tejaswi chavan ph.d. thesis.pdf

MODULATION OF PATHOPHYSIOLOGY OF

HEPATOTOXICITY WITH NUTRITIONAL AND HERBAL

INTERVENTIONS IN ANIMAL MODELS

THESIS SUBMITTED TO

M

RS

. TE

JAS

WI C

HA

ND

RA

KA

NT

CH

AV

AN

BHARATI VIDYAPEETH DEEMED UNIVERSITY, PUNE

FOR THE AWARD

OF

DOCTOR OF PHTLOSOPHY (Ph.D.)

IN

BIOTECHNOLOGY

UNDER

FACULTY OF SCIENCE

BY

MRS. TEJASWI CHANDRAKANT CHAVAN

UNDER THE GUIDANCE

OF

DR. ANIKET A. KUVALEKAR

BHARATI VIDYAPEETH DEEMED UNIVERSITY

INTERACTIVE RESEARCH SCHOOL FOR HEALTH AFFAIRS (IRSHA)

PUNE, MAHARASHTRA, INDIA

September 2016

Ph.D. THESIS

September 2016

MODULATION OF PATHOPHYSIOLOGY OF

HEPATOTOXICITY WITH NUTRITIONAL AND HERBAL

INTERVENTIONS IN ANIMAL MODELS

Thesis submitted to

Bharati Vidyapeeth Deemed University, Pune

for award of

DOCTOR OF PHILOSOPHY (Ph.D.)

in Biotechnology

Under

Faculty of Science

By

Mrs. Tejaswi Chandrakant Chavan (M.Sc.)

Under the Guidance of

Dr. Aniket A. Kuvalekar

Bharati Vidyapeeth Deemed University,

Interactive Research School for Health Affairs (Irsha),

Pune, Maharashtra, India

September 2016

Certificate

This is to certify that the work incorporated in the thesis entitled “Modulation of

Pathophysiology of Hepatotoxicity with Nutritional and Herbal Interventions in

Animal Models” for the degree of ‘Doctor of Philosophy’ in the subject of

Biotechnology under the faculty of Science has been carried out by Mrs. Tejaswi

Chandrakant Chavan in the Diabetes Laboratory, Interactive Research School for

Health Affairs at Bharati Vidyapeeth Deemed University, Pune, during the period

from November 2012 to July 2016, under the guidance of Dr. Aniket A. Kuvalekar.

Place: Pune Dr. A.C.Mishra

Date: Director

Certification of Guide

This is to certify that the work incorporated in the thesis entitled “Modulation of

Pathophysiology of Hepatotoxicity with Nutritional and Herbal Interventions in

Animal Models” submitted by Mrs. Tejaswi Chandrakant Chavan for the degree

of ‘Doctor of Philosophy’ in the subject of Biotechnology under the faculty of

Science has been carried out in the Diabetes Laboratory, Interactive Research School

for Health Affairs, Bharati Vidyapeeth Deemed University, Pune, during the period

from November 2012 to July 2016, under my direct supervision/guidance.

Place: Pune Dr. Aniket A. Kuvalekar

Date: Research Guide

Declaration by the Candidate

I hereby declare that the thesis entitled “Modulation of Pathophysiology of

Hepatotoxicity with Nutritional and Herbal Interventions in Animal Models”

submitted by me to the Bharati Vidyapeeth University, Pune for the degree of Doctor

of Philosophy (PhD) in Biotechnology under the faculty of Science is original piece

of work carried out by me under the supervision of Dr. Aniket A. Kuvalekar. I

further declare that it has not been submitted to this or any other university or

institution for the award of any degree or diploma.

I also confirm that all the material which I have borrowed from other sources and

incorporated in this thesis is duly acknowledged. If any material is not duly

acknowledged and found incorporated in this thesis, it is entirely my responsibility. I

am fully aware of the implications of any such act which might have been committed

by me advertently or inadvertently.

Place: Mrs. Tejaswi Chandrakant Chavan

Date: Research Student

Acknowledgements

“Research is to see what everybody has seen but think what nobody has

thought.” Research involves collaborative efforts by team of persons striving to

conquer new horizons in the different fields of sciences. This study would not have

been completed without the encouragement, guidance and co-operation of my

teachers, parents, friends, well-wishers and relatives. Getting such help, I feel, is

comparable to our humanity. Almighty has given us a wonderful body in which

organs and systems work together, synchronously to lead a healthy life. Similarly, my

thesis work was like a human body where many people helped me in some or the

other way for its successful completion. I would like to take this opportunity to

express my deep gratitude towards all of them.

I wish to express my sincere thanks and profound respect towards my guide

Dr. Aniket Kuvalekar (Diabetes Laboratory, Bharati Vidyapeeth University,

IRSHA, Pune) with a deep sense of my gratitude for his guidance, invaluable advice,

constant support, intellectual supervision and professional expertise he has bestowed

upon me for the timely completion of my research work. I would also like to thank

him for giving me freedom of thoughts and guiding me on the right path with the

trust. I am proud to have him as my guide.

I am thankful to Dr. Akhilesh Mishra (Director, BVDU-IRSHA, Pune), Dr.

Prabhakar Ranjekar (Former Director, BVDU-IRSHA, Pune) and Dr. Sahebrao

Mahadik (Professor, Medical college of Georgia, Augusta, U.S.A.) for their support

and invaluable guidance during my entire research work. I would like to thank Dr.

Digambar Mokat (Assistant Professor, Savitribai Phule Pune University) for

providing plant material. I am also thankful to the Medicinal Plants

Conservation Centre, Pune (MPCC) for identification and authentication of plant

material. I would like to thank Dr. Omkar Kulkarni for providing ayurvedic

expertise for preparation of satwa for my research work. I am also thankful to Mr.

Ravi Mulik for his help in executing animal experiments.

My sincere thanks also go to Dr. Vijaya Pandit (Head, Department of

Pharmacology, Bharati Vidyapeeth Medical College, Pune) for reviewing animal

experiment protocols and Dr. Manjiri Karandikar (Associate Professor, Department

of Pathology, Bharati Vidyapeeth Medical College, Pune) for her support in

histopathological study in my research work.

A special note of thanks to my friend Mr. Abhijit Ghadge for his help and

encouragement throughout the tenure of my thesis.

It will be very unfair if I forget my labmates Mr. Suresh Khadke and Mrs.

Shubhangi Harke for giving their helping hands whenever and wherever required

and maintaining lively atmosphere in lab. Their invaluable interactions always

strengthened me emotionally and encouraged me for my work. I am grateful to my

juniors Mrs. Amruta Mandhare, Mr. Suyash Pawar, Miss. Amruta Kakde for

their cordial support throughout my research work.

I am fortunate to have parents like mine. My sincere thanks and appreciation

to my mother Mrs. Kamal Patil, my father Mr. Bhimarao Patil, my mother-in-law

Mrs. Sindhu Chavan and father-in-law Mr. Shivaji Chavan for their unconditional

true love, affection, blessing, sacrifice and support which paved my path in life and

the encouragement I needed to succeed.

I have no words to show my hearty gratitude towards my lovely son Atharva

and husband Mr. Chandrakant who are my inspiration, support and true love.

My apologies as it is not possible to mention everyone’s name here but their

blessings helped for successful completion of my work.

Last but not the least, I wish to express my gratitude towards “God almighty”, who

gave me the strength and courage to fulfill my dream and bestowed his best

blessings upon me always.

Thankful I ever remain.

List of Abbreviations

Abbreviations Full Form

ALP - Alkaline phosphate

ANOVA - Analysis of variance

APAP - Acetaminophen

b.w. - Body weight

BIL - Bilirubin

BSA - Bovine serum albumin

CAT - Catalase

CPCSEA - Committee for the purpose of control and

supervision of experiments on animals

cDNA - Complementary DNA

CHO - Total cholesterol

D.P.X. - Diphenyl phthalate xylene

DEPC - Diethylpyrocarbonate

DMSO - Dimethyl sulfoxide

dNTPs - Deoxynucleotides

DTNB - Dithionitrobenzoic acid

DTT - Dithiothreitol

EDTA - Ethylene diamine tetra acetic acid

FABP - Fatty acid binding protein

GAPDH - Glyceraldehyde 3-phosphate dehydrogenase

GSH - Reduced glutathione

HCl - Hydrochloric acid

HDL - High density lipoprotein

KCl - Potassium chloride

LDL - Low density lipoprotein

MDA - Malondialdehyde

NaOH - Sodium hydroxide

NFkβ - Nuclear factor kappaβ

PBS - Phosphate buffered saline

PPARγ - Peroxisome proliferator-activated receptor

gamma

RNA - Ribonucleic acid

RNaseOUT - Recombinant ribonuclease inhibitar

RT - Reverse transcriptase

SD - Standard deviation

SE - Standard error

SGOT - Serum glutamic oxaloacetic transaminase

SGPT - Serum glutamic pyruvic transaminase

SOD - Superoxide dismutase

SREBP - Sterol regulatory element binding protein

TGL - Triglycerides

TNFα - Tumor necrosis factor alpha

INDEX

Sr.No Particular Page

No

Chapter 1 Introduction

1.1 Liver 1

1.2 Hepatotoxicity 2

1.3 Prevalence of Liver Disease 3

1.3.1 International Scenario 3

1.3.2 National Scenario 4

1.4 Risk Factor for Liver Disease 5

1.4.1 Age and Gender 5

1.4.2 Genetic Factors 6

1.4.3 Obesity 6

1.4.4 Arsenic 6

1.4.5 Aflatoxins 6

1.4.6 Dietary Supplements 6

1.4.7 Industrial Toxins 7

1.4.8 Diabetes 7

1.4.9 Alcoholism 7

1.4.10 Long Term Use of Certain Medicinal Drugs 7

1.5 Biochemical Markers 8

1.5.1 Liver Function Tests 8

1.5.2 Total Protein 9

1.5.3 Lipid Profile 9

1.5.4 Oxidative Stress Marker 9

1.6 Management of Hepatic Diseases 10

1.6.1 Surgical Procedures 10

1.6.2 Hepatoprotective Agents 11

1.6.2.1 Silymarin 11

1.6.3 Herbal Formulations 11

1.6.4 Medicinal Plants 12

1.6.4.1 Tinospora forms 13

(a) Tinospora cordifolia 14

Sr. No Particular Page

No

(b) Tinospora sinensis 15

(c) Neem-giloe 16

1.6.5 Satwa 16

1.6.6 Nutritional Supplements 18

1.6.6.1 Polyunsaturated Fatty Acid (Omega-3 Fatty

Acids) 18

1.7 Animal Models for Hepatotoxicity 18

1.7.1 Carbon Tetrachloride Induced Hepatotoxicity 19

1.7.2 Acetaminophen Induced Hepatotoxicity 19

1.7.3 Alcohol Induced Hepatotoxicity 20

1.8 Genesis of the Thesis 21

Hypothesis 22

Chapter 2 Objectives

Objectives 23

Chapter 3 Review of Literature

3.1 Acetaminophen Induced Hepatotoxicity 24

3.2 Alcohol Induced Hepatotoxicity 28

3.3 Treatments 32

3.4 Alternative Treatments 33

3.4.1 Hepatoprotective Herbal Formulations 33

3.4.2 Hepatoprotective Medicinal Plants 41

3.4.2.1 Tinospora cordifolia 49

3.4.2.2 Tinospora sinensis 50

3.4.2.3 Neem-giloe 51

3.4.3 Guduchi Satwa 51

3.4.4 Nutritional Supplements 53

3.4.4.1 Polyunsaturated Fatty Acid (Omega-3

Fatty Acids) 57

Chapter 4 Materials and Methods

4.1 Collection of Plant Material 60

4.2 Identification and Authentication of Plant Material 61


No

4.3 Preparation of Satwa from Three Tinospora forms 61

4.4 Nutritional Analysis of Satwa 64

4.4.1 Protein 64

4.4.2 Total Carbohydrates 65

4.4.3 Starch 67

4.4.4 Total Lipids 68

4.4.5 Crude Fibre 69

4.4.6 Total Ash 70

4.5 Hepatoprotective Activty of Satwa from Three Different

Tinospora forms 72

4.6 Heaptorpotective Activity of Flax Oil and Fish Oil 77

4.7 Hepatoprotective Activity of Combination of Best

Performing Herbal and Nutritional Intervention

81

4.8 Biochemical Parameters 86

4.8.1 Serum Biochemical Parameters 86

4.9 Methods for Estimation of Serum Biochemical Markers 86

4.9.1 Estimation of Serum Glutamic Oxaloacetic

Transaminase (SGOT) 86

4.9.2 Estimation of Serum Glutamic Pyruvic Transaminase

(SGPT) 87

4.9.3 Estimation of Alkaline Phosphatase (ALP) 89

4.9.4 Estimation of Total Bilirubin 90

4.9.5 Estimation of Total Cholesterol 91

4.9.6 Estimation of Triglycerides 91

4.9.7 Estimation of HDL-D Cholesterol 93

4.9.8 Estimation of LDL-D Cholesterol 94

4.10 Liver Biochemical Parameters 95

4.10.1 Estimation of Lipid Peroxidation 95

4.10.2 Estimation of Superoxide Dismutase (SOD) 96

4.10.3 Estimation of Catalase 98

4.10.4 Estimation of Total Protein 100


No

4.10.5 Estimation of Reduced Glutathione 100

4.10.6 Estimation of Total Cholesterol 102

4.10.7 Estimation of Triglycerides 102

4.10.8 Estimation of HDL-D Cholesterol 103

4.10.9 Estimation of LDL-D Cholesterol 103

4.11 Liver Histology 104

4.11.1 Fixation 104

4.11.2 Tissue Processing 104

4.11.3 Section Cutting 105

4.11.4 Observation 105

4.12 Gene Expression Study 106

4.12.1 RNA Extraction 106

4.12.2 RNA Quantification and Quality Check 106

4.12.3 cDNA Synthesis 107

4.12.4 Semi-Quantitative Polymerase Chain Reaction 107

4.13 Statistical Analysis 109

Chapter 5 Results

5.1 Organoleptic Characteristics 110

5.2 Nutritional Analysis of Three Tinospora forms 111

5.3 Hepatoprotective Activity of Satwa from Three different

forms of Tinospora

115

5.3.1 Hepatoprotective Activity of Satwa against

Acetaminophen Induced Hepatotoxicity

115

5.3.2 Hepatoprotective Activity of Satwa against Ethanol

Induced Hepatotoxicity

122

5.4 Hepatoprotective Activity of Flax Oil and Fish Oil 129

5.4.1 Hepatoprotective Activity of Flax Oil and Fish Oil

against Acetaminophen Induced Hepatotoxicity

129

5.4.2 Hepatoprotective Activity of Flax Oil and Fish Oil

against Ethanol Induced Hepatotoxicity

136


No

5.5 Hepatoprotective Activity of Combination of Best

Performing Herbal and Nutritional Intervention

142

5.5.1 Effects of Protective Treatment of Combination of

Neem-giloe Satwa and Fish Oil against


142

5.5.2 Effects of Corrective Treatment of Combination of

Neem-giloe Satwa and Fish Oil against


149

5.5.3 Effects of Prophylactic Treatment of combination of

T. sinensis Satwa and Flax Oil against Ethanol


155

Chapter 6 Discussion

6.1 Guduchi Satwa (T. cordifolia, T. sinensis and Neem-giloe) 162

6.1.1 Organoleptic Characteristics of Three Tinospora

forms 162

6.1.2 Nutritional Analysis of Three Tinospora forms 163

6.2 Drug Induced Liver Injury 164

6.3 Hepatoprotective Activity of Satwa against


165

6.3.1 Biochemical Parameters 165

6.3.2 Histological Analysis 167

6.3.3 Gene Expression 168

6.4 Hepatoprotective Activity of Satwa against Ethanol


176




6.5 Hepatoprotective Activity of Flax Oil and Fish Oil against

Acetaminophen Induced Hepatotoxicity 182





No

6.6 Hepatoprotective Activity of Flax Oil and Fish Oil against

Ethanol Induced Hepatotoxicity

187




6.7 Protective and Corrective Effect of Combination of Neem-

giloe Satwa and Fish Oil against Acetaminophen Induced

Hepatotoxicity

191




6.8 Prophylactic Effect of Combination of Tinospora sinensis

Satwa and Flax Oil against Ethanol Induced

Hepatotoxicity

195




Summary and Conclusions 198

Future Direction 201

Bibliography 202

Annexure I Publications 273

Annexure II Awards 275

Annexure III Presentations 276

LIST OF TABLES

Table

No

Title Page

No

1 Classification of T. cordifolia 14

2 Classification of T. sinensis 15

3 Differences between T. cordifolia and T. sinensis 17

4 List of Hepatoprotective Herbal Formulations 34

5 List of Hepatoprotective Medicinal Plants 42

6 List of Hepatoprotective Nutritional Supplements 54

7 List of Primers Used for the Study 108

8 Organoleptic Characteristics of Guduchi Satwa from Three different

forms of Tinospora

110

9 Nutritional Analysis of Satwa from Three Tinospora forms 112

10 Effect of Satwa from Three Tinospora forms on Liver Function Markers

and Serum Lipid Profile in Animals with Acetaminophen Induced

Hepatotoxicity

118

11 Effect of Satwa from Three Tinospora forms on Hepatic Oxidative Stress

Markers, Total Protein and Hepatic Lipid Profile in Animals with


119

12 Effect of Satwa from Three Tinospora forms on Liver Function Markers

and Serum Lipid Profile in Animals with Ethanol Induced Hepatotoxicity 125

13 Effect of Satwa from Three Tinospora forms on Hepatic Oxidative Stress

Markers, Total Protein and Hepatic Lipid Profile in Animals with


126

14 Effect of Flax Oil/Fish Oil on Liver Function Markers and Serum Lipid

Profile in Animals with Acetaminophen Induced Hepatotoxicity

132

15 Effect of Flax Oil/Fish Oil on Hepatic Oxidative Stress Markers, Total

Protein and Hepatic Lipid Profile in Animals with Acetaminophen


133

16 Effect of Flax Oil/Fish Oil on Liver Function Markers and Serum Lipid

Profile in Animals with Ethanol Induced Hepatotoxicity

138

17 Effect of Flax Oil/Fish Oil on Hepatic Oxidative Stress Markers, Total

Protein and Hepatic Lipid Profile in Animals with Ethanol Induced

Hepatotoxicity 139

18 Protective Effect of Combination of Neem-giloe Satwa and Fish Oil on

Liver Function Markers and Serum Lipid Profile in Animals with

Hepatotoxicity Induced with A Single High Dose of Acetaminophen

145

19 Protective Effect of Combination of Neem-giloe Satwa and Fish Oil on

Hepatic Oxidative Stress Markers, Total Protein and Hepatic Lipid

Profile in Animals with Hepatotoxicity Induced with A Single High Dose

of Acetaminophen

146

20 Corrective Effect of Combination of Neem-giloe Satwa and Fish oil on


Hepatotoxicity Induced with A Single High Dose of Acetaminophen

151

21 Corrective Effect of Combination of Neem-giloe Satwa and Fish Oil on


Profile in Animals with Hepatotoxicity Induced with A Single High Dose

of Acetaminophen

152

22 Prophylactic Effect of Combination of T. sinensis Satwa and Flax Oil on



157

23 Prophylactic Effect of Combination of T. sinensis Satwa and Flax oil on


Profile in Animals with Ethanol Induced Hepatotoxicity

158

LIST OF FIGURES

Figure

No

Title Page

No

1 Structure of Liver 1

2 Risk Factors for Liver Disease 5

3 Three different forms of Tinospora A. T. cordifolia, B T. sinensis,

C. Neem-giloe 16

4 Schematic Representation Depicting Activation of Acetaminophen

and NAPQI-Mediated Acetaminophen Toxicity 26

5 Alcohol Metabolism in Hepatocytes and Acetaminophen of

Reactive Oxygen Species (ROS) Leading to Liver Diseases 29

6 Stem Pieces of Guduchi 62

7 Medium Size Stem Diameter (1.6-2.0 cm diameter) 62

8 Preparation of Guduchi Satwa 63

9 Guduchi Satwa from Three different forms of Tinospora 111

10 Levels of Lipids in T. cordifolia, T. sinensis and Neem-giloe 113

11 Levels of Carbohydrate in T. cordifolia, T. sinensis and Neem-giloe 113

12 Levels of Protein in T. cordifolia, T. sinensis and Neem-giloe 114

13 Levels of Starch in T. cordifolia, T. sinensis and Neem-giloe 114

14 Effect of T. cordifolia, T. sinensis and Neem-giloe Satwa on Liver

on Liver Histology in Animals with Acetaminophen Induced

Hepatotoxicity

120

15 Gene Expression from Liver Tissues of Experimental Animals 121

16 Effect of T. cordifolia, T. sinensis and Neem-giloe Satwa on Liver

on Liver Histology in Animals with Ethanol Induced

Hepatotoxicity

127


18 Effect of Polyunsaturated Fatty Acids (Flax oil and fish oil) on

Liver Histology in Animals with Acetaminophen Induced

Hepatotoxicity

134


20 Effect of Polyunsaturated Fatty Acids (Flax oil and fish oil) on

Liver Histology in Animals with Ethanol Induced Hepatotoxicity 140


22 Protective Effect of Combination of Neem-giloe and Fish oil on

Liver in Rats Treated with A Single High Dose of Acetaminophen

147


24 Corrective Effect of Combination of Neem-giloe and Fish oil on

Liver in Rats Treated with A Single High Dose of Acetaminophen

153


26 Prophylactic Effect of Combination of T. sinensis Satwa and Flax

Oil on Liver Histology in Animals with Ethanol Induced

Hepatotoxicity

159

27 Gene Expression from Liver Tissues of Experimental Animals. 160

28 A Model for Probable Molecular Mechanism of Action of Satwa

from Three different forms of Tinospora

175

29 A Model for Probable Molecular Mechanism of Action of Satwa

from Three different forms of Tinospora

181

30 A Model for Probable Molecular Mechanism of Action of Flax Oil

and Fish Oil Interventions.

186

31 A Model for Probable Molecular Mechanism of Action of Flax Oil

and Fish Oil Interventions

190

32 A Model for Probable Molecular Mechanism of Action of Neem-

giloe and Fish Oil

194

33 A Model for Probable Mechanism of Action of T. sinensis Satwa

and Flax Oil

197

CHAPTER 1

INTRODUCTION

Awards

Second prize in oral

Presentation Award

Best Poster Presentation

Award

1

1.1 Liver

Liver is the largest gland in the body weighing about 1500g in an adult and

accounts for approximately 2.5% of total body weight (Singh et al., 2012; Juza and

Pauli, 2014). Liver is called as the metabolic “engine-room of the body” (Vishal,

2013). The liver is divided into two lobes, right and left, a large right lobe and a

smaller left lobe are separated by falciform ligament. Liver has 50,000-100,000

lobules (Caruthers, 1997). Each lobule consists of a central vein surrounded by tiny

cells (Fig. 1) like hepatocytes, endothelial cells, kupffer cells and stellate cells

grouped in sheets or bundles (Jacobs et al., 2010; Ho et al., 2013). The liver

metabolizes xenobiotics and drugs into toxic intermediates (Bhattacharjee and Sil,

2006).

Liver performs vital role in wide range of functions such as metabolism of

nutrients like amino acids, carbohydrates, lipids, minerals, vitamins; it also helps in

blood clotting through synthesis and secretion of plasma proteins; eliminates dead red

blood cells from blood circulation; eliminates bacteria; detoxifies chemicals, drugs,

xenobiotics, helps in digestion and fat metabolism by excretion of bile salts; and

excretion of end products of metabolism through urine (Singh et al., 2012). Liver

plays role in both metabolism as well as biochemical transformation (Sahu, 2007).

Fig. 1. Structure of Liver

(From Ho et al., 2013)

2

1.2 Hepatotoxicity

Toxicity mentions to the unsafe impacts of substances on an entire living

being, in a creature, bacterium or plant, and in addition the substructure of life forms,

for example, a cell (Cytotoxicity), or organ (Organotoxicity) and liver Hepatotoxicity

(Bahar et al., 2013).

Hepatotoxicity is most commonly seen in the form of malfunction or damage

to the liver due to excess amount of drugs or xenobiotics (Navarro, 2006; Singh et al.,

2011). Hepatotoxicants are exogenous substances of clinical relevance which may

include an overdose of certain medicinal drugs (acetaminophen, nimesulide,

antitubercular drugs like isoniazid, rifampicin etc.), industrial chemicals (alcohol,

CCl4, beta galactosamine, thioacetamide) etc., which causes liver injury (Willett et al.,

2004; Bigoniya et al., 2009; Papay et al., 2009; Singh et al., 2011; Pandit et al., 2012).

The exact mechanism of drug induced liver injury remains largely unknown, but it

appears to involve two pathways – direct hepatotoxicity (Type A or DILI1 (drug

induced liver injury1), intrinsic or predictable drug reaction) and indirect

hepatotoxicity (Type B or DILI2 (drug induced liver injury2), unpredictable or

idiosyncratic drug reaction,) or adverse immune reaction (Bigoniya et al., 2009). The

most common direct hepatotoxins are carbon tetrachloride, thioacetamide,

acetaminophen, galactosamine, fulvine, phalloidin, ethyl alcohol, aflatoxins etc. Some

examples of indirect hepatotoxins are methyl testosterone, chlorpropamide,

tetracycline, halothane, phenytoin, methyldopa, sulphonamides, allopurinol,

rifampicin etc (Bigoniya et al., 2009). Hepatotoxicity is manifested by different types

of injuries, depending on the nature and dose of the chemical. Hepatotoxicity may

result into cytotoxic effects (necrosis, apoptosis), cholestasis, steatosis, fibrosis,

cirrhosis, hepatitis and liver tumors (Lee, 2003). Hepatotoxicity related symptoms

may include jaundice or icterus appearance causing yellowing of the skin, eyes and

severe abdominal pain, nausea or vomiting, weakness, severe fatigue, continuous

bleeding, skin rashes, generalized itching, swelling of the feet and/or legs, abnormal

and rapid weight gain in a short period of time, dark urine and light coloured stools

(Chang and Schaino, 2007).

3

1.3 Prevalence of Liver Disease

Liver diseases are fatal and leading cause of illness and deaths worldwide

(Wang et al., 2014a). As per study, liver disorders cause about 18000 to 20000 deaths

every year globally (Fatma and Uphadhyay, 2015; Akila and Prasanna, 2014). In

United States, about 2-5 % of hospital admissions are due to liver injury out of which

10% results in acute liver failures (Pandit et al., 2012). In United States, rate of liver

transplantation is more than 75% for Type B drug reactions (Ostapowicz et al., 2002).

The crude incidence of liver disorder is 14 per 100000 per annum globally, whereas

the standard incidence is 8.1 per 100000 per annum (Bedi et al., 2016). Acute liver

failure rate is up to 13% of the cases in developed nations like USA whereas it is less

(5%) in tropical countries like India (MeMahon, 2005).

1.3.1 International Scenario

Acetaminophen or paracetamol is easily available as over the counter drug and

has become major reason of self-poisoning in recent years (Ghaffar and Tadvi, 2014).

About 50% of self-poisoning cases are due to paracetamol causing 100-200 deaths per

annum in UK (Kumar et al., 2005). In USA, 61.8% of paracetamol overdose cases

were found unintentional and 30.5% were related with suicidal attempts (Serper et al.,

2016). It has been reported as the most common drug overdose either accidentally or

unintentionally resulting into acute liver failures (ALF) in the United Kingdom (UK,

60-75% of ALF aetiology), Europe (2% of ALF aetiology in France), Canada, United

States (US, approximately 20% of ALF aetiology), and Australia (Ostapowicz and

Lee, 2000; Robinson et al., 2000; Ostapowicz et al., 2002; Ayonrinde et al., 2005;

Marzilawati et al., 2012). However, the incidence of acetaminophen-induced acute

liver failure cases in US has increased exponentially (Larson, 2005). In year 2009,

401 deaths were reported due to acetaminophen overdose by American Association of

Poison Control Centers (Sreejith et al., 2015). According to a recent report, about

42% of acute liver failure cases out of more than 80000 emergency visits and 30000

hospitalizations are reported in US (Lancaster et al., 2015).

Alcohol is one of the main causes of end stage liver disease and leading cause

of morbidity and mortality worldwide (WHO, 2011; Wang et al., 2014a). Deaths due

to alcohol liver diseases are increased since last decade (Mandayam et al., 2004) and

4

have become common reason for cirrhosis in western countries (WHO, 2011). In

USA, second leading cause for liver transplantation is alcoholic cirrhosis (Varma et

al., 2010). The mortality rate for alcoholic liver disease (ALD) was 7.9 per 100000 in

the United States (Roizen et al., 1999). In 2006, 22,073 deaths in the United States

(excluding accidents/homicides) were related to alcohol, with approximately 13,000

deaths specifically due to ALD (Beier et al., 2011). The Global Status Report on

Alcohol and Health by WHO reported highest alcohol consumption in developed

world including Western and Eastern Europe (WHO, 2011). European countries

reported 1 in 7 male deaths and 1 in 13 female deaths due to ALD in 2004 (WHO,

2012). Nearly 88,000 people (approximately 62,000 men and 26,000 women) die

from alcohol-related causes annually, making it the fourth leading preventable cause

of death in the United States (CDC, 2013; Stahre et al., 2014). A WHO study in 2012

reported about 3.3 million deaths worldwide, of which 5.9% were caused by alcohol

consumption (WHO, 2014). About 3.8% of global mortality is accounted for alcohol

consumption (Li et al., 2015).

1.3.2 National Scenario

In India, 33.2% patients were reported with acetaminophen overdose in the

study on 1024 patients (Median age 23 years, 82.0% female) from January 2005 to

December 2009 (Marzilawati et al., 2012). The data on acetaminophen self-poisoning

in India is highly insufficient as compared to that of western countries (Nambiar,

2012).

In India, the prevalence rate of liver injury due to alcohol is higher than that of

acetaminophen which is largely attributable to utilization of illegal alcohol (Malik et

al., 2015). In India, 5% of all deaths are because of liver diseases for which the most

critical culprit is alcohol (Vasudevan, 2011). Alcoholism is the most common cause

of fatty liver and cirrhosis in India (Vasudevan, 2011). The prevalence of alcohol

consumption ranges from 7% in Gujarat, to 75% in Arunachal Pradesh (Murthy et al.,

2010; Punia, 2014). The per capita consumption is 2 litres per adult per year which

accounts for 50% of chronic liver diseases (Punia, 2014). In India, mortality rates due

to ALD for males (11/100000) are reported to be higher than that of females

(6/100000) (Nagaraju, 2014). Significantly higher alcohol consumption has been

5

recorded among tribal, country and lower socio-economic urban sections (Benegal,

2005; Punia, 2014).

1.4 Risk Factors for Liver Disease

The risk for developing liver disease varies, depending on cause and co-

occurrence of other medical conditions. Different risk factors for liver disease (Fig. 2)

include age and gender, genetic factors, obesity, arsenic, aflatoxins, dietary

supplements, industrial toxins, diabetes, alcoholism, and long-term use of certain

medicinal drugs (Acetaminophen) (Lin et al., 2013; Purnak and Yilmaz , 2013;

Mehta, 2014; Singh et al., 2016).

Fig. 2. Risk Factors for Liver Diseases

(Figure modified from Singh et al., 2016)

1.4.1 Age and Gender

Hepatic drug reactions are rare in children except accidental exposure. Risk of

hepatic injury is higher in adults due to reduced hepatic blood flow, drug-to-drug

interactions, variations in drug binding, and lower hepatic volume. Hepatic drug

reactions are more common in females, though the reasons are unknown (Mehta,

2014).

http://www.ncbi.nlm.nih.gov/pubmed/?term=Yilmaz%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=24090946

6

1.4.2 Genetic Factors

P-450 protein is in charge of the metabolism of most of the medications.

Hereditary varieties in the P-450 compounds can lead to abnormal reactions to drug

including peculiar adverse reactions. Variations in the P-450 can be recognized by

amplification in polymerase chain reaction of mutant genes. This has prompted the

likelihood of future identification of persons who can have anomalous responses to a

medication (Mehta, 2014).

1.4.3 Obesity

Risk of liver disease increases with weight and obese individuals are more

likely to develop liver complications than non-obese individuals. The fat cells which

aggregate in liver of obese persons cause liver damage and scarring (sclerosis)

(Lieber, 2003). With increasing weight, the likelihood of liver disease advancement,

cirrhosis, and NASH (Non Alcoholic Steato-Hepatitis) goes on increasing

(Tolman and Dalpiaz, 2007). Fat accumulation in liver consequently leads to NAFLD

(Non Alcoholic Fatty Liver Disease) (Kneeman et al., 2012).

1.4.4 Arsenic

An increased risk in development of some form of liver cancers has been

reported due to chronic exposure to naturally occurring arsenic through drinking

water (Contaminations in some wells) (Lin et al., 2013).

1.4.5 Aflatoxins

Aflatoxin is a substance made by fungus that contaminates mouldy wheat,

corn, soybeans, rice, and some types of nuts which causes cancer. Storage of the food

stuff in a moist, warm environment causes this kind of contamination and is more

common in warmer and tropical countries (Egner et al., 2001).

1.4.6 Dietary Supplements

Poor nutrition and fasting involves risk of liver disease (Malik et al., 2015).

Liver plays key role in regulating the nutritional state and the energy balance in the

body. Development of malnutrition is common in patients with hepatic disorders

7

(Purnak and Yilmaz, 2013). Deficiency of vitamin A and E may aggravate effects of

alcohol induced liver damage by preventing regeneration of hepatocytes (Addagudi et

al., 2013). This is a particular concern as alcoholics are usually malnourished due to

poor diet, anorexia and encephalopathy (Narayanan Menon et al., 2001).

1.4.7 Industrial Toxins

Many chemicals and organic solvents used in different industrial processes

may be associated with hepatotoxicity. Industrial toxins include dimethylformamide,

trichloroethylene, tetrachloroethylene, xylene, toluene, carbon tetrachloride, and vinyl

chloride (Malaguarnera et al., 2012).

1.4.8 Diabetes

Risk of developing chronic liver disease and hepatocellular carcinoma is

higher in diabetes patients than in normoglycemic individuals (El-Serag et al., 2004).

1.4.9 Alcoholism

Alcohol consumption is common reason for liver cirrhosis which increases

risk of liver cancer (Tome and Lucey, 2004; Galicia-Moreno and Gutierrez-Reyes,

2014). The quantity and duration of alcohol intake increases risk of liver disease

(Bruha et al., 2012). One in five heavy drinkers develops alcoholic hepatitis, and one

in four develops cirrhosis (Bruha et al., 2012). It is estimated that, individuals

consuming more than 200mL alcohol per day for more than 14 years may develop

liver disease (Malik et al., 2015). Alcohol also causes increased hepatotoxicity of

several xenobiotics (Radhika et al., 2011). Alcohol induced hepatic injury is due to

accumulation of reactive oxygen species and consequent lipid peroxidation of cellular

layers, proteins and DNA oxidation (Zhou et al., 2002; Galicia-Moreno and Gutierrez-

Reyes, 2014).

1.4.10 Long-Term Use of Certain Medicinal Drugs

Long term use of analgesics and antipyretics cause hepatic injury and on

prolonged conditions it leads to centrilobular hepatic necrosis (Pandit et al., 2012;

Bebnista and. Nowak, 2014). Consumption of acetaminophen like drugs may lead to

hepatocellular carcinoma (Singh et al., 2012). Oxidative stress plays a central role in


https://en.wikipedia.org/wiki/Carbon_tetrachloride

8

the hepatic damage caused by acetaminophen and antioxidants have been tested as

alternative treatment against acetaminophen toxicity (Li et al., 2015). Paracetamol

(Acetaminophen) overdose is the most frequent reason for medication induced liver

failure in the United States and in Great Britain (Jaeschke and Bajt, 2006). Long-

acting medications might bring about more harm than short-acting medications

(Mehta, 2014).

1.5 Biochemical Markers

1.5.1 Liver Function Tests

The hepatotoxin causes certain histological changes with typical clinical signs

which indicate liver injury (Singh et al., 2011). Clinical assessment of liver damage

and injury include assessment of serum biochemical markers like serum glutamic

oxaloacetic transaminase (SGOT), serum glutamate-pyruvate transaminase (SGPT),

alkaline phosphatase (ALP) and bilirubin, which are known as liver function test

markers.

Based on the mechanism of injury, hepatotoxicity can be broadly classified

into hepatocellular and cholestatic injury (Lee, 2003; Singh et al., 2011). Increase in

SGOT, SGPT, and ALP levels is an indication of cellular leakage. It further causes

decreased functional integrity of hepatic cell membranes resulting into hepatocellular

damage (Basu et al., 2012). Total bilirubin indicates functional status of the hepatic

cells (Basu et al., 2012). The obvious signs of hepatocellular injury primarily involve

increase in SGOT, SGPT preceding increase in total bilirubin level and small increase

in ALP level (Singh et al., 2011). In cholestatic injury, elevation in ALP level is more

prominent as compared to that in SGOT and SGPT. Generally mixed type of injuries,

involving both hepatocellular and cholestasis injuries, are observed clinically

(Teschke, 2009). The ratio of SGOT: SGPT plays an important role in deciding the

type of liver damage by hepatotoxins (Mishra, 2012). SGOT: SGPT ratio in

hepatocellular damage is greater than or equal to five while it is less than or equal to

two during cholestatic liver damage (Singh et al., 2011). The ratio ranges between two

and five for mixed type of liver damage (Singh et al., 2011).

9

1.5.2 Total Protein

The liver is the major source of most of the serum proteins and amount of

serum total proteins indicate the functional status of the hepatic cells (Gupta et al.,

2007; Thapa and Walia, 2007; Basu et al., 2012). Liver cells synthesize albumin,

fibrinogen, prothrombin, alpha-1-antitrypsin, hepatoglobin, ceruloplasmin, transferrin,

alpha foetoproteins etc., while damaged liver shows decreased levels of these plasma

proteins (Thapa and Walia, 2007; Mishra, 2012).

1.5.3 Lipid Profile

The liver is a major organ regulating lipid metabolism and it synthesizes and

metabolizes cholesterol, bile acids and phospholipids (Werner et al., 2000). Liver

synthesizes nearly 80% of the cholesterol produced in the body from Acetyl-CoA via

a pathway that connects metabolism of carbohydrates with that of lipid. Liver can

synthesize, store and export triglycerides (Dean et al., 2009; Rui, 2014).

Acetaminophen intoxicated rats show elevated levels of cholesterol and

triglycerides, indicating hepatic damage and consequent impairment in fat metabolism

(Haldar et al., 2011). The liver controls cholesterol and triglyceride levels in the body

by assembling, secreting, and taking up various lipoprotein particles (Cox and Garcia-

Palmieri, 1990). During these functions, loss of lipid and protein components causes

change in structure of VLDL particles (Marais, 2004). The resulting LDL particles are

then returned to the liver through LDL receptors on hepatocytes (Marais, 2004).

Greater increase of LDL and VLDL may also cause a greater decrease of HDL,

disturbing lipid metabolism in liver (Al-Assaf, 2013).

1.5.4 Oxidative Stress Markers

Oxidative stress is one of the important reasons for pathogenesis of hepatic

dysfunction in humans (Rahman et al., 2012) and animals (Abd Ellah et al., 2009;

Abd Ellah, 2010). Toxicity of a xenobiotic is also affected by the generation of

reactive oxygen species (ROS) by a few different mechanisms including

mitochondrial damage, activation of cytochrome P450 2E1 (CYP2E1), and infiltration

of Kupffer cells and granulocytes (Arteel, 2003; Smathers, 2006; Albano, 2008).

Oxidative stress within the cells by partially reduced free oxygen species such as, O2-,

H2O2 and OH. Causes damage to hepatic parenchymal cells leading to hepatic injury

10

(Jothy et al., 2012; Novo and Parola, 2012). Liver releases free radicals during

detoxification of chemicals, drugs and other toxic materials (Abd Ellah, 2011). The

elevation in free radicals and decreased scavenging potential of the cells is observed

in hepatic injury (Jothy et al., 2012). Liver cells have endogenous antioxidant system

comprising of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase

(GPx), reduced glutathione (GSH) and malondialdehyde (MDA) which offers

protection against oxidative stress (Haldar et al., 2011). Elevated levels of antioxidant

enzymes and decrease in MDA helps in hepatoprotection while decrease in

antioxidant enzyme activities and increased MDA results in hepatocellular damage

which leads to degradation of cellular macromolecules in liver by chemical induced

toxicity (Janero, 1990; Durairaj et al., 2007; Palanivel et al., 2008; Haldar et al.,

2011).

1.6 Management of Hepatic Diseases

In today’s world, liver diseases are a major serious health problem. Despite

considerable progress in modern medicine, the drugs or agents which can stimulate

liver function or help regeneration of hepatic cells or offer protection to the liver

damage are still wanted (Vishal, 2013). In addition, the synthetic drugs used in

modern medicine have been reported to have many undesirable side effects. Hence,

there is a recent renewal of interest in search for the natural resources like medicinal

plants which have promising potential to offer several herbal medicines with less side

effects (Sudaroli, 2013). Thousands of medicinal plants are used worldwide to prevent

as well as cure many diseases, but the mode of their protective and curative actions

still remains unclear (Pan et al., 2014).

The therapies available to treat liver diseases include surgical procedures,

hepatoprotective agents, herbal formulations, medicinal plants, nutritional

supplements etc.

1.6.1 Surgical Procedures

Liver transplantation has become an acceptable means with excellent long-

term outcomes to treat end-stage liver diseases, but it is very costly. Liver failure due

to viral hepatitis (especially hepatitis B and C) is a common indication for liver

transplantation (Terrault et al., 2005).

