PHYTOCHEMICAL INVESTIGATION, ANTIOXIDANT ACTIVITIES AND
HEPATOPROTECTIVE EFFICACY OF INDIGOFERA TIRUNELVELICA
SANJAPPA AGAINST CCL4 INDUCED WISTAR ALBINO RATS.

A THESIS

SUBMITTED BY
S. SUBBURAYALU
(Reg. No. 8176)

BIOCHEMISTRY

in partial fulfillment of the requirements for the award of degree of

DOCTOR OF PHILOSOPHY

MANONMANIAM SUNDARANAR UNIVERSITY
TIRUNELVELI - 627012.
TAMILNADU, INDIA.

DECEMBER – 2018


MANONMANIAM SUNDARANAR UNIVERSITY
TIRUNELVELI - 627 012

CERTIFICATE
The

research

work

embodied

in

the

present

thesis

entitled

“PHYTOCHEMICAL INVESTIGATION, ANTIOXIDANT ACTIVITIES AND
HEPATOPROTECTIVE EFFICACY OF INDIGOFERA TIRUNELVELICA
SANJAPPA AGAINST CCL4 INDUCED WISTAR ALBINO RATS.” has been
carried out in the BIOCHEMISTRY, Manonmaniam Sundaranar University,
Tirunelveli. The work reported herein is original and does not form part of any other
thesis or dissertation on the basis of which a degree or award was conferred on an
earlier occasion or to any other scholar.
I understand the University’s policy on plagiarism and declare that the thesis
and publications are my own work, except where specifically acknowledged and has
not been copied from other sources or been previously submitted for award or
assessment.

S. SUBBURAYALU
(Reg.No. 8176)
RESEARCH SCHOLAR


Dr.A.PALAVESAM

Dr. K.R. T. ASHA

CO-SUPERVISOR

SUPERVISOR

Prof. and Head

Assistant professor

Department of Animal Science

Department of Biochemistry

Manonmaniam Sundaranar University

Government Arts College

Tirunelveli

Paramakudi


ACKNOWLEDGEMENT
I am most thankful to God Almighty for sustaining and keeping me in His
grace and providential protection throughout my life.
I feel highly Privilege to acknowledge my sincere thanks to my research
supervisor Dr.K.R.T.Asha, Assistant Professor, Department of Biochemistry, K.R

College of Arts & Science, Government Arts College, Paramakudi, Ramnad Dist., for
her encouraging and continuous support as a guide throughout the entire period of
research investigation. Her dynamic, vibrant personality and Immunse knowledge has
been a source of insipiration to me for achieving more miles in life.
I deeply acknowledge the constant support, encouragement and invaluable
guidance at every step of my project by Dr.A.Palavesam, Prof. and Head,
Department of Animal Science, Manonmaniam Sundaranar University, Tirunelveli
for his assistance throughout this work were unparallel without which this work
would not have been possible.
I would like to acknowledge my approbation and humility to Kalvi Thanthai.
Thiru K. Ramasamy, Chairman,Thiru K.R. Arunachalam, Managing Director ,
Dr.KNKSK.Chockalingam, Director, National Engineering College,Kovilpatti and
Dr.An.Kannappan, Principal of K.R College of Arts & Science, Kovilpatti inspired
me to start this research.
I express my heartful thanks to Dr.S.Chockalingam and Dr.R.Gopal,
Formerly Principals of K.R College of Arts & Science, Kovilpatti for their advice and
suggestions to carry out my work successfully.
I express my sincere thanks to the management of KRCAS for all their
support in proving the lab facilities and library facilities to me throughout my studies.
I also thankful to the Staff Members, and Non Teaching Staff members of K.R
College of Arts & Science, Kovilpatti inspired me to start this research.
My sincere thanks to my family members and all my friends who all behind
the successful completion of this thesis.

(S.SUBBURAYALU)


TABLE OF CONTENT
1


INTRODUCTION ............................................................................................................ 1
1.1 HERBAL MEDICINE .......................................................................................................................... 1
1.2 SECONDARY METABOLITES........................................................................................................... 2
1.3 ANTIOXIDANTS ................................................................................................................................. 4
1.4 LIVER DISEASE ................................................................................................................................. 6
1.4.1 Epidemiology......................................................................................................................... 6
1.4.2 Hepatocytes............................................................................................................................ 7
1.4.3 Hepatitis.................................................................................................................................. 8
1.4.4 Alcohol related liver diseases........................................................................................... 9
1.4.5 Jaundice .................................................................................................................................. 9
1.4.6 Liver tumors........................................................................................................................... 9
1.4.7 Reye’s syndrome ................................................................................................................. 10
1.4.8 Hepatotoxins ........................................................................................................................ 10
1.5 STAGES IN THE LIVER DISEASES ................................................................................................ 10
1.5.1 Inflammation ....................................................................................................................... 10
1.5.2 Fibrosis ................................................................................................................................. 11
1.5.3 Cirrhosis ............................................................................................................................... 11
1.5.4 Liver failure ......................................................................................................................... 11
1.6 DRUG INDUCED LIVER DISEASES .............................................................................. 12
1.6.1 Drugs that causes Liver Injury ....................................................................................... 12
1.7 LIVER FUNCTION TESTS ................................................................................................................ 15
1.8 HERBS FOR LIVER DISEASES ....................................................................................................... 16
1.9 EXTRACTION PROCEDURES:-....................................................................................................... 21
1.10 CHROMATOGRAPHIC TECHNIQUES ........................................................................................... 22

