6´, 5´
CH3 1.57, s 29.3 CH3
OCH3 3.96, s 61.8 CH3
OCH3 3.89, s 56.5 CH3 1´, 2´, 3´
Chapter – 3 RESULTS & DISCUSSION
120
3.3.2 Known Compounds from Viola serpens
3.3.2.1 5-Hydroxy-7-methoxy flavone (tectochrysine) (4)
The compound 4 was isolated as colorless solid from the sub fraction FMC-3. The HREI-
MS showed the M+ ion peak at m/z corresponding to formula C16H13O4 (calcd. for
C16H13O4, 268.2011). The UV spectrum displayed 268.2013 the maxima at 325 and 267
nm. These values indicated that compound 4 is a flavone (Bernard, 1983). The IR
spectrum showed the absorption bands at 3400, 3000, 1649, 1475, and 1460 cm-1.
O
OOH
H3CO
9 2
34
56
78
10
Tectochrysine (4)
1'
2' 3'4'
5'6'
Figure 3.31: Structure of compound Tectochrysine (4)
The 1H-NMR spectrum showed in the downfield region a singlet at δ 6.65 corresponding
to the H-3 position, while two doublet at δ 6.48 (H, J = 2.2) and δ 6.39 (d, J = 2.2 Hz),
were assigned to H-6, and H-8. The multiplets at δ 7.87 and 7.52 were due to H-2′, H-6′,
H-3′, and H-4′, H-5′. A sharp singlet of three protons integration at δ 3.94 was assigned to
the methoxyl group present at ring A. The 13C-NMR spectrum (BB and DEPT) of
compound 4 corroborated the presence of one methyl, eight methine and seven quaternary
carbons. The signal at δ 165.6 corresponded to C-7, while the signal at δ 106.0 was due to
C-3. The signal at δ 60.3 was due to the presence of methoxyl group.
Chapter – 3 RESULTS & DISCUSSION
121
By comparison with the literature data, compound 4 was identified as 5-hydroxy-7-
methoxy flavone (4), previously reported from Boesenbergia pandurata plant (Debral et
al., 1994).
3.3.2.2 4́, 5-Dihydroxy-7-methoxy-6, 8-dimethylflavone (Sideroxylin) (5)
Compound 5 was isolated as yellow needles from the sub fraction FMC-5, showed the
UV absorption at max 330 and 275 nm. The IR spectrum displayed absorption bands at
3500 and 1655 cm-1 due to the presence of hydroxyl and ketonic functionalities. The EI-
MS showed the M+ at m/z 312. The molecular formula was confirmed as C18H16O5,
through M+ ion peak in HREI-MS at m/z 312.0125 (calcd. for C18H16O5, 312.0123).
O
OOH
H3C
H3CO
CH3
OH1'
2
345
6
78
9
10
2' 3'
4'
5'6'
Sideroxyline (5)
Figure 3.32: Structure of compound Sideroxyline (5)
The 1H-NMR spectrum showed a singlet at δ 6.87 corresponding to the H-3 position,
while the two doublets at δ 7.97 (J = 8.8 Hz) and 6.94 (J = 8.7 Hz) were assigned to H-2′,
H-6′, and H-3′, H-5′, respectively. A downfield one-proton singlet at δ 13.07 was assigned
to the hydroxyl group at C-5 position. The signal at δ 3.94 was assigned to methoxyl
group at C-7 position. Two singlets each of three protons integration at δ 2.08 and δ 2.32
were assigned to the methyl group present at C-6 and C-8 in ring A. The 13C-NMR
spectrum (BB and DEPT) of compound 5 corroborated the presence of three methyl, five
methine and ten quaternary carbons. The signal at 108.6 corresponded to C-8, while the
Chapter – 3 RESULTS & DISCUSSION
122
signal at 102.8 was due to C-3. In the up field region the signals at δ 60.3, 8.29, and 8.07
were due to the presence of one methoxyl and two methyl groups in ring A.