11

1.6.2 Hepatoprotective Agents

Hepatoprotective agents have recently been given attention due to their roles

in the additional treatment of liver disease (Flatland, 2003; Sartor and Trepanier,

2003; Twedt, 2004). These products include both prescription drugs and

nutraceuticals. In order for a compound to be used as a drug, it must be harmless and

effective for its intended use. The drug can be released in the market only after

undergoing an extensive Food and Drugs Administration’s (FDA) drug approval

process which is lengthy and costly. Apart from modern drugs, there are several

hepatoprotective agents like L-carnitine (Yapar et al., 2007), Vitamin C (Adikwu and

Deo, 2013), N-acetylcysteine (Maheswari et al., 2014), and Milk Thistle (Silymarin)

(Vargas-Mendoza et al., 2014). Silymarin is a unique flavonoid complex derived from

milk thistle and is commonly used for regeneration of liver cells, decongestion of

liver, complementary treatment to the patients of liver cirrhosis and viral hepatitis etc.

It is also used for hepatoprotection against industrial chemicals and pharmaceuticals

(Das et al., 2011).

1.6.2.1 Silymarin

Silymarin is a standardized extract from the seeds of a plant called milk thistle

(Silybum marianum L.; Family: Asteraceae). In rural areas, it has been used as a

natural remedy to treat liver diseases (Saller et al., 2001). Silymarin helps to protect

and enhance the regeneration of liver cells in most of the liver diseases like cirrhosis,

hepatitis and jaundice (Flora et al., 1998). Silymarin possess membrane stabilizing,

anti-oxidative, anti-lipid peroxidative (Pascual et al., 1993), anti-fibrotic (Jia et al.,

2001), and immune-modulatory properties and helps in liver regeneration (Pradhan

and Girish, 2006). Studies on human beings demonstrated that about 20-40%

silymarin is excreted as sulphates and glucuronide conjugates in bile (Saller et al.,

2001). There are a few reports of low level of silymarin toxicity causing allergic skin

rashes and gastrointestinal disturbances (Saller et al., 2001).

1.6.3 Herbal Formulations

Numerous medicinal plants and their formulations are used to treat liver

disorders in ethno medicine practice as well as traditional system of medicine in India.

There are about 600 commercial herbal formulations available in market all over the

12

world, which are claimed to have hepatoprotective activity (Bedi et al., 2016). In

India, about 40 anti-hepatotoxic, patented, polyherbal formulations representing a

variety of combination of 93 medicinal plants from 44 families are available (Sharma

et al., 1991). More than 700 mono and poly-herbal hepatoprotective preparations from

more than 100 plants are in clinical use in the form of decoction, tincture, tablets and

capsules. Recent global increase in the utilization of herbal drugs has also been

reported in the literature (Girish et al., 2009).

1.6.4 Medicinal Plants

Eastern countries have been using herbal drugs to treat liver diseases since

ancient time (Rajaratnam et al., 2014). The ancient Chinese and Egyptian written

records are available which describe medicinal uses of plants (Rajaratnam et al.,

2014). In ancient India (Vedic period) and China (Xia dynasty), records on use of

herbal medicines track back to 2100 BC. The first written reports date back to 600

B.C. with the Charka Samhita of India and the early notes of the Eastern Zhou

dynasty of China around 400 B.C (Onyije and Avwioro, 2012).

Ayurveda, an indigenous system of medicine in India, has a long tradition of

treating liver disorders with plant drugs. Minimizing side effects and increasing

therapeutic efficacy of medicines is the basic need of today. Alternative system of

medicine like Ayurveda, Unani etc. has been proved to be effective with minimum

side effects. With rich diversity of plants, over 45,000 diverse plant species are found

in India out of which about 15,000-20,000 plants have medicinal and therapeutic

properties. Of these, only about 7,000-7,500 are being used by traditional practitioners

(Bedi et al., 2016). As per WHO report, around three quarters of the world’s

population uses herbs and other traditional medicines to cure various diseases,

including liver disorders (Chaudhury and Refei, 2001). The medicinal plant such as

Guduchi (Sharma and Pandey, 2010), Elephantopus scaber (Ho et al., 2012),

Aquilegia vulgaris (Adamska et al., 2003), Strychnos potatorum (Sanmugapriya &

Venkataraman, 2006), Tridax procumbens (Ravikumar et al., 2006), Picrorhiza

kurroa (Mohd et al., 2012), Silybum marianum (Hermenean et al., 2015),

Andrographis paniculata (Nasir et al., 2013), Azadirachta indica (Johnson et al.,

2015) and Glycyrrhiza glabra (Sharma and Agrawal, 2014) has proven

hepatoprotective properties and are used to treat liver disorders. Guduchi (Tinospora

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

13

sp.) is one of the most versatile rejuvenating shrubs, also known as ’Giloya’ in Indian

vernacular, and is reported to have many therapeutic applications (Pandey et al.,

2012). Guduchi, as it is most commonly called, has been described as “one which

protects the body” (Gawhare, 2013).

1.6.4.1 Tinospora forms

Tinospora (Guduchi) is one of the most commonly used plants for preparation

of hepatoprotective ayurvedic formulations. Tinospora belongs to family

Menispermaceae. Tinospora is a climbing or twining shrub (Choudhary et al., 2013;

Tripathi et al., 2015) and is found mostly in tropical and subtropical areas of India

with different names (Nidhi et al., 2013). More than 32 species of Guduchi are found

all over the world (Choudhary et al., 2013). Four different species of Tinospora occur

in India viz. Tinospora cordifolia (Wild.) Miers ex Hook. F. & Thoms, Tinospora

sinensis (Lour.) Merr., Tinospora crispa (L.) Miers ex Hook. f. & Thoms and

Tinospora glabra (Burm f.) Merrill (Pramanik and Gangopadhyay, 1993). Other

common names for Guduchi are Gilo (Arabic), Amarlata (Assamese), Gadancha,

Guluncha, Giloe (Bengali), K’uan chu Hsing (Chinese), Culancha (French), Tinospora

(English), Gado, Galo, Gulo (Gujerati), Giloe, Gulbel, Gurcha (Hindi), Amrytu,

Sittamrytu (Malayalam), Ambarvel, Giroli, Gulvel (Marathi), Garjo (Nepali), Gulancha

(Oriya), Gulbel (Persian), Gilo (Punjabi, Kashmiri), Amrita, Guduchi, (Sanskrit), Gurjo

(Sikkikim), Amridavalli, Niraidarudian (Tamil), Guduchi, Iruluchi (Telugu) and Guruch

(Urdu) (Choudhary et al., 2013; Tripathi et al., 2013). In this study we have selected

three difference forms of guduchi (Fig. 3):

(a) Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms.

(b) Tinospora sinensis (Lour.) Merrill.

(c) Neem-giloe (Tinospora cordifolia plant growing on Azadirachta indica (Neem

tree).

14

(a) Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms

Table 1. Classification of T. cordifolia

Distribution

Global: India, Sri Lanka, Bangladesh, China Malaysia, Indonesia, Pakistan

and Thailand (Raghu et al., 2006). National: Plant is distributed throughout the

tropical region of India up to 800-1200 m above sea level, extending from Himalayas

down to the southern part of peninsular India (Geetha et al., 2007; Nidhi et al., 2013).

Vernacular Names

Guduchi, Madhuparni, Amrita, Amritavallari, Chhinna, Chhinnaruha,

Chhinnodbhava, Vatasadani, Tantrika, Kundalini, Chakralakshanika, Somavalli,

Dhira, Vishalya, Rasayani, Chandrahasa, Vayastha, Mandali, Devanirmita (Sharma

et al., 2005).

Medicinal Properties

Tinospora cordifolia contains alkaloids, glycosides, diterpenoid lactones,

sesquiterpenoids, steroids, phenolics, aliphatic compounds and polysaccharides and

are rich in protein, calcium and phosphorus (Singh et al., 2013). T. cordifolia has been

used in Ayurvedic Rasayana due to its immune-modulatory and hepatoprotective

activity (Bishayi et al., 2002; Upadhyay et al., 2010). T. cordifolia is one of the major

constituents of several Ayurvedic preparations and is used preferably for general

Classification

Botanical name Tinospora cordifolia

Synonyms Menispermum cordifolium (Willd)

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Ranunculales

Family Menispermaceae

Genus Tinospora

Species Cordifolia

15

debility, dyspepsia, fever, urinary diseases, jaundice, skin diseases, diabetes, anaemia,

cancer, liver disorder, heart disease, Parkinson’s disease, emaciations, and hepatitis B

and E (Sinha et al., 2004; Ganguly and Prasad, 2011).

(b) Tinospora sinensis (Lour.) Merrill

Table 2. Classification of T. sinensis

Distribution

Global: India, Sri Lanka, Nepal, Bangladesh, Myanmar, China, Thailand,

Vietnam and Cambodia (Ravikumar and Ved, 2000). National: Plant is distributed

throughout the subtropical evergreen or mixed deciduous forests, scrub jungles and

forests, on sandy loam, hedges and occasionally in rocky valleys, up to 800 m (eFlora

of India, 2014). In India, it occurs in Assam, Bihar, Orissa, Maharashtra, Andhra

Pradesh, Karnataka, Kerala and Tamilnadu (Ravikumar and Ved, 2000).

Vernacular Names

Malabar gulbel, Chinese tinospora, Hoguni-lota, Giloy, Gulancha, Gurch,

Sudarsana balli, Pee-amerda, Kattu amrita, Gulval, Vhadlli-amrutvel, Guruj

Vatsadani, Sudarsana, Amarta, Potchindil, Tippategu (Nidhi et al., 2013).

Classification

Botanical name Tinospora sinensis (Lour.) Merrill

Synonyms Tinospora malabarica (Lam.) Miers;

Campylus sinensis Lour.

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Ranunculales

Family Menispermaceae

Genus Tinospora

Species Sinensis

16

Medicinal Properties

T. sinensis contains starch and traces of berberin (Jain et al., 2010). T. sinensis

has been used to treat piles, ulcerated wounds, liver complaints, chronic rheumatism,

tuberculosis, debility (weakness), burning sensations during urination, ear pain, body

pain, diabetes and is also used as a muscle relaxant (Jain et al., 2010; Udayan, 2004).

(c) Neem-giloe

Neem-giloe (Neem-guduchi) is Tinospora cordifolia plant growing on

Azadirachta indica Guss. (Meliaceae) (Neem) which is also mentioned in ayurvedic

literature. Neem-giloe has been used anti-inflammatory, immunosuppressive and

hepatoprotective (Pendse, et al., 1977; Sinha et al., 2004).

Fig.3. Three different forms of Tinospora A. T. cordifolia; B. T. sinensis; C. Neem-

giloe

1.6.5 Satwa

According to the ayurvedic formulary of India, ‘satwa’ is aqueous extractable

solid substance collected from herbal plant (Anonymous, 2003a). In current study, we

have used satwa of three Tinospora forms to study their hepatoprotective activity.

A B C

Tinospora cordifolia Tinospora sinensis Neem-giloe

17

Table 4. Differences between Tinospora cordifolia and Tinospora sinensis (Adapted

from Cooke, 1901; Narkhede et al., 2014)

No Charatcter Tinospora cordifolia Tinospora sinensis

1 Habit Extensive climber Straggling shrubs

2 Stem With lenticels With lenticels

3 Bark Green and corky Dirty green, worty

4 T. S. of stem Wheel like shape,

Shows white exudates

Wheel like shape,

Shows yellow exudates

5 Taste Bitter Bitter

6 Leaves size 5.0-8.5 cm. One leaf

arise from one node

8-12 cm. More than one

Leaf arises from one node

7 Leaf proportion Broad as long Long as broad or broader

than long

8 Leaves shape Ovate reniform Ovate of cordate

9 Leaf hair Non hairy Dense hairy

10 Leaf width Thin papery Thick leathery

11 Leaf colour Dark green Yellowish green

12 Leaves number More (up to 10 per feet) Less (Up to 4 per feet)

13 Petiole 3-4 cm long 8-11 cm long

14 Branches Wiry long Thickly short

15 Flowers Greenish –yellow Greenish-yellow

16 Flower size 5-8 mm across 5-7 mm across

17 Flowers male Fascicled Petals obovate, cuneate,

rounded at the apex, not

embracing the stamens

18 Flowers female Solitary or in raceme In raceme from bare branches

19 Drupe size 5-6mm across 0.9-1.2mm across

20 Drupe colour Drupe orange–red when

Ripe

Drupe orange–red when ripe

21 Drupe shape Globose Ellipsoid

22 Flowerand

fruiting time

January –August January-May

23 First botanically

identified in India

1806 1934

24 Strach content More Comparatively poor

18

1.6.6 Nutritional Supplements

It is very important for patients with liver disease, to have balanced diet with

suitable calories, carbohydrates, fats and proteins (Worman, 1999). Balanced diet with

good nutritive value helps in regeneration of liver cells. Dietary supplements contain

herbal products, vitamins, minerals, and any product that is not a drug (medication)

(American Cancer Society, 2015). Several Asian nations use numerous food and

nutrition supplements, in routine diet that possess hepatoprotective activity. Several

phytochemicals present in nutritional supplements possess potential ability to prevent

or reverse different kinds of liver injuries (Shukla and Kumar, 2013). Omega-3 fatty

acids are also known to offer significant benefits as nutritional supplement or dietary

supplement for hepatoprotection.

1.6.6.1 Polyunsaturated Fatty Acid (Omega-3 Fatty Acids)

Ancient people consumed food containing a lot more omega-3 fatty acids than

we do today. Omega-3 fatty acids are characterized by double bond (C=C) between

third and fourth carbon atom from methyl end of the carbon chain (Scorletti and

Byrne, 2013). α-linolenic acid (ALA), found in plant oils and eicosapentaenoic acid

(EPA), docosahexaenoic acid (DHA), both commonly found in marine oils, are three

physiologically important omega-3 fatty acids (Rodriguez-Leyva et al., 2010). Plant

oils mainly contain ALA and it is obtained from walnut, edible seeds, clary sage seed

oil, algal oil, flaxseed oil, Sacha Inchi oil, Echium oil, and hemp oil (Simopoulos,

2008). EPA and DHA are chief components of fish oils, egg oil, squid oils, and krill

oil (Demark-Wahnefried et al., 2001). Omega-3: Omega-6 fatty acids ratio in wild

animals is 1:1 which is ideal for normal biological processes (Simopoulos, 2008).

1.7 Animal Models for Hepatotoxicity

In in vivo experimental models, animals are administered with repeated dose

of hepatotoxin to induce liver damage. To evaluate hepatoprotective ability of drugs,

various hepatotoxins (chemical agents) such as carbon tetrachloride, acetaminophen,

alcohol, are used in experimental models.

19

1.7.1 Carbon Tetrachloride Induced Hepatotoxicity

Formerly, carbon tetrachloride (CCl4) was used in fire-extinguishers, as a

grain fumigant and also as a refrigerant (El-Sayed et al., 2015). Carbon tetrachloride

(CCl4) is one of the commonly used model drug (hepatotoxin) to induce

hepatotoxicity in experimental models to study acute and chronic liver failures (Singh

et al., 2012; Olatosin et al., 2014). In endoplasmic reticulum and mitochondria,

cytochrome P-450 metabolizes CCl4 and forms a reactive oxidative free radical called

CCl3O which induces lipid peroxidation (Weber et al., 2003). Within 3 hours of single

dose of CCl4 administration, poison reaches its maximum concentration, and within

24 hours centrilobular necrosis and fatty changes are observed in experimental rats

(Singh et al., 2012). Different methods and concentrations of doses used in

experimental models are: repetitive dose of 0.1 to 3ml CCl4/kg bw, I.P. for 7 days; 1

ml CCl4/kg bw, I.P in liquid paraffin (30% v/v); 1ml CCl4/kg bw, I.P. single dose to

induce acute hepatotoxicity (Adewale et al., 2014; Sarkar et al., 2014).

1.7.2 Acetaminophen Induced Hepatotoxicity

Acetaminophen (international name used in USA and Japan) and Paracetamol

(international name used in Europe) are two official names of the same chemical

compound derived from its chemical name: N-acetyl-para-aminophenol (Benista and

Nowak, 2014). Acetaminophen is over-the-counter analgesic and anti-pyretic

medicine. Acetaminophen is a dynamic metabolite of phenacetin used to get relief

from fever, migraine, muscle hurts, joint inflammation, spinal pain, toothache and

frosty (Vidhya Malar and Bai, 2012). Overdose of acetaminophen leads to

‘Acetaminophen hepatotoxicity,’ causes liver injury and is one of the most common

causes of poisoning all over world (Vidhya Malar and Bai, 2012). Therapeutic dose of

acetaminophen is safe but, its overdose can cause hepatotoxicity and acute liver

failure (Michaut et al., 2014). Higher dose of acetaminophen causes hepatotoxicity in

human and animal models (Jaeschke et al., 2014). Events of acetaminophen

hepatotoxicity lead to liver cirrhosis, hepatitis etc. (Fontana, 2008).

It is primarily metabolized by sulfation and glucuronidation to unreactive

metabolites, and then activated by cytochrome P450 system. Bioactivation of

acetaminophen produces a toxic electrophile, N-acetyl p- benzoquinone imine

20

(NAPQI). NAPQI binds covalently to tissue macromolecules and also induces lipid

oxidation. Apart from oxidizing lipids, NAPQI also oxidizes sulfhydryl groups in

protein thiols. NAPQI is also known to alter the homeostasis of calcium (Lin et al.,

1997). Different reactive metabolites are produced during acetaminophen metabolism,

which covalently modify proteins (Bernareggi, 1998), impose oxidative stress (Ritter

and Giganti, 1998) and results in mitochondrial injury (Mingatto et al., 2000). Several

studies in animals and human have demonstrated that paracetamol overdose causes

liver damage primarily due to enhanced production and/or decreased glutathione

conjugation of NAPQI that eventually results in increased covalent binding of NAPQI

to cell proteins (Leung et al., 2012; Sharoud, 2015). Several experimental studies

reveal that daily treatment of acetaminophen to Wistar albino rats [at a dose of

600mg/kg for 14 days (Ita et al., 2009) or a single dose of 500 mg/kg to 3 gm/kg

(Murugesh et al., 2005; Juraj et al., 2004) or a single dose of 2g/kg (Meganathan et

al., 2011; Prabu et al., 2011) or a dose of 400 mg/kg for seven days (Kanchan and

Sadiq, 2011)] leads to liver injury and consequent increase in the levels of liver

marker enzymes.

1.7.3 Alcohol Induced Hepatotoxicity

Liver is among the organs most susceptible to the toxic effects of ethanol.

Alcoholic liver disease (ALD) is considered a major health and economic problem

worldwide (Bruha et al., 2012; Cui et al., 2013). Alcohols are hydroxy derivatives of

aliphatic hydrocarbons and commonly consumed alcohol is ethyl alcohol or ethanol

(Tripathi, 2013). Alcohol is a psychoactive and addictive substance which is quickly

absorbed by the body and detoxified by liver (Hadzic et al., 2013). Alcohol (Ethanol)

is one of the most important and commonly used hepatotoxic agents in the

experimental study of liver related disorders. The alcohol over dose leads to liver

damages (Arulkumaran et al., 2009) caused by complex mechanisms involving

metabolites of ethanol with ability to form protein adducts with several proteins of

hepatocytes (Zimmerman, 1999). Further, it has direct cytotoxicity which leads to

increase in reduced form of nicotinamide adenine dinucleotide (NADH) causing fat

accumulation (Zimmerman, 1999). Many pathways are reported to be involved in

ALD, including oxidative stress and mitochondrial damage (Stewart et al., 2001; Wu

and Cederbaum, 2003; Gramenzi et al., 2006). Ethanol is metabolized in the body by

enzyme catalyzed oxidative processes into acetaldehyde. The acetaldehyde is further

21

oxidized to acetate which is then converted to carbon dioxide via the citric acid cycle

(Samundeeswari et al., 2013). Alcohol metabolites also cause induction of free

radicals leading to peroxidation and inflammatory response (Jarvelainen, 2000).

Various different concentrations of alcohol at different doses have been reported to be

hepatotoxic in animal studies. Alcohol (15% to 40%) at a dose of 2ml-25 ml/kg bw or

1.5gm-24gm/kg bw (Mahendran and Devi, 2001; Ghosh et al., 2007; Arulkumaran et

al., 2009; Patel et al., 2010; Nigam and Paarakh, 2011; Radhika et al., 2011; Singh

and Gupta, 2011; Vetriselvan et al., 2011; Sudhir et al., 2012) is reported to induce

hepatic damage in Wistar rats.

In recent years, herbal medicine and nutritional supplementation have been

used as alternative medicine to treat liver disorder (Lee et al., 2007; Abo El-Magd et

al., 2015). Tinospora is known to be used in many ailments in alternative medicine.

Omega 3 fatty acids are also known to have several health benefits (Wu et al., 2012;

Li et al., 2014). In current study, protective, corrective and prophylactic effects of

Guduchi satwa and omega-3 fatty acids against acetaminophen and alcohol induced

hepatotoxicity were analyzed.

1.8 Genesis of Thesis

Herbal medicines and nutrition are known to play an important role in

management of various health ailments. Acetaminophen and alcohol induced

hepatotoxicity may be modulated through interventions with herbal and nutritional

supplements. The efficacy of the interventions can be enhanced by their simultaneous

delivery during progression of hepatotoxicity and may serve as curative as well as

preventive therapies against liver toxicity.

In this study three different forms of Tinospora i.e. T. cordifolia, T.

sinensis and Neem-giloe were analyzed for the hepatoprotective efficacy of their

satwa. T. cordifolia is easily available in the fields and hence it is used frequently. But

it is observed that the description of Guduchi in Ayurvedic literature matches with T.

sinensis. Hence, to ascertain the most potent variant of Guduchi, hepatoprotective

activity of all three satwa (from T. cordifolia, T. sinensis and Neem-giloe) was

assessed against acetaminophen and alcohol induced hepatotoxicity in rats.

22

In this study, flax oil and fish oil were used as nutritional supplements (rich

source of omega-3 fatty acids) to assess their hepatoprotective activity against

acetaminophen and alcohol induced liver toxicity. The protective and corrective

effects of nutritional and herbal interventions against acetaminophen and alcohol

induced hepatotoxicity in rats were also studied.

Hypothesis

Pathophysiology of acetaminophen and alcohol induced hepatotoxicity may be

modulated through interventions with herbal and nutritional supplements. The

efficacy of the interventions can be enhanced by their simultaneous delivery during

progression of hepatotoxicity and may have corrective/preventive roles in liver

toxicity.

LITERATU

CHAPTER 2

OBJECTIVES

Awards


Presentation Award


Award

23

Objectives

1. To study the hepatoprotective efficacy of residual marc of aqueous extract of

Tinospora cordifolia, Tinospora sinensis and Neem-giloe (T. cordifolia

growing on Azadirachta indica A. Juss.) on acetaminophen and alcohol

induced hepatotoxicity in rats at biochemical, histological and molecular level.

2. To study the effect of polyunsaturated fatty acid supplementation against

acetaminophen and alcohol induced hepatotoxicity in rats at biochemical,

histological and molecular level.

3. To study the combinatorial protective and corrective effect of nutritional and

herbal interventions against acetaminophen and alcohol induced

hepatotoxicity in rats at biochemical, histological and molecular level.

LITERATU

CHAPTER 3

REVIEW OF LITERATURE

24

Hepatotoxicity implies chemical driven or drug induced liver damage. The

agents responsible for such liver damage are called hepatotoxicants. Such

hepatotoxicants are employed as inducers of hepatotoxicity in several experimental

animal models and such induction of hepatotoxicity is indispensable for testing of

novel hepatoprotective agents and their safety. Several herbal formulations, medicinal

plants and nutritional supplements are recommended for treatment of liver diseases. In

the present study, satwa from three different forms of Tinospora (Guduchi satwa) and

omega-3 fatty acids were analyzed for their hepatoprotective activities. This chapter

reviews literature about drug (acetaminophen and alcohol) induced hepatotoxicity and

hepatoprotective effects of different interventions.

3.1 Acetaminophen Induced Hepatotoxicity

Acetaminophen is the most popular over-the-counter, analgesic and antipyretic

drug commonly used to treat mild to moderate pain. It was discovered in 1893 and

first introduced in Canada in 1950’s (Meredith and Goulding., 1980; Jackson et al.,

1984; Anker and Smiikstein, 1994; Bessems and Vermeulen, 2001; James et al.,

2003). The therapeutic dose (650-1000 mg every 4 to 6 hours) of acetaminophen is

considered to be safe (Jackson et al., 1984). First case of acetaminophen

hepatotoxicity was reported in UK in 1966 (Davidson and Eastham, 1966) followed

by in North America in 1971 (Boyer and Rouff, 1971; Hinson et al., 2010).

Acetaminophen-induced hepatotoxicity has been studied in experimental animals

(Mitchell et al., 1973; Lim et al., 1995; Hinson et al., 2010) as well as in clinical cases

(Boyd and Bereczky, 1966; McJunkin et al., 1976; Golden et al., 1981; Whitcomb and

Block, 1994). Higher dose of acetaminophen produces centrilobular hepatic necrosis,

fulminant hepatic failure and renal tubular necrosis in humans and laboratory animals

(Boyd and Bereczky, 1966; Boyer and Rouff, 1971; Jollow et al., 1973).

Mechanism of Acetaminophen Induced Hepatotoxicity

Several studies reported mechanism of acetaminophen induced hepatotoxicity

(Jollow et al., 1973; Potter et al., 1973; Cohen and Khairallah, 1997; Bessems and

Vermeulen, 2001; Irwin et al., 2004). Acetaminophen is metabolized in liver via

glucuronidation, sulfation and the hepatic cytochrome P450 enzyme system. The

cytochrome P450 enzyme system metabolically activates acetaminophen and forms a

reactive metabolite N-Acetyl-P-benzo-quinoneimine (NAPQI) (Prescott, 1980;

Awards


Presentation Award


Award

25

Vidhya Malar and Bai, 2012). NAPQI covalently binds to proteins and forms

acetaminophen-protein adducts (James et al., 2003) NAPQI is formed by direct two-

electron oxidation of acetaminophen by cytochrome P450 (Dahlin et al., 1984).

Several reports demonstrated that acetaminophen is oxidized into the reactive

metabolite by the cytochromes 2E1, 1A2, 3A4, and 2A6 (Patten et al., 1993;

Thummel et al., 1993; Chen et al., 1998). Toxic dose of acetaminophen leads to

almost 90% depletion of total hepatic Glutathione (GSH) (Mitchell et al., 1973). The

mechanism is shown in Fig. 4. Acetaminophen mainly undergoes glucuronidation

(47%-62%) and sulfation (25%-36%) in the liver when taken in therapeutic dose

(Prescott, 1980; Pacifici and Allegaert, 2015). The hepatic cytochrome P450 enzyme

system (specifically CYP2E1) metabolizes about 5 to 10% of therapeutic

acetaminophen resulting into production of toxic product (NAPQI) which is

detoxified by combining with the sulfhydryl groups of glutathione and then excreted

through urine (Pacifici and Allegaert, 2015). Acetaminophen overdose causes

increased formation of toxic product NAPQI and utilization of substantial amounts of

GSH from liver. This leads to inability of the liver to detoxify NAPQI which

accumulates in liver to toxic concentrations (Dahlin et al., 1984; Raucy et al., 1989;

Pacifici and Allegaert, 2015). The formation of NAPQI is dependent on cytochrome

P450 such as CYP1A1, CYP1A2, and CYP2E1 (Patten et al., 1993; Laine et al., 2009;

Cederbaum, 2015) in rats; CYP1A2 and CYP3A4 in mice (Hazai et al., 2002); and

CYP2E1, CYP1A2 and CYP3A4 in humans (Raucy et al., 1989; Thummel et al.,

1993; Prescott, 2000). Acetaminophen also obstructs intracellular processes in

nucleus (Ray et al., 1996), plasma membrane (Moore et al., 1985), and cytoplasm

(Pumford et al., 1989). Acetaminophen toxicity has been attributed to the interference

of many metabolic processes including depletion of glutathione, impairment of

mitochondrial respiration, and interference with Ca2+

homeostasis (Boulares and Ren,

2004). In addition to formation of NAPQI, high levels of reactive oxygen species

(ROS) also contribute to cell injury process (Shon and Nam, 2002).

26

Fig. 4. Schematic Representation Depicting Activation of Acetaminophen and

NAPQI-Mediated Acetaminophen Toxicity

(From James et al., 2003)

Covalent binding of acetaminophen to protein (Jollow et al., 1973) and the

role of specific adducts in acetaminophen toxicity (Bartolone et al., 1988, 1989;

Cohen and Khairallah, 1997) was evident from the specific immunochemical assays

developed for acetaminophen covalently bound to cysteine groups on proteins

(Roberts et al., 1991). Molecular nature of the acetaminophen protein adducts have

been determined using western blot assays (Bartolone et al., 1988) and these adducts

have been detected in the blood samples of individuals with severe acetaminophen

toxicity (James et al., 2003). The high performance liquid chromatography with

electrochemical detection has been used to report presence of such adducts in the

blood samples from patients with acetaminophen toxicity (Muldrew et al., 2002). The

appearance of acetaminophen-protein adducts in serum correlated with elevations of

serum glutamic oxaloacetic transaminase (SGOT) and serum glutamate-pyruvate

transaminase (SGPT) in acetaminophen treated mice (Pumford et al., 1989). Several

reports indicate elevations in SGOT, SGPT, alkaline phosphatase (ALP) and bilirubin

levels following administration of toxic doses of acetaminophen in rats (Mahesh et al.,

2009; Rasool et al., 2010; Galal et al., 2012). Acetaminophen overdose causes

27

decrease in antioxidant enzyme activities such as superoxide dismutase, catalase and

glutathione peroxidase and increased levels of lipid peroxidation in animal models

(Wendel, 1979; Nakae et al., 1990; Arnaiz et al., 1995; Chen and Lin, 1997).

Hepatoprotective activity of different medicinal plants or herbal medicines has been

extensively studied against acetaminophen induced hepatotoxicity in animals

(Manokaran et al., 2007; Rajkapoor et al., 2008; Kumar et al., 2011; Kanchana and

Sadiq, 2011; Manivannan et al., 2011; Mahmood et al., 2014). Histological analyses

revealed necrosis and apoptosis of hepatocytes in acetaminophen treated mice (Ray et

al., 1996).

Several studies reported gene expression analysis of acetaminophen induced

hepatotoxicity in rodents (Reilly et al., 2001; Ruepp et al., 2002; Irwin et al., 2004;

Ishida et al., 2004; Kim et al., 2007; Germoush and Mahmoud, 2014; Gong et al.,

2014; Mahmoud et al., 2014; Mohar et al., 2014). Critical role of kupffer cells in

development of acetaminophen hepatotoxicity was demonstrated by Laskin et al.

(1995) through pretreating rats with compounds suppressing kupffer cell function

which leads to secretion of multiple cytokines in acetaminophen toxicity (Blazka et

al., 1995; Bourdi et al., 2002). Elevation in serum levels of tumor necrosis factor

alpha (TNF-α), and Interleukin-1 alpha (IL-1α) was reported by Blazka et al. (1995)

and Ishida et al. (2004) in mice treated with acetaminophen. Some studies reported

that treatment of acetaminophen intoxicated mice with either anti-TNF-α or anti-IL-

1α antibody partially prevented hepatotoxicity (Blazka et al., 1996). Other genes

involved in the production of inflammatory cytokines, chemokines, cell adhesion

molecules, and growth factors, involved in acetaminophen induced hepatotoxicity, are

in turn regulated by nuclear factor κβ (Richmond, 2002). It has also been reported that

NFκβ protects numerous cell types from TNF-α induced cell death (Beg and

Baltimore, 1996; Baichwal and Baeuerle, 1997; Boulares et al., 2000). NFκβ is known

to activate expression of different apoptotic as well as cell proliferation proteins

(Richmond, 2002). Overdose of acetaminophen is known to increase the DNA

binding activity of NFκβ while protective effect of NFκβ against acetaminophen

induced hepatotoxicity is associated with reduced NFκβ (Meraz et al., 1996; Kim et

al., 2013). Gong et al. (2014) also showed the protective role of fatty acid binding

protein 1 (FABP1) in in vitro model of acetaminophen induced oxidative stress.

28

3.2 Alcohol Induced Hepatotoxicity

Alcoholic liver disorders are frequently observed all through the world,

including India (Bruha et al., 2012), where alcohol is mostly consumed in the form of

country made liquor (CML) (Kumar et al., 2007). Alcohol is one of the direct

hepatotoxic agents, and has intoxicating effects which produces oxidative stress and

impairs tissues when consumed in high doses (Askay et al., 2009; Beier and McClain,

2010; Pompili et al., 2010). Carson and Pruett (1996) proposed animal model for

acute ethanol-induced hepatotoxicity which was later modified by Song et al. (2003).

DeCarli and Liber (1976) reported one of the first successful rodent model for

alcoholic liver disease (ALD). Studies on variety of systems, cells and species

including humans have demonstrated that acute and chronic ethanol treatment

increases production of reactive oxygen species like superoxide, hydroxyl radical and

hydrogen peroxide in the hepatic cells that oxidize the glutathione (Arteel, 2003; Lu

and Cederbaum, 2008), increase oxidative stress (Sharma et al., 2011), enhance

peroxidation of lipids, oxidation of protein and DNA (Albano, 2006; Conde de la

Rosa et al., 2008) resulting in hepatic damage. Metabolism of alcohol primarily

occurs in liver (Shanmugam et al., 2010) through oxidation reactions through a

pathway known as ADH (alcohol dehydrogenase pathway) (Crabb et al., 2004).

Mechanism of Alcohol Induced Hepatotoxicity

Absorbed alcohol is detoxified by the liver through oxidation and expelled

from the blood, preventing alcohol accumulation in cells and organs (NIAAA, 1997).

Around 80-90% of ingested alcohol is metabolized in liver in a two stage oxidative

procedure, first to acetaldehyde by alcohol dehydrogenase and to acetate by

acetaldehyde dehydrogenase (Horton and Mills, 1979). The hepatocytes contain three

fundamental pathways for ethanol digestion system, each situated in an alternate sub

cell compartment: the alcohol dehydrogenase (ADH) pathway of the cytosol, the

microsomal ethanol oxidizing system (MEOS) located in the endoplasmic reticulum

and catalase located in the peroxisomes (Lieber, 1991; Lieber, 1997). Each of these

pathways produces a particular metabolic and toxic unsettling influence and generates

acetaldehyde, a highly toxic metabolite (Lieber, 1997). In the second oxidation step,

acetaldehyde is quickly metabolized to acetate by mitochondrial acetaldehyde

29

dehydrogenase (ALDH) (Harada, 2001; Caballeria, 2003). All these biochemical

pathways produce acetaldehyde as a toxic product (Fig. 5).

Fig. 5. Alcohol Metabolism in Hepatocytes and Accumulation of Reactive Oxygen

Species (ROS) Leading to Liver Disease

(From Li et al., 2015)

Alcohol Dehydrogenase (ADH) System

The principle pathway for ethanol oxidation in the liver is by means of

alcohol dehydrogenases (ADH) to acetaldehyde, which is associated with reduction of

nicotinamide adenine dinucleotide (NAD) to NADH. NADH increases xanthine

oxidase activity, which elevates generation of superoxide (Lieber, 1997; Zima et al.,

2001). The excess of reduced equivalents, mainly NADH, produces a change in the

redox system of the cytosol, which is demonstrated, by a change in the

lactate/pyruvate ratio (Caballeria, 2003). This redox imbalance is responsible for a

series of metabolic alterations, which favor liver damage (Caballeria, 2003).

Acetaldehyde causes nucleic acid oxidation and lipid peroxidation by formation of

hybrid-adducts with reactive residues (e.g. malondialdehyde adduct) which acts on

proteins or small molecules (e.g. cysteines) (Gramenzi et al., 2006). ADH and

oxidation of ethanol to acetaldehyde and subsequent formation of acetate alters

30

NADH to NAD+ ratio which leads to fatty liver via inhibition of gluconeogenesis and

fatty acid synthesis in alcoholic persons (Lieber, 1994; Sahekia et al., 2005; Gramenzi

et al., 2006).

Microsomal Ethanol Oxidizing System (MEOS) and Catalase

The microsomal ethanol oxidizing framework (MEOS), generally situated in

the endoplasmic reticulum, constitutes a second system that can oxidize alcohol

(Caballeria, 2003; Siegmund and Brenner, 2005). An increase in the MEOS action is a

consequence of chronic alcohol consumption which influences CYP2E1, an ethanol

inducible part of cytochrome P450, capable of activating different hepatotoxins

(Lieber, 1999). At high concentration of alcohol and in chronic alcoholism, the

MEOS is included, unlike non-inducible ADH (Caballeria, 2003).

Peroxisomal catalase oxidizes alcohol in vitro in presence of hydrogen

peroxide. Under physiological conditions peroxisomal catalase has a little role in

alcohol metabolism (Lieber, 1997). Catalase might add to the oxidation of fatty acids

compensating, in part, the lower oxidation because of mitochondrial damage

(Caballeria, 2003). This leads to impaired carbohydrate and lipid metabolism, finally

causing impairment of gluconeogenesis and diversion of metabolism to ketogenesis

and fatty acid synthesis (Gramenzi et al., 2006). Several studies suggest that the

activity of alcohol dehydrogenases is a key determinant of ethanol oxidation

(Lumeng et al., 1980; Braggins and Crow, 1981; Vonlanthen et al., 2000). The rate of

alcohol disposition is known to be dependent upon the ratio of reduced to oxidized

nicotinamide adenince dinucleotide (NAD) as well as the acetaldehyde concentration

in the cells (Cheema-Dhadli et al., 1987; Cronholm et al., 1988; Zorzano and Herrera,

1990; Vonlanthen et al., 2000).

Acetaldehyde Dehydrogenase

Acetaldehyde dehydrogenase (ALDH) is located in the soluble and insoluble

fractions of hepatocytes and it rapidly metabolizes acetaldehyde to acetate (Harada,

2001).

The possible mechanism of hepatotoxicity of ethanol is CYP2E1-dependent

ethanol metabolism which produces oxidative stress through generation of reactive

31

oxygen species (ROS) (Bondy, 1992). Cytochrome P4502E1 induction by ethanol is a

central pathway by which ethanol generates oxidative stress. Induction of CYP2E1

causes significant alcohol liver injury in the intragastric model of ethanol feeding in

rats (Nanji et al., 1999). Elevation in the non-heme iron content of the liver was

observed in rodents treated with ethanol (Tsukamoto et al., 1995; Lu and Cederbaum,

2008). It has been reported that long-term consumption of ethanol produces liver

apoptosis in mice or rats (Cohen et al., 2009).

Elevated levels of SGOT, SGPT, ALP and bilirubin were observed in rats

treated with alcohol whereas treatment with medicinal plants helps to reduce these

levels which are indicative of hepatoprotective activity of the medicinal plants (Flora

et al., 1998; Kumar et al., 2007; Singanan et al., 2007; Parmar et al., 2008;

Arulkumaran et al., 2009; Arun and Balasubramanian, 2011; Nigam and Paarakh,

2011; Singh and Gupta, 2011; Sharma et al., 2012; Sudhir et al., 2012; Sharma, 2013).

There are many reports on hepatic gene expression analysis in chronic ethanol

treatment models which use administration of alcohol-containing liquid diets or

intragastric alcohol infusion (Tadic et al., 2002; Deaciuc et al., 2004; Yin et al., 2007).

Modulation of pro-inflammatory cytokines such as TNF-α and IL-1, produced by

Kupffer cells due to endotoxin stimulation, has been widely studied in ethanol

induced hepatotoxicity (Hoek and Pastorino, 2002; Wheeler, 2003; Shaw et al., 2010).

Tilg and Diehl (2000) have reviewed several studies depicting correlation between

endotoxin levels and alcohol-induced liver damage. Enhanced formation of cytokines,

especially TNF-α by hepatic Kupffer cells, has a significant role in liver injury

(Thurman et al., 1998; Zhou et al., 2003). McClain and Cohen (1989) reported that

the increased serum TNF-α has been observed in patients with alcoholic hepatitis.

Studies have shown that ethanol increases NF-κβ activation via both ROS-dependent

(Hill et al., 1999) and ROS-independent pathways (Roman et al., 1999). NF-κβ is

transcription factor of the genes related to proinflammatory response and its

expression is one of the important events linking proinflammatory response to

alcoholic liver disease (Mandrekar et al., 2009). Recently, You et al. (2002) have

reported that the metabolism of ethanol increased hepatic lipogenesis by activating

SREBP-1, and that this effect of ethanol may contribute to the development of an

alcoholic fatty liver. Several reports showed that the expression levels of SREBP-1

32

were upregulated in ethanol induced hepatotoxicity in mice (Yin et al., 2007; Cui et

al., 2013). Pelsers et al. (2002) have recommended FABP as a promising biomarker

for early detection of liver damage. Number of reports showed that expression levels

of FABP1 were downregulated in animal models (Rat and mice) of ethanol induced

hepatotoxicity (Nanji et al., 2004; Kim et al., 2007; Smathers, 2011).