2

AIM OF THE WORK .................................................................................................... 24

3


REVIEW OF LITERATURE........................................................................................ 25
3.1 LIVER DISEASES .............................................................................................................................. 25
3.2 HERBAL MEDICINE ......................................................................................................................... 32
3.3 QUALITY OF EXTRACTS ................................................................................................................ 36
3.4 SECONDARY METABOLITES......................................................................................................... 39
3.5 ANTIOXIDANTS ............................................................................................................................... 45
3.6 GC-MS STUDIES ............................................................................................................................ 52
i


3.7 HEPATOPROTECTIVE PLANTS ...................................................................................................... 57
3.7.1 Fabaceae Family plants for Hepatic diseases ........................................................... 75
4

MATERIALS AND METHODS ................................................................................... 83
4.1 PLANT MATERIAL .......................................................................................................................... 83
4.1.1 Taxonomy ............................................................................................................................. 83
4.1.2 Habitat................................................................................................................................... 83
4.1.3 Description........................................................................................................................... 83
4.2 IDENTIFICATION AND AUTHENTICATION OF I. TIRUNELVELICA ........................................... 85
4.3 PHARMACOGNOSTIC METHODS .................................................................................................. 85
4.3.1 Physiological and Organoleptic Characteristic evaluation of I. tirunelvelica 85
4.3.2 Fluorescence Analysis of I. tirunelvelica .................................................................... 85
4.4 PHYSICOCHEMICAL PROPERTIES OF I. TIRUNELVELICA .......................................................... 85
4.4.1 Determination of Foreign Matter .................................................................................. 85
4.4.2 Determination of Moisture Content (Loss on Drying) ............................................ 85
4.4.3 Determination of Total Ash ............................................................................................. 86
4.4.4 Determination of Acid Insoluble Ash ........................................................................... 86
4.4.5 Determination of Water Soluble Ash ............................................................................ 86

4.4.6 Determination of extractive values ............................................................................... 86
4.4.7 Determination of Alcohol Soluble Extractive Value ................................................ 87
4.4.8 Determination of Water Soluble Extractive Value ................................................... 87
4.5 PHYTOCHEMICAL SCREENING ..................................................................................................... 87
4.5.1 Extraction of plant materials- Cold extraction method .......................................... 87
4.5.2 Behaviour of drug powder with various chemical reagents .................................. 87
4.6 QUANTITATIVE ANALYSIS OF PHYTOCHEMIAL CONSTITUENTS ........................................ 89
4.6.1 Estimation of Total Alkaloids ......................................................................................... 89
4.6.2 Estimation of Total Flavonoids ...................................................................................... 89
4.6.3 Estimation of Phenol ......................................................................................................... 90
4.7 IN-VITRO ANTIOXIDANT ASSAY .................................................................................................. 90
4.7.1 DPPH Radical Scavenging Assay ................................................................................. 90
4.7.2 ABTS Radical Scavenging Assay ................................................................................... 91
4.7.3 Ferric Reducing Antioxidant Power (FRAP) Assay ................................................ 92
4.7.4Superoxide Radical Scavenging Activity ...................................................................... 93
4.7.5 H2O2 Radical Scavenging Activity................................................................................. 94

ii


4.7.6 Hydroxyl Radical Scavenging Activity......................................................................... 94
4.8 STUDIES ON HEPATOPROTECTIVE SCREENING........................................................................ 95
4.8.1 In-vitro Hepatoprotective Studies ................................................................................. 95
4.8.2 MTT ASSAY ........................................................................................................................ 95
4.9 IN-VIVO STUDIES ............................................................................................................................. 97
4.9.1 Experimental design .......................................................................................................... 97
4.9.2 Experimental animals ....................................................................................................... 97
4.9.3 Experimental Design for Toxicity Studies................................................................... 98
4.9.4 Carbon Tetrachloride (CCl4) induced Hepatotoxicity ............................................ 98
4.9.5 Experimental Design for Hepatoprotective Studies ................................................. 99

4.9.6 Body Weight of the Animals .......................................................................................... 100
4.9.7 Determination of Red Blood Cells Count ................................................................. 100
4.9.8 Determination of White Blood Cells Count .............................................................. 100
4.9.9 Determination of Haemoglobin ................................................................................... 100
4.9.10 Enumeration of Platelets Count ................................................................................ 101
4.9.11 Determination of ESR................................................................................................... 104
4.9.12 Determination of PCV .................................................................................................. 105
4.9.13 Estimation of Blood Glucose ...................................................................................... 105
4.9.14 Estimation of Protein .................................................................................................... 106
4.9.15 Estimation of Blood Urea ............................................................................................ 106
4.9.16 Estimation of Serum Uric Acid .................................................................................. 106
4.9.17 Estimation of Creatinine.............................................................................................. 107
4.9.18 Estimation of Liver Glycogen..................................................................................... 107
4.9.19 Estimation of Serum Bilirubin ................................................................................... 108
4.9.20 Extraction of Lipids....................................................................................................... 108
4.9.21 Estimation of Serum Cholesterol .............................................................................. 108
4.9.22 Estimation of Serum Triglycerides ........................................................................... 109
4.9.23 Estimation of Phospholipids ....................................................................................... 109
4.9.24 Estimation of Free Fatty Acid .................................................................................... 109
4.9.25 Assay of Serum HDL Cholesterol ............................................................................. 110
4.9.26 Assay of Aspartate Transaminase (AST) ................................................................ 111
4.9.27 Estimation of Alanine Transaminase (ALT) .......................................................... 111
4.9.28 Estimation of Serum Alkaline Phosphatase ........................................................... 111