By comparison with the data published in literature, compound 5 was identified as 4′, 5-
dihydroxy-7-methoxy-6,8-dimethylflavone (5), previously reported from Eucalyptus
sideroxylone plant (Guimaraes et al., 1975).
3.3.2.3 2, 5-Dihydroxy-4-methoxybenzophenone (Cearoin) (6)
Compound 6 was isolated as yellow amorphous powder from sub fraction FMC-5 ethyl
acetate soluble part of crud methnol extract. The HREI-MS exhibited M+ at m/z 244.1322
corresponding to the molecular formula C14H12O4 (calcd. for C14H12O4, 244.1334). The
IR spectrum showed absorption bands at 3448 (OH), 1743 (C = O) cm-1.
O
OH
OCH3
OH
12
3
456
1'2'3'
4'5' 6'
AB
Cearoin (6)
Figure 3.33: Structure of compound Cearoin (6)
The 1H-NMR spectrum showed singlets at δ 6.88 and 6.59 corresponding to the H-6 and
H-3 position, while the multiplets at δ 7.61 and 7.54 were due to aromatic proton of ring
B, respectively. There were downfield signals at δ 11.95 and 8.89 due to hydroxyl group
at C-2 and C-5 positions. The 13C-NMR spectrum (BB and DEPT) of cearoin (6)
corroborated the presence of one methyl, seven methine, and six quaternary carbons. The
signal at δ 198.7 corresponded to carboxylic carbon, while the signal at δ 157.8 was due
Chapter – 3 RESULTS & DISCUSSION
123
to C-2 having the hydroxyl group. The signal at δ 60.3 was assigned to the methoxyl
carbon at C-4 position in ring A.
By comparison with the data published in literature, compound 6 was identified as 2, 5-
dihydroxy-4-methoxybenzophenone (cearoin) previously reported from Dalbergia
melanoxylon plant (Lounasmaa et al., 1977).
.
CONCLUSION
124
CONCLUSION
Over the years, medicinal plants have played historical role in new drug discovery.
For this reason, traditional uses of these plants need to be explored in well established
scientific paradigms. V. seprens, an important medicinal plant, is traditionally used for
the treatment of various ailments including jaundice, asthma, throat cancer, dermatitis
and constipation. These uses are purely based on empirical knowledge from
generations without any scientific rationale. In this regard, various in vivo and in vitro
pharmacological activities of V. serpens have been carried out in order to provide
scientific background to its folkloric uses.
The in vitro activities were included antimicrobial, DPPH free radical scavenging
assay, larvicidal and enzyme inhibition (acetylcholine esterase). The results showed
marked therapeutic potential of the crude extracts and subsequent solvent fractions in
various already reported tests. Similarly, the in vivo activities like acute toxicity,
antinociceptive, anti-inflammatory, hepatoprotective and nephroprotective were
carried in different recommended protocols. The animal based studies showed
profound effects in specific assays.
The column chromatography technique used led to the isolation of six compounds
including three new flavonoids. Among the six, three compounds were new (not
reported before) and the other three were already reported from the other sources but
first time from V. serpens. Commulin-A, Commuline-B and Commuline-C were the
new compounds whereas, tectochrysine, Sideroxylin and Cearoin were the already
reported compounds. The chemical structures of these isolated compounds were
elucidated using various spectroscopic techniques. When these isolated compounds
were tested for antibacterial activity against various pathogenic bacteria, most of them
CONCLUSION
125
were found susceptible. Additionally, when these isolated compounds were tested for
free radical scavenging effect against DPPH, they exhibited strong antioxidant action.
In short, we have provided scientific foundation to different traditional uses of this
plant in various in vitro and in vivo protocols. Similarly, the isolation of pure
secondary metabolites explored the molecular background of the plant. Keeping in
view the outstanding pharmacological activities of the crude extract and extracted
fractions, which were supported by the isolated compound, suggests further
mechanistic detail studies to discover new effective therapeutic agents for clinical
uses.
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126
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