3.3 Treatments

Treatment options available for common liver diseases, such as cirrhosis, fatty

liver and chronic hepatitis are inadequate in modern medicine (Padmanabhan and

Jangle, 2014). A liver disease treatment is dependent upon the causative agent, the

degree of liver dysfunction, and the age and general health condition of the individual

(Mehta, 2014). There is no effective treatment other than preventing the causative

medication or removal from the exposure to the causative agent and providing general

supportive care (Singh et al., 2011). The best mode is to discontinue the use of any

medicinal drug that may put excess stress on the liver and use an alternate medication

that helps to manage the side effects of hepatotoxicity (Singh et al., 2011). N-

acetylcysteine, vitamins (A, C, E), S-adenosylmethionine/ursodiol/ursodeoxycholic

acid and zinc are used as hepatoprotective agents (Prescott, 1979; Smilkstein et al.,

1988; Keays et al., 1991; Perry and Shannon, 1998; Flatland, 2003; Sartor and

Trepanier, 2003; Center, 2004; Twedt, 2004; Polson and Lee, 2005).

Hepatoprotective agents have been recently given attention due to their role in

the treatment of liver disease (Flatland, 2003; Sartor and Trepanier, 2003; Center,

2004; Twedt, 2004). Several treatment regimens for acetaminophen hepatotoxicity

have been proposed, but N-acetylcysteine (NAC) is a most frequent treatment for

severe acetaminophen toxicity (Prescott, 1979; Smilkstein et al., 1988; Keays et al.,

1991; Perry and Shannon, 1998; Center, 2004; Polson and Lee, 2005). Studies

demonstrated the ability of NAC to prevent hepatotoxicity in animals (Piperno et al.,

1978). Intravenous carnitine in valproate-induced hepatotoxicity has been reported for

the treatment of acute liver injury (Lheureux and Hantson, 2009). Vitamin C has been

reported to have protective activity towards hepatic damage against some drugs and

chemicals including ethanol (Shalan et al., 2007; Awodele et al., 2010; Bashandy and

Alwasel, 2011; Adikwu and Deo, 2013; Hassanin et al., 2013). S-adenosylmethionine

(SAMe) has been shown to have protective effect on the erythrocytes in cats with

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hantson%20P%5BAuthor%5D&cauthor=true&cauthor_uid=19280426

33

experimental acetaminophen toxicity (Webb et al., 2003). Ursodiol or

ursodeoxycholic acid and Zinc have also been used as hepatoprotective agents

(Flatland, 2003; Sartor and Trepanier, 2003).

3.4 Alternative Treatments

Alternative treatments in the form of herbal medicines for the treatment of

liver diseases are now frequently sought instead of currently used drugs of doubtful

efficacy and safety (Vetriselvan et al., 2010; Padmanabhan and Jangle, 2014). Herbal

formulations, medicinal plants and nutritional supplements against liver diseases have

long term benefits without major side effects. Now-a-days, herbal remedies are

gaining popularity in commercial and developing economies of the world (Pan et al.,

2014). Number of medicinal plants have been reported to have promising effects,

either experimentally in animal models or in cell cultures (in vitro models) (Saad et

al., 2006; Mukazayire et al., 2010), or even in clinical trials (Teschke and Eickhoff,

2015).

3.4.1 Hepatoprotective Herbal Formulations

Dahanukar et al. (2000) have reviewed the experimental and clinical research

related to hepatoprotective effects of various formulations available in the Indian

market. The usage of alternative medicine, including herbal preparations, has

increased due to limited therapeutic options and disappointing success of modern

medicine in treating liver ailments (Stickel and Schuppan, 2007). Most frequently

used plants in liver disorders are, Adhatoda vasica Nees., Aloe barbadensis Mill.,

Andrographis paniculata Nees, Lawsonia alba Lam., Tinospora cordifolia,

Picrorhiza kurroa Royle ex Benth., Silybum marianum Linn. These plants have been

scientifically validated in experimental animal models and are also used by ayurvedic

industries as constituents of herbal formulations for liver disorders. There are many

scientific reports about favorable results of some hepatoprotective herbal

formulations, (mostly in the form of extracts) such as G-LIV-DS syrup, Livex, HD-

03, Hepatomed, Live 100, Hepatoguard, Liv-52, Hepta-B, Livotone, Liv up, Stimuliv,

Tefroli etc. These formulations have also been found to be effective against chemical

induced hepatotoxicity. The details of the frequently used formulations and their

constituent plants are shown in Table 4.

34

Table 4. List of Hepatoprotective Herbal Formulations

Sr.

No

Name of the

formulation

Name of the Plant (Family; parts used in the formulation) Hepatotoxicity

inducing agents

Reference

1 Herbal

formulation F1

Andrographis paniculata (Acanthaceae; leaves),

Boerhavia diffusa (Nyctaginaceae; root), Eclipta alba (Asteraceae;

whole plant) and Picrorhiza kurroa (Scrophulariaceae; rhizome)

Carbon

tetrachloride and

ethanol

Kumar et al., 2014

2 Herbal Preparation

(HP-4)

Aloe vera (Liliaceae; leaves), Bacopa monnieri (Scrophulariaceae;

leaves), Moringa oleifera (Moringaceae; Leaves) and Zingiber

officinale (Zingiberaceae; rhizome)

Alcohol Padmanabhan and

Jangle, 2014

3 Polyherbal

formulation

Curcuma longa (Zingiberaceae; rhizomes), Emblica officinalis

(Phyllanthaceae; fruit), Terminalia chebula (Combretaceae; fruit),

Terminalia bellirica (Combretaceae; fruit) and Myrica nagi

(Myricaceae; fruit) and bees wax.

Paracetamol Arote et al., 2014

4 DHC-1

(Himalaya Drug

Company)

Bacopa monnieri (Scrophulariaceae; whole),

Emblica officinalis (Euphorbiaceae; fruit),

Glycyrrhiza glabra (Papilionaceae; roots),

Mangifera indica (Anacardiaceae; bark),

Syzygium aromaticum (Myrtaceae; flower bud)

Carbon

tetrachloride

Bafna and Balaraman,

2013

http://en.wikipedia.org/wiki/Asteraceae

35

Sr.

No

Name of the

formulation


inducing agents

Reference

5 Clearliv

polyherbal

formulation

(Apex

Laboratories Ltd.,

Chennai)

Phyllanthus niruri (Phyllanthaceae; whole plant), Eclipta alba

(Asteraceae; whole plant), Boerhavia diffusa (Nyctaginaceae; root),

Tinospora cordifolia (Menisperemaceae; stem), Tribulus terrestris

(Zygophyllaceae; fruit), Tephrosia purpurea (Fabaceae; root),

Indigofera tinctoria (Fabaceae; leaves), Aconitum heterophyllum

(Ranunculaceae; stem and root), Andrographis paniculata

(Acanthaceae; leaves), Rubia cordifolia (Rubiaceae; root),

Terminalia chebula (Combretaceae; fruit), Curcuma longa

(Zingiberaceae;rhizome), Ricinus cummunis (Euphorbiaceae;

leaves)

Thioacetamide, D-

Galactosamine and

carbon tetrachloride,

Kumar et al., 2013a

6 Polyherbal

formulation

Emblica officinalis (Euphorbiaceae; fruit), Phyllanthus acidus

(Phyllanthaceae; leaves), Moringa oleifera (Moringaceae; leaves)

Paracetamol Sabbani et al., 2013

7 Polyherbal

formulation tablets

(TulsiAmrit Pvt.

Ltd., Indore, India)

Andrographis paniculata (Acanthaceae; leaves), Phyllanthus niruri

(Phyllanthaceae; whole plant), Phyllanthus emblica

(Phyllanthaceae; fruit)

Paracetamol carbon

tetrachloride and

alcohol

Tatiya et al., 2012

8 Livergen,

(Standard

Pharamcuticals)

Andrographis paniculata (Acanthaceae; leaves), Apium graveolens

(Apiaceae;celery), Berberis lycium (Berberidaceae; whole plant),

Carum copticum (Apiaceae; fruit), Cichorium intybus (Asteraceae;),

Cyperus rotundus (Cyperaceae; rhizomes), Eclipta alba

(Asteraceae), Ipomoea turpethum (Convolvulaceae; root),

Oldenlandia corymbosa (Rubiaceae; leaves), Picrorhiza kurroa

(Scrophulariaceae; rhizome), Plumbago zeylanica

(Plumbaginaceae; root), Solanum nigrum (Solanaceae; leaves),

Carbon tetrachloride Arsul et al., 2011

http://en.wikipedia.org/wiki/Apiaceae

36

Sr.

No

Name of the

formulation


inducing agents

Reference

9

Polyherbal

formulation

tablets

Tephrosia purpurea (Fabaceae; roots), Terminalia arjuna

(Combretaceae; leaves), Terminalia chebula (Combretaceae; fruit),

Trigonella foenum-graecum (Fabaceae; fruit)

Phyllanthus niruri (Phyllanthaceae; whole plant), Eclipta alba

(Asteraceae; whole plant), Cichorium intybus (Asteraceae; seed),

Boerhavia diffusa (Nyctaginaceae; root), Embelia ribes

(Myrsinaceae; leaves, root), Berberis aristata (Berberidaceae; root),

Picrorhiza kurroa (Scrophulariaceae; rhizome)

Carbon tetrachloride

Arsul et al., 2010

10 Liv 52

(Himalaya Drug

Company)

Achillea millefolium (Asteraceae; aerial parts), Capparis spinosa

(Capparaceae; whole plant), Cassia occidentalis (Fabaceae; leaves),

Cichorium intybus (Asteraceae; seed), Solanum nigrum (Solanaceae;

whole plant), Tamarix gallica (Tamaricaceae; leaves), Terminalia

arjuna (Combretaceae; leaves)


and

Paracetamol

Girish et al., 2009

11 Livergen

(Standard

Pharamcuticals

Serampore, West

Bengal)


(Apiaceae; celery), Asteracantha longifolia (Acanthaceae; leaves),

Cassia angustifolia (Fabaceae; leaves), Trachyspermum ammi

(Apiaceae; leaves and fruit), Trigonella foenum-graecum (Fabaceae;

fruit)


and paracetamol

Girish et al., 2009

12 Livokin

(Herbo-med,

Kolkata)


(Apiaceae; celery), Berberis lycium (Berberidaceae; whole plant),

Carum copticum (Apiaceae; fruit), Cichorium intybus (Asteraceae;

flower), Cyperus rotundus (Cyperaceae; leaves), Eclipta alba

(Asteraceae; whole plant), Ipomoea turpethum (Convolvulaceae; root


and paracetamol

Girish et al., 2009

37

Sr.

No.

Name of the

formulation


inducing agents

Reference

13

Octogen

(Plethico

Pharamcuticals

Ltd., Indore)

bark, leaves), Oldenlandia corymbosa (Rubiaceae; whole plant),

Picrorhiza kurroa (Scrophulariaceae; rhizome) Hygrophila spinosa

(Acanthaceae; whole plant), Plumbago zeylanica (Plumbaginaceae;

leaves), Solanum nigrum (Solanaceae; leaves), Tephrosia purpurea

(Fabaceae; aerial and root), Terminalia arjuna (Combretaceae;

leaves), Terminalia chebula (Combretaceae; fruti), Trigonella

foenum-graecum (Fabaceae; fruit)

Arogyavardhini rasa, Phyllanthus niruri (Phyllanthaceae; whole

plant)


and paracetamol

Girish et al., 2009

14 Stimuliv

(Franco-Indian

Pharamcuticals

Pvt. Ltd.,

Mumbai)

Andrographis paniculata (Acanthaceae; leaves),

Eclipta alba (Asteraceae; whole plant), Phyllanthus niruri

(Phyllanthaceae; whole plant), Justicia procumbens (Acanthaceae;

leaves)


and paracetamol

Girish et al., 2009

15 Tefroliv

(TTK Pharma

Pvt.Ltd.,

Chennai)

Andrographis paniculata (Acanthaceae; leaves), Eclipta alba

(Asteraceae; whole plant), Ocimum sanctum (Lamiaceae; leaves),

Phyllanthus niruri (Phyllanthaceae; whole plant), Picrrorhiza kurroa

(Scrophulariaceae; rhizome, Piper longum (Piperaceae; fruit),

Solanum nigrum (Solanaceae; whole plant), Tephrosia purpurea

(Fabaceae; leaves), Terminalia chebula (Combretaceae; fruit)


and paracetamol

Girish et al., 2009

http://en.wikipedia.org/wiki/Solanaceae

38

Sr.

No

Name of the

formulation


inducing agents

Reference

16 Polyherbal

formulation

Acacia catechu (Fabaceae; stem bark), Allium sativum

(Amaryllidaceae; bulb), Andrographis paniculata (Acanthaceae;

leaves), Azadirachta indica (Meliaceae; leaves), Boerhavia diffusa

(Nyctaginaceae; root), Curcuma longa (Zingiberaceae; rhizome),

Eclipta alba (Asteraceae), Emblica officinalis (Euphorbiaceae),

Luffa echinata (Cucurbitaceae; fruit), Picrorhiza kurroa

(Scrophulariaceae; rhizome), Phyllanthus amarus (Phyllanthaceae;

whole plant)


and paracetamol

Kamble et al., 2008

17 Panchagavya

ghrit (GoVigyan

Anusandhan

Kendra,

Deolapar)

Cow milk, ghee, urine, dung and curd in equal proportions

Carbon tetrachloride Achliya et al., 2003

18 HD-03

(Himalaya Drug

company.)

Solanum nigrum (Solanaceae; whole plant),

Cichorium intybus (Asteraceae; seeds), Picrorhiza kurroa

(Scrophulariaceae; rhizome), Tephrosia purpurea (Fabaceae; whole

plant), Andographis paniculata (Acanthaceae; leaves)

D-Galactosamine Mitra et al., 2000

19 Liv. 100

(Himalaya Drug

company)


whole plant), Phyllanthus amarus (Phyllanthaceae; whole plant),

Picrorrhiza kurroa (Scrophulariaceae; rhizome) Emblica officinalis

(Euphorbiaceae; fruit)

Isoniazid,

rifampicin, and

Pyrazinamide

Saraswathy et al., 1998

http://en.wikipedia.org/wiki/Zingiberaceae

http://en.wikipedia.org/wiki/Asteraceae

39

Sr.

No

Name of the

formulation


inducing agents

Reference

20 Rhinax

(Hindustan

Antibiotics

Limited, Pune)

Withania somnifera (Solanaceae; root), Asparagus racemosus Wild.

(Liliaceae; root), Mucuna pruriens (Papilionaceae; root), Phyllanthus

emblica Gasertn. (Euphorbiaceae; fruit), Glycyrrhiza glabra

(Papilionaceae; root), Terminalia chebula (Combretaceae; fruit),

Myristica fragrans Houtt. (Myristicaceae; seed)

Carbon tetrachloride Dhuley and Naik, 1997

21 Blood wort

(Herb pharma,

Nigeria)

Rumex acetosa (Polygonacease; bark), Cinchona succirubra

(Rubiaceae; bark)

Carbon tetrachloride Okonkwo and Msonthi,

1995

22 Jigrine

(Unani

polypharmaceuti

cal herbal

formulation)

Cichorium intybus (Asteraceae; seed), Tamarix dioica Roxb

(Tamaricaceae), Solanum nigrum (Solanaceae; whole plant), Rheum

emodi (Polygonaceae; whole plant), Rubia cordifolia Linn

(Rubiaceae; root), Vitex negundo (Lamiaceae; flower), Cassia

occidentalis (Fabacea; leaves), Foeniculum vulgare (Apiaceae;

bulb), Cuscuta reflexa (Convolvulaceae; whole plant), Careya

arborea (Lecythidaceae; leaves and stem), Phy1lanthus niruri

(Phyllanthaceae; whole plant), Plantago major (Plantaginaceae;

whole plants), Rosa damascena (Rosaceae; flower), Solanum

xanthocarpum (Solanaceae; whole plant)

Alcohol, carbon

tetrachloride and

paracetamol

Kapur et al., 1994

23 Liv. 52

(Himalaya Drug

Company)

Achillea millefolium (Asteraceae; aerial parts), Capparis spinosa

(Capparaceae; flower buds), Cassia occidentalis (Fabaceae; leaves),



Pandey et al., 1994

40

Sr.

No

Name of the

formulation


inducing agents

Reference

whole plants), Tamarix gallica (Tamaricaceae; leaves), Terminalia

arjuna (Combretaceae; leaves)

24 BR-16A

(Mentat)

Himalaya Drug

Company

Bacopa monnieri (Scrophulariaceae; whole plants), Asparagus

racemosus (Asparagaceae; root), Acorus calamus (Acoraceae; root),

Withania somnifera (Solanaceae; stem), Tinospora cordifolia

(Menispermaceae; stem), Emblica officinalis (Euphorbiaceae; fruit),

Evolvulus alsinoides (Convolvulaceae; whole plant), Saussurea

lappa (Compositae; root), Terminalia chebula (Combretaceae; fruit),

Terminalia bellirica (Combretaceae; fruit)

Ethanol Kulkarni and Verma,

1993

25 Mandur bhasma

(Himalaya’s

herbs)

Ferric oxide/hematite or red iron oxide Carbon tetrachloride Devarshi et al., 1986

41

The synchronous activity of ingredients from polyherbal formulation is believed to be

responsible for hepatoprotective activities of these formulations (Range et al., 2003;

Arsul et al., 2011). Individual or polyherbal drugs are used for treatment of various

liver disorders caused by toxic chemicals, viruses and excess alcohol intake (Kashaw

et al., 2011). Herbs and herbal preparations are generally viewed as safe. But along

with the beneficial components, they may contain many bioactive compounds which

could impose potentially deleterious effects (Lal et al., 2007).

3.4.2 Hepatoprotective Medicinal Plants

Medicinal plants play an important role in human health care. Management of

liver diseases is still a challenge for modern medicine (Deepthi, 2015). Even the

developed countries are now looking for time-tested traditional and alternative

medicines as a remedy for liver diseases (Sheth, 2005). As per WHO report, more

than 80% of global population uses medicines derived from plants or plant products

(Sheth, 2005; WHO, 2011). The traditional medicine system specifies a broad range

of natural health care practices including folk/tribal practices as well as Ayurveda,

Siddha and Unani (WHO, 2011). To a large extent these medical practices, originated

from time immemorial, have developed gradually based on practical experiences

(Shaik et al., 2012; Deepthi, 2015). Many medicinal plants exhibit significant

hepatoprotective activity in animal models. These plants and their parts used for

treatment are arranged systemically in Table 5.

42

Table 5. List of Hepatoprotective Medicinal Plants

Sr.

No

Name of the plant Plant

parts used

Hepatotoxicity inducing

agents

Extracts

studied

Reference

1 Acalypha racemosa Wall

(Euphorbiaceae)

Leaves Carbon tetrachloride

Methanol Iniaghe et al., 2008

2 Achillea millefolium

(Asteraceae)

Aerial parts

Carbon tetrachloride,

Paracetamol

Aqueous,

methanol,

chloroform

Gadgoli and Mishra, 1995

3 Actinidias deliciosa

(Actinidiaceae)

Roots Carbon tetrachloride Ethanol Bai et al., 2007

4 Adina cordifolia Roxb

(Rubiaceae)

Leaves Ethanol Acetone,

aqueous

Sharma et al., 2012

5 Aegle marmelos Corr

(Rutaceae)

Leaves 30%Ethanol Ethanol Singanan et al., 2007

6 Aerva lanata Linn

(Amaranthaceae)

Fresh bulbs Paracetamol Ethanol:

Water

Manokaran et al., 2007

7 Aloe barbadensis Mill

(Liliaceae)

Aerial Carbon tetrachloride Aqueous Chandan et al., 2007

8 Andrographis paniculata

(Burm.f.) Wall. Nees.

(Acanthaceae)

Ethanol Aqueous Vetriselvan et al., 2011

43

Sr.

No


parts used


agents

Extracts

studied

Reference

9 Anisochilus carnosus (L.f)

Wall (Lamiaceae)

Stems Carbon tertrachloride Ethanol Venkatesh et al., 2011

10 Annona squamosa Linn

(Annonaceae)

Leaves Isoniazid,

Rifampicin

Ethanol Mohamed et al., 2008

11 Apium graveolens

L(Apiaceae)

Seeds Paracetamol,

thioacetamide

Methanol Singh and Handa, 1995

12 Artemisia maritime Linn

(Asteraceae)

Whole Acetaminophen,

carbon tetrachloride

Aqueous-

methanolic

Janbaz and Gilani, 1995

13 Asparagus racemosus Linn

(Liliaceae)

Roots Paracetamol Ethanol Om et al., 2011

14 Azadirachta indica A. Juss

(Meliaceae)

Leaves Acetaminophen Fresh juice Yanpallewara et al., 2002

15 Azadirachta indica A. Juss

(Meliaceae)

Leaf Paracetamol Aqueous Bhanwra et al., 2000

16 Berberis tinctoria Lesch

(Berberidaceae)

Leaves Acetaminophen Methanol Murugesh et al., 2005

44

Sr.

No


parts used


agents

Extracts

studied

Reference

17 Cajanus scarabaeoides

Linn (Fabaceae)

Whole plant

Paracetamol n-butanol,

ethanol

Pattanayak et al., 2011

18 Calotropis procera Ait

(Asclepiadaceae)

Aerial Paracetamol Petroleum

ether,

chloroforma

cetone,

alcohol

Manivannan et al., 2011

19 Carissa carandas Linn

(Apocynaceae)

Root


paracetamol,

ethanol

Ethanol Balakrishanan et al., 2011

20 Cichorium intybus Linn (Asteraceae)

Seeds


Paracetamol

Aqueous,

methanol,

chloroform


21 Capparis spinosa Linn

(Capparaceae)

Fruit Carbon tetrachloride,

Paracetamol

Aqueous,

methanol,

chloroform


22 Cassia occidentalis Linn

(Caesalpiniaceae)

Leaves Paracetamol ethyl

alcohol

Aqueous-

ethanol

Jafri et al., 1999

23 Cassia roxburghii DC

(Fabaceae)

Seeds Ethanol,

carbon tetrachloride

Methanol Arulkumaran et al., 2009

45

Sr.

No


parts used


agents

Extracts

studied

Reference

24 Chamomile capitula Linn

(Asteraceae)

Flower Acetaminophen

50% ethanol Gupta and Misra, 2006

25 Chenopodium album Linn.

(Chenopodiaceae)

Aerial Alcohol Petroleum

ether

Nigam and Paarakh, 2011

26 Cichorium intybus

(Asteraceae)

Seeds Carbon tetrachloride Aqueous Jamshidzadeh et al., 2006

27 Clitoria ternatea (Fabaceae)

Linn

Leaves Acetaminophen Methanol Nithiananthama et al., 2013

28 Cryptolepis buchanani

Roem (Berberidaceae)

Leaves Acetaminophen Ethanol Padmalochana et al., 2013

29 Cyperus articulatus

(Cyperaceae)

Whole

rhizome

Paracetamol Methanol Datta et al., 2013

30 Hyptis suaveolens Linn

(Lamiaceae)

Leaves Acetaminophen Aqueous Babalola et al., 2011

31 Ichnocarpus frutescens Linn

(Apocynaceae)

Whole Acetaminophen

Chloroform

Methanol

Dash et al., 2007

32 Lawsonia inermis Linn

(Lythraceae)

Roots Paracetamol and anti-

tubercular drugs

Chloroform-

water

Ravishah et al., 2012

46

Sr.

No


parts used


agents

Extracts

studied

Reference

33 Mentha arvensis Linn

(Lamiaceae)

Whole Alcohol and carbon tetra

chloride

Aqueous Radhika et al., 2011

34 Phyllanthus longiflorus

Heyne ex Hook

(Euphorbiaceae)

Leaves Acetaminophen Ethanol Muthulakshmi and

Mariammal, 2013

35 Phyllanthus

maderaspatensis Linn

(Phyllanthaceae)

Whole Acetaminophen

n-Hexane Asha et al., 2004

36 Phyllanthus niruri Linn

(Phyllanthaceae)

Leaves Acetaminophen

Alcohol Tabassum et al., 2005

37 Phyllanthus polyphyllus

Willd (Euphorbiaceae)

Leaves Acetaminophen

Methanol Rajkapoor et al., 2008

38 Pergularia daemia (Forsk.)

Chiov. (Asclepiadaceae)

Root


paracetamol,

ethanol

Ethanol Balakrishanan et al., 2011

39 Platycodon grandiflorum

Linn (Campanulaceae)

Roots Acetaminophen Aqueous Lee et al., 2001

47

Sr.

No


parts used


agents

Extracts

studied

Reference

40 Saccharum officinarum

Linn (Poaceae)

Cane 20% Ethyl alcohol Juice Patel et al., 2010

41 Sida cordifolia Linn

(Malvaceae)

Leaves Ethanol Petroleum

ether,

chloroform,

acetone,

ethanol

Sharma, 2012

42 Swertia longifolia Boiss

(Gentianaceae)

Aerial Acetaminophen

Ethanol Hajimehdipoor et al., 2006

43 Syzgium aromaticum Linn

(Myrtaceae)

Flowers buds Acetaminophen

Ethanol Nassar et al., 2007

44 Tecomella undulate Seem

(Bignoniaceae)

Leaves Alcohol and paracetamol Methanol Singh and Gupta, 2011

45 Terminalia belerica

(Gaertn.) Roxb (Combretaceae)

Fruits Alcohol Aqueous

Ethanol

Jain et al., 2008

46 Tinospra cordifolia Wild

(Menispermaceae)

Roots Isoniazid,

rifampicin and

Ethanol Adhvaryu et al., 2007

48

Sr.

No


parts used


agents

Extracts

studied

Reference

47 Ocimum sanctum Linn

(Labiatae)

Roots Isoniazid,

rifampicin and

pyrazinamide


48 Zizyphus mauritiana Lam

(Rhamnaceae)

Roots Isoniazid,

rifampicin and

pyrazinamide


49 Vetiveria zizanioides Linn

(Poaceae)

Root Paracetamol Methanol Parmar et al., 2013

50 Vetiveria zizanioides Linn

(Poaceae)

Roots Alcohol Methanol Parmar et al., 2008

51 Xylopia phloiodora Mildbr.

(Annonaceae)

Stem bark,

Leaves


acetaminophen

Methylene

chloride:

Methanol

Moundipa et al., 2007

49

Various herbs and plant extracts have significant hepatoprotective activity as

indicated from studies in animal models (Chaudhary et al., 2010). The

hepatoprotective activity is probably due to the presence of active principles which

are mostly in the form of secondary metabolites in medicinal plants (Singh et al.,

2011). The literature till date indicates that extracts of leaves, stems, fruits, roots or

even whole plants have significant potential towards treatment of hepatic diseases

(Ramalingum and Mahomoodally, 2014).

3.4.2.1 Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms.

T. cordifolia has been reported to possess strong hepatoprotective and

antioxidant activities. Root and stem of T. cordifolia exhibit anti-inflammatory

(Patgiri et al., 2014), antipyretic (Ashok et al., 2010), immunomodulatory (Upadhyaya

et al., 2011) and anti-cancer activities (Ahmad et al., 2015). The immunomodulatory

activity of T. cordifolia is attributed to G1-4A, an arabinogalactan, found in fresh

stem of T. Cordifolia (Desai et al., 2007).

Bishayi et al. (2002) and Kavitha et al. (2011b) reported hepatoprotective and

immunostimulatory effects of T. cordifolia in mature rats intoxicated with carbon

tetrachloride (CCl4). Administration of T. cordifolia extract (100 mg/kg body weight

for 15 days) was found to protect the liver in CCl4 intoxicated rats, as indicated by

significant reduction in serum levels of SGOT, SGPT, ALP, and bilirubin. Extracts of

aerial roots of T. cordifolia reported hepatoprotective effects and immunosuppression

activity against isoniazid, rifampicin and pyrazinamide induced liver injury in guinea

pigs (Adhvaryu et al., 2007). A patented polyherbal formulation with T. cordifolia

extract also exhibited in vitro inactivation of Hepatitis B and E surface antigens

within 48-72 hrs of treatment (Mehrotra et al., 2000). The aqueous extract of roots of

T. cordifolia showed anti-oxidant action in alloxan induced diabetic rats. The

treatment of root extract of T. cordifolia (25, 50 mg/kg body weight) for 6 weeks in

alloxan induced diabetic rats showed significant reduction in serum and tissue

cholesterol, phospholipids and free fatty acids (Stanely et al., 1999). Treatment of

alloxan induced diabetic rats with alcoholic extracts of root (at a dose of 100 mg/kg

orally for six weeks) is reported to be highly effective in normalizing antioxidant

status of heart, brain, liver and kidney (Prince et al., 2004). Being major determinant

in outcome of liver injury, Kupffer cells activity has been studied in chronic liver

50

disease model. Carbon clearance test after T. cordifolia treatment showed significant

improvement in Kupffer cell function and normalization of SGOT, SGPT, ALP and

bilirubin levels (Nagarkatti et al., 1994). Sharma and Pandey (2010) reported

hepatoprotective activity of aqueous extracts of stem and leaves (400 mg/kg body

weight, orally) against lead nitrate induced toxicity in Swiss albino male mice.

Increase in SOD and CAT activity while decrease in the levels of SGOT, SGPT and

ALP enzymes were reported in mice treated with aqueous stem and leaves extract

along with lead nitrate, depicting hepatoprotective activity of T. cordifolia. Kumar et

al. (2013b) also reported hepatoprotective activity of aqueous extract of aerial parts of

T. cordifolia against CCl4 induced hepatotoxicity in wistar albino rats by reducing

levels of serum total bilirubin, SGPT, SGOT, and ALP. Treatment of T. cordifolia

aqueous extract also showed radio-protective activity against a sub-lethal dose of

gamma radiation in mice (Kapur et al., 2010; Mittal et al., 2014). A clinical study has

reported that T. cordifolia plays an important role in normalization of altered liver

functions (ALT, AST) (Karkal and Bairy, 2007).

3.4.2.2 Tinospora sinensis (Lour.) Merrill

The alcoholic and aqueous extract of T. sinensis has been reported to possess

anti-inflammatory (Li et al., 2003), anti-diabetic (Yonemitsu et al., 1993),

hepatoprotective (Chavan et al., 2013a), immunomodulatory (Manjrekar et al., 2000)

adaptogenic (Gupta et al., 2012) and other biological potentials (Yonemitsu et al.,

1993) with different animal models. It has also been shown to have anti-cancer

activity (Punitha et al., 2012) in human malignant cancer cell lines.

Yonemitsu et al. (1993) isolated a new phenolic glycoside, tinosinen from the

fresh stems of T. sinensis Merr. The structure was established on the basis of acid

hydrolysis and spectral data (IR, NMR, mass) as (E)-l-(3-hydroxy-1-propenyl)-3, 5-

dimethoxyphenyl 4-0-13-napiofuranosyl-(l-->3)-J3-D-glucopyranoside. Maurya et al.

(2009) reported isolation of two new compounds 4-methyl-heptadec-6-enoic acid

ethyl ester and 3-hydroxy-2, 9, 11-trimethoxy-5, 6-dihydro isoquino [3, 2-a]

isoquinolinylium from ethanolic extract of the stems of T. sinensis, along with other

six known compounds. Detailed spectroscopic studies were performed to establish

structures of these new compounds. Activity guided purification of ethanol extract of

T. sinensis yielded two compounds with significant in vitro inhibitory activity against

51

protein tyrosine phosphatase 1B (PTP1B) (Gupta et al., 2012). Naik et al. (2013)

reported hepatoprotective activity of ethanolic root extract of T. sinensis against

carbon tetrachloride (CCl4) induced liver damage in rats at a dose of 150mg/kg body

weight and 300mg/kg body weight orally in rats. The animals exhibited reduction in

levels of SGOT, SGPT and ALP indicating hepatoprotective activity of ethanolic

extract of T. sinensis roots. Treatment of ethanolic extract of T. sinensis leaf at a dose

of 300 mg/kg body weight showed anti-arthritic activity against adjuvant (Freud’s)

induced arthritis in rats (Sumathy et al., 2014). Analgesic activity of ethanolic extract

of T. sinensis leaves at a dose of 250 mg/kg and 500 mg/kg has also been reported in

rats (Sandhyarani and Kumar, 2014).

3.4.2.3 Neem-giloe

Neem-giloe (Neem-guduchi) is a traditionally used medicine from Ayurveda. It

is a Guduchi plant growing on Neem tree (Azadirachta indica). It is bitter to taste and

is believed to contain the medicinal virtues of Neem also and hence supposed to be

more efficacious (Sinha et al., 2004). Chemical investigations of water extract of the

stem of Neem-guduchi revealed that it contains glycoside giloin and non-glycosides,

bitter gilenin and glosteral (Bhide et al., 1941; Pendse et al., 1977). Oral and

intraperitoneal treatment of the water extract of the stem of Neem-guduchi at dose of

60 mg/100g displayed anti-inflammatory anti-arthritic effects (Pendse et al., 1977). It

also significantly reversed formation of antibody by typhoid "H" antigen (Pendse et

al., 1977). These reports indicate anti-inflammatory, immunosuppressive and

analgesic properties of the Satwa (Pendse, et al., 1977; Upadhyay, 2010). Nagarkar et

al. (2013) reported that Neem-guduchi showed hepatoprotective activity against a

single high-dose induced acetaminophen toxicity in rats. As per Bhalerao et al.

(2012), immunostimulatory effect produced by Neem-guduchi may be due to cell

mediated and humoral activation of T and B cells. Administration of Neem-guduchi

extract at a dose of 200mg/kg body weight showed radical scavenging and

immunomodulatory activity in rats (Bhalerao et al., 2012).

3.4.3 Guduchi Satwa

Satwa is an aqueous extractable solid substance collected from herbal drug

(Patil and Chaudhary, 2013). Guduchi satwa is the most common example of

52

herbal satwa and is very commonly prescribed in Ayurveda (Sharma et al., 2013b).

Rasendra Mangalam mentions Guduchi satwa for the first time in the literature

(Nagarjuna, 2008; Sharma et al., 2012). Guduchi satwa preparation has been

mentioned in Yoga Ratnakara (Yogaratnakar, 2002) and then Siddha Yoga Sangraha

(Tzrikamaji, 1954), Rasa Yoga Sagar (Sharma, 2004) etc. All these texts have

mentioned different methods for satwa preparation. Due to its usefulness in diseases

like fever and high nutritive and digestive values (Sinha et al., 2004), it is known as

Indian Quinine (Sharma et al., 2012) and is used as a general tonic (Thakur et al.,

1989).

Earlier studies reported the pharmaceutical aspect of guduchi satwa with

quantitative variations in the final product (Sharma et al., 2012). Guduchi Satwa (a

whitish starch like material extracted from T. cordifolia) is commonly recommended

in conditions like Jwara (fever), Daha (burning sensation) and Pitta predominant

disorders (Mishra and Vaishya, 2002; Sharma et al., 2013b). As per the report of

Pandey (2012), Guduchi Satwa is highly valued for many ailments like fever, chronic

diarrhea, chronic dysentery, burning sensation, secondary syphilis, chronic gonorrhea,

leucorrhoea, jaundice, rheumatism, urinary disorders etc. Prasad et al. (2014) have

reported scientific validation and standardization of the pharmaceutical procedure of

Guduchi Satwa for treating Kshaya (Phthisis), Raktapitta (Bleeding Disorder) and

Pada daha (Burning sensation of feet).

The standardization of Guduchi satwa includes parameters like organoleptic

characters, physicochemical parameters, phyto-analysis and development of TLC as

recommended by WHO and pharmacopoeia committee (Anonymous, 1987; Prasad et

al., 2014). Guduchi satwa has been reported to contain arabinogalactan with

immunological activity (Chintalwar et al., 1999). Ayurvedic system has also

recommended the use of Guduchi satwa in different stages of prameha (Shastri, 2002;

Panashikar et al., 2011; Sharma et al., 2013a). Efforts have been made to evaluate the

acceptability of Guduchi incorporated food products and thus the utilization of its

proven ethno-medicinal properties in improving nutritional status of vulnerable

sections of the society (Geeta and Kumari, 2013).

https://www.google.co.in/search?biw=1242&bih=615&q=parameters&spell=1&sa=X&ved=0CBkQvwUoAGoVChMIzvyRvOfIyAIVxwiOCh2T5QV8

53

3.4.4 Nutritional Supplements

Nutrition or dietary supplements help the regeneration of liver cells forming a

basis for treatment of some liver disorders (Shukla and Kumar, 2013). Nutritional

supplements contain herbal products, vitamins, minerals, and any product that is not a

food or drug (medication) (American Cancer Society, 2015). There are very few

reports available on hepatoprotective activity of nutritional supplements (Shukla and

Kumar, 2013).

The list of nutritional supplements used in liver disorders is given in Table 6.

54

Table 6. List of Hepatoprotective Nutritional Supplements

Sr.

No

Name Nutritional Supplement Sources Hepatotoxicity inducing

agents

Extracts/material

studied

Reference

1 Allium flavum Linn (Amaryllidaceae) Peels Carbon tetrachloride Ethanol Chyun et al., 2013

2 Camel Milk Paracetamol Milk Al-Fartosi et al., 2011

3 Cocos nucifera Linn (Arecaceae) Coconut Paracetamol Dried- and

Fermented-

Processed

Virgin Coconut Oil

Zakaria et al., 2011

4 Honey Honey Paracetamol Honey Ayyavu et al., 2009

Galal et al., 2012

5 Linum usitatissimum Linn (Linaceae) Flaxseed Carbon tetrachloride n-butanol Kasote et al., 2012

6 Linum usitatissimum Linn (Linaceae) Seeds Carbon tetrachloride Flaxseed chutney Shakir and Madhusudhan,

2007

7 Malus domestica Borkh (Rosaceae) Peel

Carbon tetrachloride Aqueous Yang et al., 2010

8 Myristica fragrans

Houtt(Myristicaceae)

Seeds Carbon tetrachloride 70% methanol Al- Jumaily et al., 2012

9 Nigella sativa Linn (Ranunculaceae) Seeds Isoniazid,

rifampicin,

pyrazinamide

Oil Jadhav, 2013

55

Sr.

No

Name Nutritional Supplement Sources Hepatotoxicity inducing

agents

Extracts/material

studied

Reference

10 Omega -3- polyunsaturated fatty

acids

Fish Cisplatin Oil Naqshbandi et al., 2011

11 Omega -3- polyunsaturated fatty

acids

Fish,

Sunflower

Galactosamine Oil Roy et al., 2007

12 Omega-3-fatty acids Fish Paracetamol (Cod) liver oil Kalra et al., 2012

13 Punica granatum Linn (Lythraceae) Fruits Carbon tetrachloride Peel powder Ashoush et al., 2013

14 Spirulina platensis Spirulina

Carbon tetrachloride Powder extract Gad et al., 2011

15 Vigna radiata Linn (Fabaceae) Seed Ethanol Aqueous Ali et al., 2013

16 Vitis vinifera Linn (Vitaceae) (Grape) Seed Carbon tetrachloride Oil Maheswari and Rao,

2005

57

3.4.4.1 Polyunsaturated Fatty Acid (Omega-3 Fatty Acids)

In recent years, there has been increased research interest in the use natural

sources to develop hepatoprotective, anticancer, cardio-protective and several other

pharmacological therapies (Varkey and Vahab, 2015). Several studies using different

animal models have been reported on beneficial effects of omega-3 long-chain

polyunsaturated fatty acids (PUFA) supplementation on liver injury (Lee et al., 2007;

Wu et al., 2012; Li et al., 2014). Bang and Dyerberg (1972) pioneered

epidemiological studies in early 1970s and hypothesized that consumption of fish oil

and other marine animals containing long chain, highly unsaturated omega-3 fatty

acids, produced beneficial effects in Eskimos. Diet supplemented with omega-3 fatty

acids has profound beneficial health effects against various pathologies (Simopoulos,

1991). Fish oil showed hepatoprotective activity against different hepatotoxins (Ruiz-

Gutierrez et al., 1999; Aguilera et al., 2003; Asaad and Aziz, 2012; Abdel-Dayem et

al., 2014). Flax seed oil is one of the rich sources of PUFA containing about 51% to

55% alpha-linolenic acid (ALA) (Prasad, 2000; Vijaimohan et al., 2006). ALA

content of flaxseed oil may be helpful in preventing clinical conditions such as

cardiovascular disease, blood pressure, cancer, skin diseases and immune disorders

like renal failure, rheumatoid arthritis, multiple sclerosis etc. (Kelley et al., 1991;

Thompson et al., 1996; Prasad et al., 1998; Cohen et al., 2005; Morris et al., 2005;

Rajesha et al., 2006; Vijaimohan et al., 2006; Sekine et al., 2008; Zanwar et al.,

2011).

Alwayn et al. (2005) reported the use of both enteral and parenteral ω3-PUFA

to treat nonalcoholic fatty liver disease (NAFLD) in a mouse model. There have been

numerous studies reporting the use of fish oil in humans and animals to treat

parenteral nutrition-associated liver disease (PNALD) (Gura et al., 2006; Gura et al.,

2008). Treatment of rodents with nonalcoholic fatty liver disease with EPA and DHA

showed improvements in liver health by reducing steatosis (Capanni et al., 2006).

Omega-3 polyunsaturated fatty acid (Fish oil) treatment in albino mice showed

hepatoprotective activity against galactosamine induced toxicity (Roy et al., 2007).

Flaxseed chutney demonstrated hepatoprotective activity against CCl4 induced

hepatotoxicity in rats (Shakir and Madhusudhan, 2007). Flaxseed contains ether

insoluble phenolic components of n-butanol fraction which plays important role in

58

mitigating hepatotoxicity due to carbon tetrachloride (CCL4) intoxication (Kasote et

al., 2012). Masterton et al. (2010) showed promising effect of omega-3 fatty acids in

non-alcoholic fatty liver disease in the form of reduced hepatic steatosis, improved

insulin sensitivity and reduced inflammatory markers. Tillman and Helms (2011)

showed some success in treatment of parenteral nutrition associated liver

disease (PNALD) using parenteral fish oil–based intravenous fat emulsion (IVFE)

(Omegaven, Fresenius Kabi AG, Bad Homburg VDH, Germany). Meganathan et al.