iii


4.9.29 Assay of Lactate Dehydrogenase (LDH) ................................................................ 112
4.9.30 Assay of Gamma- Glutamyl Transferase ................................................................ 112
4.9.31 Determination of Glucose- 6- Phosphatase. .......................................................... 112

4.9.32 Assay of Glucose-6- Phosphatase Dehydrogenase .............................................. 113
4.9.33 Assay of Hexokinase ..................................................................................................... 113
4.9.34 Estimation of Lipid Peroxides .................................................................................... 113
4.9.35 Assay of Reduced Glutathione ................................................................................... 114
4.9.36 Assay of Superoxide Dismutase ................................................................................. 114
4.9.37 Assay of Catalase........................................................................................................... 114
4.9.38 Assay of Glutathione Peroxidase (GPx) ................................................................. 115
4.9.39 Assay for Glutathione S Transferase (GST)........................................................... 115
4.9.40 Determination of Ascorbic Acid (Vitamin -C). ..................................................... 115
4.9.41 Determination of D-Tocopherol (Vitamin E)......................................................... 116
4.9.42 Activity of Na+/K+ Adenosine Triphosphatase ...................................................... 116
4.9.43 Activity of Magnesium Adenosine Triphosphatase .............................................. 117
4.9.44 Activity of Calcium Adenosine Triphosphatase .................................................... 117
4.9.45 Histopathological Studies ........................................................................................... 117
4.10 ISOLATION AND PREDICTION OF MARKER COMPOUND ...................................................... 117
4.11 BIOINFORMATIC STUDIES........................................................................................................... 118
4.11.1 Docking............................................................................................................................. 118
4.11.2 Ligand preparation ....................................................................................................... 118
4.11.3 Active site prediction .................................................................................................... 119
4.11.4 Docking protocol ........................................................................................................... 119
4.12 STATISTICAL ANALYSIS ........................................................................................................... 119
5

RESULTS AND DISCUSSION ................................................................................... 121
5.1 PHYSIOLOGICAL AND ORGANOLEPTIC CHARACTERISTICS ................................................ 121
5.2 PHYSICOCHEMICAL ANALYSIS ................................................................................................. 121
5.3 FLUORESCENCE ANALYSIS ......................................................................................................... 123
5.4 PHYSICOCHEMICAL PARAMETERS ........................................................................................... 124
5.5 PRELIMINARY PHYTOCHEMICAL SCREENING ....................................................................... 126
5.6 QUANTITATIVE ANALYSIS OF SECONDARY METABOLITES ............................................... 128

5.7 ANALYSIS OF IN-VITRO ANTIOXIDANT ACTIVITY .................................................................. 131
5.7.1 DPPH free radical scavenging Activity..................................................................... 131

iv


5.7.2 ABTS free radical scavenging activity ....................................................................... 134
5.7.3 FRAP Free Radical Scavenging Activity .................................................................. 138
5.7.4 SO Free Radical Scavenging Activity ........................................................................ 141
5.7.5 H2O2 Free Radical Scavenging Activity .................................................................... 144
5.7.6 Hydroxyl Free Radical Scavenging Activity ............................................................ 147
5.8 IN-VITRO HEPATOPROTECTIVE SCREENING ............................................................................ 150
5.8.1 MTT Assay.......................................................................................................................... 150
5.9 IN-VIVO TOXICITY STUDIES........................................................................................................ 154
5.9.1Effect of ItW-Et on weight of body, liver and kidney of Wistar Albino
Rats . .............................................................................................................................................. 166
5.9.2 Effect of ItW-Et on hematological parameters of Wistar Albino Rats. ............ 166
5.9.3 Effect of ItW-Et on biochemical parameters of Wistar Albino Rats. ................ 167
5.9.4 Effect of ItW-Et on hepatic enzymes of Wistar Albino Rats. ............................... 167
5.9.5 Effect of ItW-Et on histological study of Wistar Albino Rats. ............................. 167
5.10 IN-VIVO HEPATOPROTECTIVE STUDIES ................................................................................... 169
5.10.1 Effect of ItW-Et on the Body weight ......................................................................... 200
5.10.2 Effect of ItW-Et on Hematological Parameters .................................................... 202
5.10.3 Effect of ItW-Et on Biochemical Parameters ........................................................ 203
5.10.4 Effect of ItW-Et on Lipid Profile ............................................................................... 207
5.10.5 Effect of ItW-Et on Hepatic enzymes........................................................................ 209
5.10.6 Effect of ItW-Et on Carbohydrates Metabolising Enzymes ............................... 211
5.10.7 Effect of ItW-Et on Antioxidants................................................................................ 213
5.10.8 Effect of ItW-Et on Non enzymatic Antioxidants .................................................. 218
5.10.9 Effect of ItW-Et on Membranous ATPase enzymes ............................................. 219