(2011) reported hepatoprotective activity of fish oil in paracetamol induced

hepatotoxicity at dose levels of 300 mg/Kg/day. Significant increase in the levels of

superoxide dismutase (SOD), reduced glutathione (GSH), and catalase (CAT)

indicated protective effect of fish oil. Fish oil (4ml/kg, i.p for 1 week) administration

to the single high dose acetaminophen intoxicated albino rats resulted in decreased

level of malondialdehyde (MDA) indicating hepatoprotective effect (Kalra et al.,

2012). Treatment of pure lignan (25 mg/ml) and partial lignan (25 mg/ml) of flaxseed

lignin against paracetamol intoxicated (200 mg/kg, oral) male rabbit showed decrease

in serum levels of liver markers like SGOT, SGPT, ALP and bilirubin, confirming

hepatoprotective activity of omega-3 fatty acids (Al-Jumaily and AL-Azawi, 2013).

Abdel-Dayem et al. (2014) reported hepatoprotective effect of DHA (120 and 200

mg/kg body weight) treatment against valproate (VPA) (500 mg/kg body weight for

14 days) induced liver damage. Varkey and Vahab (2015) studied the

hepatoprotective activity of Stolephorus commersonnii fish extracts against isoniazid

induced hepatotoxicity in albino rats. Treatment of fish extract at a dose of 300 mg/kg

body weight showed improvements in liver histology and liver function markers. The

beneficial effect of omega-3 fatty acids treatment on methotrexate induced

hepatotoxicity in children with acute lymphoblastic leukemia (Elbarbary et al., 2016:

Clinical Trial Registry Number: NCT02373579) has also been

reported. Administration of omega-3 fatty acids (100 mg/kg bw.p.o) in rats with

cyclosporine-A induced liver apoptosis protected liver injury by reducing apoptotic

cell count (Erol et al., 2011).

Several reports demonstrated that omega-3 fatty acids are involved in

regulation of expression of several genes such as adhesion molecules, lipid

metabolism, inflammatory cytokines etc. involved in endothelial activation in

response to inflammatory and pro-atherogenic stimuli and also nuclear transcription

59

factors, peroxisome proliferator-activated receptor, sterol regulatory element binding

protein-1, cyclooxygenase-2, etc. (Price et al., 2000; De Caterina and Massaro, 2005;

Deckelbaum et al., 2006). Omega-3 fatty acids affect the expression of several key

proteins related to inflammation, lipid metabolism and they help to reduce

inflammation and lipid oxidation (Deckelbaum et al., 2006). Omega-3 fatty acids have

also been shown to act through modulation of cytokine expression (Lee et al., 2007).

Omega-3 fatty acids have been shown to downregulate TNFα which is supposed to be

a direct role of omega-3 fatty acids in hepatoprotection (Babcock et al., 2000).

Omega-3 fatty acids also show anti-inflammatory properties (Asaad and Aziz, 2012),

they act as natural ligands for activation of PPARγ (Trombetta et al., 2007; Yamazaki

et al., 2007; Selvaraj et al., 2010), they down regulate SREBP-1c (Kim et al., 1999;

Yahagi et al., 1999; Xu et al., 1999; Botolin et al., 2006; Wada et al., 2008; Kim et al.,

2014) and up regulate FABP-1 (Dutta-Roy, 2000; Gaca et al., 2012).

Based on the review of literature, it was evident that the comparative efficacy

of the satwa from three Tinospora forms has not yet been studied. In addition, the

mechanism of action of the satwa on hepatotoxicity is not yet clear. Hence it was

decided to undertake studies on hepatoprotective activities of satwa of three

Tinospora forms (T. cordifolia, T. sinensis, Neem-giloe) and nutritional supplements

(Flax oil/Fish oil) and their comparative study against acetaminophen and alcohol

induced hepatotoxicity in rats.

CHAPTER

1TRODUCTION

CHAPTER 4

MATERIALS AND METHODS

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The present study was designed to assess the effects of an ayurvedic

formulation, Guduchi satwa and omega-3 fatty acids (Flax oil and fish oil) on

acetaminophen and alcohol induced hepatotoxicity in rats. The elaborate process for

preparation of Guduchi satwa is described in Ayurvedic literature, which requires

collection of fresh plant material. The omega-3 fatty acids (Alpha linoleic acid

(ALA), Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA)) are available

commercially. The detail procedures for preparation of satwa, its nutritional analysis

and design and methodology of animal experiments with satwa and omega-3 fatty

acid interventions, is explained here. 4.1 Collection of Plant Material

As Guduchi is advocated to be collected in fresh condition, stems of

Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms., Tinospora sinensis

(Lour.) Merr. and Neem-giloe (Tinospora cordifolia (Willd.) Miers ex Hook. F. &

Thoms., growing on neem (Azadirachta indica A. Juss.) tree) were collected during

February to April 2012 from Research Farm of Dr. Balasaheb Sawant Konkan Krishi

Vidyapeeth, Dapoli, Maharashtra, India (Lat Long 17.45°N, 73.10°E) (Map 1 and 2).

The matured stem was separated from other parts like roots,

leaves, flowers, fruits and washed thoroughly with water for three times.

Map. 1. Google Earth image of Maharashtra showing the Site of Collection of

Plant Material

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Map. 2. Google Earth image of Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, the

Site of Collection of Plant Material in Dapoli, Maharashtra

4.2 Identification and Authentication of Plant Material

The plant material was identified by an expert plant taxonomist and voucher

specimens of three species were deposited at the herbarium of Medicinal Plants

Conservation Centre (MPCC), Pune [Tinospora cordifolia (Willd.) Miers ex Hook. F.

& Thoms. (MPCC 3483), Tinospora sinensis (Lour.) Merr. (MPCC 3529) and Neem-

giloe (T. cordifolia (Willd.) Miers ex Hook. F. & Thoms., growing on Azadirachta

indica A. Juss. (Neem tree) (MPCC 3526)].

4.3 Preparation of Satwa from Three Tinospora forms

Fresh stems of three Tinospora forms were used for preparation of Guduchi

satwa (Residual marc of aqueous extract). The preparation, as defined in Ayurvedic

literature is a sediment extract predominantly starchy in nature. The preparation of

satwa was done as per the procedure described by Khandal (1992).

Five kilograms of freshly collected stem pieces were washed thoroughly with

water. The stem peel was removed and the stem was cut into pieces of 1.5-2 inches

(Fig. 6) having 1.6-2.0 cm diameter (Fig. 7). The stem pieces thus obtained (Fig. 8 A,

B) were pounded slightly (Fig. 8C). The crushed stem pieces of three forms were

62

separately suspended in a quantity of water 4 times of their weight (Fig. 8D). This

mixture was kept undisturbed for 24 hours. Next day, Guduchi was hand-rubbed till it

was slimy with appearance of foam on water (Fig. 8E). This homogenized mixture

was then filtered through several layers of sterile muslin cloth and filtrate was left

undisturbed for 24 hours. On the next day, the water was decanted carefully without

disturbing the sediment (Fig. 8F). The sediment was again suspended in half liter

water and kept undisturbed for two hours. The water was then carefully decanted (Fig.

8G) and the sediment was collected and sun dried for 48 hours (Fig. 8H). The sun-

dried residue thus obtained, is termed as Satwa. Satwa was stored in air-tight

containers till further use (Fig. 8I and 8J). In the present study, satwa from three

species of Guduchi (Tinospora cordifolia, Tinospora sinensis and Neem-giloe) was

prepared and was further subjected to nutritive analysis and intervention studies in

animals.

Fig. 6. Stem Pieces of Guduchi (1.5-2 inches)

Fig. 7. Medium Size Stem Diameter (1.6-2.0 cm diameter)

63

Fig. 8. Preparation of Guduchi Satwa

A-Guduchi stem, B-Guduchi stem with outer brownish white coloured peel removed, C-

Pounding, D-Overnight soaking, E-Rubbing of slimy, crushed stem pieces, F- Sedimentation,

G-Removal of supernatant, H-Collection and drying of white sediment, I-Completely dried

Guduchi satwa of three Guduchi forms and J-Guduchi satwa stored in air tight containers

64

4.4 Nutritional Analysis of Satwa

Estimation of proteins, carbohydrates, starch, total lipids, crude fibres and total

ash content of satwa from three Tinospora forms was done by following standard

techniques.

Chemicals/Reagents

Folin-Ciocalteu’s phenol reagent (Sigma-Aldrich Chemie GmbH),

Chloroform, Methanol, Anthrone reagent (Sigma-Aldrich Chemical, Bangalore,

India), Sodium carbonate, Sulphuric acid, Phenol (Qualigens Fine Chemicals,

GlaxoSmithKline Pharmaceuticals Limited, Mumbai, India), Perchloric acid, Sodium

hydroxide, Hydrochloric acid (Merck Specialities Private Limited, Mumbai, India),

Alkaline copper sulphate, Glucose (Hi-Media Laboratory Private Limited, Mumbai,

India), Bovine serum albumin (Sisco Research Laboratory Private Limited, Mumbai,

India), Ethanol (Changshu Yangyuan Chemical, China), Distilled water.

4.4.1 Protein

There are different methods for protein and total nitrogen content estimation.

Hydrolysing the protein and estimating the amino acid alone will give the exact

quantification (Sadasivam and Manickam, 1991a). The method developed by Lowry

et al. (1951) is largely followed as it is sensitive enough to give moderately constant

value and was used for estimation of proteins from satwa.

Principle

The blue colour developed by the reduction of the phosphomolybdic-

phosphotungstic components in the Folin-Ciocalteu reagents by the amino acid

tyrosine and tryptophan present in the protein and the colour developed by Biuret

reaction of the protein with alkaline cupric tartrate are measured in the Lowry’s

method (Sadasivam and Manickam, 1991a).

Materials

1. Reagent A: 2% (w/v) Sodium Carbonate in 0.1 N Sodium Hydroxide

2. Reagent B: 0.5% (w/v) Copper Sulphate (CuSO4.5H2O) in 1% (w/v)

Potassium Sodium Tartrate

65

3. Reagent C: Alkaline Copper solution: Reagent A (50mL) and Reagent B

(1mL) was mixed prior to use

4. Reagent D: Folin-Ciocalteu’s Phenol Reagent commercially available reagent

was diluted 1:1 in distilled water and used

5. Protein Solution (Stock Standard)

Accurately weighed 50mg bovine serum albumin (Fraction V) was dissolved in

distilled water and volume of the solution was made up to 50mL.

Extraction of Protein from Satwa

The satwa from three Guduchi forms were used for extraction of proteins. Pre-

weighed (500mg) satwa from each Tinospora forms was homogenized with 5mL

10mM phosphate buffer (pH 7.4), separately. The homogenates were centrifuged at

2000 rpm for 10 min at room temperature. The supernatants were collected and used

for protein estimation.

Procedure

Known volumes of standard protein solution (0.2, 0.4, 0.6, 0.8 and 1.0mL)

were pipetted in a test tube and the volume was made up to 1mL with sterile distilled

water in all tubes. The reaction with 1mL distilled water (without standard protein)

served as a blank. The protein extract from satwa (0.1mL) was taken in a separate test

tube and the volume was made up to 1mL with sterile distilled water. The contents of

the tubes were mixed well, 5.0mL alkaline copper sulphate (Reagent C) was added to

the tubes and the contents were mixed thoroughly. The tubes were incubated for 10

minutes at room temperature and then 0.5mL Folin-Ciocalteu’s Phenol (Reagent D)

was added to the tubes. The contents were mixed well and the reactions were

incubated at room temperature, in dark for 30 minutes. The blue colour developed

was read in a microplate reader at 660 nm (Bio-Rad Laboratories Inc., Berkeley,

California, U.S.A.) against blank. Intensity of the colour developed is directly

proportional to protein content in the sample.

4.4.2 Total Carbohydrates

Carbohydrates are the main components of storage and structural materials in

the plants and they occur as free sugars and polysaccharides. Monosaccharaides are

the basic units of carbohydrates which cannot be split by hydrolysis into simpler

66

sugars. The carbohydrate content from the samples can be measured by acid

hydrolysis of polysaccharides to monosaccharides followed by estimation of

monosaccharaides (Sadasivam and Manickam, 1991b)

Principle

Glucose undergoes dehydration reaction in hot acidic medium resulting in the

formation of hydroxymethyl furfural which forms a green coloured product with

phenol with absorption maximum at 490 nm. The intensity of colour developed is

directly proportional to the amount of carbohydrates in the sample (Sadasivam and

Manickam, 1991b).

Materials

1. 2.5 N HCl

2. Sodium Carbonate

3. Phenol 5%: Redistilled (Reagent grade) phenol (5gm) dissolved in water and

diluted to 100mL.

4. Sulphuric acid 96% reagent grade.

5. Standard Glucose Stock- 1mg/mL glucose solution

6. Working standard: 10mL stock diluted to 100mL with distilled water

(100µg/mL working stock concentration)

Extraction of Carbohydrates from Satwa (Sadasivam and Manickam, 1991b)

The satwa (100mg) from each forms were suspended in 5mL 2.5N

Hydrochloric acid and were hydrolyzed in a boiling water bath for 3 hours. After

cooling to room temperature, it was neutralized with solid sodium carbonate until

there was no effervescence and the volume was made to 20mL with distilled water. It

was then centrifuged at 2000 rpm for 10 minutes. Volume of the supernatant was

measured and adjusted to 20mL and used for carbohydrate estimation.

Procedure

Total carbohydrates were estimated by following phenol sulphuric acid

method (Dubois et al., 1956). The known volume of working glucose standard (0.2,

0.4, 0.6, 0.8 and 1.0 mL) was taken in separate test tubes and the volume was made up

to 1mL with sterile distilled water. Blank was prepared by taking 1mL distilled water

67

instead of standard glucose solution or extract. Satwa extract (0.1mL) was taken in a

test tube and volume was made up to 1mL with sterile distilled water. Phenol solution

(1mL) and 96% sulphuric acid (5mL) was added to all the tubes and mixed. The tubes

were incubated at room temperature for 10 minutes with intermittent mixing of the

contents. The tubes were then placed in water bath for 20 minutes at 25-30°C. The

green colour developed was read at 490 nm in a microplate reader.

4.4.3 Starch

Starch is an important polysaccharide and is the storage form of carbohydrates

in plants and is richly found in roots, tubers, stems, fruits and cereals. Starch is a

mixture of two types of components namely amylose and amylopectin which are

composed of several glucose molecules. Dilute acids are used to hydrolyse starch into

simple sugars and the quantity of simple sugars is measured colorimetrically

(Sadasivam and Manickam, 1991c).

Principle

The sample is treated with 80% alcohol to remove sugars and the starch is

extracted with perchloric acid. In hot acidic medium, starch is hydrolysed to glucose

and dehydrated to hydroxymethyl furfural. This compound forms a green coloured

product with anthrone which can be measured at 630 nm (Sadasivam and Manickam,

1991c).

Material

1. Anthrone: Dissolve 200mg Anthrone in 100mL of ice-cold 95% Sulphuric

acid

2. 80% (v/v) Ethanol

3. 52% (v/v) Perchloric Acid

4. Standard Glucose: 1mg/mL Glucose Solution.

5. Working Standard: 10mL of stock diluted to 100mL with water (giving

100µg/mL working stock).

Extraction of Starch from Satwa (Sadasivam and Manickam, 1991c)

Each satwa was separately processed for extraction of starch. Known amount

of satwa (500mg) was homogenized in hot 80% ethanol to remove sugars. The residue

was retained after centrifugation at 2000 rpm for 10 minutes. The residue was washed

68

with hot 80% ethanol till the washings did not give colour with anthrone reagent. The

residue was dried well and 5mL water and 6.5mL 52% Perchloric acid was added to

the residue. Starch was extracted at 0°C for 20 minutes. The residue was retained after

centrifugation at 2000 rpm at 4°C for 10 minutes and the supernatant was collected in

separate test tube. The extraction of the residue was repeated with fresh 52%

Perchloric acid. The supernatants were pooled after centrifugation and the volume

was made up to 100mL with sterile distilled water.

Procedure for Estimation of Starch

Anthrone method (Hodge and Hofreiter, 1962) was used for starch estimation.

Known volume of standard glucose solution (0.2, 0.4, 0.6, 0.8 and 1.0mL) was

pipetted in separate test tubes and the volume was made up to 1mL with sterile

distilled water. Satwa extract (0.1mL) was pipetted in a separate test tube and volume

was made up to 1mL with sterile distilled water. Blank was prepared by taking 1mL

sterile distilled water instead of standard glucose solution or extract. Anthrone reagent

(4mL) was added to all the tubes. The reaction mixture was incubated in boiling water

bath for 8 minutes and cooled rapidly. The green colour developed was read at 630

nm in a microplate reader. Content of starch was estimated from standard graph.

4.4.4 Total Lipids

Lipids are structural components of cell membranes and play a critical role in

gene transcription, signaling and metabolism. Several lipid species exist in biological

systems, including phospholipids, cholesterol, triglycerides and fatty acids (Fahy et

al., 2005)

Principle

Various solvents or solvent combinations are suggested for extraction of lipid

fractions from the material depending upon the polarity of the lipids. But chloroform:

methanol extraction of lipids as suggested by Folch (Folch et al., 1957) extracts

maximum lipids from the material. The partitioning of the extract with water removes

the non-lipid contaminants from the extract. The organic phase can be used for lipid

estimation after phase separation.

69

Material

1. Chloroform

2. Methanol

3. Chloroform: methanol 2:1 (v/v)

Procedure

Total lipids were estimated by a method modified from Folch (Folch et al.,

1957) as described by Alam (Alam et al., 2008). Satwa (1gm) of three Tinospora

forms were suspended separately in 10mL chloroform: methanol (2:1 v/v) mixture,

mixed thoroughly and were left undisturbed at room temperature for 3 days. The

solutions were filtrated through a muslin cloth and centrifuged at 3000 rpm for 5

minutes. The upper layer of methanol was removed and the interface was washed

three times with chloroform: methanol: water (3:48:47) without disturbing the lower

phase. This has the effect of removing any 'fluff' at the interface. The suspensions

were then centrifuged at 3000 rpm for 5 minutes. The remaining was the crude lipid.

The lower layer (“lipid extract”) was transferred to pre-weighed petri plate. The

extract in a petri plate was dried in incubator and the petri plate with dried lipids was

weighed again. Difference between the weights was recorded as the weight of total

lipids.

4.4.5 Crude Fibre

Crude fibre contains 60% to 80% of the cellulose and 4% to 6% of the lignin

and some mineral matter. Content of crude fibre is a measure of nutritive value of

poultry and livestock feed. It is also used in the analysis of various foods and food

products to detect adulteration, quality and quantity (Sadasivam and Manickam,

1991d).

Principle

During the acid and subsequent alkali treatment, oxidative hydrolytic

degradation of the native cellulose and considerable degradation of lignin occurs. The

residue obtained after final filtration is weighed, incinerated, cooled and weighed

again. The loss in weight indicates the crude fibre content (Sadasivam and Manickam,

1991d).

70

Materials

1. Sulphuric acid solution (0.255 ± 0.005N): 1.25gm concentrated sulphuric acid

diluted to 100mL distilled water.

2. Sodium hydroxide solution (0.313 ± 0.005N): 1.25gm sodium hydroxide in

100mL distilled water.

3. Alcohol

4. Crucible

Procedure

Crude fiber contents in the satwa of T. cordifolia, T. sinensis and Neem-giloe

were estimated according to the method described by Maynard (1970). Known

amounts (1gm) of each satwa were taken separately in a beaker and 100mL of (0.255

± 0.005N) sulphuric acid was added. The mixture was boiled for 30 minutes keeping

the volume constant by the addition of water at frequent intervals. The mixture was

filtered through a muslin cloth and the residue washed with hot water till free from

acid. The material was then transferred to the same beaker and 100mL of boiling

(0.255 ± 0.005N) sodium hydroxide solution was added. After boiling for 30 minutes

(keeping the volume constant as before) the mixture was filtered through a muslin

cloth and residue washed with hot water till free from alkali, followed by single

350mL of water and 25mL alcohol wash. The residue was then removed and

transferred to a crucible (Preweighed dish W1) and dried for 2 hours at 130±2ºC. The

crucible was cooled in a desiccator and weighed again (W2). The crucible was heated

in a muffle furnace at 600±150ºC for 5 hours, cooled and weighed again (W3). Crude

fibre was determined using fat-free samples.

Crude fibre content was determined using the following Equation:

% crude fibre in ground sample

Loss in weight on ignition (W2-W1) - (W3-W1)/Weight of sample) * 100

4.4.6 Total Ash

The ash content is a measure of the total amount of minerals present within a

food, whereas the mineral content is a measure of the amount of specific inorganic

components present within a food, such as Ca, Na, K and Cl (Zhang and Dotson,

1994).

71

Principle

Ash is the inorganic or mineral component of the sample left after complete

ignition of the sample at 450ºC in muffle furnace (The Ayurvedic Pharmacopoeia of

India, Vol II, 2008).

Material

1. Crucible

Procedure

Total ash content was determined as described in The Ayurvedic

Pharmacopoeia of India (2008). One gram of each satwa from three Tinospora forms

was weighed separately into different pre-weighed crucibles. The crucibles were

placed on a clay pipe triangle and heated first over a low flame till all the material was

completely charred, followed by heating in a muffle furnace for 4 hours at 450ºC. The

crucibles were then cooled in desiccators and reweighed. The procedure was repeated

till two consecutive weights were same and the ash was almost white or grayish

white.

The ash content was determined by the equation as follows:

Ash Content (g/100gm sample) = weight of ash x 100 / weight of sample taken

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4.5 Hepatoprotective Activity of Satwa from Three different forms of Tinospora

Liver diseases are a major health problem. The allopathic drugs prescribed for

liver disorders are known to have major side effects. Hence, time tested and reliable

herbal medicines are attractive candidates for management of various liver disorders

(Dash et al., 2007; Suryawanshi et al., 2011 and Kavitha et al., 2011a). Number of

medicinal plants and their formulations are common for the treatment of liver diseases

(Kavitha et al., 2011a). Modern medicine has little to suggest for improvement of

hepatic diseases and it is essentially the plant based preparations which are employed

for the treatment of liver disorders (Chatterjee, 2000). The present study was

undertaken to assess the hepatoprotective activity of satwa from three different forms

of Tinospora against two experimentally induced hepatotoxicity models, namely,

acetaminophen and ethanol induced hepatotoxicity in rats.

Drugs / Chemicals

Acetaminophen (Paramol; Ranbaxy Laboratories Ltd.) was purchased from

the local pharmacy and dissolved in sterile water to make the stock solution

convenient for animal administration as inducing agent for hepatotoxicity. Alcohol

(Ethanol) was obtained from Changshu Yangyuan Chemical, China. Ethanol

(Alcohol) (30%) was used as hepatotoxicant for animal administration. Silymarin

(Silybon-140; Micro Labs) was purchased from local pharmacy and dissolved in

sterile water to make the stock solution convenient for animal administration.

Formalin, ethylenediamine tetra-acetic acid (EDTA) and sodium dihydrogen

phosphate were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.).

Animals

Three months old Male Albino Wistar rats weighing between 150-200 gm

were procured for the study from institutional animal house.

Housing of Animals

The animals were acclimatized for seven days and were maintained under

standard husbandry conditions (Temperature 25±2°C, 12-h light: 12-h dark cycle)

throughout the experimentation. The animals were fed with standard pellet diet

(Nutrivet life science, Pune, M.S., India) and water was supplied ad-libitum. The

73

studies were carried out as per the CPCSEA guidelines and after approval of the

Institutional Animal Ethical Committee (Ref. No. BVDUMC/443/2012-2013).

Selection and Preparation of Dose

The dose of satwa was finalized on the basis of previous studies carried out in

the Laboratory. The quantity of satwa for administration to each animal was

calculated based on the weight of the animal. The required quantity of satwa was

weighed and suspended in water for administration to animals.

Standard Drug, Hepatotoxicants and Interventions

Silymarin (100mg/kg b.w./day, p.o.) was used as standard.

Acetaminophen (1000mg/kg b.w./day, p.o.) and 30% Ethanol (Alcohol) (1ml/100g

b.w./day, p.o.) was used to induce hepatotoxicity.

The satwa of three Tinospora forms (200mg/kg b.w./day, p.o.) was administered to

rats to study their hepatoprotective activity.

Assessment of Hepatoprotective Activity

Male Albino Wistar rats were administered acetaminophen or 30% ethanol

daily for 15 days to induce hepatotoxicity. Satwa from three forms of Guduchi (T.

cordifolia, T. sinensis and Neem-giloe) was orally administered to the rats, daily for

15 days. The satwa was administered 30 minutes before the administration of the

hepatotoxicant. The animals were sacrificed after completion of the experiment and

blood and liver were collected. Blood samples were collected by cardiac puncture and

were subjected to liver function tests (like SGOT, SGPT, Bilirubin, Alkaline

phosphatase etc.) and lipid profile (Total cholesterol, HDL-D cholesterol, LDL-D

cholesterol, and Triglycerides). Part of the liver tissues were stored in 10% neutral-

buffered formalin for histopathology while the remaining part was snap frozen in

liquid nitrogen for assessment of liver biochemical parameters (Lipid peroxidation,

Superoxide dismutase activity, Catalase activity, Reduced glutathione content, Total

protein and Lipid profile) and for molecular analysis.

74

Evaluation of Hepatoprotective Activity of Satwa against Acetaminophen


The animals were divided into six groups by random assignment of six

animals per group. The variation in the average weight of the animals in and between

the groups was less than 20%.

The treatment protocol to assess the hepatoprotective potential of satwa of

three different forms of Tinospora (T. cordifolia, T. sinensis and Neem-giloe) against

acetaminophen induced liver injury is outlined below:

1. Group I: Healthy Control (n=6); received feed and water normally for 15

days

2. Group II: Negative Control (n=6); rats were administered acetaminophen

(1000mg/kg b.w./day, p.o.), daily for 15 days

3. Group III: Positive Control (n=6); the rats in this group were treated daily

with acetaminophen (1000mg/kg b.w./day, p.o.), 30 minutes after

administration of silymarin (100mg/kg b.w./day, p.o.), for 15 days

4. Group IV: Treatment group 1 (n=6); the rats in this group were treated daily


administration of Tinospora cordifolia satwa (200mg/kg b.w./day, p.o.), for 15

days

5. Group V: Treatment group 2 (n=6); the rats in this group were treated daily


administration of Tinospora sinensis satwa (200mg/kg b.w./day, p.o.), for 15

days

6. Group VI: Treatment group 3 (n=6); the rats in this group were treated daily


administration of Neem-giloe satwa (200mg/kg b.w./day, p.o.), for 15 days

During the period of experiment, animals were observed daily for signs of

infection and/or discomfort. After completion of the experiment (15 days), all animals

were fasted overnight and were humanely sacrificed under light ether anaesthesia.

Blood samples were collected by cardiac puncture for evaluating the serum

biochemical parameters. Blood samples were allowed to clot at room temperature for

75

30 minutes and serum was collected by centrifugation at 2000 rpm for 15 minutes.

Aliquots of sera were stored in eppendorfs at -20°C till further use. Liver was excised

from the dissected animals immediately, blotted off blood, washed with saline,

weighed and the left medial lobe was stored in 10% neutral-buffered formalin for

histopathological evaluation. The remaining liver tissues were collected and snap

frozen in liquid nitrogen. Frozen tissues were stored at -80°C till further use.

Evaluation of Hepatoprotective Activity of Satwa against Ethanol Induced

Hepatotoxicity

The animals were divided into six groups by random assignment of six



The treatment protocol to assess the hepatoprotective potential of satwa of

three different forms of Tinospora (T. cordifolia, T. sinensis and Neem-giloe) against

ethanol induced liver injury is outlined below:


days

2. Group II: Negative Control (n=6); administrated 30% ethanol (1ml/100g

b.w./day, p.o.), for 15 days


with 30% ethanol (1ml/100g b.w./day, p.o.), 30 minutes after administration of

silymarin (100mg/kg b.w./day, p.o.), for 15 days



Tinospora cordifolia satwa (200mg/kg b.w./day, p.o.), for 15 days



Tinospora sinensis satwa (200mg/kg b.w./day, p.o.), for 15 days

6. Group VI: Treatment group 3 (n=6); the rats in this group were treated daily


Neem-giloe satwa (200mg/kg b.w./day, p.o.), for 15 days

76

During the period of experiment, animals were observed daily for any signs of











77

4.6 Hepatoprotective Activity of Flax Oil and Fish Oil

In previous experiment, the hepatoprotective activity of satwa from three

Tinospora forms was studied. Apart from herbal medicines; nutrition and various

dietary supplements are also known to play an important role in management of

various health ailments. Omega-3 fatty acids or polyunsaturated fatty acids (PUFA)

are one of such dietary supplements (Ruxton et al., 2004). Omega-3 fatty acids have

been shown to have beneficial effects against number of different pathologies

(Simopoulos et al., 1991). Fish oil and Flax oil are the richest sources of omega-3

fatty acids and are potent antioxidants. Hence, in the present study, we analyzed the

hepatoprotective activity of polyunsaturated fatty acids or omega-3 fatty acids (Fish

oil and Flax oil) against acetaminophen and ethanol induced hepatotoxicity.

Chemicals/reagents

Flax oil (Alpha Lite) was purchased from EnSigns Diet Care Pvt. Ltd. (Pune,

MS, India) and contained 50% alpha-linolenic acid (ALA), 20% Oleic acid (OA) and

12% Linoleic acid (LA). Fish oil (Maxepa) was purchased from Merck Limited (Goa,

India) and contained 60% EPA and 40% DHA.

Animals



Housing of Animals






Institutional Animal Ethical Committee (Ref. No. BVDUMC/443/2012-2013).


The dose of flax oil and fish oil was finalized on the basis of previous studies

carried out on the references (Meganathan et al., 2011; Kasote et al., 2012).

78

Commercially available flax oil and fish oil were used directly for intervention

studies.



Acetaminophen (1000mg/kg b.w. /day, p.o.) or 30% Ethanol (Alcohol) (1ml/100g


The flax oil (500mg/kg b.w./day, p.o.) or fish oil (500mg/kg b.w./day, p.o.) was

administered to study their hepatoprotective activity.


Male Albino Wistar rats were administered acetaminophen or 30% ethanol

daily for 15 days to induce hepatotoxicity. Flax oil and fish oil were administered to

rats daily, 30 minutes before the administration of the hepatotoxicant, for 15 days.

The animals were sacrificed after the treatment and blood and liver were collected.

Blood samples were collected by cardiac puncture and were subjected to liver

function tests (like SGOT, SGPT, Bilirubin, Alkaline phosphatase etc.) and lipid

profile (Total cholesterol, HDL-D cholesterol, LDL-D cholesterol, and Triglycerides).

Part of the liver tissues were stored in 10% neutral-buffered formalin for

histopathology while the remaining part was snap frozen in liquid nitrogen for

assessment of liver biochemical parameters (Lipid peroxidation, Superoxide

dismutase activity, Catalase activity, Reduced glutathione content, Total protein and

lipid profile) and for molecular analysis.

Evaluation of Hepatoprotective Activity of Flax Oil and Fish Oil against


The animals were divided into five groups by random assignment of six



The treatment protocol to assess the hepatoprotective potential of

polyunsaturated fatty acids against acetaminophen induced liver injury is outlined

below:

79


days.

2. Group II: Negative Control (n=6); rats were administered acetaminophen

(1000mg/kg b.w./day, p.o.), for 15 days.



administration of silymarin (100mg/kg b.w./day, p.o.), for 15 days.



administration of flax oil (500mg/kg b.w./day, p.o.), for 15 days.



administration of fish oil (500mg/kg b.w./day, p.o.), for 15 days.












Evaluation of Hepatoprotective Activity of Flax Oil and Fish Oil against Ethanol


The animals were divided into five groups by random assignment of six



The treatment protocol to assess the hepatoprotective potential of

polyunsaturated fatty acids against ethanol induced liver injury is outlined below:

80

1. Group I: Healthy Control (n=6); fed on a normal diet and water for 15 days.

2. Group II: Negative Control (n=6); rats were administered 30% ethanol

(1ml/100g b.w./day, p.o.), for 15 days.



silymarin (100mg/kg b.w./day, p.o.), for 15 days.



flax oil (500mg/kg b.w./day, p.o.), for 15 days.



fish oil (500mg/kg b.w./day, p.o.), for 15 days.












The effect of satwa and omega 3 fatty acids on acetaminophen induced

hepatotoxicity was studied in the same experiment (with common healthy, positive

and negative control groups) while the effect of satwa and omega 3 fatty acids on

alcohol induced hepatotoxicity was studied in another experiment (with common

healthy, positive and negative control groups).

81

4.7 Hepatoprotective Activity of Combination of Best Performing Herbal and

Nutritional Intervention

In previous experiments, the hepatoprotective activity of satwa of three

Tinospora forms and polyunsaturated fatty acids was studied, separately, against

acetaminophen and alcohol induced hepatotoxicity in rats. Based on the results of

these experiments, the combination of Neem-giloe and fish oil was used for

intervention in acetaminophen induced hepatotoxicity while combination of

Tinospora sinensis and flax oil was used for intervention in ethanol induced

hepatotoxicity in rats.

Animals



Housing of Animals






Institutional Animal Ethical Committee (Ref. No.: BVDUMC/2679/2012-2013).


The dose of best performing satwa and omega-3 fatty acids were based on the

results of previous experiments on acetaminophen and alcohol induced liver injury.



Acetaminophen (1000mg/kg b.w. /day, p.o.) and 30% Ethanol (Alcohol) (1ml/100g


Combination of Neem-giloe (200mg/kg; b.w/day, p.o.) and fish oil (500mg/kg;

b.w/day, p.o.) was used for screening its hepatoprotective activity against

acetaminophen induced hepatotoxicity.

82

Combination of Tinospora sinensis satwa (200mg/kg b.w./day, p.o.) and flax oil

(500mg/kg b.w/day, p.o.) was used for screening its hepatoprotective activity against

ethanol induced hepatotoxicity.


Male Albino Wistar rats were administered single dose of acetaminophen on

8th

day or 1st day or 30% ethanol daily for 15 days to induce hepatotoxicity. The best

performing Guduchi and omega-3 fatty acids at the selected dosage (As per the results

from experiment 4.5 and 4.6, respectively) were administered simultaneously, every

24 hours, to rats treated with hepatotoxicants. The combination of interventions was

administered to rats for assessing their protective or corrective effects in

acetaminophen induced liver injury. In case of alcohol induced liver damage, the

hepatotoxicant and combination of interventions were administered simultaneously to

assess the prophylactic effect of the intervention. The animals were sacrificed after

the treatment and blood and liver were collected. Blood samples were collected by

cardiac puncture and were subjected to liver function tests (like SGOT, SGPT,

Bilirubin, Alkaline phosphatase etc.) and lipid profile (Total cholesterol, HDL-D

cholesterol, LDL-D cholesterol, and Triglycerides). Part of the liver tissues were

stored in 10% neutral-buffered formalin for histopathology while the remaining part

were snap frozen in liquid nitrogen for liver biochemical parameters (Lipid

Peroxidation, Superoxide dismutase, Catalase, Reduced glutathione, Total protein

and Lipid profile) and for molecular analysis.

Effects of Protective and Corrective Treatment of Combination of Neem-giloe

Satwa and Fish oil against Acetaminophen Induced Hepatotoxicity

The animals were divided into seven groups by random assignment of six



The treatment protocol to assess the combinatorial protective and corrective

effects of Neem-giloe satwa and fish oil interventions against acetaminophen induced

liver injury is outlined below:

83

1. Group I: Healthy Control (n=6); received feed and water normally for 8 days

2. Group II (Negative Control for Protective Treatment): Negative Control

(n=6); rats were administered a single dose of acetaminophen (1000mg/kg;

b.w./day, p.o.), on 8th

day

3. Group III (Negative Control for Corrective Treatment): Negative Control

(n=6); rats were administered a single dose of acetaminophen (1000mg/kg;

b.w./day, p.o.), on 1st day

4. Group IV (Positive Control for Protective Treatment): (n=6); the rats in

this group were administered with standard drug silymarin (100mg/kg;

b.w./day, p.o.), for 7 days and administered a single dose of acetaminophen

(1000mg/kg; b.w/day, p.o.), on 8th

day

5. Group V (Positive Control for Corrective Treatment): (n=6); the rats in

this group were treated on 1st day with a single dose of acetaminophen

(1000mg/kg; b.w/day, p.o.) and administered standard drug silymarin

(100mg/kg; b.w./day, p.o.), for next 7 days

6. Group VI (Experimental Group for Protective Treatment): (n=6); the rats

in this group were administered with the combined intervention of Neem-giloe

satwa (200mg/kg; b.w/day, p.o.) and fish oil (500mg/kg; b.w/day, p.o.) for 7

days and a single dose of acetaminophen (1000mg/kg; b.w/day, p.o.), on 8th

day

7. Group VII (Experimental Group for Corrective Treatment): (n=6); the

rats in this group were treated on 1st day with single dose of acetaminophen

(1000mg/kg; b.w/day, p.o.) and administered combined interventions (satwa

of Neem-giloe (200mg/kg; b.w/day, p.o.) and fish oil (500mg/kg; b.w/day,

p.o.), for next 7 days.

During the period of the experiment, animals were observed daily for signs of

infection and/or discomfort. After completion of the experiment, all animals were

fasted overnight and were humanely sacrificed under light ether anaesthesia. Blood

samples were collected by cardiac puncture for evaluating the serum biochemical

parameters. Blood samples were allowed to clot at room temperature for 30 minutes

and serum was collected by centrifugation at 2000 rpm for 15 minutes. Aliquots of

sera were stored in eppendorfs at -20°C till further use. Liver was excised from the

dissected animals immediately, blotted off blood, washed with saline, weighed and

84

the left medial lobe was stored in 10% neutral-buffered formalin for histopathological

evaluation. The remaining liver tissues were collected and snap frozen in liquid

nitrogen. Frozen tissues were stored at -80°C till further use.

Prophylactic Effect of Combination of Tinospora sinensis Satwa and Flax Oil

against Alcohol Induced Hepatotoxicity

The animals were divided into four groups by random assignment of six

animals per group. The variation in the average weight of the animals, in and between

the groups, was less than 20%.

The treatment protocol to assess the prophylactic effect of combination of

Tinospora sinensis satwa and flax oil against ethanol induced liver injury is outlined

below:

1. Group I: Healthy Control (n=6); received feed and water normally, for 15

days

2. Group II: Negative Control (n=6); rats were administrated 30% ethanol

(1ml/100g b.w./day, p.o.), for 15 days



silymarin (100mg/kg b.w./day, p.o.), for 15 days

4. Group IV: Treatment group (n=6); the rats in this group were treated daily


Tinospora sinensis satwa (200mg/kg b.w./day, p.o.) and flax oil (500mg/kg

b.w/day, p.o.), for 15 days

During the period of the experiment, animals were observed daily for signs of

infection and/or discomfort. After 15 days of the protocol, all animals were fasted

overnight and were humanely sacrificed under light ether anaesthesia. Blood samples

were collected by cardiac puncture for evaluating the serum biochemical parameters.

Blood samples were allowed to clot at room temperature for 30 minutes and serum

was collected by centrifugation at 2000 rpm for 15 minutes. Aliquots of sera were

stored in eppendorfs at -20°C till further use. Liver was excised from the dissected

animals immediately, blotted off blood, washed with saline, weighed and the left

medial lobe was stored in 10% neutral-buffered formalin for histopathological

85

evaluation. The remaining liver tissues were collected and snap frozen in liquid

nitrogen. Frozen tissues were stored at -80°C till further use.

86

4.8 Biochemical Parameters

Liver function tests, total protein, lipid profile and antioxidant parameters

were evaluated from serum and/or liver homogenate.

4.8.1 Serum Biochemical Parameters

Liver function tests, total protein and lipid profile were evaluated as per the

manufacturer’s instructions provided with the kit (Coral clinical system, Goa, India).

Serum Glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic

Transaminase (SGPT), Alkaline Phosphatase (ALP), Total Bilirubin and Lipid Profile

(Total Cholesterol, HDL-D Cholesterol, LDL-D Cholesterol, and Triglycerides), were

estimated using commercial kits (Coral clinical system, Goa, India). VLDL

Cholesterol was calculated by using the formula: Triglycerides/5. Absorbance values

of the reactions were measured using the iMarkTM

micro plate absorbance reader

(168-1135) (Bio-Rad Laboratories, Inc. Berkeley, California, U.S.A.).

4.9 Methods for Estimation of Serum Biochemical Markers:

4.9.1 Estimation of Serum Glutamic Oxaloacetic Transaminase

(SGOT) (Reitman & Frankel's Method) (Reitman and Frankel, 1957 and Tietz,

1970; Instruction Manual of SGOT Kit, Catalog Number GOT 010) (Coral Clinical

Systems, Goa, India).

The Serum Glutamic Oxaloacetic Transaminase (aspartate aminotransferase) was

estimated by the method of Reitman and Frankel (1957) using SGOT test kit (Coral

Clinical Systems, Goa, India).

Principle

SGOT converts L-Aspartate and α-Ketoglutarate to Oxaloacetate and

Glutamate. The Oxaloacetate formed reacts with 2, 4, Dinitrophenyl hydrazine to

produce a hydrazone derivative, which in an alkaline medium produces a brown

coloured complex whose intensity is measured. The reaction does not obey Beer's law

and hence a calibration curve is plotted using a Pyruvate standard. The activity of

SGOT (ASAT) is read off this calibration curve.

87

SGOT

L-Alanine + α Ketoglutarate Oxaloacetate + L-Glutamate pH 7.4

Alkaline

Oxaloacetate + 2,4, DNPH 2, 4, Dinitrophenyl Hydrazone Medium (Brown coloured complex)

Reagents

L1: Substrate reagent

L2: DNPH (2, 4- Dinitrophenyl hydrazine) reagent

L3: NaOH reagent (4N))

S: Pyruvate standard (2mM)

Working NaOH Solution I: Volume of 1mL of Reagent III was made up to 10mL

with distilled water.

Procedure

All the test tubes were marked properly as blank (B), standard (S1, S2, S3, S4

and S5), and test (T).

L1 Reagent (0.50, 0.45, 0.40, 0.35, 0.30 mL) was added in all the tubes of

standard. Pyruvate standard (0.05, 0.1, 0.15 and 0.2) was added in tubes S2, S3, S4

and S5 respectively. Distilled water (0.10mL) and L2 (0.5mL) was added in all tubes

of standard. The contents in the standard tubes were mixed well and incubated at

room temperature for 20 minutes. Reagent L3 (5.0mL) was added to all tubes, the

contents were mixed and tubes were incubated at room temperature for 10 minutes.

The absorbance of the standard tubes S2 to S5 was measured against blank S1.

L1 Reagent (0.50mL) was added in B and T tubes. The tubes were incubated at

37ºC for 3 minutes. Serum (0.10mL) was added in the test (T). The contents of B and

T tubes were mixed well and incubated at 37ºC for 60 minutes. Reagent L2 (0.50mL)

was added in tubes B and T. The contents were mixed well and allowed to stand at

room temperature for 20 minutes. Distilled water (0.10mL) was added in the blank

(B) tube. Reagent L3 (5mL) (Working NaOH Solution I) was added to B and T tubes,

contents were mixed well and allowed to stand at room temperature for 10 min. The

absorbance of test sample was read against blank at 505 nm.

4.9.2 Estimation of Serum Glutamic Pyruvic Transaminase (SGPT)

(Reitman & Frankel's method) (Reitman and Frankel, 1957 and Tietz, 1970;

88

Instruction Manual of SGPT Kit, Catalog Number GPT 010) (Coral Clinical Systems,

Goa, India).

The Serum Glutamic Pyruvic Transaminase (alanine aminotransferase) was estimated

by the method of Reitman and Frankel (1957) using SGPT test kit (Coral Clinical

System, Goa, India).

Principle

SGPT converts L-Alanine and α-Ketoglutarate to Pyruvate and Glutamate.

The Pyruvate formed reacts with 2, 4, Dinitrophenyl hydrazine to produce a

hydrazone derivative, which in an alkaline medium produces a brown coloured

complex whose intensity is measured. The reaction does not obey Beer's law and

hence a calibration curve is plotted using a pyruvate standard. The activity of SGPT

(ALAT) is read off this calibration curve.

SGPT

L-Alanine + α Ketoglutarate Pyruvate + L-Glutamate pH 7.4 Alkaline

Pyruvate + 2,4, DNPH 2, 4, Dinitrophenyl Hydrazone Medium (Brown coloured complex)

Reagents


L2: DNPH (2, 4- Dinitrophenyl hydrazine) reagent

L3: NaOH reagent (Sodium hydroxide, (4N))

S: Pyruvate standard (2mM)

Working NaOH Solution I: Volume of 1mL of Reagent III was made up to 10mL

with distilled water.

Procedure

All the test tubes were marked properly as blank (B), standard (S1, S2, S3, S4

and S5), and test (T).

L1 Reagent (0.50, 0.45, 0.40, 0.35, 0.30 mL) was added in all the tubes of

standard. Pyruvate standard (0.05, 0.1, 0.15 and 0.2) was added in tubes S2, S3, S4

and S5 respectively. Distilled water (0.10mL) and L2 (0.5mL) was added in all tubes

of standard. The contents in the standard tubes were mixed well and incubated at

room temperature for 20 minutes. Reagent L3 (5.0mL) was added to all tubes, the

89

contents were mixed and tubes were incubated at room temperature for 10 minutes.

The absorbance of the standard tubes S2 to S5 was measured against blank S1.

L1 Reagent (0.50mL) was added in B and T tubes. The tubes were incubated at

37ºC for 3 minutes. Serum (0.10mL) was added in the test (T). The contents of B and

T tubes were mixed well and incubated at 37ºC for 30 minutes. Reagent L2 (0.50mL)

was added in tubes B and T. The contents were mixed well and allowed to stand at

room temperature for 20 minutes. Distilled water (0.10mL) was added in the blank

(B) tube. Reagent L3 (5mL) (Working NaOH Solution I) was added to B and T tubes,

contents were mixed well and allowed to stand at room temperature for 10 min. The

absorbance of test sample was read against blank at 505 nm.

4.9.3 Estimation of Alkaline Phosphatase (ALP) (Mod. Kind & King's

Method) (Kind and King, 1954 and Varley, 1975; Instruction Manual of ALP Kit,

Catalog Number ALP 010) (Coral Clinical Systems, Goa, India).

Alkaline Phosphatase activity was estimated by the method of Kind and King (1954)

using ALP test kit (Coral Clinical Systems, Goa, India).

Principle

ALP at an alkaline pH hydrolyses di Sodium Phenylphosphate to form phenol.

The Phenol formed reacts with 4-Aminoantipyrine in the presence of Potassium

Ferricyanide, as an oxidising agent, to form a red coloured complex. The intensity of

the colour formed is directly proportional to the activity of ALP present in the sample.

ALP

Di Na Phenylphosphate + H2O Phenol + di Na Hydrogen Phosphate pH 10.0 Alkaline medium Phenol + 4-Aminoantipyrine Red Coloured Complex K3Fe (CN) 6

Reagents

L1: Buffer reagent


L3: Colour reagent

S: Phenol standard (10 mg/dL)

Procedure

All the test tubes were marked properly as blank (B), standard (S), control (C),

and test (T). Distilled water (1.05mL in B and 1.0mL in S, C and T) was added in the

90

tubes. L1 Reagent (1mL) and L2 Reagent (0.10mL) were added in all the tubes. The

contents were mixed well and incubated at 37°C for 3 min. Serum (0.05mL) was

added in test (T) and Phenol standard (0.05mL) was added in standard (S). Contents

of the tubes were mixed well and incubated at 37°C for 15 min. L3 Reagent (1mL)

was added in all the tubes. Serum (0.05mL) was added in control (C). Contents of the

tubes were mixed well and absorbance was read at 510 nm against distilled water as

blank. Serum alkaline phosphatase activity is expressed as KA units.

Abs.T-Abs.C

Total ALP activity in K.A. Units = X 10

Abs.S-Abs.B

4.9.4 Estimation of Total Bilirubin (Mod. Jendrassik and Grof’s Method)

(Jendrassik and Grof’s, 1938; Sherlock, 1951; Instruction Manual of BIL Kit, Catalog

Number BIL 010) (Coral Clinical Systems, Goa, India).

Total Bilirubin was estimated by the method of Jendrassik and Grof’s (1938) using

BIL test kit (Coral Clinical System, Goa, India).

Principle

Bilirubin reacts with diazotized sulphanilic acid to form a coloured

azobilirubin compound. The unconjugated bilirubin couples with the sulphanilic acid

in presence of a caffein-benzoate accelerator. The intensity of the colour formed is

directly proportional to the amount of bilirubin present in the sample.

Bilirubin + Diazotized Sulphanilic acid Azobilirubin Compound

Reagents

L1: Total bilirubin reagent

L2: Total nitrite reagent

Procedure

All the test tubes were marked properly as blank (B) and test (T). L1 Reagent

(1mL) was added in blank (B) and test (T) tubes. L2 Reagent (0.05mL) was added in

test (T). Serum (0.1mL) was added in test (T) and blank (B) tubes. Contents of the

tubes were mixed well and incubated at room temperature for 10 minutes and

absorbance of test sample T was read against blank B at 546 nm.

Total bilirubin in mg/dL = Abs. T × 13

91

4.9.5 Estimation of Total Cholesterol (CHOD / PAP Method) (Trinder,

1969; Allain et al., 1974; Instruction Manual of CHO Kit, Catalog Number CHO 010)

(Coral Clinical Systems, Goa, India).

Total Cholesterol levels were estimated by enzymatic Cholesterol Oxidase Peroxidase

(CHOD-PAP) endpoint method using Cholesterol test kit (Coral Clinical Systems,

Goa, India).

Principle

Cholesterol esterase hydrolyses esterified cholesterols to free cholesterol. The

free cholesterol is oxidised to form hydrogen peroxide which further reacts with

phenol and 4-aminoantipyrine by the catalytic action of peroxidase to form a red

coloured quinoneimine dye complex. Intensity of the colour formed is directly

proportional to the amount of cholesterol present in the sample.

Cholesterol Esterase

Cholesterol esters + H2O Cholesterol + Fatty Acids Cholesterol Oxidase

Cholesterol+ O2 Cholestenone + H2O Peroxidase

H2O2 + 4 Aminoantipyrine + Phenol Red Quinoneimine Dye + H2O

Reagents

L1: Cholesterol reagent

S: Cholesterol standard (200 mg/dL)

Procedure

All the test tubes were marked properly as blank (B), standard (S) and test (T).

L1 Reagent (1mL) was added in standard (S), blank (B) and test (T) tubes. Distilled

water (0.01mL) was added in blank (B) and cholesterol standard (0.01mL) was added

in standard (S) tube. Serum (0.01mL) was added in test (T). Contents of the tubes

were mixed well and incubated at room temperature for 15 minutes and absorbance of

standard (S) and test (T) was read against blank (B) at 505 nm within 60 minutes.

Abs.T

Cholesterol in mg/dL = X 200

Abs.S

4.9.6 Estimation of Triglycerides (GPO / PAP Method) (Trinder, 1969;

Bucolo and David, 1973, and Fossati and Prencipe, 1982; Instruction Manual of TGL

Kit, Catalog Number TGL 010) (Coral Clinical Systems, Goa, India).

92

Serum Triglyceride (TG) was estimated by enzymatic Glycerol Phosphate Oxidase

Peroxidase GPO/POD, endpoint method using Triglyceride test kit (Coral Clinical

Systems, Goa, India).

Principle

Lipoprotein lipase hydrolyses triglycerides to glycerol and free fatty acids.

The glycerol formed with ATP in the presence of glycerol kinase forms glycerol 3

phosphate, which is oxidised by the enzyme glycerol phosphate oxidase to form

hydrogen peroxide. The hydrogen peroxide further reacts with phenolic compound

and 4-aminoantipyrine by the catalytic action of peroxidase to form a red coloured

quinoneimine dye complex. Intensity of the colour formed is directly proportional to

the amount of triglycerides present in the sample.

Lipoprotein Lipase

Triglycerides Glycerol + Free Fatty Acids Glycerol Kinase

Glycerol+ ATP Glycerol 3 Phosphate + ADP Glycerol 3 PO

Glycerol 3 Phosphate + O2 Dihydroxyacetone Phos. + H2O2

Peroxidase

H2O2 + 4 Aminoantipyrine + Phenol Red Quinoneimine dye + H2O

Reagents

L1: Enzyme reagent 1

L2: Enzyme reagent 2

S: Triglycerides standard (200 mg/dL)

Working reagent: Working reagent was prepared by mixing together 4 parts of L1

(Enzyme reagent I) and 1 part L2 (Enzyme reagent II). Alternatively, 0.8mL of L1and

0.2mL of L2 may also be used instead of 1ml of the working reagent.

Procedure


Working reagent (1mL) was added in standard (S), blank (B) and test (T) tubes.

Distilled water (0.01mL) was added in blank (B) and triglycerides standard (0.01mL)

was added in standard (S) tube. Serum (0.01mL) was added in test (T). Contents of

the tubes were mixed well and incubated at room temperature for 15 minutes and

absorbance of standard (S) and test (T) tubes was read against blank (B) at 505 nm.

Abs.T

Triglycerides in mg/dL = X 200

Abs.S

93

4.9.7 Estimation of HDL-D Cholesterol (Direct Enzymatic Method) (Tietz

et al., 1995; Young, 1995; Burtis et al., 1999 and Young, 2001; Instruction Manual of

HDL Kit, Catalog Number HDL 030) (Coral Clinical Systems, Goa, India).

Serum High Density Lipoprotein Cholesterol estimated by direct enzymatic method

using HDL-D test kit (Coral Clinical Systems, Goa, India).

Principle

The method relies on direct determination of serum HDLc (high-density

lipoprotein cholesterol) levels without the need for any pre-treatment or centrifugation

of the sample. The method depends on the properties of a detergent which solubilizes

only the HDL so that the HDLc is released to react with the cholesterol esterase,

cholesterol oxidase and chromogens to give colour. The non HDL lipoproteins LDL,

VLDL and chylomicrons are inhibited from reacting with the enzymes due to

adsorption of the detergents on their surfaces. The intensity of the colour formed is

proportional to the HDLc concentration in the sample.

Reagents

L1: HDL-D reagent 1

L2: HDL-D reagent 2

C: Calibrator reagent

Procedure

All the test tubes were marked properly as blank (B), calibrator (C) and test

(T). L1 Reagent (375µL) was added in tubes B, C and T. Calibrator reagent (5.0µL)

was added in the calibrator (C) tube. Serum (5.0µL) was added in test (T). Contents

were mixed well and incubated at 37ºC for 5 minutes and absorbance A1 of calibrator

and test was read at 578nm against blank (B). Thereafter, 125µL of L2 Reagent was

added to all the tubes, mixed well and incubated at 37ºC for 5 minutes. The

absorbance A2 of calibrator and test was read at 578 nm against blank (B).

For calibrator ΔAC = A2C – A1C

For Test ΔAT = A2T-A1T

ΔAT

HDLc in mg/dL = X 200

ΔAC

94

4.9.8 Estimation of LDL-D Cholesterol (Direct Enzymatic Method) (Tietz

et al., 1995; Young, 1995; Burtis et al., 1999 and Young, 2001; Instruction Manual of

LDL Kit, Catalog Number LDL 040) (Coral Clinical Systems, Goa, India).

Serum Low Density Lipoprotein Cholesterol estimated by direct enzymatic method

using LDL-D test kit (Coral Clinical Systems, Goa, India).

Principle

The method directly determines serum LDLc (low-density lipoprotein

cholesterol) levels without the need for any pre-treatment or centrifugation steps. The

assay takes place in two steps. First, lipoprotein non-LDL-D cholesterol is eliminated

and then LDLc is measured. The intensity of the colour formed is proportional to the

LDLc concentration in the sample.

Elimination of non-LDL Cholesterol


Cholesterol esters + H2O Cholesterol + Fatty acids Cholesterol Oxidase

Cholesterol + O2 4 - Cholestenone + H2O Catalase

2H2O2 2H2O + O2

Measurement of LDL Cholesterol


Cholesterol esters Cholesterol + Fatty acids Cholesterol Oxidase

Cholesterol + O2 4 - Cholestenone + H2O2 Peroxidase

2H2O2 + TODS + 4-Aminoantipyrine Quinonimine + 4 H2O

Reagents

L1: LDL-D reagent 1

L2: LDL-D reagent 2

C: Calibrator

Procedure

All the test tubes were marked properly as blank (B), calibrator (C) and test

(T). L1 Reagent (375µL) was added in tubes B, C and T. Calibrator reagent (5.0µL)

was added in the calibrator (C) tube. Serum (5.0µL) was added in test (T). Contents

were mixed well and incubated at 37ºC for 5 minutes. Thereafter, 125µL of L2

reagent was added to all the tubes, mixed well and incubated at 37ºC for 5 minutes

and absorbance of calibrator (C) and test (T) was read against blank (B) at 595 nm.

95

Abs.T

LDLc in mg/dL = X Conc of calibrator

Abs.C

4.10 Liver Biochemical Parameters

Cell free supernatant from liver homogenate was used to estimate Lipid

Peroxidation (OxisResearchTM

, USA), Catalase (CAT) (Sigma-Aldrich, USA),

Superoxide Dismutase (SOD) (Sigma-Aldrich, USA), Reduced Glutathione (Cayman

Chemical Compan, USA), Total Protein and Lipid Profile (Total Cholesterol, HDL-D

Cholesterol, LDL-D Cholesterol, Triglycerides) using commercial kits (Coral clinical

systems, Goa, India). VLDL Cholesterol was estimated by using the formula:

Triglycerides/5. Absorbance of the reactions was read using iMarkTM

micro plate

absorbance reader (168-1135) (Bio-Rad Laboratories, Inc. Berkeley, California,

USA).

Preparation of Liver Homogenate

Liver tissues from rats of all groups were excised, blotted and homogenized

with phosphate buffered saline (1/10 weight/volume), (pH 7.0) and the homogenate

was centrifuged at 30,000 rpm for 15 minutes at 4ºC. Cell free supernatant was

collected and stored in a freezer (-80ºC) until further use.

4.10.1 Estimation of Lipid Peroxidation (Colorimetric Method) (Bull and

Marnett, 1985; Esterbauer et al., 1991; Botsoglou, 1994; Instruction Manual of MDA

Kit, Catalog Number 21044).

Measurement of MDA was used as an indicator of Lipid Peroxidation. Bioxytech®

MDS-586 Lipid Peroxidation Kit (OxisResearchTM

, USA) was used to estimate MDA

from liver homogenate.

Principle

The MDA-586 method is based on the reaction of a chromogenic reagent N-

methyl-2-phenylindole (NMPI), with MDA at 45°C. One molecule of MDA reacts

with 2 molecules of NMPI to yield a stable carbocyanine dye.

Reagents

Reagent R1: N-methyl-2-phenylindole in acetonitrile

Reagent R2: Concentrated hydrochloric acid

96

MDA Standard: 1, 1, 3, 3-Tetramethoxypropane (TMOP) in Tris-HCl

Diluent: Methanol

Probucol: Probucol in methanol

Dilution of the R1 solution for use in the assay: One volume (6 mL) of diluent

(100% methanol) was added to three volumes (18 mL) of reagent R1.

Procedure

The MDA Standard (10mM stock solution) was diluted 1/500 (v/v) in

deionized water to yield a 20µM stock solution and used in the assay. The tubes were

marked properly (A to F). MDA Standards (0, 25, 50, 100, 150, 200µl) were added in

tubes A to F. Deioninzed water was added to these tubes to make a final volume of

200µl. Separate tubes were allocated to samples to be tested. Probucol (10µl) was

added in all assay tubes. Cell free supernatant sample (200µl) or MDA Standards

(200µl) were added to these tubes. Diluted R1 Reagent (640µl) was added in each

assay tube. The mixture was gently vortexed. Thereafter, 150µl of R2 Reagent was

added in each assay tube. Contents of the tubes were mixed by vortexing. The tubes

were incubated at 45°C for 60 minutes. Samples were centrifuged at 10,000g for 10

minutes. Clear supernatant was collected and transferred to each well of microplate.

The absorbance was read at 586 nm. The regression line for standard was plotted and

the values of regression coefficient and y-intercept were noted.

Calculate the concentration of analyte in a sample:

A586 - b

[MDA] = x df

a

Where, [MDA] = Concentration of MDA in the sample

A586 = Absorbance at 586 nm of sample

a = Regression coefficient (slope)

b = Intercept

df = Sample dilution factor

4.10.2 Estimation of Superoxide Dismutase (SOD) (Colorimetric Method)

(Instruction Manual of SOD Kit, Catalog Number SOD 19160)

The Superoxide Dismutase was estimated by colorimetric method using the SOD

assay test kit (Sigma-Aldrich, USA)

97

Principle

Superoxide dismutase (SOD) is one of the most important antioxidative

enzymes. It catalyses the dismutation of the superoxide anion (O2.-) into hydrogen

peroxide and molecular oxygen. An indirect method using nitroblue tetrazolium

(NBT) is commonly used due to its convenience and ease of use. The procedure from

this kit allows convenient SOD assaying by utilizing Dojindo’s highly water-soluble

tetrazolium salt, WST-1(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-

2H-tetrazolium, monosodium salt) producing a water-soluble formazan dye after

reduction with a superoxide anion. The rate of the reduction with O2 are linearly

related to the xanthine oxidase (XO) activity, and is inhibited by SOD. Therefore, the

IC50 (50% inhibition activity of SOD or SOD-like materials) can be determined by a

colorimetric method.

Reagents

Reagent I: WST solution

Reagent II: Enzyme solution

Reagent III: Buffer solution

Reagent IV: Dilution buffer

WST working solution: Dilute 1mL of WST Solution (Reagent I) with 19mL of

Buffer Solution (Reagent II)

Enzyme working solution: Dilute 15µl of Enzyme Solution (Reagent III) with

2.5mL of Dilution Buffer (Reagent IV)

Procedure

The microplate wells were marked as test (T), blank 1, blank 2 and blank 3.

Sample (20µl) was added in test (T) and blank 2 wells, and double distilled water

(20µl) was added to blank 1 and blank 3 wells. WST (200µl) working solution was

added in each well and mixed properly. Dilution buffer (20µl) was added to blank 2

and blank 3 wells. Enzyme working solution (20µl) was added to test and blank 1

well. The contents were mixed well and the plate was incubated at 37°C for 20

minutes. The absorbance was read at 450 nm using a micro plate reader.

SOD activity (inhibition rate %) was calculated using the following equation:

SOD activity (inhibition rate %) =

{[(Ablank 1 - Ablank 3) - Asample - Ablank 2)]/ (Ablank 1 - Ablank 3)} x 100

98

4.10.3 Estimation of Catalase (Colorimetric Method) (Deisseroth and

Dounce, 1970; Zamocky and Koller, 1999 and Zhou and Kang, 2000; Instruction

Manual of CAT kit, Catalog Number CAT100).

The Catalase was estimated by colorimetric method using the CAT assay test kit

(Sigma-Aldrich, USA).

Principle

Catalase is an antioxidant enzyme universally present in mammalian and non-

mammalian aerobic cells containing a cytochrome system. Catalase is able to

decompose hydrogen peroxide by two different reaction pathways. In the first, known

as the “catalatic” pathway, 2 molecules of hydrogen peroxide are converted to water

and oxygen (catalatic activity)

Protein-Fe3+

+ H2O2 → Protein-Fe3+

-OOH (Primary Complex) + H2O

Protein-Fe3+

-OOH + H2O2 → Protein-Fe3+

-OH + H2O + O2

The overall reaction gives: 2 H2O2 → 2 H2O + O2

The primary complex can also decompose by another pathway (peroxidatic

decomposition): Protein-Fe3+

-OOH + AH2 → Protein-Fe3+

-OH + H2O + A

Where AH2 is an internal or external donor of hydrogen. Low molecular weight

alcohols can serve as electron donors. The catalatic pathway is predominant when the

hydrogen peroxide concentration is greater than 0.1mM and the peroxidatic pathway

is dominant when the hydrogen peroxide concentration is less than 0.1mM or the

substrate is alkyl peroxide.

Reagents

Reagent I: Assay buffer 10× (500mM potassium phosphate buffer, pH 7.0)

Reagent II: Chromogen reagent

Reagent III: Stop solution (15mM sodium azide in water)

Reagent IV: 3% (w/w) Hydrogen peroxide solution

Reagent V: Enzyme dilution buffer (50mM potassium phosphate buffer, pH 7.0, and

containing 0.1% TritonX-100)

1X Assay Buffer: Dilute 2mL of 10X assay buffer (reagent I) 10-folds to 20mL with

distilled water

Colorimetric Assay Substrate Solution (200mM H2O2): Dilute 200µl of 3% H2O2

to 1mL with 1X assay buffer. Dilute 50µl of the above solution to 1mL (20-fold) with

1X assay buffer

Catalase Colorimetric Enzymatic Reaction Scheme

99

Sample Volume 1X Assay Buffer 200mM H2O2

Solution

Blank 0 75µl 25µl

Test 10µl 65µl 25µl

Procedure

The microcentrifuge tubes were marked properly as test (T), blank (B) and

standard. Series of standard solutions of H2O2 was prepared by pipetting 0, 125, 250,

500, and 750µl of 10mM H2O2 solution in micro-centrifuge tubes and 1X Assay

Buffer was added to a final volume of 1.0mL. The contents were mixed properly. An

aliquot (10µl) of each solution was transferred to separate labeled wells of the

microplate and 1mL of reagent II was added to each sample. The plate was incubated

at room temperature for 15 minutes and the absorbance was read at 520nm and

standard curve of the absorbance at 520nm against the final amount of hydrogen

peroxide in the reaction mixture was plotted. The cell free supernatant 10µl was

added to separate labeled micro-centrifuge tube. 1X Assay buffer 65µl was added in

test (T) and 75µl 1X assay buffer was added in blank (B) tubes. The reaction was

started by addition of 25µl of colorimetric assay substrate solution (200mM H2O2).

The contents were mixed by inversion and the tubes were incubated at room

temperature for 5 minutes. Reagent III (900µl) was added to each test and blank tubes

and contents were mixed by inverting the tubes. An aliquot (10µl) of the catalase

enzymatic reaction mixture was added to another micro centrifuge tube. Reagent II

(1mL) was added to each test and blank. Then they were mixed well and allowed to

stand at room temperature for 15 minutes for colour development and then the

absorbance was measured at 520 nm.

Catalase activity:

Activity (µmoles/min/ml) = Δµmoles (H2O2) x d x 100/ V x t

Where, Δµmoles (H2O2) = Difference in amount of H2O2 added to the colorimetric reaction

between the blank and given sample

d= Dilution of original sample for catalase reaction

t= Catalase reaction duration (minutes)

V= Sample volume in catalase reaction (10ul= 0.01 ml)

100= Dilution of aliquot from catalase reaction in colorimetric reaction (10µl from 1ml )

100

4.10.4 Estimation of Total Protein (Biuret Method) (Gornall et al., 1949

and Doumas, 1975; Instruction Manual of Total Protein Kit, Catalog Number TPR

010) (Coral Clinical Systems, Goa, India).

The Total Protein was estimated by modified Biuret method using the total protein

test kit (Coral Clinical System, Goa, India).

Principle

Proteins, in an alkaline medium, bind with the cupric ions present in the biuret

reagent to form a blue-violet coloured complex. The intensity of the colour formed is

directly proportional to the amount of Proteins present in the sample.

Proteins + Cu+ +

Blue Violet Coloured Complex

Reagents

L1: Biuret reagent

S: Protein standard (8g/dL)

Procedure


L1 Reagent (1.0mL) was added to all the test tubes. Thereafter, Distilled water

(0.02mL) was added in blank (B) and protein standard (0.02mL) was added in the

standard test tube (S). Cell free supernatant sample (0.02mL) was added in the tube

for test sample (T). Contents of the tubes were mixed well and incubated at 37ºC for

10 minutes and absorbance of standard (S) and test (T) was read against blank (B) at

550 nm within 60 minutes.

Abs. T

Total Proteins in g/dl = x 8

Abs. S

4.10.5 Estimation of Reduced Glutathione (End Point Method) (Griffith,

1980; Lash et al., 1985 and Baillie and Slatter, 1991 and Instruction Manual of

Reduced Glutathione (GSH) kit, Catalog Number GSH 703002).

The assay was estimated by end point method using the GSH assay test kit (Cayman

Chemical Company).

Estimation of reduced glutathione was done from deproteinized samples.

101

Procedure for Deproteination of Samples

Part of the homogenate was deproteinated with metaphosphoric acid (MPA)

reagent and 4M triethanolamine (TEAM) reagent and used for estimation of reduced

glutathione (GSH) content in the tissue by using commercially available kits (Cayman

Chemical Company, USA).

MPA Reagent: Dissolve 5g of metaphosphoric acid (Sigma-Aldrich) in 50mL water.

The MPA solution was stable for 4 hours at 25°C. Equal volumes of MPA reagent

was added to the sample and mixed by vortexing. The mixture was allowed to stand at

room temperature for 5 minutes and centrifuged at 2000 rpm for 2 minutes, and the

supernatant was collected.

TEAM Reagent: A 4M solution of triethanolamine (Sigma-Aldrich) in water was

prepared by mixing 531µl of the triethanolamine with 469µl of water. The TEAM

solution was stable for 4 hours at 25°C. 50µl of TEAM reagent was added per 1mL of

the supernatant and vortexed immediately. The TEAM reagent increased the pH of

the sample. The sample was ready for assay of the total GSH.

Principle

Glutathione (GSH) is a tripeptide (γ-glutamylcysteinylglycine) widely

distributed in both plants and animals. GSH serves as a nucleophilic co-substrate to

glutathione transferases in the detoxification of xenobiotics and is an essential

electron donor to glutathione peroxidase in the reduction of hydroperoxides. GSH is

also involved in amino acid transport and maintenance of protein sulfhydryl reduction

status. The sulfhydryl group of GSH reacts with DTNB (5, 5’- dithio-bis-2-

(nitrobenzoic acid), Ellman’s reagent) and produces a yellow coloured 5-thio-2-

nitrobenzoic acid (TNB). The mixed disulfide GSTNB (between GSH and TNB) that

is concomitantly produced, is reduced by glutathione reductase to recycle the GSH

and produce more TNB. The rate of TNB production is directly proportional to this

recycling reaction, which is in turn directly proportional to the concentration of GSH

in the sample.

Reagents

Reagent I: GSH MES buffer (2X)

The buffer consists of 0.4M 2-(N-morpholino) ethanesulphonic acid, 0.1M phosphate,

and 2mM EDTA. PH 6.0

102

This GSH MES buffer was added to equal volume of HPLC-grade water (60mL of the

buffer with 60mL of HPLC-grade water) to make a 1X buffer for assay.

Reagent II: GSSG standard

Ready to use, 2mL of 25µM GSSG in MES buffer was supplied with the kit.

Reagent III: GSH Co-factor mixture

The vial contained a lyophilized powder of NADP+ and glucose-6-phosphate. The

contents of the vial were reconstituted with 0.5mL water and mixed well.

Reagent IV: GSH enzyme mixture

The vials contained glutathione reductase and glucose-6-phosphate dehydrogenase in

0.2mL buffer. 1X MES buffer (2mL) was added to the vial before use, the cap was

replaced, and mixed well.

Reagent V: GSH DTNB vial contained a lyophilized powder of DTNB (5, 5’-dithio-

bis-(2-nitrobenzoic acid). The contents of the vial were reconstituted with 0.5mL of

water and mixed well.

Assay cocktail: MES buffer (11.25mL), reconstituted cofactor mixture (0.45mL),

reconstituted enzyme mixture (2.1mL), water (2.3mL) and reconstituted DTNB

(0.45mL).

Procedure

The tubes were marked properly (A to H). Reagent II (0, 5, 10, 20, 40, 80,

120, 160 µl) was added in tubes A to H. Reagent I was added to these tubes to make a

final volume of 500µl. The contents of the tubes were mixed well. The microplate

wells were accordingly labelled and 50µl of standard was added in labelled wells.

Deproteinated samples (50µl) were added to separate labelled wells of the micro

plate. Freshly prepared assay cocktail (150µl) was mixed with the standard wells (A-

H) and each well of samples. The plate was covered and incubated in dark, on an

orbital shaker, for 25 minutes. The absorbance was measured at 415 nm.

GSSG or Total GSH (µM):

[Total GSH] or [GSSG] =

[(Absorbance at 415nm) - (Y-intercept)/Slope] × 2 × Sample Dilution

4.10.6 Estimation of Total cholesterol (CHOD / PAP Method)

As described in chapter 4, section 4.9.5

4.10.7 Estimation of Triglycerides (GPO / PAP Method)


103

4.10.8 Estimation of HDL-D Cholesterol (Direct Enzymatic Method)


4.10.9 Estimation of LDL-D Cholesterol (Direct Enzymatic Method)


104

4.11 Liver Histology

Portion of the liver tissues (left medial lobe) from rats of all groups were used

for the histopathological analysis. The tissues were transferred in 10% neutral-

buffered formalin solution for fixation and later on processed for histopathological

studies following the standard procedure. The sections were cut on microtome

(Microm, HM 315), processed, and stained with Mayer’s haematoxylin and eosin

(H&E staining) (Mayer, 1891) for examination. The stained tissues were observed

under a light microscope at 20X magnifying power and photographed using Image

Pro Plus (v5.1.2.59).

4.11.1 Fixation

Fixation is the process of preserving, hardening and preventing changes in the

tissues. The tissues were excised out immediately after sacrificing, washed with

saline, blot dried and cut into pieces of such thickness that the fixative readily

penetrated throughout the tissue to be fixed. Tissue was transferred to the 10%

formalin solution till further processing.

4.11.2 Tissue Processing

Tissue processing involves dehydration, clearing and infiltration of the tissue

with paraffin. The usual dehydrating agent is ethyl alcohol; acetone and isopropyl

alcohol can also be used. Following dehydration, the tissue was transferred to a

paraffin solvent, which is miscible with the dehydrating agent as well. These are

known as clearing agents such as chloroform and xylene. Tissues were thoroughly

washed by placing them under running tap water and then conveyed through a series

of following solvents as per schedule for dehydration, clearing and paraffin

infiltration:

Solvent Grades Time

Alcohol 70% 20 minutes




Isopropyl alcohol 20 minutes

Acetone 20 minutes

Chloroform 20 minutes

Melted paraffin wax

(60°C)

20 minutes

105

The tissues were then embedded in paraffin wax to prepare tissue blocks,

which were oriented so that sections could be cut in desired plane of the tissue.

Tissues were then fixed in cassette after trimming them to suitable size.

4.11.3 Section Cutting

A smear of 5% Mayer’s egg albumin was prepared and smeared onto the slide

and dried. The tissue sections of 4µM thickness were cut with the help of spencer type

rotating microtome. The tissue sections were put on slide and then sections were

floated in water on slide at 55-60°C, water was drained off and slides were dried on

hot plate at 50°C for 30 minutes. The sections were thus ready for staining.

Staining Procedure

Reagents

1) Mayer’s hematoxylin stain

2) Eosin stain, 2% w/v in alcohol

After fixing the sections on slides, they were stained by serially passing them through

following reagents:

Reagents Time

Xylol 3 minutes

Acetone 3 minutes


Haematoxylin stain 20 minutes

Running water 20 minutes

Eosin stain 5 minutes

Alcohol 95% (3 changes) 3 minutes each

Acetone (2 changes) 3 minutes each

Xylol (2 changes) 3 minutes each

After passing through all the above reagents and stains, the slides were

mounted with D.P.X. (Diphenyl Phthalate Xylene) and cover slip was placed. Care

was taken to avoid trapping air bubbles while mounting the slide.

4.11.4 Observation

Changes in histopathological characteristics were observed for all the slides

and photographs were taken.

106

4.12 Gene Expression Study

4.12.1 RNA Extraction

RNA extraction was performed by TRIzol method (Chomczynski and Sacchi,

1987). Total RNA extraction was performed from liver tissues from rats of all groups

after completion of the experiment (8th

or 15th

day liver tissue). Total RNA was

extracted using TRIzol reagent (Sigma-Aldrich). Each frozen liver sample (~100mg

of tissue) was crushed in liquid nitrogen with mortar and pestle and made into a fine

powder. The powdered tissue was added in 1mL of TRIzol reagent before thawing

and vortexed vigorously for 15 seconds. Chloroform (200µl) was added in these tubes

and the contents were gently mixed by inverting the tubes. The tubes were incubated

for 2 to 3 minutes at room temperature. The mixture was centrifuged at 12000 rpm for

15 minutes at 4°C. The aqueous phase was transferred carefully to a new tube,

without disturbing the interphase. Chilled isopropyl alcohol was added to the aqueous

phase in a new tube and incubated overnight at -20°C. Next day, the mixture was kept

for 10 minutes at room temperature. The mixture was centrifuged at 12000 rpm for 15

minutes at 4°C. The pellet was washed with 500µl 75% chilled ethanol (Freshly

prepared) and centrifuged at 7500 rpm for 5 minutes at 4°C. The supernatant was

discarded and pellet was suspended in 30µl diethylpyrocarbonate-treated water

(DEPC) water. RNA samples (2µl) were loaded on 0.8% agarose gel.

4.12.2 RNA Quantification and Quality Check

Quantification of RNA was performed with UV spectrophotometer

(NanoDrop; Eppendorf). RNA quantification and purity was checked at 260 nm, 230

nm and 280 nm using a spectrophotometer. RNA concentration was determined by

the measurement of absorbances at 260 nm, while the ratios 260/280 and 260/230

were recorded to detect contamination by phenols, proteins and other organic

compounds – ideally in pure RNA preparations both of these ratios would give a

value of 2. The isolated RNA with 260nm/280nm ratio between 1.5 to 2.0 is a

dimensionless parameter for RNA purity. Quality of RNA was checked on agarose

gel by ethidium bromide staining.

https://en.wikipedia.org/w/index.php?title=Piotr_Chomczy%C5%84ski&action=edit&redlink=1

107

4.12.3 cDNA Synthesis

Total RNA was reverse transcribed using the Super Script First-Strand cDNA

synthesis kit (Invitrogen, USA) according to the manufacturer’s instructions. First

strand synthesis of complementary DNA (cDNA) was done by reverse transcription.

Briefly, 4µg RNA was mixed with 3µl of random hexamer (50ng/µl) and 1µl of dNTP

(10mM) in a total volume of 12µl. The mixture was incubated at 65°C for 5 minutes.

After the incubation, the reaction was cooled rapidly on ice for 1 minute, followed by

addition of 4µl 5x first strand buffer (Promega), [250 mM Tris-HCl (pH 8.3), 375mM

KCl, 15mM MgCl2], 2µl 0.1M DTT (Invitrogen, USA) and 1µl RNaseOUT™

Recombinant ribonuclease inhibitor (40 units/µl, Invitrogen, USA). The tubes were

incubated at 37°C for 2 minutes followed by addition of 0.5μl M-MLV RT (200 units,

Promega). The contents of the reaction were mixed gently by pipetting up and down.

Reverse transcription was carried out using random priming and Moloney murine

leukaemia virus (MMLV) reverse transcriptase (RT) (Promega, USA), in a reaction

volume of 20μl. This RT also has an endogenous RNase H function which ensures

degradation of the RNA. Reverse transcription included the following three phases:

The reaction was incubated at 25°C for 10 minutes for RT enzyme activation

followed by 50 minutes at 37°C for reverse transcription and the reaction was

inactivated by heating at 70°C for 15 minutes. Products of the reaction were

appropriately diluted and subjected to end point PCR using house-keeping gene

GAPDH. The synthesized cDNA was stored at -80°C.

4.12.4 Semi-Quantitative Polymerase Chain Reaction

The cDNA was diluted with 1:40 Tris buffer (T10E1 buffer) (10 mM, Tris (pH

8.0), 1mM EDTA (pH 8.0) and used for semi-quantitative polymerase chain reaction

(PCR). The diluted cDNA was used for normalization using gene specific primers for

amplification of house-keeping gene GAPDH. For semi-quantitative PCR, normalized

concentration of 1:40 diluted cDNA was used for amplification in a 25µl reaction

consisting of 2.5µl 10X PCR buffer (Sigma- Aldrich, USA), 2μl 2.5mM dNTPs

(GeNei, India), 0.3μl Taq DNA polymerase (5U/μl, Sigma-Aldrich, USA), 0.5μl each

of forward and reverse KiCqStart® primers (10pM/μl stock) (Sigma-Aldrich, USA).

The temperature profile for semi-quantitative PCR was as below:

108

Initial denaturation at 94°C for 10 minutes, followed by 25 cycles, each

comprising 1-minute denaturation at 94°C, 30 seconds annealing temperature at 60°C

and 1-minute extension at 72°C with final extension at 72°C for 5 minutes followed

by incubation at 4°C. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene

was used as a control (endogenous or housekeeping gene) for normalization.

Expression analysis of fatty acid binding protein 1 (FABP1), peroxisome proliferator-

activated receptor gamma (PPARγ), sterol regulatory element binding protein

1 (SREBP1), nuclear factor kappaβ (NF-κβ) and tumor necrosis factor alpha (TNF-α)

was done from all the samples. Sigma KiCqStart® primers were used to study

modulation of gene expression. The primer sequences are listed in Table 7. The

amplified 25μl PCR products were resolved by electrophoresis on 1.5% agarose gel

(Low EEO Genei). The image was captured under a UV-transilluminator (Image

LabTM

software 4.1, Bio-Rad Laboratories, Inc). The bands were quantified or

compared by densitometry using ‘Image J’ analysis software V 1.41o (National

Institute of Health, Washington). Gene expression levels were normalized to those of

GAPDH.

Table 7. List of Primers Used for the Study

Gene Primer sequence Amplified

fragment

Annealing

temperature

GAPDH

F 5’-AGTTCAACGGCACAGTCAAG-3’

R 5’-TACTCAGCACCAGCATCACC-3’

136 60⁰C

FABP1

F 5’-TGGAGGGTGACAATAAAATG-3’

R 5’-TCATGGTATTGGTGATTGTG-3’

86 60⁰C

PPARγ

F 5’-AAGACAACAGACAAATCACC-3’

R 5’-CAGGGATATTTTTGGCATACTC-3’

195 60⁰C

SREBP1

F 5’- AAACCTGAAGTGGTAGAAAC-3’

R 5’-TTATCCTCAAAGGCTGGG-3’

142 60⁰C

NF-κβ

F 5’- AAAAACGAGCCTAGAGATTG-3’

R 5’-ACATCCTCTTCCTTGTCTTC-3’

157 60⁰C

TNF-α F 5’- CTCACACTCAGATCATCTTC-3’

R 5’-GAGAACCTGGGAGTAGATAAG-3’

194 60⁰C

109

4.13 Statistical Analysis

The data were presented as Mean ± Standard Error (SE). The Dunnett

Multiple Comparison Test and One Way Analysis of Variance (ANOVA) was done to

estimate the statistical significance between groups. GraphPad Instat (Trial Version

3.06, GraphPad Software, San Diego, CA, USA) was used for statistical analysis

while graphs were plotted using GraphPad Prism (Trial Version 5.0, GraphPad

Software, San Diego, CA, USA).