5.10.10 Effect of ItW-Et on histopathological studies of Liver Cells. ......................... 220
5.11 MARKER COMPOUND ISOLATION ............................................................................................. 222
5.12 IN-SILICO ANALYSIS .................................................................................................................. 228
5.12.1Docking studies of a Identified compounds from I. tirunelvelica .. 228
6

SUMMARY ................................................................................................................... 243

7

CONCLUSION ............................................................................................................. 246

8

REFERENCES ............................................................................................................. 247

v


LIST OF TABLES
Table 5.1: Physiological and organoleptic characteristics of whole plant dry powder of
Indigofera tirunelvelica Sanjappa .......................................................................... 121
Table 5.2: Fluorescence analysis of whole plant dry powder of Indigofera tirunelvelica
Sanjappa treated with various reagents ................................................................... 122
Table 5.3: Fluorescence analysis of whole plant extracts of Indigofera tirunelvelica
Sanjappa in different solvent systems ..................................................................... 122
Table 5.4: Physiochemical analysis of whole plant of Indigofera tirunelvelica Sanjappa ....... 124
Table 5.5: Extractive values of different extracts of whole plant dry powder of
Indigofera tirunelvelica Sanjappa ........................................................................... 125
Table 5.6: Preliminary Phytochemical screening of Indigofera tirunelvelica Sanjappa .......... 126

Table 5.7: Quantitative analysis of important organic constituents of Indigofera
tirunelvelica Sanjappa ............................................................................................. 128
Table 5.8: Effect of various concentrations of whole plant extracts of Indigofera
tirunelvelica Sanjappa on the percentage of DPPH activity in different
solvent systems ....................................................................................................... 132
Table 5.9: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of ABTS activity in Different
Solvent Systems ...................................................................................................... 136
Table 5.10: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of FRAP activity in Different
Solvent Systems ...................................................................................................... 138
Table 5.11: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of SO activity in Different Solvent
Systems ................................................................................................................... 141
Table 5.12: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of H2O2 activity in Different
Solvent Systems ...................................................................................................... 144
Table 5.13: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of Hydroxyl activity in Different
Solvent Systems ...................................................................................................... 147
Table 5.14: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Hep G2
cell line (MTT Assay) ............................................................................................. 151

vi


Table 5.15: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on weight
of body, liver and kidney of Wistar Albino Rats .................................................... 154
Table 5.16: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Hematological and Biochemical parameters of Wistar Albino Rats ...................... 158

Table 5.17: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Hepatic
enzymes of Wistar Albino Rats .............................................................................. 162
Table 5.18: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on the
Body weight (g) of CCl4 treated Wistar Albino Rats ............................................. 169
Table 5.19: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Hematological Parameters of CCl4 treated Wistar Albino Rats .............................. 172
Table 5.20: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Biochemical Parameters of CCl4 treated Wistar Albino Rats ................................. 176
Table 5.21: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Lipid
Profile of CCl4 treated Wistar Albino Rats ............................................................. 182
Table 5.22: Effect of ItW-Et on enzymes of CCl4 treated Wistar Albino Rats ......................... 185
Table 5.23: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Carbohydrates Metabolising Enzymes of CCl4 treated Wistar Albino Rats........... 188
Table 5.24: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Antioxidants of CCl4 treated Wistar Albino Rats ................................................... 190
Table 5.25: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Non
enzymatic Antioxidants of CCl4 treated Wistar Albino Rats.................................. 193
Table 5.26: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Membranous ATPase enzymes of CCl4 treated Wistar Albino Rats ...................... 196
Table 5.27: List of compounds Identified from ItW-Et ............................................................ 225
Table 5.28: Docking Score on the interaction of the high affinity potential of plant
compounds with target protein - With Hepatitis B X (1QGT) ............................... 240
Table 5.29: Docking Score on the interaction of the high affinity potential of plant
compounds with target protein - Heme Oxygenase I (1N3U) ................................ 241

vii


LIST OF FIGURES
Figure 1.1: Structure of Liver .................................................................................................... 7

Figure 5.1: Quantitative analysis of important organic constituent of Indigofera
tirunelvelica Sanjappa in various solvents ........................................................ 129
Figure 5.2: Effect of various concentrations of whole plant extracts of Indigofera
tirunelvelica Sanjappa on the percentage of DPPH activity in different
solvent systems ................................................................................................. 133
Figure 5.3: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of ABTS activity in Different
Solvent Systems ................................................................................................ 137
Figure 5.4: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of FRAP activity in Different
Solvent Systems ................................................................................................ 139
Figure 5.5: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of SO activity in Different
Solvent Systems ................................................................................................ 142
Figure 5.6: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of H2O2 activity in Different
Solvent Systems ................................................................................................ 145
Figure 5.7: Effect of Various Concentrations of Whole Plant Extracts of Indigofera
tirunelvelica Sanjappa on the percentage of Hydroxyl activity in
Different Solvent Systems ................................................................................ 148
Figure 5.8: Effect of whole plant ethanol extract of I. tirunelvelica on Hep G2 cell line
(MTT Assay) ..................................................................................................... 151
Figure 5.9: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
bodyweight of Wistar Albino Rats ................................................................... 155
Figure 5.10: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on the
weight of Liver of Wistar Albino Rats ............................................................. 156
Figure 5.11: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on the
weight of Kidney of Wistar Albino Rats .......................................................... 157
Figure 5.12: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Hematological parameters of Wistar Albino Rats ............................................ 159