CHAPTER 5

RESULTS

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110

In the present study, satwa from three Tinospora forms were investigated with

reference to organoleptic characteristics, nutritional analysis and their

hepatoprotective potential. The hepatoprotective potential of satwa was assessed

against acetaminophen and alcohol induced hepatotoxicity in rats. Alteration in the

expression levels of the genes related to inflammation, fatty acid metabolism and

transcription factors, was also studied from liver tissues of the animals. Results

obtained in the study are presented in this chapter.

5.1 Organoleptic Characteristics

Satwa from three Tinospora forms was prepared as previously described under

“Materials and Methods”. The organoleptic characters, which correspond to the

panchagyanedriya pariksha (perception by five sense organs) as explained in

Ayurveda, were analysed for satwa from three different forms of Tinospora. The

organoleptic characters like rupa (colour), rasa (taste), gandha (smell/odour), and

sparsha (touch) were evaluated (Table 8.). In present study, satwa from T. cordifolia

was found tasteless. The satwa from T. sinensis and Neem-giloe were found to be

bitter in taste. The satwa from T. cordifolia was gray in colour while that of T.

sinensis and Neem-giloe was grayish white and yellowish white respectively (Fig. 9).

The details of organoleptic characteristics are given in Table 8.

Table 8. Organoleptic Characteristics of Guduchi Satwa from Three different forms

of Tinospora

Sr.No. Parameter T. cordifolia T. sinensis Neem-giloe

1 Fresh weight of

Guduchi stem (kg) 5 5 5

2 Total yield (gm) 74 80 74

3 % Yield 1.48 1.60 1.48

4 Rupa (Colour) Gray Grayish White YellowishWhite

5 Rasa (Taste) Tasteless Bitter Bitter

6 Gandha (Smell) No specific No specific No specific

7 Sparsha (Touch) Coarse Coarse Coarse

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111

Fig. 9. Guduchi Satwa from Three different forms of Tinospora

5.2 Nutritional Analysis of Three Tinospora forms

Several nutritional parameters were measured from the satwa of three

Tinospora forms and the results are shown in Table 9. Satwa from T. sinensis showed

significantly higher amount of protein (P≤0.001), starch, crude fiber and ash (P≤

0.01). The content of lipid was significantly low in T. sinensis satwa as compared

with of satwa from T. cordifolia (Fig. 10.). As compared to satwa of T. cordifolia, T.

sinensis satwa showed 72.45% more proteins, 153.21% more starch and 115% more

content of crude fibre. Among three satwa, highest amount of protein, starch, crude

fiber was seen in T. sinensis whereas maximum amount of carbohydrates and ash (P≤

0.01) content were found in satwa from Neem-giloe. Neem-giloe satwa was found to

have around 3 times more carbohydrates and 1.1 times higher content of ash as

compared to T. cordifolia. In the present study, satwa from Neem-giloe was found to

be significantly rich in carbohydrates (Fig. 11.) and ash, while satwa from T. sinensis

was found to be rich in protein (Fig. 12.), and starch (Fig. 13.).

112

Table 9. Nutritional Analysis of Satwa from Three Tinospora forms

The values are a mean of three replicates and are represented as Mean ± SE. Dunnett test was performed to test the significance of difference between

the nutritional parameters of three satwa. T. cordifolia was taken as a control for comparison. *P≤0.05; **P≤ 0.01; *** P ≤ 0.001 ns: Non-significant.

Species Proteins

(mg/g)

Carbohydrates

(mg/g)

Lipids

(mg/g)

Starch

(µg/g)

Crude Fiber

(gm%)

Ash

(mg/gm)

T. cordifolia 21.06±0.02 272.28±27.67 18.67±2.01 16.33±1.20 0.2±0.04 88.43±0.01

T. sinensis 36.31±0.66*** 333.16±8.11ns

11±0.97** 41.35±2.95** 0.43±0.05** 96.07±0.02**

Neem-giloe 18.48±0.45** 797.11±11.30** 21.33±0.92ns

10.88±2.58 ns

0.22±0.01ns

98.21±0.01**

113

Fig. 10. Levels of Lipids in T. cordifolia, T. sinensis and Neem-giloe

The data were expressed as Mean ± SE. T. cordifolia was taken as a control for comparison.

**P≤ 0.01.

Fig. 11. Levels of Carbohydrate in T. cordifolia, T. sinensis and Neem-giloe


**P≤ 0.01.

114

Fig. 12. Levels of Protein in T. cordifolia, T. sinensis and Neem-giloe


**P≤ 0.01; *** P≤ 0.001.

Fig. 13. Levels of Starch in T. cordifolia, T. sinensis and Neem-giloe


**P≤ 0.01.

115

5.3 Hepatoprotective Activity of Satwa from Three different forms of Tinospora

5.3.1 Hepatoprotective Activity of Satwa against Acetaminophen Induced

Hepatotoxicity

In the present study, comparative hepatoprotective potential of T. cordifolia, T.

sinensis and Neem-giloe satwa was evaluated by assessing serum levels of SGOT,

SGPT, ALP and total bilirubin. The liver tissues were also processed for biochemical,

histological and molecular studies.

The animals of acetaminophen treated group showed elevated levels of SGOT,

SGPT, ALP and bilirubin, as compared to healthy animals (Table 10.) indicating

successful induction of hepatotoxicity. Satwa of T. cordifolia, T. sinensis and Neem-

giloe indicated differential hepatoprotective activity in rats treated with

acetaminophen. T. cordifolia was found to normalize lipid profile in acetaminophen

treated animals. T. cordifolia treated group exhibited improvement in the contents of

total cholesterol (46.57±6.1 mg/dL) with approx. 47% decrease (P≤0.01) as compared

to acetaminophen treated group. The alterations in the content of HDL (11.86±2.4

mg/dL; with 45% increase) and LDL (16.07±8.6 mg/dL; with approx. 70% decrease)

were not found to be statistically significant as compared to acetaminophen treated

group. The cholesterol, HDL and LDL in T. cordifolia treated group were comparable

with the group treated with silymarin. Animals treated with satwa of T. sinensis

exhibited improvements in ALP (P≤0.01), VLDL and triglyceride levels as compared

to acetaminophen treated group. The animals treated with the satwa of Neem-giloe

showed improvement in ALP (P≤0.05). The levels of VLDL and triglycerides in T.

sinensis treated group were found to be comparable to positive control.

The liver tissues of the animals treated with satwa also showed improvements

at biochemical, histological and molecular levels. Statistically non-significant

decrease in hepatic lipid peroxidation (MDA levels) was found in animals treated with

T. cordifolia (47.47% reduction), T. sinensis (47.47% reduction) and Neem-giloe

(43.65% reduction) satwa, as compared to acetaminophen treated animals. The levels

of reduced glutathione (GSH) were found to be significantly increased in T. cordifolia

(P≤0.05) as compared to that of negative control. T. sinensis and Neem-giloe

improved SOD and catalase activities (P≤0.01) as compared to acetaminophen treated

116

animals (Table 11).The total protein and lipid content of liver tissues showed

significant improvement (P≤0.01) in Neem-giloe treated group as compared to

acetaminophen treated animals (Table 11). T. cordifolia, T. sinensis and Neem-giloe

satwa treated animals showed 15.2%, 19.2% and 42.4% decrease in LDL (P≤0.01)

and 56.84%, 58.93% and 64.73% decrease in VLDL (P≤0.01) respectively as

compared to animals from negative control group. T. cordifolia, T. sinensis and Neem-

giloe treated group showed 56.83%, 58.99% and 64.94% decrease in triglycerides

respectively as compared to the negative control group (P≤0.01) (Table 11).

Liver from healthy (Fig. 14A) group showed normal architecture. Sections of

liver from acetaminophen treated group (Fig. 14B) showed mild conjunction of

central vein and apoptotic death of hepatocytes. Intra and extracellular hyaline

globules were seen around central vein. The hepatocytes surrounding the central vein

showed ballooning and degeneration along with mild nucleomegaly. Liver from

silymarin treated group (Fig. 14C) showed near normal liver architecture with a few

swollen hepatocytes. The differential hepatoprotective effects of Guduchi satwa

prepared from three Tinospora forms were also evident from liver histology (Fig.

14D-F). The liver histology of the animals treated with T. cordifolia satwa exhibits

improvements over acetaminophen treated group (Fig. 14D) but intermittently

swollen centrilobular hepatocytes were visible in liver sections which are known to be

more prone to ischemic injury. The periportal hepatocytes were normal in animals

treated with satwa of T. cordifolia. The liver histology of the group treated with T.

sinensis exhibited near normal histology (Fig. 14E) with prominent hepato-

regeneration as evident from distribution of normal hepatocytes among degenerating

swollen hepatocytes. This group also showed normal periportal hepatocytes. The liver

histology of Neem-giloe satwa treated group (Fig. 14F) was strikingly normal without

any histologically detectable anomalies.

Fig. 15A-E depicts the modulation of expression levels of the genes from liver

tissues of animals treated with the satwa of three different forms of Tinospora.

Expression levels of FABP1 (Fig. 15A) and PPARγ (Fig. 15B) were found to be

decreased in acetaminophen induced hepatotoxicity. Treatment with the satwa of

Neem-giloe significantly improved (P≤0.05) the expression of FABP1. The increase

in the expression level of PPARγ observed in satwa treated groups was statistically

not significant. NF-κβ, SREBP1 and TNF-α were up-regulated in acetaminophen

117

induced hepatotoxicity. NF-κβ and SREBP1 were significantly down-regulated

(P≤0.01 or P≤0.001) in groups treated with T. cordifolia, T. sinensis and Neem-giloe

(Fig. 15C-D). Expression in TNF-α, though down-regulated in treatment groups, was

not found to be statistically significant than that of negative control.

From these results, it is evident that from the three satwa, treatment with

Neem-giloe exhibited improvements in several parameters like liver function markers,

serum and hepatic lipid profile, hepatic oxidative stress and it also had beneficial

effects on normalizing liver histology and expression of genes from lipid metabolism

and inflammatory pathways.

118

Table 10. Effect of Satwa from Three Tinospora forms Species on Liver Function Markers and Serum Lipid Profile in Animals with


Group I is healthy control while Group II and III served as negative and positive control respectively. Group IV, V and VI were treated with 200mg/kg b.w. T.

cordifolia, T. sinensis and Neem-giloe satwa (p.o.) respectively. Results are a mean of three replicates and are represented as mean ± standard error (SE). The

data were subjected to Dunnett Multiple Comparison Test. *P≤0.05; **P≤0.01; Degrees of freedom for one variable: Treatments 5; Residuals 24; Total 29;

SGOT: serum glutamic oxaloacetic transaminase; SGPT: serum glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL: High

density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group

SGOT

(U/mL)

SGPT

(U/mL)

ALP

(U/mL)

BIL

(mg/dL)

Lipid Profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 129.38±11.8* 151.64±6.3 29.94±5.7

** 0.84±0.2

* 59.00±4.6

** 22.75±5.2 20.38±7.7 15.87±2.1 79.36±10.5

II 186.10±9.7 183.40±3.9 53.96±4.4 1.56±0.1 87.45±7.9 6.49±0.7 53.33±4.9 27.62±3.6 138.12±18.1

III 128.14±20.4* 175.12 ±3.7 26.31±4.1

** 1.12±0.3 42.65±5.4

** 9.16±0.9 18.11±3.2 15.38±4.1 76.89±20.6

IV 148.98±11.3 178.10±4.6 36.77±2.9 1.39±0.1 46.57±6.1**

11.86±2.4 16.07±8.6 18.64±3.3 93.22±16.3

V 150.71±8.5 176.60±4.4 22.13±6.5**

1.55±0.2 56.40±2.6**

11.57±1.2 28.39±4.8 16.43±2.6 82.15±13

VI 147.43±18.9 182.10±1.1 33.56±2.9* 1.05±0.1 54.37±4.5

** 9.29±0.5 25.39±6.7 19.68±2.5 98.43±12.3

119

Table 11. Effect of Satwa from Three Tinospora forms on Hepatic Oxidative Stress Markers, Total Protein and Hepatic Lipid Profile in Animals with


Group I is healthy control while Group II and III served as negative and positive control respectively. Group IV, V and VI were treated with 200mg/kg b.w. T. cordifolia, T.

sinensis and Neem-giloe satwa (p.o.) respectively. Results are a mean of three replicates and are represented as mean ± standard error (SE). The data were subjected to

Dunnett multiple comparison test. *P≤ 0.05; **P ≤0.01; Degrees of freedom for one variable: Treatments 5; Residuals 24; Total 29; SOD: Superoxide dismutase; CAT:

Catalase; MDA: Malondialdehyde; GSH: Reduced glutathione; TC: Total cholesterol; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low

density lipoprotein; TG: Triglycerides.

Group SOD

(U/mg)

CAT

(mU/g Tissue)

MDA

(μM)

GSH

(μM)

Total

Protein

(mg/g Tissue)

Lipid Profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 68.24±0.8**

0.66±0.5**

5.5±1.0* 0.26±0.03

** 5.36±0.2 24.61±2.2

* 15.04±0.8

** 6.4±0.1

** 1.34±0.1

** 6.7±0.6

**

II 49.60±1.1 0.57±1.1 12.3±1.6 0.09±0.00 2.97±0.31 34.35±0.4 8.54±1.1 12.50±0.2 6.21±0.2 31.09±1.1

III 72.36±1.4**

0.68±0.3**

8.36±1.1 0.05±0.04 2.93±0.08 20.91±3.8**

6.49±0.34 6.5±0.2**

2.10±0.1**

10.5±0.8*

IV 73.25±1.4**

0.65±0.2**

6.46±1.4 0.2±0.02*

4.51±1.1 23.31±1.5**

7.00±0.7 10.6±0.3**

2.68±0.1**

13.42±0.6**

V 73.08±1.17**

0.64±0.7**

6.46±2.8 0.12±0.01 4.72±0.6 31.59±2.1 11.28±1.2 10.1±0.1**

2.55±0.1**

12.75±0.9**

VI 74.05±2.1**

0.66±0.6**

6.93±1.3 0.11±0.04 9.41±0.9**

26.36±0.5 12.3±1.7**

7.2±0.4**

2.19±0.1**

10.9±0.9**

120

Fig. 14. Effects of T. cordifolia, T. sinensis and Neem-giloe Satwa on Liver Histology

in Animals with Acetaminophen Induced hepatotoxicity

Cross sections of paraffin embedded liver tissues (4µm thick) of rats from control and

experimental groups were stained with hematoxylin and eosin and observed under light

microscope (20X) and photographed. Liver sections from A: Healthy animals, B:

Acetaminophen treated animals, C: Silymarin treated animals, D: T. cordifolia satwa treated

animals, E: T. sinensis treated animals and F: Neem-giloe satwa treated animals.

121

Fig. 15. Gene Expression from Liver Tissues of Experimental Animals

Densitometric Analysis of Expression was done by using GAPDH and gene specific

expression data. *P≤0.05; **P≤0.01; ***P≤0.001 compared with negative control rats. Lanes:

Healthy control (Group I) -1; Negative control (Group II) -2; Positive control (Group III) -3;

T. cordifolia satwa treated rats (Group IV) -4; T. sinensis satwa treated rats (Group V) -5;

Neem-giloe satwa treated rats (Group VI) -6. GAPDH (Internal standard). A: FABP1, B:

PPARγ, C: NF-κβ, D: SREBP1, E: TNF-α.

A B

I II III IV V VI

0.0

0.2

0.4

0.6

0.8

1.0 *F

AB

P1

/GA

PD

H

I II III IV V VI0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 ***

PP

AR

/GA

PD

H

C D

I II III IV V VI0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

******

** **

NF

-

/G

AP

DH

I II III IV V VI0.0

0.2

0.4

0.6

0.8

1.0

** ** **

SR

EB

P1

/GA

PD

H

E Data 5

I II III IV V VI0.0

0.5

1.0

1.5

2.0

TN

F-

/GA

PD

H

122

5.3.2 Hepatoprotective Activity of Satwa against Ethanol Induced

Hepatotoxicity

In the present study, comparative hepatoprotective potential of T. cordifolia, T.

sinensis and Neem-giloe satwa were estimated by evaluating serum SGOT, SGPT,

ALP and total bilirubin. The animals from negative control (ethanol treated) group

showed significant increase in the levels of SGOT, SGPT, ALP, bilirubin and lipid

profiles as compared to healthy control group indicating successful induction of liver

injury (Table 12). The satwa of T. cordifolia, T. sinensis and Neem-giloe exhibited

hepatoprotective activity against ethanol induced hepatotoxicity. Significant

improvements in lipid profile were observed in the animals treated with the satwa of

Neem-giloe. The experimental group treated with Neem-giloe showed significant

decrease in total cholesterol, LDL, VLDL and triglycerides (P≤0.01) and increase in

HDL (P≤0.01) over 30% ethanol treated group. The lipid profile from Neem-giloe

treated animals was comparable with the animals treated with standard drug

silymarin. Animals treated with the satwa of T. sinensis showed marked

improvements in liver function markers (P≤0.01) as compared to the animals with

30% ethanol treated group. The lipid profile in the animals treated with T. sinensis

satwa also exhibited improvements (P≤0.05 or P≤0.01) as compared to the animals

treated with 30% ethanol. The animals treated with the satwa of T. cordifolia

exhibited improvements in lipid profile and liver function test markers as compared to

the animals treated with 30% ethanol.

Liver tissues of animals treated with three different satwa exhibited alterations

at biochemical, histological and molecular levels. The level of lipid peroxidation

(MDA levels) was significantly decreased in animals treated with T. sinensis satwa

(84.56% reduction) as compared to 30% ethanol treated animals (P≤0.01). The levels

of reduced glutathione (GSH) were also significantly increased in Neem-giloe

(P≤0.01; increase by 11.11%) and T. sinensis (P≤0.05; increase by 8.33%) than

ethanol treated animals (Table 13). T. cordifolia, T. sinensis and Neem-giloe improved

SOD and catalase activities (P≤0.01) as compared to ethanol treated animals (Table

13). The total protein content of liver tissue significantly improved (P≤0.01) in T.

cordifolia, T. sinensis and Neem-giloe satwa treated groups as compared to negative

control. Hepatic lipid profile (Table 13) displayed significant improvements in

123

animals treated with three different satwa. Total cholesterol, LDL, VLDL and TG

contents improved significantly (P≤0.01) by the treatments as compared to negative

control. Significant increase in HDL was seen only in animals treated with T. sinensis

satwa.

The differential hepatoprotective effects of satwa prepared from these three

Tinospora forms were also evident from liver histology (Fig. 16A-F). Liver from

healthy (Fig. 16A) group exhibited normal architecture. Sections of liver from alcohol

treated group (Fig. 16B) had swollen hepatocytes with granular cytoplasm.

Collections of few polymorphs were seen in hepatic parenchyma, suggesting small

foci of necrosis. Liver from silymarin treated group (Fig. 16C) also displayed near

normal liver architecture. The liver tissues of animals treated with T. cordifolia satwa

(Fig. 16D) showed collection of lymphocytes within the hepatic parenchyma. Kupffer

cells were seen prominent in this group and portal triad showed lymphocyte

infiltration. The liver histology of the group treated with T. sinensis satwa was found

to be absolutely normal (Fig. 16E). The liver histology of Neem-giloe satwa treated

group (Fig. 16F) had some portal triads and sinusoids showing collection of

lymphocytes.

Fig 17A-E shows the effect of three different forms of Tinospora on FABP1,

PPARγ, NF-κβ, SREBP1 and TNF-α expression in ethanol induced hepatotoxicity in

rats. The expression of FABP1 (Fig. 17A) and PPARγ (Fig. 17B), were down-

regulated while expression levels of NF-κβ (Fig. 17C), SREBP1 (Fig. 17D) and TNF-

α (Fig. 17E) were increased in ethanol induced hepatotoxicity as compared to healthy

control group. FABP1 expression was significantly up-regulated (P≤0.001) by

treatment with T. sinensis and Neem-giloe (Fig. 17A). Treatment with Neem-giloe

leads to significant down-regulation while treatment with T. sinensis showed

significant up-regulation of PPARγ expression (Fig. 17B). Expression of NF-κβ was

not altered in the positive control group while all treatment groups exhibited

significantly reduced (P≤0.001 or P≤0.001) expression of NF-κβ. Similarly,

expression of TNF-α was marginally reduced in positive control group while the three

treatment groups showed significant decrease (P≤0.001) in the expression levels of

TNF-α (Fig. 17E). Treatment with Neem-giloe lead to significant down-regulation

(P≤0.05) of SREBP1 (Fig. 17D).

124

From the results, it is evident that from the three satwa, treatment with T.

sinensis exhibited improvements in several parameters like liver function markers,

serum and hepatic lipid profile, hepatic oxidative stress and it also had beneficial

effects on normalizing liver histology and expression of genes from lipid metabolism


125

Table 12. Effect of Satwa from Three Tinospora forms on Liver Function Markers and Serum Lipid Profile in Animals with Ethanol Induced

Hepatotoxicity

Group I is healthy control while Group II and III served as negative and positive control respectively. Group IV, V and VI were treated with 200mg/kg b.w. T.

cordifolia, T. sinensis and Neem-giloe satwa (p.o.) respectively. Results are a mean of three replicates and are represented as Mean ± Standard Error (SE). The

data were subjected to Dunnett Multiple Comparison Test. *P≤0.05; **P≤0.01; Degrees of freedom for one variable: Treatments 5; Residuals 30; Total 35;

SGOT: serum glutamic oxaloacetic transaminase; SGPT: serum glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL: High


Group

SGOT

(U/mL)

SGPT

(U/mL)

ALP

(U/mL)

BIL

(mg/dL)

Lipid profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 134.49±3.0**

154.67±2.4**

30.40±1.3**

1.13±0.1**

85.89±1.6**

19.31±0.5**

17.1±1.3**

27.71±0.5**

138.54±2.5**

II 162.31±0.8 182.50±2.5 55.89±2.7 2.11±0.2 203.36±7.2 13.51±1.0 40.2±1.0 74.37±0.8 371.87±3.9

III 137.56±1.5**

174.77±1.0 33.33±0.7**

1.14±0.1**

110.09±2.6**

14.02±0.5 20.1±0.6**

39.9±0.7**

199.48±3.3**

IV 138.85±1.1**

144.08±2.0**

36.52±1.4**

0.91±0.1**

128.84±2.7**

13.84±0.5 18.6±0.8**

48.28±0.8**

241.40±3.9**

V 131.92±2.7**

131.58±2.9**

32.90±0.9**

0.71±0.02**

132.05±3.3**

16.41±0.7* 19.2±0.9

** 46.09±1.0

** 230.47±5.0

**

VI 139.49±2.9**

150.83±2.4**

36.62±0.5**

0.93±0.04**

110.89±2.1**

16.58±0.4**

17.4±1.0**

39.27±0.7**

196.36±3.3**

126

Table 13. Effect of Satwa from Three Tinospora forms on Hepatic Oxidative Stress Markers, Total Protein and Hepatic Lipid Profile in Animals

with Ethanol Induced Hepatotoxicity

Group I is healthy control while Group II and III served as negative and positive control respectively. Group IV, V, and VI were treated with 200mg/kg b.w.

T. cordifolia, T. sinensis and Neem-giloe satwa (p.o.) respectively. Results are a mean of three replicates and are represented as Mean ± Standard Error (SE).

The data were subjected to Dunnett Multiple Comparison Test. *P≤0.05; **P≤0.01; Degrees of freedom for one variable: Treatments 5; Residuals 30; Total

35. SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; GSH: Reduced glutathione; TC: Total cholesterol; HDL: High density lipoprotein;

LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group SOD

(U/mg)

CAT

(mU/g Tissue)

MDA

(μM)

GSH

(μM)

Total

Protein

(mg/g Tissue)

Lipid Profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 66.23±0.4**

0.84±0.03**

5.70±0.4**

0.82±0.02**

4.51±0.1**

27.42±1.4**

14.36±0.4 6.30±0.4**

4.64±0.1**

23.17±0.7**

II 14.56±0.5 0.56±0.03 17.49±0.6 0.72±0.01 3.38±0.07 39.18±1.7 11.79±0.3 16.50±0.5 10.47±0.1 49.27±2.6

III 15.45±0.8**

0.48±0.03 14.00±0.5**

0.72±0.02 4.27±0.01**

29.81±0.4**

13.67±0.5 5.70±0.7**

7.13±0.01**

35.68±0.4**

IV 25.20±0.6**

0.71±0.01**

13.44±0.7**

0.52±0.01**

4.18±0.08**

30.00±1.4**

14.02±0.6 8.40±0.3**

7.24±0.1**

36.2±0.4**

V 26.20±0.1**

0.72±0.02**

2.49±0.5**

0.78±0.01* 4.67±0.1

** 28.17±0.3

** 15.72±1.2

** 6.90±0.7

** 4.58±0.1

** 22.91±0.6

**

VI 27.20±0.3**

0.73±0.01**

12.25±0.7**

0.8±0.01**

5.34±0.1**

30.74±1.6**

13.65±0.3 6.60±0.3**

7.03±0.1**

35.15±0.7**

127

Fig. 16. Effects of T. cordifolia, T. sinensis and Neem-giloe Satwa on Liver Histology

in Animals with Ethanol Induced Hepatotoxicity



microscope (20X) and photographed. Liver sections from A: Healthy animals, B: Ethanol

treated animals, C: Silymarin treated animals, D: T. cordifolia satwa treated animals, E: T.

sinensis satwa treated animals and F: Neem-giloe satwa treated animals.

128


Densitometric analysis of expression was done by using GAPDH and gene specific

expression data. *P≤0.05; **P≤0.01; ***P≤0.001 compared with negative control rats; Lanes:


T. cordifolia satwa treated rats (Group IV) - 4; T. sinensis satwa treated rats (Group V) -5;

Neem-giloe satwa treated rats (Group VI) -6. GAPDH (Internal standard). A: FABP1, B:

PPARγ, C: NF-κβ, D: SREBP1, E: TNF-α.

A B

I II II IV V VI0.0

0.2

0.4

0.6

0.8

1.0***

******

FA

BP

1/G

AP

DH

I II III IV V VI0.0

0.2

0.4

0.6

0.8

1.0

1.2

PP

AR/

GA

PD

H

***

**

***

**

C D

I II III IV V VI0.0

0.4

0.8

1.2

1.6

***

** *** ***

NF

-

/GA

PD

H

I II III IV V VI0.0

0.4

0.8

1.2

1.6

* *

**

SR

EB

P1

/GA

PD

H

E

I II III IV V VI0.0

0.4

0.8

1.2

1.6

*** *** ***

**

**

TN

F-

/GA

PD

H

129

5.4 Hepatoprotective Activity of Flax Oil and Fish Oil (Polyunsaturated fatty

acids)

In the present study, nutritional interventions (Polyunsaturated fatty acids)

were assessed for their hepatoprotective activity. The hepatoprotective potential of

flax oil and fish oil against acetaminophen and alcohol induced hepatotoxicity in rats

was studied. Alteration in the expression levels of the genes related to inflammation,

fatty acid metabolism, and transcription factors, were also studied from liver tissues of

the animals. Results obtained in the study are presented below.

5.4.1 Hepatoprotective Activity of Flax Oil and Fish Oil against


In the present study, serum SGOT, SGPT, ALP, bilirubin and lipid profile

from negative control group showed a sharp and significant shift as compared to a

healthy control group, indicating successful induction of hepatotoxicity by repeated

dosing of acetaminophen (Table 14). Administration of flax oil and fish oil at a dose

of 500mg/kg b.w./day had a beneficial effect on the levels of these biochemical

markers in the treatment groups. SGOT, SGPT and ALP activities were found to be

elevated in the negative control group as compared to healthy control. Animals treated

with the fish oil showed significant improvement in SGOT (P≤0.01) and SGPT

(P≤0.05) and flax oil showed significant decrees in SGOT (P≤0.05) as compared with

negative control group. Treatment with silymarin helped to lower the activities of

these enzymes. As compared to negative control group, flax oil and fish oil displayed

remarkable benefits in relation to improvements of lipid profiles (Table 14). Flax oil

and fish oil treated groups showed decrease in total cholesterol (P≤0.01), LDL

(P≤0.01) as compared to the negative control group. Significant (P≤0.05) increase in

HDL was seen only in the animals treated with fish oil (Table 14). Silymarin treated

groups showed decreased in total cholesterol (P≤0.01), LDL (P≤0.01), VLDL

(P≤0.01), triglycerides (P≤0.01).

Flax and fish oil supplementation was found to be significantly effective in

ameliorating the oxidative damage through decrease in the extent of lipid peroxidation

and increase in the contents of reduced glutathione which acts as a principal

antioxidant compound in liver (Table 15). The extent of lipid peroxidation (MDA

130

levels) was significantly decreased in flax oil and fish oil (P≤0.001) treated animals as

compared to acetaminophen treated animals while the levels of reduced glutathione

(GSH) were significantly increased in flax oil and fish oil supplemented groups

(P≤0.05) as compared to acetaminophen treated animals. Flax oil and fish oil

supplementation improved SOD and catalase activities (P≤0.01 or P≤0.001) as

compared to acetaminophen treated animals (Table 15). The total protein content of

liver tissues significantly improved in flax oil and fish oil treated groups as compared

to acetaminophen treated group (P≤0.01). The lipid profile of liver also displayed

significant improvements in flax oil and fish oil treated groups when compared with

negative control group. Flax oil and fish oil treated groups displayed decrease in total

cholesterol (P≤0.01), LDL (P≤0.001), and VLDL (P≤0.01). The treatment of animals

with flax oil and fish oil also decreased triglycerides (P≤0.01) as compared to the

negative control group. Significant (P≤0.01) increase in HDL was seen only in the

animals treated with flax oil (Table 15).

The hepatoprotective effects of flax oil and fish oil were also evident from the

prominent variations in liver histology (Fig. 18A-E). The healthy control group (Fig.

18A) displayed normal liver histology while the acetaminophen treated group (Fig.

18B) showed swollen or occasionally apoptotic hepatocytes with coarse granular

cytoplasm and compressed sinusoids. The group also displayed a few nucleated cells

indicating spontaneous regeneration of hepatocytes. The group treated with the

standard drug silymarin (Fig. 18C) had near normal liver histology with mild

congestion of central vein and mildly swollen hepatocytes. Both flax oil and fish oil

treated groups (Fig. 18D-E) exhibited strikingly normal liver histology without any

anatomically detectable anomalies.

Fig. 19A-E show the effect of flax and fish oil on the expression of FABP1,

PPARγ, NF-κβ, SREBP1 and TNF-α. The expression of FABP1 (Fig. 19A) and

PPARγ (Fig. 19B) was decreased in acetaminophen treated animals than in healthy

animals. Though treatment with flax oil and fish oil up-regulated the expression of

FABP1 (Fig. 19A), it was found to be statistically insignificant. PPARγ (Fig. 19B)

was significantly up-regulated (P≤0.05) in animals treated with fish oil. NF-κβ (Fig.

19C), SREBP1 (Fig. 19D) and TNF-α (Fig.19E) were up-regulated in acetaminophen

treated animals. Treatment with flax oil (Fig. 19C) normalized the expression of NF-

κβ (P≤0.05) (Fig. 19C) while the SREBP1 (Fig. 19D) and TNF-α (Fig. 19E)

131

expressions were significantly down regulated by both flax and fish oil (P≤0.05 or

P≤0.01).

From the results, it is evident that treatment with fish oil exhibited

improvements in several parameters like liver function markers, serum and hepatic

lipid profile, hepatic oxidative stress and it also had beneficial effects on normalizing

liver histology and expression of genes from lipid metabolism fatty acid metabolism


132

Table 14. Effect of Flax Oil/Fish Oil on Liver Function Markers and Serum Lipid Profile in Animals with Acetaminophen Induced

Hepatotoxicity

Group I is healthy control while Group II and III served as negative and positive control respectively. Group IV and V were treated with 500mg/kg b.w. flax

oil and fish oil (p.o.) respectively. Results are a mean of three replicates and are represented as Mean ± Standard Error (SE). The data were subjected to

Dunnett Multiple Comparison *P≤0.05; **P≤0.01; Degrees of freedom for one variable: Treatments 4; Residuals 25; Total 29. SGOT: serum glutamic

oxaloacetic transaminase; SGPT: serum glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; TC: Total cholesterol; HDL: High


Group

SGOT

(U/mL)

SGPT

(U/mL)

ALP

(U/mL)

BIL

(mg/dL)

Lipid Profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 132.83±8.5 150.46±4.3**

30.17±3.3 0.71±0.1 56.86±8.8 22.32±9.2 18.71±5.0 15.83±3.9 79.17±8.1*

II 191.84±6.9 181.63±2.9 53.75±3.20 1.59±0.08 91.06±0.7 6.32±1.9 57.20±1.7 27.54±0.4 137.72 ±0.8

III 131.88±14.1**

173.15±2.3 26.04±2.7**

1.15±0.2 43.61±8.8**

9.29±2.2 19.16±6.0**

15.16±4.6**

75.82±9.5**

IV 146.60±11.9* 177.56

±0.6 42.61±1.5 1.23±0.3 54.51 ±3.8

** 11.79±4.0 16.67 ±6.9

** 26.06±3.3 130.28 ±6.7

V 100.83±3.7**

168.13±2.7* 41.90±3.8 0.78±0.0 67.47±6.1

** 15.14±2.2

* 25.69 ±9.4

** 26.64±5.9 133.22±12.1

133

Table 15. Effect of Flax Oil/Fish Oil on Hepatic Oxidative Stress Markers, Total Protein in Animals with Acetaminophen Induced

Hepatotoxicity


oil and fish oil (p.o.) respectively. Results are a mean of three replicates and are represented as Mean ± Standard Error (SE). The data were subjected to

Dunnett Multiple Comparison *P≤0.05; **P≤0.01; *** P≤0.001; Degrees of freedom for one variable: Treatments 4; Residuals 25; Total 29. SOD:

Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; GSH: Reduced glutathione; TC: Total cholesterol; HDL: High density lipoprotein; LDL:

Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group SOD

(U/mg)

CAT

(mU/g Tissue)

MDA

(μM)

GSH

(μM)

Total

Protein

(mg/g Tissue)

Lipid Profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 68.5±1.0**

0.84±0.0**

11.7±0.5* 0.88±0.0

* 5.9±1.2

** 23.5±0.7 15.6±0.3 4.54±1.0

*** 3.37±0.5 16.87±2.7

II 42.1±3.10 0.38±0.03 22.1±0.6 0.33±0.10 3.7±1.1 26.09±0.8 8.21±0.2 10.7±0.9 7.12±1.04 35.5±5.1

III 20.2±0.6**

0.69±0.03**

17.09±0.4**

0.61±0.1 5.6±0.8**

18.90±0.3**

11.61±0.1**

1.54 ±0.5***

5.74±0.5 28.71±2.8

IV 50.7±1.5**

0.90±0.02**

7.69±0.7***

0.81±0.01* 5.3±1.2

** 16.67±0.6

** 11.1±0.5

** 2.41±0.5

*** 3.10±0.6

** 15.4±3.1

**

V 68.5±0.0**

1.38±0.05***

3.40±0.2 ***

0.86±0.1* 5.4±1.8

** 17.55±0.4

** 9.7 ±0.4 5.07±0.7

*** 2.74±0.5

** 13.6±2.6

**

134

Fig. 18. Effects of Polyunsaturated Fatty Acids (Flax oil and fish oil) on Liver

Histology in Animals with Acetaminophen Induced Hepatotoxicity



microscope (20X). Liver from A: Healthy animals, B: Alcohol treated animals, C: Silymarin

treated animals, D: Flax oil treated animals, E: Fish oil treated animals.

135



expression data. *P≤0.05; **P≤0.01 compared with negative control rats. Lanes: Healthy

control (Group I) -1; Negative control (Group II) -2; Positive control (Group III) -3; Flax oil

treated rats (Group IV) -4; Fish oil treated rats (Group V) -5; GAPDH (Internal standard). A:

FABP1, B: PPARγ, C: NF-κβ, D: SREBP1, E: TNF-α.

A B

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

1.2

FA

BP

1/G

AP

DH

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

1.2*

PP

AR/

GA

PD

H

C D

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

*

*

NF

-

/GA

PD

H

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

****

*

SR

EB

P1

/GA

PD

H

E

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

** *

TN

F-

/GA

PD

H

136

5.4.2 Hepatoprotective Activity of Flax Oil and Fish Oil against Ethanol Induced

Hepatotoxicity

In the present study, activities of serum SGOT, SGPT, ALP, bilirubin and

lipid profile from negative control group showed a sharp and significant increase as

compared to a healthy control group indicating successful induction of hepatic

damage by repeated dosing of 30% alcohol (Table 16). Administration of flax oil and

fish oil at a dose of 500mg/kg b.w./day had a favorable effect on the levels of these

biochemical markers in the treatment groups.

Animals treated with flax oil and fish oil exhibited improvements in SGOT,

SGPT, ALP and bilirubin (P≤0.01) levels but the animals treated with the fish oil had

statistically insignificant improvement in SGOT as compared to ethanol treated group.

Treatment with silymarin helped to decrease the activities of these enzymes (Table

16). As compared to negative control group, flax oil and fish oil treatment exhibited

improvement in lipid profiles in the animals with ethanol induced liver damage. Flax

oil and fish oil treated groups displayed decrease in total cholesterol (P≤0.01), LDL

(P≤0.01), VLDL (P≤0.01), and triglycerides (P≤0.01) as compared to the negative

control group. Significant (P≤0.01) increase in HDL was seen in the animals treated

with flax oil and fish oil (Table 16).

The lipid peroxidation (MDA levels) was significantly reduced in flax oil and

fish oil (P≤0.05 or P≤0.01) treated animals. Flax oil and fish oil treatment did not

show any effect in improving the levels of reduced glutathione (GSH) as compared to

alcohol treated animals (Table 17). SOD and catalase activities were found to be

significantly increased in flax oil and fish oil treated animals (P≤0.01) as compared to

negative control group (Table 17). The total protein content of liver tissues was

improved only in flax oil (P≤0.01) treated group as compared to ethanol group. The

lipid profile of liver also displayed improvements in flax and fish oil treated groups

when compared with negative control. Flax and fish oil treated groups displayed

decrease in total cholesterol, LDL, VLDL and triglycerides (P≤0.01 for all lipid

profile parameters) as compared to negative control. Significant increase in HDL

(P≤0.01) was seen in the animals treated with flax oil and fish oil (Table 17).

137

The effects of treatment with flax oil and fish oil were also visible at

histological levels in animals. Liver from healthy (Fig. 20A) group showed normal

architecture. Sections of liver from ethanol treated group (Fig. 20B) exhibited

coarsely condensed cytoplasm in some cells with prominent coarse granularity in

other cells, the sinusoid appeared compressed and hence the trabecular pattern of

arrangement appeared disrupted. The hepatocytes were found to be swollen.

Cytoplasmic borders were distinct. Liver from silymarin treated group (Fig. 20C) had

near normal liver architecture. The liver histology of the animals treated with flax oil

(Fig. 20D) exhibited absolutely normal histology. The liver histology of the group

treated with fish oil (Fig. 20E) exhibited improvements in the architecture except a

few portal triad areas with collection of lymphocytes.

Fig. 21A-E, shows effect of flax oil and fish oil on the expression of FABP1,

PPARγ, NF-κβ, SREBP1 and TNF-α. Expression of FABP1 (Fig. 21A) and PPARγ

(Fig. 21B) decreased in ethanol treated animals than in healthy animals. Treatment

with flax oil showed marginal improvement in the expression of FABP1 (Fig. 21A)

but it was found to be statistically insignificant. PPARγ (Fig. 21B) was significantly

up-regulated (P≤0.001, 2.16 fold) in animals treated with fish oil and in animals

treated with flax oil (P≤0.05). NF-κβ (Fig. 21C), SREBP1 (Fig. 21D) and TNF-α (Fig.

21E) were up-regulated in ethanol treated animals than in healthy animals. Treatment

with flax oil normalized the expression of NF-κβ (P≤0.05) (Fig. 21C) while

expression of SREBP1 (Fig. 21D) was significantly down regulated in both flax oil

and fish oil treated groups (P≤0.05). Treatment with fish oil exhibited 2.18-fold

decrease in SREBP1 (Fig. 21D). TNF-α (Fig. 21E) expressions were significantly

down regulated by both flax oil and fish oil (P≤0.001 and P≤0.01, respectively).

Treatment with flax oil showed 2.56-fold decrease in TNF-α (Fig. 21E).

From the results, it is evident that treatment with flax oil exhibited

improvements in several parameters like liver function markers, serum and hepatic

lipid profile, hepatic oxidative stress and it also had beneficial effects on normalizing

liver histology and expression of genes from lipid metabolism and inflammatory

pathways.