viii


Figure 5.13: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Blood glucose, Blood Protein, Blood Urea of Wistar Albino Rats .................. 160
Figure 5.14: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Bilirubin, Creatinine and Uric acid of Wistar Albino Rats ............................... 161
Figure 5.15: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Hepatic enzymes of Wistar Albino Rats ........................................................... 163
Figure 5.16: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Body
weight of CCl4 treated Wistar Albino Rats ....................................................... 170
Figure 5.17: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Liver
weight of CCl4 treated Wistar Albino Rats ....................................................... 171
Figure 5.18: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Hematological Parameters of CCl4 treated Wistar Albino Rats ....................... 173
Figure 5.19: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Platelets of CCl4 treated Wistar Albino Rats .................................................... 174
Figure 5.20: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Packed Cell Volume of CCl4 treated Wistar Albino Rats................................. 175
Figure 5.21: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Blood Glucose of CCl4 treated Wistar Albino Rats .......................................... 177
Figure 5.22: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Blood Protein of CCl4 treated Wistar Albino Rats ........................................... 178
Figure 5.23: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Blood Urea and Liver Glycogen of CCl4 treated Wistar Albino Rats .............. 179
Figure 5.24: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Uric
Acid and Creatinine of CCl4 treated Wistar Albino Rats ................................. 180
Figure 5.25: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Direct and Indirect Bilirubin of CCl4 treated Wistar Albino Rats .................... 181

Figure 5.26: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on Lipid
Profile of CCl4 treated Wistar Albino Rats ....................................................... 183
Figure 5.27: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Lipoprotein of CCl4 treated Wistar Albino Rats ............................................... 184
Figure 5.28: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Hepatoenzymes of CCl4 treated Wistar Albino Rats ........................................ 186
Figure 5.29: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on LDH
of CCl4 treated Wistar Albino Rats ................................................................... 187
ix


Figure 5.30: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Carbohydrates Metabolising Enzymes of CCl4 treated Wistar Albino
Rats ................................................................................................................... 189
Figure 5.31: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on LPO
of CCl4 treated Wistar Albino Rats ................................................................... 191
Figure 5.32: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Antioxidants of CCl4 treated Wistar Albino Rats ............................................. 192
Figure 5.33: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Vitamin C of CCl4 treated Wistar Albino Rats ................................................. 194
Figure 5.34: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Vitamin E of CCl4 treated Wistar Albino Rats ................................................. 195
Figure 5.35: Effect of whole plant ethanol extract of I. tirunelvelica (ItW-Et) on
Membranous ATPase enzymes of CCl4 treated Wistar Albino Rats ................ 197
Figure 5.36: Identification of bioactive compounds from whole plant ethanolic extract
of I. tirunelvelica ............................................................................................... 223
Figure 5.37: GC-MS spectra of isolated compound from ItW-Et .......................................... 224

x



LIST OF PLATES
Plate 4.1: Indigofera tirunelvelica Sanjappa ............................................................................ 84
Plate 5.1: Cytotoxic effects of whoel plant ethanol extracts of I. tirunelvelica on
HepG2 cell lines (MTT Assay) ........................................................................... 152
Plate 5.2: Photomicrographs of histopathological studies of Liver of Wistar Albino
Rats ...................................................................................................................... 164
Plate 5.3: Photomicrographs of histopathological studies of Kidney of Wistar Albino
Rats ...................................................................................................................... 165
Plate 5.4: Photomicrographs of histopathological studies of Liver Cells of CCl4
treated Wistar Albino Rats ................................................................................... 198
Plate 5.5: 3D view of Target Protein – Hepatitis B (1QGT) Receptors ................................ 230
Plate 5.6: 3D view of Target Protein –Heme Oxygenase I (1N3U) Receptors ..................... 231
Plate 5.7: Different views of the interaction of the high affinity potential Phytol with
Hepatitis B (1QGT) Receptors............................................................................. 232
Plate 5.8: Different views of the interaction of the high affinity potential
Hexadecanoic Acid with Hepatitis B (1QGT) Receptors .................................... 233
Plate 5.9: Different views of the interaction of the high affinity potential Methyl-Z,Z3,13-octadecadienol with Hepatitis B (1QGT) Receptors ................................... 234
Plate 5.10: Different views of the interaction of the high affinity potential of
Heptadecenal with Hepatitis B (1QGT) Receptors .............................................. 235
Plate 5.11: Different views of the interaction of the high affinity potential of Phytol
with Heme Oxygenase I (1N3U) Receptors ........................................................ 236
Plate 5.12: Different views of the interaction of the high affinity potential of
Hexadecanoic Acid with Heme Oxygenase I (1N3U) Receptors ........................ 237
Plate 5.13: Different views of the interaction of the high affinity potential of MethylZ,Z-3,13-octadecadienol with Heme Oxygenase I (1N3U) Receptors ................ 238
Plate 5.14: Different views of the interaction of the high affinity potential of
Heptadecenal with Heme Oxygenase I (1N3U) Receptors.................................. 239

xi



ABBREVIATIONS
%

: Percentage

+

: Present / Positive

±

: Plus or Minus

°

: Degree

µ

: Micro

µg

: microgram

µM

: microMole


1D

: Single Dimension

2D

: Two Dimension

3D

: Three Dimension

5-LOX

: 5-lipoxygenase

Å

: Angstrom

ABTS

: 2, 2’-azinobis-3-ethylbenzothiozoline- 6-sulphonic acid

AD

: Anno Domini

AE


: Adverse Event

AF

: Aqueous Fractions

ALD

Alcoholic liver disease

ALP

: Alkaline Phosphatase

ALT

: Alanine transaminase

AMP

: Adenosine Mono Phosphate

Annexin V-/PI-

: Annexin V -/Propidium iodide

Annexin-V FITC

: Annexin-V with Fluorescein Isothiocyanate


ANOVA

: Analysis of Variance

ANSA

: 1-amino-2-naphthol-6-sulphonilic acid

AST

: Aspartate aminotransferase

ATP

: Adenosine triphosphate

ATPase

: Adenosine triphosphatase

BC

: Before Christ

BDS

: Base Deactivated Silanol

BHT


: Butylated hydroxy toluene

BSA

: Bovine Serum Albumin

xii


BuOH

: Butanol

bw /BW

: body weight

C

: Centigrade

CAM

: Complementary and Alternative Medicine

CAT

: Catalase

CCl4


: Carbon tetrachloride

CDMT

: Combination-drugs-multi-targets

CE

: Catechin Equivalents / Chloroform Extract

CEE

: Crude Ethanolic Leaf Extract

CHCl3

: Chloroform

CI

: Confidence Interval

CIOMS

: Council for International Organizations of Medical Sciences

cm

: Centimeter


CMV

: Cytomegalovirus

CO2

: Carbon Dioxide

CT

: Computed Tomography

CTAB

: Cetyl trimethylammonium bromide

CTC50

: 50% Cytotoxic Concentrations

CYP2E1

: Cytochrome P450 2E1

DAM

: Diacetyl monoxime

DENA


: Diethylnitrosamine

dl

: Deciliter

DMEM

: Dulbecco's Modified Eagle's Medium

DMSO

:

DNA

: Deoxyribo Nucleic Acid

DNPH

: Dinitro Phenyl Hydrazine

dpf

: Docking Parameter File

dpi

: dots per inch


DPPH

: 1,1-diphenyl-2-picrylhydrazyl/2,2-diphenyl-1-picrylhydrazyl

DS

: Dietary supplements

DSILI

: DS-induced liver injury

DTNB

: 5,5 dithiobis(2-nitrobenzoic acid)

EAE

: Ethyl Acetate Extract

Dimethyl sulfoxide

xiii


EBV

: Epstein-Barr virus


EC50

: 50% Effective Concentration

EDTA

: Ethylene diamine tetra acetate

EGCG

: Epigallocatechin-gallate

ESI-MS

: Electrospray Ionisation Mass Spectrometry

ESIMS

: Electrospray Ionization Mass Spectrometry

et al.

: et alia

EtAC

: Ethyl Acetate

FBS


: Fetal Bovine Serum

Fe2+

: Ferrous ion

Fe3+

: Ferric ion

FeSO4.7H2O

: Iron(II) Sulfate Heptahydrate

FFA

: Free fatty acid

FRAP

: Ferric reducing antioxidant power

FTIR

: Fourier transformed infrared spectroscopy

g

: Gram


GAE

: Gallic Acid Equivalent

GC/O

: GC/olfactometry

GC-FID

: Gas Chromatography with Flame Ionization Detection

GC-MS

: Gas chromatography-Mass spectroscopy

GGT

: Gamma Glutamyl Transferase

GlmU

: N-acetylglucosamine-1-phosphate uridyltransferase

GPx

: Glutathione Peroxidase

GSH


: Reduced glutathione

GSSH

: Oxidized glutathione

GST

: Glutathione-S-transferase

h

: Hour

H&E

: Hematoxylin and Eosin

H2O2

: Hydrogen peroxide

H2SO4

: Sulphuric acid

HBsAg

: Hepatitis B surface antigen


HBV

: Hepatitis B Virus

HCC

: Hepatocellular Carcinoma

xiv


HCl

: Hydrochloric acid

HCT-116

: An Orthotopic Model Of Colon Cancer

HCV

Hepatitis C Virus

HDL

: High density lipoprotein

HDS

: Herbal and dietary supplements


He

: Helium

HeLa

: Cervical Carcinoma Cell line

Hep G2

: Human, Hepatocellular Carcinoma

HEP-2

: Human epithelial type 2 Cell Line

HepG2

: Liver Cancer Cell Lines

HPLC

: High Performance Liquid Chromatography

HPLC-ESIMS/MS

:

High-Performance Liquid Chromatography/Electrospray

Ionization Tandem Mass Spectrometry

HPLC-MS

: High Performance Liquid Chromatography-Mass Spectrometry

HPTLC

: High Performance Thin Layer chromatography

HR-EI-MS

: High-Resolution Electron Ionisation Mass Spectrometry

HRMS

: High Resolution Mass Spectrometry

HS-SPME-GC/MS

:

HSV

: Herpes Simplex Virus

i.p.