138

Table 16. Effect of Flax Oil/Fish Oil on Liver Function Markers and Serum Lipid Profile in Animals with Ethanol Induced Hepatotoxicity


oil and fish oil (p.o.) respectively. Results are a mean of measurements from six animals and are represented as Mean±Standard Error. The data were

subjected to Dunnett Multiple Comparison Test. *P≤0.05; **P≤0.01. Degree of freedom for one variable: Treatments 4; Residuals 25; Total 29. SGOT:

Serum glutamic oxaloacetic transaminase; SGPT: Serum glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL: High


Group

SGOT

(U/mL)

SGPT

(U/mL)

ALP

(U/mL)

BIL

(mg/dL)

Lipid profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 134.49±3.0**

154.67±2.4**

30.40±1.3**

1.13±0.1**

85.89±1.6**

19.31±0.5**

17.1±1.3**

27.71±0.5**

138.54±2.5**

II 162.31±0.8 182.50±2.5 55.89±2.7 2.11±0.2 203.36±7.2 13.51±1.0 40.2±1.0 74.37±0.8 371.87±3.9

III 137.56±1.5**

174.77±1.0* 33.33±0.7

** 1.14±0.1

** 110.09±2.6

** 14.02±0.5 20.1±0.6

** 39.9±0.7

** 199.48±3.3

**

IV 143.84±1.2**

143.67±1.1**

32.82±1.0**

0.95±0.1**

89.10±1.3**

19.65±0.3**

19.5±1.1**

49.42±0.7**

247.13±3.4**

V 156.15±3.7 140.67±2.2**

32.87±0.7**

0.99±0.2**

113.62±2.9**

18.80±0.3**

20.1±0.6**

63.80±0.3**

319.01±1.7**

139

Table 17. Effect of Flax Oil/Fish Oil on Hepatic Oxidative Stress Markers, Total Protein and Hepatic Lipid Profile in animals with Ethanol



oil and fish oil (p.o.) respectively. Results are a mean of measurements from six animals and are represented as Mean±Standard Error. The data were

subjected to Dunnett Multiple Comparison Test.*P≤0.05; **P≤0.01; Degrees of freedom for one variable: Treatments 4; Residuals 25; Total 29. SOD:

Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; GSH: Reduced Glutathione TC: Total Cholesterol; HDL: High density Lipoprotein; LDL:

Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group

SOD

(U/mg)

CAT

(mU/g tissue)

MDA

(μM)

GSH

(μM)

Total

Protein

(mg/gTissue)

Lipid profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 66.23±0.4**

0.84±0.03**

5.74±0.4**

0.82±0.01* 4.51±0.1

** 27.42±1.4

** 14.36±0.4

** 6.30±0.4

** 4.64±0.1

** 23.17±0.70

**

II 14.56±0.5 0.56±0.03 17.17±0.6 0.72±0.01 3.38±0.07 39.18±1.7 11.79±0.3 16.50±0.5 10.47±0.1 52.34±0.7

III 15.45±0.81 0.48±0.03 14.00±0.5**

0.72±0.01 4.27±0.01**

29.18±0.4**

13.67±0.5* 5.70±0.7

** 7.13±0.001

** 35.68±0.4

**

IV 24.29±0.30**

0.74±0.02**

14.39±0.6* 0.70±0.01 6.0±0.1

** 29.11±0.4

** 15.04±0.4

** 6.30±0.4

** 6.83±0.1

** 34.11±0.7

**

V 18.47±0.34**

0.71±0.01**

13.44±0.6**

0.61±0.02**

3.16±0.1 31.45±0.7**

14.01±0.4**

7.50±0.5**

8.54±0.1**

42.71±0.7**

140

Fig. 20. Effects of Polyunsaturated Fatty Acids (Flax oil and fish oil) on Liver

Histology in Animals with Ethanol Induced Hepatotoxicity



microscope (20X) and photographed. Liver sections from A: healthy animals, B: Ethanol

treated animals, C: Silymarin treated animals, D: Flax oil treated animals, E: Fish oil treated

animals.

141

Fig. 21 Gene Expression from Liver Tissues of Experimental Animals




Flax oil treated rats (Group IV) -4; Fish oil treated rats (Group V)-5. GAPDH (Internal

standard). A: FABP1, B: PPARγ, C: NF-κβ, D: SREBP1, E: TNF-α.

A B

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

1.2 *

FA

BP

1/G

AP

DH

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

***

***

***

*

PP

AR/

GA

PD

H

C D

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

NF

-

/GA

PD

H

I II III IV V0.0

0.2

0.4

0.6

0.8

**

* *

SR

EB

P1

/GA

PD

H

E

I II III IV V0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

***

**

*

TN

F-

/GA

PD

H

142

5.5 Hepatoprotective Activity of Combination of Best Performing Herbal and

Nutritional Intervention

The previous studies reported in this thesis, revealed that treatment with

Neem-giloe satwa and T. sinensis satwa was more effective in acetaminophen and

alcohol induced hepatotoxicity, respectively. Nutritional intervention in the form of

fish oil and flax oil was effective in acetaminophen and alcohol induced

hepatotoxicity, respectively. In the present study, the corrective and protective effects

of combination of Neem-giloe satwa and fish oil were assessed against hepatotoxicity

induced with a single high-dose of acetaminophen. The effect of prophylactic

treatment of combination of T. sinensis satwa and flax oil was studied in ethanol

induced hepatotoxicity in rats.

5.5.1 Effects of Protective Treatment of Combination of Neem-giloe Satwa

and Fish oil against Acetaminophen Induced Hepatotoxicity

In the present study, the animals treated with single dose of acetaminophen on

8th

day showed elevated levels of SGOT, SGPT, ALP and bilirubin as compared to

healthy control group indicating the induction of liver damage (Table 18). Animals

treated with combination of Neem-giloe satwa and fish oil showed protective effect by

maintaining the levels of SGOT, SGPT, ALP and bilirubin, and also a significant

protection towards imbalance in lipids, especially in comparison with the negative

control group (Table 18).

Combination of Neem-giloe satwa and fish oil treated animals had protective

effect on SGOT, SGPT, ALP and bilirubin (P≤0.05 or P≤0.01) levels as compared to

acetaminophen treated group. Combination of Neem-giloe satwa and fish oil treated

animals also protected against imbalance in lipid profile. The animals exhibited

comparable total cholesterol levels after acetaminophen dosing. The level of HDL

cholesterol was found to be decreased by 73.28% after acetaminophen dosing but the

animals treated with the combination showed 28.90% decrease, thereby maintaining

the HDL cholesterol levels. The LDL and VLDL cholesterol levels showed 73.13%

and 130.20% increase after acetaminophen dosing in negative control animals. The

animals pre-treated with the combination showed only 1.49% increase in LDL

cholesterol while the levels of VLDL cholesterol were found to be lower than the

143

healthy animals. TG levels showed 130% increase after treatment with

acetaminophen, but the animals receiving the interventions exhibited the TG content

lower than the healthy animals. The protective effect of the combination on total

cholesterol, HDL, LDL and VLDL was found to be statistically significant (P≤0.05 or

P≤0.01) as compared with negative control (Table 18).

The pre-treatment of animals with a combination of Neem-giloe satwa and fish

oil was found to be protective towards oxidative damage through decrease in the

extent of lipid peroxidation and increase in the contents of reduced glutathione (Table

19). The extent of lipid peroxidation (MDA levels) was significantly less in

combination of Neem-giloe satwa and fish oil (P≤0.01) treated animals while the

levels of reduced glutathione (GSH) were higher in the animals receiving the

intervention of Neem-giloe satwa and fish oil as compared to acetaminophen treated

animals (Table 19). Catalase activity was significantly higher in combination of

Neem-giloe satwa and fish oil treated animals (P≤0.01) as compared to negative

control group (Table 19). The total protein content of liver tissues was also higher in

combination of Neem-giloe satwa and fish oil treated animals as compared to that of

negative control. The intervention also protected against variations in the hepatic lipid

profile of the animals. The treatment group displayed lower levels of LDL (P≤0.01),

VLDL and triglycerides (P≤0.01) as compared to negative control group.

Significantly high HDL (P≤0.01) was seen in the animals treated with combination of

Neem-giloe satwa and fish oil (Table 19).

Hematoxylin and eosin stained cross sections of paraffin embedded liver

tissues of rats from control and experimental groups showed variations in the liver

architecture. Healthy group (Fig. 22A) showed normal hepatic architecture. Sections

of liver from the animals treated with single dose of acetaminophen on 8th

day (Fig.

22B) indicated swelling and degenerative changes in hepatocytes. Animals treated

with silymarin (Fig. 22C) showed hepatocytes in periportal area with degenerative

changes. Animals treated with combination of Neem-giloe satwa and fish oil (Fig.

22D) exhibited periportal hepatocytes with degenerative changes. Some foci of

hepatocytes also showed degenerative changes.

144

Fig. 23A-E, shows the protective effect of combination of Neem-giloe satwa

and fish oil on the expression of FABP1, PPARγ, NF-κβ, SREBP1 and TNF-α. The

expressions of FABP1 (Fig. 23A), PPARγ (Fig. 23B) were decreased in

acetaminophen treated animals than in healthy animals. Treatment with combination

of Neem-giloe satwa and fish oil significantly up-regulated (P≤0.001 or P≤0.01) the

expression of FABP1 and PPARγ by 2.8 fold and 1.7 fold respectively. The treatment

with combination of Neem-giloe satwa and fish oil prevented increase in the

expression of NF-κβ (Fig. 23C) which was 3.02 fold less than negative control

(P≤0.01). The expressions of SREBP1 (Fig. 23D) (P≤0.01; 2.07 fold less than

negative control) and TNF-α (Fig. 23E) (P≤0.001; 4.1 fold less than negative control)

genes were also less in the animals treated with the intervention.

145

Table 18. Protective Effect of Combination of Neem-giloe Satwa and Fish Oil on Liver Function Markers and Serum Lipid Profile in Animals

with Hepatotoxicity Induced with a Single High Dose of Acetaminophen

Group I is healthy control while Group II is negative control (single dose of acetaminophen on 8th day), Group III is positive control (silymarin (100mg/kg

b.w./day; p.o.) for 7 days and a single high dose of acetaminophen on 8th day) and Group IV is treatment group (Combination of Neem-giloe satwa

(200mg/kg; p.o.; daily) and fish Oil (500mg/kg; p.o.; daily) for 7 days and a single high dose of acetaminophen on 8th day). Results are a mean of

measurements from six animals and are represented as Mean±Standard Error (SE). The data were subjected to Dunnett Multiple Comparison Test. *P≤0.05;

**P≤0.01. Degrees of freedom for one variable: Treatments 3, Residuals 20, Total 23. SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum

glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL:

Very low density lipoprotein; TG: Triglycerides.

Group

SGOT

(U/mL)

SGPT

(U/mL)

ALP

(U/mL)

BIL

(mg/dL)

Lipid profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 135.00±5.3 123.5±8.2**

28.17±0.3**

0.72±0.1 99.47±6.1* 41.80±3.7

** 20.1±0.6

** 16.19±0.9

** 80.99±4.8

**

II 188.33±25.5 158.5±8.8 38.45±1.2 1.129±0.1 146±20.5 11.17±0.7 34.8±0.3 37.27±3.6 186.3±18.1

III 126.17±8.3* 134.5±7.3 26.16±2.5

** 0.564±0.1

* 108.03±5.5 30.08±2.3

** 27.9±0.4

** 20.04±0.4

** 100.22±2.0

**

IV 123.5±14.1* 108.29±4.3

** 20.09±1.3

** 0.494±0.1

* 97.75±6.4

* 29.72±1.4

** 20.4±1.6

** 13.9±0.5

** 69.80±3.0

**

146

Table 19. Protective Effect of Combination of Neem-giloe Satwa and Fish Oil on Hepatic Oxidative Stress Markers, Total Protein and Hepatic

Lipid Profile in Animals with Hepatotoxicity Induced with a Single High Dose of Acetaminophen

Group I is healthy control while Group II is negative control (single dose of acetaminophen on 8th day), Group III is positive control (silymarin (100mg/kg

b.w./day; p.o.) for 7 days and a single dose of acetaminophen on 8th day) and Group IV is treatment group (Combination of Neem-guduchi satwa (200mg/kg;

p.o.; daily) and fish Oil (500mg/kg; p.o.;) for 7 days and single dose of acetaminophen on 8th day). Results are a mean of measurements from six animals and

are represented as Mean±Standard Error. The data were subjected to Dunnett Multiple Comparison Test. *P≤0.05; **P≤0.01. Degrees of freedom for one

variable; Treatments 3, Residuals 20, Total 23. SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; GSH: Reduced glutathione; TC: Total

cholesterol; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group SOD

(U/mg)

CAT

(mU/g Tissue)

MDA

(μM)

GSH

(μM)

Total

Protein

(mg/g Tissue)

Lipid profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 57.11±2.8**

0.66±0.5**

27.93±1.70**

0.78±0.048**

6.64±0.4 27.79±0.5**

16.58±0.7**

5.45±0.5**

5.14±0.7**

23.71±1.6**

II 20.31±0.8 0.59±1.1 277.14±4.7 0.54±0.055 4.9±0.9 32.93±0.9 8.83±0.6 10.18±0.5 12.79±0.3 63.98±1.32

III 17.83±1.6 0.62±0.5 39.04±3.9**

0.63±0.041 8.33±0.6 26.08±0.5**

11.71±0.6**

7.64±0.7* 5.77±0.3

** 28.86±1.6

**

IV 19.83±0.5 0.65±0.5**

34.28±2.30**

0.65±0.024 8.48±2.03 29.11±0.8 16.22±0.4**

7.28±0.8**

5.37±0.2**

26.84±1.2**

147

Fig. 22. Protective Effects of Combination of Neem-giloe Satwa and Fish oil on Liver

in Rats Treated with a Single High Dose of Acetaminophen



microscope (20X) and photographed. Liver sections from A: Healthy animals, B: Animals

treated with a single high dose of acetaminophen, C: Silymarin treated animals, D:

Combination of Neem-giloe satwa and fish oil treated animals.

148



expression data. *P≤0.05; **P≤0.01; ***P≤0.001 compared with negative control. Lanes:


Combination of Neem-giloe satwa and fish oil treated rats (Group IV) -4. GAPDH (Internal


A B

I II III IV0.0

0.2

0.4

0.6

0.8

1.0

1.2

*

***

FA

BP

1/G

AP

DH

I II III IV

0.0

0.2

0.4

0.6

0.8

1.0

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**

**

PP

AR

/GA

PD

H

C D

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-

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0.4

0.6

0.8

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EB

P1

/GA

PD

H

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I II III IV0.0

0.2

0.4

0.6

0.8

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***

***TN

F-

/GA

PD

H

149

5.5.2. Effects of Corrective Treatment of Combination of Neem-giloe

Satwa and Fish Oil against Acetaminophen Induced Hepatotoxicity

The animals from negative control showed significant increase in the levels of

SGOT, SGPT, ALP and bilirubin after administration of a single high dose of

acetaminophen on 1st day. Levels of SGOT, SGPT, ALP and bilirubin increased

significantly after acetaminophen treatment in all animals as compared to healthy

control indicating successful induction of liver injury.

Animals receiving the intervention of Neem-giloe satwa and fish oil

combination showed significant decrease (P≤0.01) in SGOT, SGPT, ALP and

bilirubin levels as compared to acetaminophen treated group (Table 20). These

animals also exhibited significant improvement in lipid profile parameters as

compared to negative control group. Animals treated with combination of Neem-giloe

satwa and fish oil were found to have improvements in the contents of total

cholesterol with approx. 38.25% decrease (P≤0.01), HDL with 191.43% increase

(P≤0.01), LDL with approx. 36.19% decrease (P≤0.01) and with about 48% increase

(P≤0.01) in VLDL and triglyceride contents, as compared to negative control group.

All these variations in the biochemical parameters were found to be statistically

significant when compared to negative control group (Table 20).

Corrective treatment of animals with combination of Neem-giloe satwa and

fish oil, lead to decrease in oxidative stress, through decrease (P≤0.01) in lipid

peroxidation (by 62.80%) and increase in reduced glutathione (by about 21.56%)

content as compared to negative control group (Table 21). Significant increase

(P≤0.01) in SOD activity (by 106.86%) and catalase activity (by 16.59%) was

observed in the intervention group as against the animals from negative control group

(Table 21). The total protein content of liver tissues was also found to be significantly

improved (P≤0.01) in the treatment group. The hepatic lipid profile displayed

significant improvements in treatment group with decrease in total cholesterol

(P≤0.01), LDL (P≤0.05) and VLDL (P≤0.01) and triglycerides (P≤0.01) as compared

to negative control group. Significant (P≤0.01) increase in HDL was seen in the

animals treated with combination of Neem-giloe satwa and fish oil (Table 21).

150

The effects of treatment with combination of Neem-giloe satwa and fish oil

were also visible at histological levels in animals. Healthy group (Fig. 24A) showed

normal hepatic architecture. Sections of liver from the animals treated with

acetaminophen indicated congestion of central vein, swollen hepatocytes, compressed

sinusoids and ballooning degeneration of hepatocytes (Fig. 24B). Animals treated

with silymarin showed normal liver histology (Fig. 24C). Animals treated with

combination of Neem-giloe satwa and fish oil also exhibited normal liver architecture

(Fig. 24D).

Fig. 25A-E, shows the corrective effect of combination of Neem-giloe satwa

and fish oil on the expression of FABP1, PPARγ, NF-κβ, SREBP1 and TNF-α. The

expressions of FABP1 (Fig. 25A), PPARγ (Fig. 25B) were down-regulated in

acetaminophen treated animals. Treatment with combination of Neem-giloe satwa and

fish oil significantly up-regulated (P≤0.05) the expression of FABP1 (Fig. 25A) and

PPARγ (Fig. 25B) by 2.3 fold and 1.5 fold respectively. The expression levels of NF-

κβ (Fig. 25C), SREBP1 (Fig. 25D) and TNF-α (Fig. 25E) which were up-regulated in

acetaminophen treated animals were significantly improved (P≤0.01) in treatment

group.

151

Table 20. Corrective Effect of Combination of Neem-giloe Satwa and Fish oil on Liver Function Markers and Serum Lipid Profile in Animals

with Hepatotoxicity Induced with a Single High Dose of Acetaminophen

Group I is healthy control while Group II is negative control (single dose of acetaminophen on 1st day), Group III is positive control (single high dose of

acetaminophen on 1st day and silymarin 100mg/kg for 8 days) and Group IV served as treatment (single high dose of acetaminophen on 1

st day + Neem-giloe

satwa (200mg/kg; p.o.) and fish Oil (500mg/kg; p.o.) for 8 days). Results are a mean of six measurements and are represented as Mean±Standard Error. The

data were subjected to Dunnett Multiple Comparison test. *P≤0.05; **P≤0.01. Degrees of freedom for one variable: Treatments3, Residuals 20, and Total 23.

SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL:

High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group

SGOT

(U/mL)

SGPT

(U/mL)

ALP

(U/mL)

BIL

(mg/dL)

Lipid profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 135.00±5.3 123.50±8.2 28.17±0.3* 0.72±0.1

** 99.47±6.1

** 41.80±3.7

** 20.1±0.6

** 16.19±0.9

** 80.99±4.8

**

II 163.50±8.8 160.33±15.01 34.39±0.6 2.55±0.6 149.80±5.3 12.61±1.2 31.50±0.6 32.16±0.3 160.85±1.7

III 126.00±8.2* 82.29±1.7

* 23.43±1.6

** 0.93±0.1

** 94.86±6.2

** 34.05±2.4

** 24.30±0.6

** 18.97±1.2

** 94.85±6.0

**

IV 76.17±9.5**

69.12±1.6**

15.25±2.7**

0.49±0.1**

92.49±8.1**

36.75±3.1**

20.10±1.4**

16.50±1.7**

82.54±8.5**

152

Table 21. Corrective Effect of Combination of Neem-giloe Satwa and Fish Oil on Hepatic Oxidative Stress Markers, Total Protein and Hepatic

Lipid Profile in Animals with Hepatotoxicity Induced with a Single High Dose of Acetaminophen

Group I is healthy control while Group II is negative control (single dose of acetaminophen on 1st day), Group III is positive control (single dose of

acetaminophen on 1st day and silymarin 100mg/kg for 8

days) and Group IV served as treatment (single dose of acetaminophen on 1

st day and Neem-giloe

satwa (200mg/kg; p.o.) and fish oil (500mg/kg; p.o.) for 8 days). Results are a mean of measurements from six animals and are represented as Mean±Standard

Error. The data were subjected to Dunnett Multiple Comparison Test. *P≤0.05; **P≤0.01. Degrees of freedom for one variable: Treatments 3, Residuals 20,

and Total 23. SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; GSH: Reduced glutathione; TC: Total cholesterol; HDL: High density

lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group SOD

(U/mg)

CAT

(mU/g Tissue)

MDA

(μM)

GSH

(μM)

Total

Protein

(mg/g Tissue)

Lipid profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 57.11±2.8**

0.66±0.5**

27.93±1.7**

0.78±0.048**

6.64±0.3 27.79±0.5**

16.58±0.7**

5.45±0.5**

5.40±0.7**

23.71±1.6**

II 11.22±0.4 0.57±0.6 64.45±3.6 0.51±0.01 5.22±0.3 34.78±1.1 9.37±0.8 10.54±0.7 13.2±1.1 66.50±6.0

III 15.29±0.7 0.61±0.4**

25.15±3.6**

0.58±0.03 8.29±0.9* 26.22±0.9

** 9.55±0.4 9.46±1.3 5.99±0.3

** 29.98±1.7

**

IV 23.21±0.7**

0.66±0.7**

23.97±1.8**

0.62±0.01 10.11±0.9**

26.74±0.5**

14.59±0.4**

6.91±0.9* 4.92±0.1

** 24.61±0.7

**

153

Fig. 24. Corrective Effects of Combination of Neem-giloe satwa and Fish Oil on Liver

in Rats Treated with a Single High Dose of Acetaminophen



microscope (20X) and photographed. Liver sections from A: Healthy animals, B: Animals

treated with a single high dose of acetaminophen, C: silymarin treated animals, D: Animals

treated with a combination of Neem-giloe satwa and fish oil.

154





Combination of Neem-giloe satwa and fish oil treated rats (Group IV) -4. GAPDH (Internal


A B

I II III IV0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

FA

BP

1/G

AP

DH

I II III IV

0.0

0.2

0.4

0.6

0.8

1.0

1.2 **

*

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AR

/GA

PD

H

C D

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0.4

0.6

0.8

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**

**

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-

/GA

PD

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I II III IV

0.0

0.2

0.4

0.6

0.8

1.0

**

**

**S

RE

BP

1/G

AP

DH

E

I II III IV0.0

0.2

0.4

0.6

0.8

1.0

***

**

TN

F-

/GA

PD

H

155

5.5.3 Effects of Prophylactic Treatment of Combination of T. sinensis

Satwa and Flax Oil against Ethanol Induced Hepatotoxicity

The significant deviation in the liver function test parameters like SGOT,

SGPT, ALP and bilirubin in negative control group indicated successful liver injury

by repeated ethanol dosing (Table 22). Treatment of animals with a combination of T.

sinensis satwa and flax oil exhibited improvements (P≤0.01) in SGOT, SGPT, ALP

and bilirubin levels as compared to 30% ethanol treated group (Table 22). Total

cholesterol, triglycerides, LDL and VLDL levels showed elevation while HDL

decreased in 30% ethanol treated animals compared to healthy control group, further

indicating liver damage. Administration of combination of T. sinensis satwa at a dose

of 200mg/kg b.w./day and flax oil at a dose of 500mg/kg.b.w./day had a beneficial

effect on the levels of these biochemical markers in the treatment groups (Table 22).

Treatment of animals with a combination of T. sinensis satwa and flax oil improved

the contents of total cholesterol with approx. 39.27% decrease (P≤0.01), LDL with

approx. 61.85% decrease (P≤0.01), and about 51% decrease in VLDL and

Triglyceride levels (P≤0.01 or P≤0.05) as compared to negative control group (Table

22). Treatment with the combination also resulted in increase in the HDL levels by

about 39.38% as compared to the animals receiving only ethanol (P≤0.05).

Prophylactic treatment of animals with combination of T. sinensis satwa and

flax oil, lead to decrease in oxidative stress, through decrease (P≤0.01) in lipid

peroxidation (by 64.78%) and increase (P≤0.01) in reduced glutathione (by about

293.93%) as compared to negative control group (Table 23). Significant increase in

SOD activity (by 40.17%) (P≤0.01) and catalase activity (by 6.01%) (P≤0.05) was

observed in animals treated with a combination of T. sinensis satwa and flax oil as

compared to negative control group (Table 23). The total protein content of liver

tissues was also found to be significantly improved (P≤0.01) in treatment group. The

animals treated with the combination of satwa and flax oil also displayed significant

improvements in total cholesterol (P≤0.01), LDL (P≤0.01), VLDL (P≤0.01) and

triglycerides (P≤0.01) as compared to negative control group. Significant (P≤0.01)

increase in HDL was seen in the animals treated with combination of T. sinensis

satwa and flax oil (Table 23).

156

The hepatoprotective effects of combination of T. sinensis satwa and flax oil

were also evident from the noticeable variations in liver histology (Fig. 26A-D). The

healthy control group (Fig. 26A) exhibited normal liver histology while the 30%

ethanol treated group (Fig. 26B) showed mild swollen hepatocytes with coarse

granularity, normal periveinular hepatocytes and ballooning degeneration in some

hepatocytes. The group treated with the standard drug silymarin (Fig. 26C) had near

normal histology. Animals treated with combination of T. sinensis satwa and flax oil

(Fig. 26D) exhibited strikingly normal liver histology without any anatomically

detectable anomalies.

Fig. 27(A-E) shows the prophylactic effect of combination of T. sinensis satwa

and flax oil on the expression of FABP1, PPARγ, NF-κβ, SREBP1 and TNF-α. The

FABP1 (Fig. 27A) and PPARγ (Fig. 27B) genes were down-regulated in 30% ethanol

treated animals. Treatment with combination of T. sinensis satwa and flax oil showed

improvement in the expression of FABP1 but it was found to be statistically

insignificant. Treatment with combination of T. sinensis satwa and flax oil showed

significant improvement (P≤0.001) in the expression of PPARγ (3.0 fold) as

compared to negative control group. NF-κβ (Fig. 27C), SREBP1 (Fig. 27D) and TNF-

α (Fig. 27E) were up-regulated in 30% ethanol treated animals. The treatment with

combination of T. sinensis satwa and flax oil down-regulated (P≤0.001) in the levels

of NF-κβ by about 2.3 fold. Though not statistically significant, there was

improvement in the expression of SREBP1 and TNF-α.

157

Table 22. Prophylactic Effect of Combination of T. sinensis Satwa and Flax Oil on Liver Function Markers and Serum Lipid Profile in Animals

with Ethanol Induced Hepatotoxicity

Group I is healthy control while Group II and III served as negative and positive controls respectively. Animals in Group IV were treated with T. sinensis

satwa (200mg/kg b.w.) and flax oil (500mg/kg b.w.). Results are a mean of measurements from six animals and are represented as Mean±Standard Error. The

data were subjected to Dunnett Multiple Comparison Test. *P≤0.05; **P≤0.01; Degrees of freedom for one variable: Treatments 3; Residuals 20; Total 23.

SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL:

High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group SGOT

(U/mL)

SGPT

(U/mL)

ALP

(U/mL)

BIL

(mg/dL)

Lipid Profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 82.63±4.1**

62.26±2.2**

29.86±2.2**

0.63±0.05**

122.53±2.3**

31.35±2.1* 11.10±0.3

** 23.76±0.6

** 118.79±2.9

**

II 150.41±2.9 83.22±2.5 54.78±2.5 1.61±0.09 138.20±4.0 22.88±0.9 29.10±0.9 46.26±1.5 231.32±7.6

III 91.24±2.5**

66.31±1.7**

39.32±2.2**

0.58±0.04**

82.61±3.01**

29.18±2.1 12.30±0.7**

25.41±0.5**

127.07±2.9**

IV 84.86±2.6**

58.1±1.4**

38±3.4**

0.82±0.06**

83.92±2.5**

31.89±2.3* 11.10±0.3

** 22.37±0.4

** 111.86±2.1

**

158

Table 23. Prophylactic Effect of Combination of T. sinensis Satwa and Flax Oil on Hepatic Oxidative Stress Markers, Total Protein and Hepatic

Lipid Profile in Animals with Ethanol Induced Hepatotoxicity

Group I is healthy control while Group II and III served as negative and positive controls respectively. Animals in Group IV were treated with T. sinensis

satwa (200mg/kg b.w.) and flax oil (500mg/kg b.w.). Results are a mean of measurements from six animals and are represented as Mean±Standard Error. The

data were subjected to Dunnett Multiple Comparison test. *P≤0.05; **P≤0.01; Degrees of freedom for one variable: Treatments 3; Residuals 20; Total 23.

SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; GSH: Reduced glutathione TC: Total cholesterol; HDL: High density Lipoprotein;

LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TG: Triglycerides.

Group SOD

(U/mg)

CAT

(mU/g Tissue)

MDA

(μM)

GSH

(μM)

Total

Protein

(mg/g Tissue)

Lipid Profile

Total

Cholesterol

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

TG

(mg/dL)

I 54.88±1.7**

0.70±1.0 4.71±0.4**

0.125±0.01**

7.67±0.2* 25.23±1.6

** 16.06±1.2

** 5.1±0.3

** 2.18±0.1

** 10.961±0.8

**

II 38.38±0.4 0.66±1.5 16.78±0.9 0.033±0.01 6.84±0.2 33.56±1.1 10.77±0.4 16.2±1.3 5.85±0.3 29.30±1.6

III 47.44±1.6**

0.66±0.2 15.83±1.0 0.135±0.001**

9.28±0.2**

29.86±1.1 15.89±0.51**

9±0.01**

4.7±0.1**

23.49±0.7**

IV 53.80±1.1**

0.70±1.1* 5.91±0.7

** 0.130±0.01

** 9.3±0.1

** 26.39±1.2

** 14.87±0.9

** 7.5±0.3

** 3.78±0.2

** 18.79±1.2

**

159

Fig. 26. Prophylactic Effects of Combination of T. sinensis Satwa and Flax Oil on

Liver Histology in Animals with Ethanol Induced Hepatotoxicity



microscope (20X) and photographed. Liver sections from A: Healthy animals, B: Ethanol

treated animals, C: Silymarin treated animals, D: Animals treated with a combination of T.

sinensis satwa and flax oil.

160


Densitometric analysis was done by using GAPDH and gene specific expression data.

*P≤0.05; **P≤0.01; ***P≤0.001 compared with negative control rats. Lanes: Healthy control

(Group I) -1; Negative control (Group II) -2; Positive control (Group III) -3; Treated with

combination of T. sinensis satwa and flax oil (Group IV) -4. GAPDH (Internal standard). A:

FABP1, B: PPARγ, C: NF-κβ, D: SREBP1, E: TNF-α.

A B

I II III IV0.0

0.4

0.8

1.2

1.6

2.0

2.4

FA

BP

1/G

AP

DH

I II III IV0.0

0.4

0.8

1.2

1.6

2.0

2.4 ***

*

PP

AR

/GA

PD

H

C D

I II III IV0.0

0.4

0.8

1.2

1.6

*** ***

*

NF

-

/GA

PD

H

I II III IV0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

*

SR

EB

P1

/GA

PD

H

E

I II III IV0.0

0.4

0.8

1.2

1.6

2.0

2.4

**

TN

F-

/GA

PD

H

CHAPTER 6

Discussion Awards


Presentation Award


Award

161

Liver is the largest organ in the human body and is crucial in maintenance of

good health. It is involved in almost all biochemical pathways that allow growth,

protection from diseases, synthesis and supply of nutrients etc. The liver performs

more than 500 vital functions of metabolic importance (Naruse et al., 2007). Liver is

constantly exposed to several environmental and chemical insults to which it gives a

specific response. Chronic exposure to such insults leads to inflammation and

compromise in liver function (Sahu, 2007). Several drugs and/or chemicals are known

to cause hepatotoxicity (Bigoniya et al., 2009). Several adverse effects of liver

protective drugs in allopathic medical practices have prompted the untiring search for

herbs and nutritional supplements for management of various liver disorders. Use of

number of medicinal plants and their formulations are common for the treatment of

liver diseases in ethno-medical practices and in traditional medicine system (Dange,

2010; Kumar et al., 2011; Shaik et al., 2012). Modern medicine has little to suggest

for improvement of hepatic diseases and it is essentially the plant based preparations

which are employed for the treatment of liver disorders (Chatterjee, 2000).

Approximately 80% of the world population depends on the use of traditional

medicine system which is predominantly based on plant materials (WHO, 2002). It is

estimated that local health traditions, mostly in rural and tribal villages of India, use

about 7,500 plants. Out of these, the real medicinal value of over 4,000 plants is either

little known or yet to be known to the mainstream population. The classical systems

of medicine such as Ayurveda, Siddha, Unani, Tibetan, and Amchi use about 1,200

plants (Pandey, 2011). Various herbs and plant extracts have significant

hepatoprotective activity as indicated from studies in animal models (Malhotra and

Singh, 2006; Chaudhary et al., 2010). Nutritional supplements with omega-3 fatty

acids and diet rich in carbohydrates, fats and proteins with adequate calories and helps

in regeneration of liver cell for patients with liver diseases (Drevon, 2009; Shukla and

Kumar, 2013)

Studies in the present thesis have indicated hepatoprotective activities of

herbal and nutritional supplement(s), and the combinatorial hepatoprotective effects

of best performing herbal and nutritional supplements against acetaminophen and

alcohol induced liver damage.

Awards


Presentation Award


Award

162

6.1 Guduchi Satwa (T. cordifolia, T. sinensis and Neem-giloe)

6.1.1 Organoleptic Characteristics of Three Tinospora forms

There are very few reports available for organoleptic characteristics of T.

cordifolia and Neem-giloe satwa (Sharma et al., 2012; Patil and Chaudhary, 2013;

Sharma et al., 2013a; Chavan et al., 2014; Sharma et al., 2015). As per classics, the

taste of Guduchi satwa from T. cordifolia is swadu (palatable/pleasant) (Navare,

2011) but some recent reports describe Guduchi satwa from T. cordifolia as slight

bitter (Sharma et al., 2013a; Sharma et al., 2015) and Guduchi satwa from Neem-

guduchi as tasteless (Sharma et al., 2012; Sharma et al., 2015). However, in present

study, satwa from T. cordifolia and T. sinensis was found to be tasteless while that

from Neem-giloe was found to be bitter in taste. In Rasa Yoga Sagara (Sharma,

2004), the colour of satwa is mentioned as ‘Shubhrakhandnibha’ (clear white like

sugar cubes) (Sharma, 2004) and Yoga Ratnakara reveals it as ‘Shankhanibha’ (clear

white like conch shell) (Shastri, 2002) but some texts mention the colour of satwa as

greenish white (Reddy, 2005) or greyish white (Hiremath, 2005) and the colour of

satwa from Neem-guduchi as pale (Sharma et al., 2012) to clear white (Sharma et al.,

2015). In the present work, colour of the satwa prepared from T. cordifolia, T.

sinensis and Neem-giloe were found to be grey, greyish white and yellowish white

respectively, which resembles the recent reports. The texture and taste of satwa also

depends on the age of the plant material used for satwa preparation. A recent study by

Patil and Chaudhary (2013) have examined stem pieces of different thickness and

maturity of T. cordifolia to find best size of the stem to achieve maximum yield of

satwa. A yield of 0.48% -0.1% satwa from T. cordifolia was reported from fresh stem

(Mehra and Puri, 1969; Rao and Rao, 1981; Salunke and Pimpalgaonkar, 1997) and

1.20% (Rao and Rao, 1981) with that of dried stem. Sharma et al. (2012) have

reported that the thickness of T. cordifolia stem also has effect(s) on the yield of satwa

with 1.6-2.0 cm thick stem giving maximum yield of satwa. These variations may be

due to different ecotypes, size of the stem, collection time and levels of maturity of

the plant. In the present study stem pieces of 1.6-2.0 cm were used for preparation of

satwa and the yield of satwa ranged from 1.48%-1.60% in three different forms of

Tinospora. Sharma et al. (2012) have reported that the yield of satwa from Neem-

guduchi is highly variable and it depends upon multiple factors like size, environment,

163

nature of cellular activities etc. They have also reported that the medium sized stem

(1.5-2.0cm) of Guduchi yields maximum satwa since there is maximum accumulation

of starch at this stage during development. Sharma et al. (2015) have reported that the

yield of Neem-guduchi satwa from male and female stem was 2.25 % and 3.18 %

respectively since relatively higher starch and mucilage contents are found in female

plants, suggesting that more yield of satwa can be obtained from them.

6.1.2 Nutritional Analysis of Three Tinospora forms

The nutritional composition of medicinal plants depends heavily on

environmental and physiological conditions as well as the time of harvesting of the

plant (Kutbay and Ok, 2003; Sharma et al., 2013b). Guduchyadi varga from

Bhavprakash nighantu (1999) mentioned the time of collection of Guduchi as end of

May. Accumulation of active components of a drug is also reported to be maximum

during specific seasons in many medicinal plants (Kutbay and Ok, 2003; Nasreen et

al., 2010; Patil and Gaikwad, 2011). The variability in the active components of an

Ayurvedic drug due to these factors can be studied with the help of principles to

determine different nutritional/pharmaceutical contents in the drug (Nasreen et al.,

2010; Patil and Gaikwad, 2011; Geeta and Kumari, 2013; Mahima et al., 2013). In

case of T. cordifolia, crude protein and fiber are reported to be the principal

components of the powder from dried stem pieces (Hussain et al., 2009). There are

reports about the proximate and elemental analysis of powder prepared from fresh or

dried stem of T. cordifolia (Nile and Khobragade, 2009; Mahima et al., 2014).

Powder prepared from dried stem of T. cordifolia was found to be rich in

phytochemicals like alkaloids, glycosides, sterols and carbohydrates (Nasreen et al.,

2010; Tanwar et al., 2012). The presence of wide range of phytochemicals is also

reported in different solvent extracts (methanol, petroleum ether, water, chloroform

and ethyl acetate) of T. cordifolia stem (Sivakumar and Rajan, 2011; Pradhan et al.,

2013). Nutritive value of the medicinal plants can be determined with the help of

proximate and elemental analysis (Mahima et al., 2013). The crude protein content of

T. cordifolia is of great importance to its nutritive value (Ajibade and Fagbohun,

2010). Satwa is a potential source of nutrition and has shown beneficial effects for

boosting the immune system-response and body building (Ajibade and Fagbohun,

2010; Geeta and Kumari, 2013; Mahima et al., 2014).

164

The reports on the nutritional analysis of Guduchi satwa are available only for

T. cordifolia satwa. There are no reports of nutritional and/or comparative analysis of

T. sinensis and Neem guduchi. In view of this, the present analysis was undertaken to

identify the nutritional richness of these Tinospora forms. The present study has lead

to the first report of the nutritional analysis of satwa prepared from T. cordifolia, T.

sinensis and Neem-guduchi (Chavan et al., 2014). In present study, the lipid, ash, and

carbohydrate contents of Neem-giloe were greater than that of T. cordifolia and T.

sinensis. T. sinensis showed higher amount of protein, starch, and crude fiber than

Neem-giloe and T. cordifolia.

6.2 Drug Induced Liver Injury

The ability to prevent damage to the liver is called as hepatoprotection (Mishra

et al., 2014). Hepatic damage may be caused due to viral hepatitis, bile duct

obstruction, cholesterol overload, etc. and also due to some chemical factors such as

overdose of several drugs, alcohol intake etc. With increase in the incidence of

hepatotoxicity, there is a need to find effective ways for prevention or management of

liver damage (Wang et al., 2009). The hepatoprotective efficacy of a drug is measured

in terms of its ability to restore normal hepatic functions or to reduce damage to the

liver (Yadav and Dixit, 2003). The drugs used for treating hepatic disorders in modern

medicine have several undesirable side effects (Singh, 2013; Ananthi and Anuradha,

2015).

The two hepatotoxicants selected in this study are Acetaminophen

(Paracetamol) and Alcohol (Ethanol) which are routinely used for medicinal or other

purposes. The incidence of acetaminophen induced liver damage has been reported to

be increased from 28% in 1998 to 52% in 2003, in USA (Larson et al., 2005). A

recent Indian report has estimated that about 33% of the people consuming

acetaminophen are subjected to liver damage (Marzilawati et al., 2012). Easy and

over-the-counter availability of acetaminophen and self-medication (Kaufman et al.,

2002; Larson et al., 2005; Abay and Amelo, 2010) is believed to be a reason behind

intentional or unintentional overdose (Lee, 2004; Larson et al., 2005). Alcohol

increases the hepatotoxicity of various xenobiotics and the interaction between

alcohol and hepatotoxins is well recognized (Zimmerman, 1999). Alcoholic liver

disease (ALD) is a common consequence of prolonged and heavy alcohol abuse. In

165

India, the incidence of alcohol induced hepatotoxicity is reported to be 31% (WHO,

2011).

In the present study, animals were dosed repeatedly with hepatotoxicant

(acetaminophen and alcohol separately) and simultaneously treated with satwa from

one of the three different forms of Tinospora or Omega-3 fatty acids (Flax oil and

Fish oil) and combination of best Tinospora forms and best omega-3 fatty acids.

Biochemical, histological and molecular analyses were performed to determine the

beneficial effects of satwa from three Tinospora forms and omega-3 fatty acids and

their combination.

6.3 Hepatoprotective Activity of Satwa against Acetaminophen Induced

Hepatotoxicity

6.3.1 Biochemical Parameters

Elevated levels of serum glutamic oxaloacetic transaminase (SGOT) indicate

liver damage, which may be due to viral hepatitis, cardiac infarction and muscle

injury while serum glutamic pyruvic transaminase (SGPT) is more specific to liver,

and is thus a better parameter for detecting liver injury (Reitman and Frankel, 1957;

Thapa and Walia, 2007). Increase in serum levels of alkaline phosphate (ALP) is due

to increased synthesis of the enzyme in presence of increasing biliary pressure (Kind

and King, 1954; Thapa and Walia, 2007). Serum bilirubin level is related to function

of hepatic cells (Jendrassik and Grof, 1938; Thapa and Walia, 2007). Increased

SGOT, SGPT and ALP are indicative of cellular leakage and reduced functional

integrity of the liver cell membranes indicating hepatocellular damages (Gutierrez and

Solis et al., 2009; Basu et al., 2012). In the current study, administration of

acetaminophen caused a significant elevation of SGOT, SGPT, ALP, and total

bilirubin when compared to healthy control, which is indication of hepatic damage.