: Intraperitoneally


IBU

: Ibuprofen

IC50

: 50% Inhibitory Concentration

IL

: Interleukin

IU

: International Unit

K2S2O8

: Potassium persulfate

Kb

: Kilo-base pair

kcal/mol

: Kilocalorie per mole

Kg


: Kilogram

l.

: Litre

LC50

: 50% Lethal Concentration

LDH

: Lactate dehydrogenase

LDL

: Low-density Lipoprotein

Headspace Solid Phase Micro Extraction-Gas
Chromatography/Mass Spectrometry

xv


LPO

: Lipid peroxide

LT


: Liver Transplantation

LTB4

: Leukotriene B4

M

: Molar

m

: Minutes

MAO

: Monoamine Oxidase

MDA

:

MeCN

: Acetonitrile

MeOH

: Methanol


Mg

: Magnesium

mg

: milligram

MgCl2

: Magnesium chloride

MGL Tools

: Molecular Graphics Lab Tools

ml

: millilitre

mM

: millimolar

mm

: Millimeter

Mn


: Manganese

MRI

: Magnetic Resonance Imaging

mRNA

: messenger Ribonucleic Acid

MS

: Mass Spectrometry

MS

: Metabolic Syndrome

MS

: Microsoft

MTT

: 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide]

N

: Normality


N2

: Nitrogen

Na

: Sodium

Na2CO3

: Sodium Carbonate

NaBH4

: Sodium borohydride

NaCl

: Sodium Chloride

NAD+

: Nicotinamide adenine dinucleotide

NADH

: Reuced nicotinamide adenine dinucleotide

NADP+


: Nicotinamide adenine dinucleotide phosphate

NADPH

: Reuced nicotinamide adenine dinucleotide phosphate

Malondialdehyde

xvi


NAFLD

: Nonalcoholic fatty liver disease

NaH2PO4

: Sodium dihydrogen phosphate

NaOH

:

NASH

: Nonalcoholic Steatohepatitis

NBT

: Nitro blue tetrazolium


NCCS

: National Centre for Cell Sciences, Pune, India

NCEs

: New Chemical Entities

Ng

: Nanogram

nm

: Nanometer

nmoles

: Nanomoles

NMR

: Nuclear Magnetic resonance

NO

: Nitric Oxide

NSAID


: Nonsteroidal Antiinflammatory Drugs

OCP

: Oral Contraceptive Pill

OD

: Optical Density

-

Sodium Hydorxide

OH

: Hydroxide

ORAC

: Oxygen radical absorbance capacity

OSL

: Observed Safe Level

P

: Probability


PBS

: Phosphate Buffered Saline

PDB

: Protein Data Bank

PEE

: Petroleum Ether Extract

pH

: Hydrogen ion Concentration

PIE

: Phenolic Inositol Ester

PL

: Phospholipid

PMS

: Phenazine Methosulphate

ppm


: Parts per million

pRb

: Retinoblastoma Protein

QE

: Quercetin

QuEChERS
Method

: Quick Easy Cheap Effective Rugged Safe Method

RBC

: Red Blood Cells

Rf

: Rate of flow

xvii


RMSD

: Root-Mean Square Deviation


RNA

: Ribonucleic acid

ROS

: Reactive oxygen species

RP-18

: Reverse Phase - 18 (5µm) Column

RP-HPLC

: Reverse phase high performance liquid chromatography

rpm

: Revolutions per minute

S

: Seconds

SALP

: Serum Alkaline Phosphatase

SC50


: 50% Scavenging Capacity

SCD

: Sickle cell disease

SDS

: Sodium dodecyl sulphate

SE

: Standard Error

SEER

: Surveillance, Epidemiology and End Results

SGGTP

: Serum Gamma Glutamyl Transpeptidase

SGOT

: Serum glutamate oxaloactetate transaminase

SGPT

: Serum glutamate pyruvate transaminase


-SH group

: Thiol group

SO

: Superoxide anion

SOD

: Superoxide Dismutase

TA-G

:

TBA

: Thiobarbituric acid

TBARS

: Thiobarbituric acid reactive substances

TCA

: Tricarboxylic acid/ trichloro acetic acid

TG


: Triglyceride

TLC

: Thin-Layer Chromatography

TPC

: Total phenolic content

TPTZ

: 2,4,6-Tripyridyl-S-triazine

TPVG

: Trypsin Phosphate Versene Glucose

TritAc

: Triterpene Acetates

U/L

: Units per liter

US

: United States


USA

: United States of America

Sesquiterpene Lactone Taraxinic Acid β-D-Glucopyranosyl
Ester

xviii


UV

: Ultraviolet

UVB

: Ultraviolet-B

UV-Vis

: ultraviolet-visible spectroscopy

V

: Volts

V

: Volume


v/v

: Volume/volume

v/v

: Volume/volume

v/v/v

: Volume per volume per volume

VCEAC

: Vitamin C Equivalent Antioxidant Capacity

VLDL

: Very low density lipoprotein

VZV

: Varicella zoster virus).

W/V

: Weight per volume

W/V


: Weight/Volume

w/w

: Weight per Weight

WBC

: White Blood Cells

WE

: Water Extract

WHO

: World Health Organization

Zn

: Zinc

α

: Alpha

β

: Beta


Μg

: Microgram

Μg

: Microgram

μl

: Microlitre

Μm

: Micro Mole

μM

: Micro Mole

D

: Alpha

E

: Beta

P


: Micro

xix


Chapter I

1
1.1

INTRODUCTION

Herbal Medicine
Herbs have molded the base for remedy of diseases in conventional medicine

for thousands of years and endure to play a main part in the principal health care of
about 80% of the world’s populations. It is also worth noting that (a) 35% of drugs
contain ‘principles’ of natural origin and (b) less than 5% of the 500,000 higher plant
species have undergone pharmacological screening. Each plant has potentially 10,000
different constituents (Saad et al., 2017). The discovery and development of
efficacious therapeutic agents from natural sources provided convincing evidence that
plants could be a source of novel drugs. Western medicine use many drugs extracted
from natural products: atropine, cocaine, digitoxin, ephedrine, hyoscine, codeine,
morphine, pilocarpine, quinine, reserpine, taxol, warfarin, menthol, etc. While the
natural product isolated as the active compound might not always be suitable for
development as an effective drug, it can provide a suitable lead for conversion into a
clinically useful agent (Alamgir, 2018). Ayurveda always gives a primary importance
to the maintenance of sound health and the prevention of disease by the simple device
of raising the individual resistance of the body and providing active immunity

(Edavalath, 2018).
In many developing countries, a large proportion of the population relies on
traditional practitioners and their armamentarium of medicinal plants in order to meet
health care needs. In this modern setting, ingredients are sometimes marketed for uses
that were never contemplated in the traditional healing systems from which they
emerged. An example is the use of Ephedra for weight loss or athletic performance
enhancement (David et al., 2015).

1


Chapter I
Plants and their secondary metabolites have a long history of use in modern
‘western’ medicine and in certain systems of traditional medicine. Monographs on
selected herbs are available from a number of sources, including the ‘European
Scientific Cooperative on Phytotherapy’ ‘Natural Medicines Comprehensive
Database’, the complete German Commission E monograph’ and the ‘World Health
Organization’ (Nafiu et al., 2017). The WHO monographs, for example, describe the
‘herb’ itself by a number of criteria including synonyms and vernacular names and the
herb part commonly used, its geographical distribution, tests used to identify and
characterize the herb (including macroscopic and microscopic examination and purity
testing), the active principles, dosage forms and dosing, medicinal uses,
pharmacology, contraindications and adverse reactions. Information about other
available databases has been published (Hosseinzadeh, et al., 2015).
The medicinal plants combine three properties – curative, preventive and
nutritive which provide the human body with necessary strength and vigour to cope
with the disease and facilitate the action of the curative agents in the herbal drug. The
major prevalent killers of India are cancer, diabetes, heart and liver diseases (Debnath
et al., 2015).
1.2


Secondary Metabolites
Metabolites are organic compounds synsthesised by plants/organisms using

enzyme-mediated chemical reactions called metabolic pathyway.

They are

synthesized for essential function, such as pollinator attraction or defence against
herbivory (secondary metabolites). In contrast to primary metabolites, which are
essential to growth and development, secondary metabolites are organic compounds
that do not necessarily serve primary metabolic functions in the growth and
maintenance. They are variously distributed in the plant kingdom, and their functions
are specific to the plants in which they are found (David et al., 2015).
2


Chapter I
Unlike primary metabolites, absence of secondary metabolites does not result
in immediate death, but rather in long-term impairment of the organism’s
survivability, fecundity, or aesthetics, or metabolites varies between species or genera
and is thus, apart from appearance and size etc., an aspect of characterization of a
species. Secondary metabolites are often colored, fragrant or flavorful compounds,
and they typically mediate the interaction of plants with other organisms. Such
interactions include those of plant-pollinator, plant-pathogen and plant-herbivore
(Chandran et al., 2015)
Secondary metabolites unlike synthetic chemical have low toxicity, complete
biodegradbiilty, and availability from renewable sources and in some cases low cost.
It is because of these reasons; healthcare products and environmentally acceptable
agricultural compounds are mainly natural products or derived by modification of

natural product leads. Moreover, nature provides compounds having unique and
diverse chemical structures with potential biological properties, which are beyond the
reach of human imagination. For the past several centuries, man has been using these
secondary metabolites for human benefits (Yuan et al., 2016).
They are needed for the plant to interact with its environment and other
organisms. Secondary metabolites often play an important role in plant defence
against herbivory and other insterspecies defences.

Humans use secondary

metabolites as medicines, flavorings and recreational drugs. They are interesting for
various reasons e.g. their structural diversity, their potential as drug candidates or as
natural pesticides. The structural diversity of secondary metabolites is astonishing
and there are several examples of compounds produced in nature with structures so
complex that no chemist could invent those (Che et al., 2017).

3