Several reports indicate elevated levels of liver function markers like SGOT, SGPT,

ALP and bilirubin in rats subjected to acetaminophen induced liver injury (Duairaj et

al., 2007; Ramachandran et al., 2010; Sundari et al., 2011; Basu et al., 2012; Galal et

al., 2012). Our study reveals comparative hepatoprotective effect of T. cordifolia, T.

sinensis and Neem-giloe satwa. The previous studies have reported the

hepatoprotective effect of T. cordifolia alone against different hepatotoxicants

166

(Bishayi et al., 2002; Sharma and Pandey, 2010; Kavitha et al., 2011b;

Venkatalakshmi and Ragadevi, 2012; Kumar et al., 2013b). In the present study,

treatment of rats with Neem-giloe (200 mg/kg) decreased levels of SGOT and

bilirubin while T. sinensis showed effects on improvements in serum SGPT and ALP

against acetaminophen induced hepatotoxicity. Apart from liver protection, studies on

T. sinensis indicate anti-inflammatory (Li et al., 2003) and anti-diabetic activities

(Yonemitsu et al., 1993). Some reports also show the immunomodulatory activity of

T. cordifolia (growing on neem tree) (Bhalerao et al., 2012; Narkhede et al., 2014).

Elevated levels of total cholesterol, phospholipids, triglycerides and free fatty

acids in the plasma have been reported in acetaminophen treated rats which is an

indication of reduced or impaired fat metabolism secondary to liver damage

(Ramachandran et al., 2010; Haldar et al., 2011; Malarvizhi et al., 2012). Several

reports indicate elevated levels of serum lipids (Total cholesterol, triglyceride, and

LDL) in rats subjected to acetaminophen overdose (Duairaj et al., 2007; Sundari et al.,

2011; Basu et al., 2012; Singh, 2013; Singh et al., 2015). In present study, animals

treated with acetaminophen showed increase in the levels of cholesterol, triglyceride,

very-low-density lipoprotein cholesterol (VLDL), low-density lipoprotein cholesterol

(LDL), and decrease in high-density lipoprotein cholesterol (HDL) levels in serum

and liver homogenates as compared to healthy animals. The earlier studies revealed

that treatment with T. cordifolia root extract resulted in significant reduction in levels

of serum and tissue cholesterol, phospholipids and free fatty acids in alloxan induced

diabetic rats (Stanely et al., 1999). In present study, T. cordifolia satwa exhibited

improvements in the serum levels of total cholesterol, HDL and LDL, T. sinensis

satwa showed improvement in VLDL and triglycerides levels while Neem-giloe satwa

showed significant improvements in total protein and lipid profile (HDL, LDL,

VLDL, Triglyceride) in liver tissues. The alterations in the lipid profile due to

chemically induced liver damage also leads to generation of several other chemical

entities which may have direct or indirect effect on the liver functions (Basu et al.,

2012).

Free radicals are generated during liver injury which attack many sub-cellular

organelles and systems. Acetaminophen hepatotoxicity is mediated through oxidative

stress due to activation of acetaminophen by cytochrome P450 (Zoubair et al., 2013).

Excessive peroxidation causes increased reduced glutathione (GSH) consumption

167

(Nandy et al., 2012). GSH is a scavenger of toxic metabolites, including NAPQI,

which is a metabolite of acetaminophen (Hsu et al., 2008). Depletion in the levels of

reduced glutathione leads to compromised antioxidant capacity of the tissue.

Acetaminophen overdose causes decrease in antioxidant enzyme activities such as

superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Lores et al.,

1995; Duairaj et al., 2007; Basu et al., 2012) and increases lipid peroxidation levels in

liver (Duairaj et al., 2007; Basu et al., 2012). In the present study, the activity of SOD

and catalase as well as GSH contents in liver significantly decreased, indicating

oxidative stress while lipid peroxidation (MDA) levels increased indicating increased

lipid peroxidation. Interventions with Neem-giloe satwa showed significant increase

in the levels of SOD, CAT, and GSH. Though statistically insignificant, animals

treated with the satwa from T. sinensis showed decreased MDA levels in liver

homogenates than that in acetaminophen treated group. The administration of T.

cordifolia (50, 100, 200 gm/kg orally daily for 25 days) has been reported to exhibit

protective effect by reducing the contents of thiobarbituric acid reactive substances

(TBARS) and increasing GSH, ascorbic acid, protein, and the activities of antioxidant

enzymes viz., SOD, CAT, glutathione peroxidase, glutathione S-transferase, (GST),

and glutathione reducates (GR) in liver and kidney against aflatoxin B1 (AFB1)

toxicity in mice (Gupta et al., 2011). Prince and Menon (2001) reported that

administration of aqueous extract of the roots of T. cordifolia (5.0 g/kg for 6 weeks)

resulted in significant reduction in TBARS and an increase in GSH, CAT and SOD in

alloxan induced diabetic rats.

6.3.2 Histological Analysis

Histological study of liver sections of rats treated with acetaminophen

displayed degenerative changes in hepatocytes and cells that line the blood sinusoids.

The anatomical changes in liver due to injury or toxicity are known to be positively

correlated with increase in transaminase activities (Galal et al., 2012). Acetaminophen

induced liver injury frequently shows intense centrilobular necrosis and

vascuolisation (Manivannan et al., 2011), severe intense congestion, hydropic

degeneration, pyknosis and occasional necrosis (Kanchana and Sadiq, 2011; Prabu et

al., 2011), sinusoidal haemorrhages and dilatations with chronic inflammatory cell

infiltrate in portal tracts (Sundari et al., 2011). In present study, sections of liver from

168

acetaminophen treated group showed mild congestion of central vein, hepatocytes

exhibited apoptotic death with few intra and extracellular hyaline globules and

ballooning and degeneration around central vein. Mild nucleomegaly was also visible

under microscope. Significant recovery of hepatocellular lesions was observed in the

animals treated with the satwa of Neem-giloe which restored the hepatic histology to

near normal architecture. Such normal hepatic lobule architecture was found in the

animals treated with 2 ml/100g of T. cordifolia for 30 days against CCL4 induced

hepatotoxicity (Kumar et al., 2013b). Normal liver histology, without any detectable

necrosis and vacuolisation, was also observed in rats treated with T. cordifolia extract

(200 mg/kg) once daily for 3 days against CCl4 induced hepatotoxicity (Kavitha et al.,

2011b).

6.3.3 Gene Expression

Expression of Genes Involved in Lipid Metabolism

PPARγ (peroxisome proliferator-activated receptor-gamma) and SREBP1

(Sterol regulatory element-binding protein 1) are transcription factors and regulators

of lipid homeostasis in hepatocytes and a target for fatty acids and hypolipidemic

drugs (Eberle et al., 2004; Shan et al., 2008; Gong et al., 2014).

The proteins encoded by different PPAR genes have the ability to induce

hepatic peroxisome proliferation in response to xenobiotic stimuli (Sahu, 2007). The

three PPAR isoforms (PPARα, PPARδ and PPARγ) are believed to play a central role

in regulation of carbohydrate and lipid metabolism, fatty acid metabolism and are also

assumed to possess anti-inflammatory activity (Wang et al., 2014b). These PPAR

isoforms inhibit the induction of pro-inflammatory cytokines and stimulate the

production of anti-inflammatory molecules (Kostadinova et al., 2005). Dysregulation

of some PPAR isoforms contribute to development of a wide range of liver diseases

(Peyrou et al., 2012). The pathways for action of PPARs have been well studied.

PPARs combine with PPRE (peroxisome proliferator response element) in the

promoter region of their target genes involved in fatty acid transport and lipid

catabolism (Sears et al., 2007). Differential effects could be explained by promoter

and cell context and also by the availability of co-factors but at the same time there

are site specific conformational changes in the receptors which are induced by PPARγ

169

ligands that ultimately lead to chromatin modeling of target genes and differential

promoter activation (Olefsky, 2000). Majority of studies deal with PPARγ in diabetic

and obese mouse livers (Memon et al., 2000; Gavrilova et al., 2003; Bedoucha et al.,

2001) but the mechanistic relationship of increase of PPARγ expression in

hepatotoxicity, remains unclear till date.

Liver is a primary site of biotransformation and is critical in modulating

metabolically and chemically induced toxicity and there are reports which suggest

that PPARs modulate hepatotoxicity. Pioglitazone, a PPARγ agonist, inhibits CCl4

(carbon tetrachloride) induced hepatic fibrosis through inhibition of inflammation and

hepatic stellate cell proliferation indicating protective role of PPARγ in hepatotoxicity

(Yuan et al., 2004). An isoform of PPAR, PPARβ, enhances chemically induced liver

toxicity (Hellemans et al., 2003). Expression of PPARβ messenger RNA was

increased in hepatic stellate cells, as they undergo spontaneous activation (Hellemans

et al., 2003).

PPARγ has direct involvement in regulation of the functional expression of

drug transporters, such as the ABCG2, ABCA1 etc. ABCG2, an ATP-binding cassette

transporter is known to perform clearance of endogenous and exogenous toxic agents.

Activation of PPARγ and consecutively increased amounts of the ABCG2 transporter

protein were shown to significantly increase efflux of xenobiotics in human dendritic

cells (Szatmari et al., 2006). Peroxisomal proliferators like clofibrate, aspirin,

valproate, ethylhexanol, ciprofibrate and perfluorooctanoate might cause local toxicity

due to inhibition of mitochondrial oxygen uptake (Keller et al., 1992). In chronic liver

injury induced by carbon tetrachloride, for instance, a downregulation of PPARγ

expression was observed in hepatocytes, while increased levels of these transcription

factors were found in Kupffer cells associated with inverse correlation to levels of

activated NFκβ (Orfila et al., 2005). PPARγ also plays an antitoxic role by inducing

liver cells to deposit harmless lipids thereby preventing the accumulation of toxic

lipids (Medina-Gomez et al., 2007). Miyahara et al. (2000), and Zhang et al. (2012)

reported PPARγ deficiency in hepatic stellate cells associated with excessive

formation of fibrotic tissue in the liver. The role of PPARγ in manifestation of

inflammation is gaining momentum (Szeles, 2007) and expression in hepatoma cell

line indicates its potential role in liver function (Koga et al., 2007). Down regulation

of PPARγ mRNA expression has been reported in isoniazid induced hepatotoxicity

170

(Mahmoud et al., 2014). In accordance with the above reports, the present study also

reported down regulation of PPARγ expression in acetaminophen induced

hepatotoxicity as compared with healthy control. Several medicinal plant extracts are

reported to activate PPARγ (Local Food-Nutraceuticals Consortium, 2005;

Christensen et al., 2009; Andaloussi et al., 2010; Vogl et al., 2013; Yang et al., 2013).

Curcuminoids have also been reported to inhibit pro-inflammatory induction by

enhancing PPARγ activation (Jacob et al., 2007). The protective effects of berberine

against isoniazid-induced hepatotoxicity may be attributed to its ability to upregulate

PPARγ and subsequently suppress NF-κβ, iNOS and release of proinflammatory

cytokines (Mahmoud et al., 2014). The mechanism of action of hepatoprotection by

several secondary metabolites from plants has been shown to be through reduction in

oxidative stress which is achieved via activating PPARγ (Duval et al., 2014). Though

statistically insignificant, the present study reported marginal improvement in PPARγ

expression in the livers of rats treated with Neem-giloe satwa and T. sinensis satwa

exhibiting near normal liver architecture.

The liver plays a central role in lipid metabolism through de novo lipid

synthesis and fatty acid oxidation. SREBPs (Sterol regulatory element-binding

proteins) are the important transcription factors which activate the expression of genes

involved in the biosynthesis of TGs (Triglycerides), fatty acids and cholesterol. The

transcription factors in SREBP family are master regulators of lipid metabolism,

which control the expression of genes required for fatty acid and cholesterol

biosynthesis. In mammals, there are three isoforms of SREBPs, SREBP- 1a, 1c and 2

(Horton et al., 2002). SREBP-1c is the predominant isoform of SREBP-1 in liver and

SREBP-1c is mainly responsible for the biosynthesis of TGs. Studies have shown that

transgenic mice overexpress SREBP-1c or SREBP-1a and produce massive fatty liver

due to accumulation of TGs and cholesterylesters (Shimano et al., 1996; Shimomura

et al., 1999). SREBP1 is involved in the activation of genes associated with fatty acid

metabolism, and involved directly in cholesterol homeostasis (Horton et al., 1998; Pai

et al., 1998). SREBP1 specifically activates several of the key genes involved in

lipogenesis (Horton et al., 2002; Horton et al., 2003) like fatty acid synthase (FAS),

and Acetyl-CoA carboxylase alpha (ACACA) (Ronnebaum et al., 2008). SREBPs are

bound as precursors to the endoplasmic reticulum and nuclear envelope. When

activated, they are released from the membrane and escorted to the Golgi complex by

171

SREBP cleavage-activating protein (SCAP) and then cleaved by specific proteases

and translocated to the nucleus, where they bind to sterol regulatory element-1

(SRE1), which is a decameric sequence flanking the low density lipoprotein receptor

gene and some genes involved in sterol biosynthesis (Sozio and Crabb, 2008). Sterols

inhibit the cleavage of the precursor, and the mature nuclear form is rapidly

catabolized, thereby reducing transcription. The genes which are activated by SREBP

are in turn regulated by AMPK (AMP-Activated protein kinase) since AMPK directly

phosphorylates and binds to SREBP-2 and SREBP-1c (Li et al., 2011) inhibiting the

expression of FAS and ACC which are key lipogenic enzymes (Viollet et al., 2009).

SREBP-1 gene knockout mice show a very low basal expression of FAS

hardly possess the ability to upregulate de novo lipogenesis (Juvet et al., 2003).

SREBP1 gene expression was observed to be down-regulated in animals treated with

single high dose of acetaminophen, carbon tetrachloride, tetracycline amiodarone

(Fukushima et al., 2006). The present study however, involved daily dosing of

acetaminophen for 15 days in rats. Thorough literature search indicated that the

reports for effects of such treatment on SREBP expression are not yet available. In the

present study, SREBP1 expression was reported to be higher in the animals which

were repeatedly treated with high dose of acetaminophen for 15 days as compared to

the healthy control.

Scanty references are available on the effect of herbal interventions on SREBP

expression in animal models for hepatotoxicity. A study involving C57BL/6-Lep

ob/ob mice reported the prevention of fatty liver by carbenoxolone intervention, an

active component of Glycyrrhiza glabra. The hepatoprotective ability of the

intervention was attributed to SREBP-1c inhibitory activity and anti-apoptotic action

of the intervention (Rhee et al., 2012). The diosgenin fraction from fenugreek inhibits

triglyceride accumulation in livers of diabetic obese KK-Ay mice as well as in HepG

2 cells with simultaneous decrease in the expressions of SREBP-1c, ACC and FAS

(Uemura et al., 2011). In the present study, expression of SREBP-1 was significantly

decreased in animals treated with the satwa of T. cordifolia, T. sinensis and Neem-

giloe as compared to negative control.

Mammalian intracellular fatty acid-binding proteins (FABPs) comprise a

superfamily of lipid-binding proteins which are involved in the fatty acid uptake,

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intracellular transport and in regulating lipid metabolism, cellular signaling

pathways and other lipid ligands (Wang et al., 2007; Storch and Thumser, 2010).

FABP is highly expressed in adipocytes, liver, muscle, heart, brain and macrophages

and the expression and activation of FABP1 has been reported to contribute to the

pathogenesis of obesity, metabolic syndrome and associated inflammation (Makowski

and Hotamisligil, 2004). Fatty acids transport, in and out of the cells, is a complicated

process and is important for function and utilization of lipids (Kim et al., 2007). The

effect of acetaminophen induced hepatotoxicity on FABP1 expression has been

studied with relation to oxidative stress. A dose-dependent increase in oxidative stress

induced by acetaminophen was associated with significantly low FABP1 expression

(Gong et al., 2014). FABP1 also plays an early protective role in acetaminophen

induced mitochondrial impairment through scavenging free radicals within the

mitochondria itself as well as in the cytosol (Gong et al., 2014). The role of L-FABP

in liver disease was further assessed by Wang et al. (2007). FABP1 has been reported

to possess strong antioxidant properties (Yan et al., 2009). In accordance with this

role of FABP, the present study observed decreased expression levels of FABP1 in

acetaminophen treated group and significantly higher expression in animals treated

with Neem-giloe satwa.

Expression of Genes Involved in Inflammation

NF-κβ (Nuclear factor-κβ) is one of the most important transcription factors

and it is activated by inflammatory cytokines like TNF-α (Tumor necrosis factor-

alpha) (Richard, 2001). Both are related to injury and inflammation, hepatitis,

immunity, including various etiologies of muscle catabolism and osteoclastogenesis

(Zwart et al., 2009). The NF-κβ pathway is complex and is activated by

phosphorylation, ubiquitination, and proteolysis of the inhibitory protein IκB (I kappa

B), which nominally binds NF-κβ in the cytosol in the inactive form (Zwart et al.,

2009). Blazka et al. (1995) reported the up-regulation of TNF-α in liver of

acetaminophen treated mice. Dambach et al. (2006) and Song et al. (2014) recently

reported significantly up-regulated expression of TNF-α and NF-κβ in

acetaminophen-induced hepatotoxicity in mice. Various inflammatory cytokines

produced during drug induced liver injury have been reported to be involved in tissue

damage (Ishida et al., 2002). Ishida et al. (2004) reported that, liver injury in mice

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deficient in the 55KDa TNF receptor (TNF-Rp55), was attenuated after APAP

challenge. In addition, APAP-induced mortality was reduced in TNF-Rp55 KO mice.

Studies in mice deficient in CCR2, the primary receptor for the chemokine MCP-1

(Monocyte chemoattractant protein-1), showed that these mice had increased toxicity

of TNF-α and IFN-α (Hogaboam et al., 2000). TNF-α is reported to promote tissue

damage during acetaminophen toxicity (Boess et al., 1998). mRNA and protein

expressions of TNF-α and NF-κβ were significantly upregulated in D-galactosamine–

induced hepatotoxicity (Aristatile et al., 2013). Tu et al. (2012) observed significant

increase in TNF-α in carbon tetrachloride intoxicated rats. The serum levels of pro-

inflammatory cytokines, such as TNF-α and NF-κβ were significantly elevated in

isoniazid induced hepatotoxicity in albino rats (Mahmoud et al., 2014). Roles of NF-

κβ in suppression (Van Antwerp et al., 1996) as well as induction (Grilli et al., 1996)

of apoptosis have been reported and NF-κβ is also thought to play a major role in liver

regeneration (Ulloa et al., 2002; Tsung et al., 2005; Luedde and Schwabe, 2011). In

the present study, NF-κβ and TNF-α gene expression were higher in acetaminophen

induced hepatotoxicity as compared with healthy control group.

The diabetic rats, treated for 24 weeks with Tinospora cordifolia extract

(250 mg/kg) exhibited significantly reduced amount of inflammatory markers such as

TNF-α and IL-1β (Agrawal et al., 2012). Several chemical constituents like alkaloids,

diterpenoid lactones, steroids, glycosidesetc from different parts of T. cordifolia are

known to inhibit the activity of NF-κβ and TNF-α (Mittal et al., 2014). G1-4A, an

arabinogalatan, found in T. cordifolia, increases levels of TNF-α and IL-10 against

endotoxic shock through modulation of cytokines and nitric oxide in mice (Desai et

al., 2007). Treatment of albino rats with Berberine, an isoquinoline alkaloid, leads to

decrease in the serum levels of TNF-α and NF-κβ in isoniazid and cyclophosphamide-

induced hepatotoxicity (Germoush and Mahmoud, 2014; Mahmoud et al., 2014).

Treatment of male Sprague-dawley rats with curcumin significantly reduced the levels

of proinflammatory mediators (TNF- α, IL-6 and MCP-1) mRNA in carbon

tetrachloride induced liver toxicity (Tu et al., 2012).

In present study, NF-κβ gene expression was found to be significantly

decreased in the rats treated with the satwa of T. cordifolia, T. sinensis and Neem-

giloe while there was statistically insignificant decrease in TNF-α gene expression in

Neem-giloe satwa treated rats. Silymarin, a standard drug used in the present study,

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has been reported to suppress NF-κβ gene expression in the hepatoma cell line

HEPG2 (Saliou et al., 1998). Apart from the intervention groups in the present study,

NF-κβ gene expression was also found to be significantly decreased in the rats treated

with Silymarin (positive control).

Based on the results obtained from biochemical, histological and gene

modulation studies in animals receiving interventions of satwa of T. cordifolia, T.

sinensis and Neem-giloe, hepatoprotective activity of Neem-giloe was found to be

better than that of T. cordifolia and T. sinensis, in acetaminophen induced

hepatotoxicity.

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Fig. 28. A Model for Probable Molecular Mechanism of Action of Satwa from Three different forms of Tinospora. A: Effect of T. cordifolia, B:

Effect of T. sinensis, C: Effect of Neem-giloe

A B C

APAP: Acetamniophen; PPARγ: Peroxisome proliferator-activated receptor γ; SREBP: Sterol regulatory element binding protein; NF-κβ: Nuclear factor kappa

β; Acyl-CoA: Acetyl coenzyme A; FAS: Fatty acid synthase; TNF-α: Tumour necrosis factor α; SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum

glutamic pyruvic transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very

low density lipoprotein; TG: Triglycerides: CH: Total cholesterol.

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6.4 Hepatoprotective Activity of Satwa against Ethanol Induced Hepatotoxicity


In the present study, administration of alcohol caused a significant elevation of

SGOT, SGPT, ALP, and total bilirubin when compared to healthy control, which is

indication of hepatic damage. Several reports indicate elevated levels of liver function

markers like SGOT, SGPT, ALP and bilirubin in ethanol-induced hepatotoxicity in

rats (Yue et al., 2006; Arulkumaran et al., 2009; Vidhya and Indira, 2009; Nigam and

Paarakh, 2011; Singh and Gupta, 2011; Cui et al., 2013; Padmanabhan and Jangle

2014; Seif, 2014). In the present study, serum levels of SGOT, SGPT, ALP and

bilirubin were significantly decreased after treatment with T. sinensis (200 mg/kg) as

compared to ethanol treated group. The hepatoprotective activity of ethanolic extract

of T. sinensis roots has previously been demonstrated in carbon tetrachloride induced

hepatotoxicity in rats (Naik et al., 2013). Treatment of albino rats with acetone and

aqueous extracts of Adina cordifolia showed significant decrease in the levels of

serum markers (SGOT, SGPT, ALP and total bilirubin), indicating the protection of

hepatic cells against ethanol induced hepatocellular injury (Sharma et al., 2012).

Treatment with aqueous extract of Andrographis paniculata (50mg/kg, 100mg/kg,

200mg/kg of body weight) was found to protect the rats from hepatotoxic action of

ethanol as evidenced by significant reduction in the elevated levels of SGOT, SGPT,

ALP and bilirubin (Vetriselvan et al., 2011).

Alcohol has significant effect on lipid and lipoproteins metabolism. Alcohol

induced liver injury is characterized by lipid accumulation in affected hepatocytes

(Ontko, 1973). In the present study, animals treated with ethanol showed increase in

the levels of serum and hepatic cholesterol, triglyceride, VLDL, LDL, along with

decrease in HDL levels as compared to healthy control. Such abnormal level of lipids

is an indication of disturbed lipid metabolism. Earlier studies also have reported

increased levels of serum and tissue lipids (Triglyceride, total cholesterol, and

phospholipids) in alcohol treated rats (Tomita et al., 2004; Kim et al., 2007;

Arulkumaran et al., 2009; Cui et al., 2013; Samundeeswari et al., 2013). In the present

study, Neem-giloe satwa exhibited normalization of serum lipid profile while T.

sinensis satwa normalized the lipid profile in liver. Treatment of animals with Neem-

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giloe satwa also revealed significant increase in total protein content. An earlier study

on polyherbal formulation, Vimliv, which contains T. cordifolia, showed significant

improvement of lipid profile (Triglyceride, total cholesterol, phospholipid) in serum

and liver tissue in ethanol-induced hepatic damage in female albino wistar rats

(Samundeeswari et al., 2013). A recent report by Sharma and Dabur (2015) also

indicated improvements in serum lipid profile after treatment of alcoholic volunteers

with water extract of T. cordifolia. They also showed that the water extract helped in

enhancing intestinal vitamin absorption and preventing multivitamin deficiency in

liver due to alcohol intake.

Oxidative stress plays an important role in the development of ALD

(Alcoholic liver disease) (Yurt and Celik, 2011). Recent literature of alcohol induced

hepatotoxicity indicates decrease in the levels of SOD, GSH and catalase and increase

in malondialdehyde, hydroperoxides in the liver homogenate of alcohol treated rats

(Das and Vasudevan, 2006; Mallikarjuna et al., 2009; Shanmugam et al., 2010; Arun

and Balasubramanian, 2011; Singh and Gupta, 2011; Rejitha et al., 2012; Seif, 2014).

Administration of aqueous extract of stem and leaves (400 mg/kg body weight, orally)

of T. cordifolia increased the activities of SOD and CAT and decreased the levels of

SGOT, SGPT and ALP enzymes in lead nitrate induced hepatotoxicity in mice

(Sharma and Pandey, 2010). In the present study, contents of hepatic SOD, GSH and

catalase significantly decreased while hepatic MDA increased in the ethanol treated

group. Treatment with T. sinensis satwa resulted in significant decrease in the levels

of MDA and significant increase in the levels of SOD, CAT and GSH in the liver

homogenate as compared with ethanol treated group.


The hepatic architecture in rats treated with alcohol frequently exhibits

hepatocytic necrosis, inflammation in centrilobular region with portal triads (Arun

and Balasubramanian, 2011), congestion, macrovesicular and microvesicular steatosis

(Nigam and Paarakh, 2011), fatty changes in the hepatocytes with intense

centrilobular necrosis and vacuolization (Singh and Gupta, 2011; Seif, 2014), tubular

epithelial cell degeneration with mononuclear cell infiltration, oedema, necrosis

(Samundeeswari et al., 2013), an enlargement of the hepatocytes, higher steatosis (fat

accumulation) and inflammatory injury (Kupffer cell activation) (Cui et al., 2013). T.

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cordifolia has been reported to protect liver damage against carbon tetrachloride

induced hepatotoxicity through preventing fibrosis, stimulating hepatic regeneration

and through diminishing congestion, inflammation, vacuolation, fatty changes etc.

(Bishayi et al., 2002; Sharma and Pandey, 2010). In the present study, sections of

liver from ethanol treated group showed swollen hepatocytes with granular

cytoplasm. Collections of few polymorphs were visible in hepatic parenchyma,

suggesting foci of necrosis. Treatment with T. sinensis satwa demonstrated near

normal liver histology as compared to ethanol treated group.



Excessive ethanol consumption leads to alcoholic liver disease (ALD) through

multifactorial and complex mechanism. Several animal experiments have shown the

effect of ethanol through regulation of hepatic expression of PPARγ and PPARγ

agonists have been shown to prevent alcohol-induced liver injury (Enomoto et al.,

2003; Ohata et al., 2004; Tomita et al., 2004). A recent study has shown that PPAR-α

and PPARγ agonist treatments reduced severity of chronic alcohol induced liver

injury including hepatic architectural disorder and steatosis (De la Monte et al., 2011).

PPAR-α and PPARγ expressions at protein levels and mRNA concentrations were

upregulated in the livers of Fischer rats with alcohol feeding (Luvizotto et al., 2010).

Chronic alcohol administration significantly reduced the hepatic expression of

PPARα, which is involved in lipid metabolism (Park et al., 2014). Alcohol intoxicated

mice supplemented with Aloe vera polysaccharides exhibit marked increase in mRNA

levels of PPAR-α which otherwise is down-regulated after alcohol treatment leading

to liver damage (Cui et al., 2013). Treatment of albino rats with 8β-Glycyrrhetinic

acid has been shown to exert hepatoprotective effects against cyclophosphamide-

induced hepatotoxicity through up-regulation of PPARγ (Mahmoud and Al Dera,

2015). In present study also, PPARγ gene expression was seen to be significantly

higher in T. sinensis treated group while the ethanol treated group showed lower

PPARγ gene expression.

Accumulation of ethanol also affects expression of SREBP and SREBP target

genes, thus further increasing lipid synthesis (Yin et al., 2007). You et al. (2002) have

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reported that chronic ethanol feeding induces fatty acid synthesis pathway by

activating SREBP-1, and this effect of ethanol may contribute to the development of

alcoholic fatty liver. Acute ethanol (A single oral dose of 0.5 or 5g/kg of body weight)

affects the expression levels of SREBP-1 and many other SREBP-1 target genes,

thereby increasing fatty acid synthesis in male ICR mice (Yin et al., 2007) and male

C57BL/6 mice (Ji and Kaplowitz, 2003). Cui et al. (2013) showed that alcohol

consumption decreases AMPK-α2 expression and elevates SREBP-1c levels in mice.

Huang et al. (2010) investigated the effects of Antrodia camphorata fruiting bodies

against chronic alcohol consumption in rats and found that the expression level of

SREBP-1c, Acetyl-CoA carboxylase, 3-hydroxy-3-methylglutaryl-CoA reductase,

fatty acid synthase and malic enzyme was down-regulated. The studies on traditional

Chinese medicines like Schisandra chinensis (Park et al., 2014) and Gentiana

manshurica (Lu et al., 2012) have demonstrated prevention of alcohol induced liver

damage through decreased expression of SREBP and decrease in SREBP-1 regulated

fatty acid synthesis. The present study also reports higher expression of SREBP-1 in

ethanol treated rats while its expression was significantly reduced in animals treated

with Neem-giloe satwa.

FABP1 has been reported in many metabolic disease processes, such as

cholestatic liver disease, cancer, diabetes, obesity, and atherosclerosis (Furuhashi and

Hotamisligil, 2008). FABP1 prevents free fatty acid induced lipotoxicity and is

known to be down regulated in the pathogenesis of non-alcoholic fatty liver disease

(NAFLD) in animal models as well as in NAFLD patients (Guzman et al., 2013).

Administration of Radix Platycodi (PR), the roots of Platycodon grandiflorum

(Traditional Oriental Medicine) significantly prevented alcohol-induced elevation of

serum and liver lipids by normalizing the FABP expression in alcohol-treated rats

(Kim et al., 2007). Nanji et al. (2004) also found reduced expression of FABP in

alcohol-fed rats. Protein as well as mRNA expression of L-FABP showed significant

decrease following ethanol consumption in mice (Smathers, 2011). In present study

also, expression of FABP1 decreased in the ethanol treated group while significant

increase in FABP1 expression was found in animals treated with T. sinensis and

Neem-giloe satwa.

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Acute ethanol administration causes prominent hepatic microvesicular

steatosis with mild necrosis and increased levels of SGPT and TNF-α in mice (Song

et al., 2006). Alcoholic hepatitis is characterized by hepatic inflammation with higher

levels of TNF-α, IL-1, IL-8 in animal models as well as patients (McClain et al.,

1999). Treatment of alcohol treated rats with Sida cordifolia is reported to decrease

the hepatic expression of inflammatory markers like NF-κβ and TNF-α (Rejitha et al.,

2012). Treatment of alcohol intoxicated mice with polysaccharides from Aloe vera is

reported to decrease the expression of TNF-α (Cui et al., 2013). Roy et al. (1994)

observed high TNF-α level in carbon tetrachloride treated rats while treatment of

animals with Liv.52 (Polyherbal hepatoprotective formulation) decreased levels of the

inflammatory marker. α-D glucan, a polysaccharide found in T. cordifolia, is reported

to enhance generation of TNF-α and other cytokines from human peripheral blood

mononuclear cells (Nair et al., 2004). In the present study also, expression of NF-κβ

and TNF-α was increased in ethanol treated animals while significant decrease in the

expression levels of the markers was found in the livers of the animals treated with T.

cordifolia satwa, T. sinensis satwa and Neem-giloe satwa.


modulation studies in animals receiving interventions of satwa of T. cordifolia, T.

sinensis and Neem-giloe, hepatoprotective activity of T. sinensis was found to be

better than that of T. cordifolia and T. sinensis, in ethanol induced hepatotoxicity.

181

Fig. 29. A Model for Probable Molecular Mechanism of Action of Satwa from Three different forms of Tinospora. A: Effect of T. cordifolia, B:

Effect of T. sinensis, C: Effect of Neem-giloe

A B C

PPARγ: Peroxisome proliferator-activated receptor γ; SREBP: Sterol regulatory element binding protein; NF-κβ: Nuclear factor kappa β; Acyl-CoA: Acetyl

coenzyme A; FAS: Fatty acid synthase; TNF-α: Tumour necrosis factor α; SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum glutamic pyruvic

transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density

lipoprotein; TG: Triglycerides: CH: Total cholesterol.

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6.5 Hepatoprotective Activity of Flax Oil and Fish Oil against Acetaminophen



Omega-3 fatty acid supplementation is known to improve liver function

markers in chronic diseases (Heller et al., 2004; Lee et al., 2007; Wu et al., 2012; Li et

al., 2014). Omega-3 fatty acids are essential dietary nutrients for normal growth and

development (Innis, 2004). Fish oil has been clinically and experimentally evaluated

in earlier studies for its beneficial effects in cardiovascular diseases, cancer,

rheumatoid arthritis, bone disease, psychiatric and immune disorders (Simopoulos,

1991; De Caterina et al., 1994; Liu et al., 2001). Khanchandani et al. (2014) revealed

that the administration of omega-3 fatty acid (Fish oil) at a dose of 600 mg/kg b.w. to

albino rabbits reduced the elevated serum liver enzymes like SGOT, SGPT, ALP,

bilirubin when compared with CCl4 administered group which indicates

heptoprotective nature of omega-3 fatty (Fish oil) acids. Treatment of albino rats with

fish oil showed significant decrease in serum SGOT, SGPT and creatinine levels in

ifosfamide induced toxicity (Asaad and Aziz, 2012). Fish oil at a dose of 300 mg/kg

revealed significant lowering of enzymes like SGOT, SGPT but had a less marked

effect on the levels of ALP and total bilirubin (Meganathan et al., 2011). Roy et al.

(2007) reported that dietary supplementation of fish oil exhibited strong

hepatoprotective activity against galactosamine induced liver damage in mice. In the

present study also, treatment of rats with fish oil (500 mg/kg bw) showed significant

reduction in the serum levels of SGOT, SGPT, ALP and total bilirubin against

acetaminophen induced hepatotoxicity.

Fish oil predominantly contains EPA (Eicosapentaenoic acid) and DHA

(Docosachexaenoic acid) (Egert et al., 2009; Skulas-Ray et al., 2011). Several studies

have reported the triglyceride lowering effect of dietary fish oil (Rivellese et al., 1996;

Montori et al., 2000; De Caterina et al., 2007; Devarshi et al., 2013; Lorente-Cebrian

et al., 2013; Bremer et al., 2014). A clinical study has shown a significant increase in

HDL and a decrease in cholesterol, triglyceride levels in hyperlipidemic individuals

after receiving 1.3g fish oil in bread daily for 2 to 4 weeks (Liu et al., 2000). Flax oil

and fish oil treatment reduced serum triglycerides and VLDL levels in STZ and STZ-

NIC induced diabetic rats (Kaithwas and Majumdar, 2012; Devarshi et al., 2013).

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Treatment with fish oil and flax oil showed reduction in the plasma levels of

triglycerides and cholesterol in mice fed on high fat diet (Riediger et al., 2008). In the

present study, flax oil treatment exhibited significant reduction in the serum levels of

total cholesterol, LDL, triglycerides and VLDL as compared to acetaminophen treated

group while fish oil treatment lead to significant increase in the HDL level. The

hepatic lipid profile in the present study was also found to be normalized by flax and

fish oil treatment.

Naqshbandi et al. (2011) reported antioxidant properties of fish oil through

increase in the levels of SOD, CAT, GSH and decrease in the levels of MDA in liver

homogenates of cisplatin-induced hepatotoxic rats. The antioxidant property of fish

oil has also been demonstrated by decrease in the hepatic MDA level in

acetaminophen induced hepatotoxicity by pre-treatment of rats with fish oil (Kalra et

al., 2012). The animals fed on fish oil showed significant higher activities of catalase,

glutathione peroxidase and superoxide dismutase in rat liver (Ruiz-Guiterrez et al.,

1999). Besides improvement in antioxidant status, fish oil also decreases the hepatic

hydroperoxide contents in atherosclerotic rabbits (Aguilera et al., 2003). Fish oil and

flax oil are the richest sources of omega-3 fatty acids and the present study indicated

that they are also potent antioxidants which significantly improve the antioxidant

markers in acetaminophen treated rats (Chavan et al., 2013b). In the present study,

fish oil exhibited higher antioxidant properties than flax oil, as indicated by

significant increase in the levels of SOD, CAT and GSH and significant decrease in

MDA levels in liver homogenates in acetaminophen induced hepatotoxicity.


Damage to the liver due to acetaminophen induced hepatotoxicity at

histological level is discussed earlier in the thesis. In the present study, acetaminophen

treated group showed swollen or occasionally apoptotic hepatocytes with coarse

granular cytoplasm and compressed sinusoids. Treatment with both fish and flax oil

exhibited strikingly normal liver histology without any anatomically detectable

anomalies. The hepatoprotective activity of omega-3 fatty acids has been

demonstrated by pre-treatment of albino mice with fish oil which reduces liver

damage caused by galactosamine (Roy et al., 2007). Meganathan et al. (2011)

observed healthy liver histology in rats treated with fish oil in acetaminophen induced

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184

liver injury. Omega-3 fatty acid (Fish oil) treated groups exhibited protection of

hepatic lobules with mild fatty changes and localized necrosis when compared with

carbon tetrachloride treated group (Khanchandani et al., 2014). As described above,

the present study also reports near normal liver histology in the animals treated with

flax oil and fish oil.



Dietary omega-3 fatty acids play various physiological roles and serve as

biological regulators. Polyunsaturated fatty acids are a fundamental part of cell

membrane and they also act as signaling molecules triggering cascade of intra-cellular

signaling (Hwang and Rhee, 1999; Merendino et al., 2013). In recent years, direct

involvement of omega-3 fatty acids in regulation of gene expression has been

established (Price et al., 2000; De Caterina and Massaro, 2005; Deckelbaum et al.,

2006). Omega-3 fatty acids have been shown to decrease the transcriptional activation

of many genes like adhesion molecules, chemoattractants, and inflammatory

cytokines involved in endothelial activation in response to inflammatory and pro-

atherogenic stimuli (De Caterina and Massaro, 2005). EPA and DHA, the

predominant omega 3 fatty acids present in fish oil, have been reported to act as

natural ligands for activation of PPARγ (Trombetta et al., 2007). Increase in the

dietary polyunsaturated fatty acids upregulates PPARα and PPARγ expression in the

spleen, liver and bursa of chickens (Selvaraj et al., 2010). In the present study, hepatic

expression of PPARγ was significantly increased in animals treated with fish oil as

compared to acetaminophen treated group which exhibited decreased levels of PPARγ

expression.

Treatment of rats with flax oil and fish oil (Omega-3 fatty acid) showed down

regulation of hepatic SREBP-1 expression along with decreased serum triglyceride

levels (Davidson, 2006; Devarshi et al., 2013). There are numerous reports on omega-

3 supplementation in animals indicating down regulation of SREBP-1c (Kim et al.,

1999; Xu et al., 1999; Yahagi et al., 1999; Botolin et al., 2006; Kim et al., 2014). Fish

oil supplementation up regulates FABP-1 mRNA, involved in DHA uptake and

decreases the expression of SREBP-1 (Dutta-Roy, 2000; Gaca et al., 2012). In present

study also, significant decrease in the expression of SREBP-1 was observed in rats

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185

treated with flax and fish oil and FABP1 was found to be up-regulated in flax oil as

compared with acetaminophen treated animals.


Omega-3 fatty acids such as EPA and DHA are bioactive dietary compounds.

Omega-3 fatty acids intervention in rats prevented inflammation and severe hepatic

steatosis when fed on methionine/choline deficient diets (Marsman et al., 2013).

Omega-3 fatty acids decrease the production of inflammatory proteins which may be

mediated by altered activation of key transcription factors regulating NF-κβ and TNF-

α (Babcock et al., 2000; Novak et al., 2003; Kang and Weylandt, 2008; Scorletti and

Byrne, 2013). TNF-α has been linked to hepatotoxicity, sepsis, and the inflammatory

response (Wielockx et al., 2001; Lee, 2007). In a mouse model, TNF-α induction was

observed in apoptosis and necrosis of hepatocytes leading to liver failure (Wielockx et

al., 2001). In the present study, expression of TNF-α and NF-κβ was higher in

acetaminophen treated group, indicating inflammatory response in the hepatocytes.

The TNF-α expression was significantly reduced in animals treated with flax and fish

oil while reduction in NF-κβ was observed in rats treated with flax oil.


modulation studies in animals receiving interventions of polyunsaturated fatty acids

(flax oil and fish oil), hepatoprotective activity of fish oil was found to be better than

that of flax oil, in acetaminophen induced hepatotoxicity.

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Fig. 30. A Model for Probable Molecular Mechanism of Action of Flax Oil and Fish Oil Interventions. A: Effect of Flax Oil, B: Effect of Fish Oil

A B


β; Acyl-CoA: Acetyl coenzyme A; FAS: Fatty acid synthase; TNF-α: Tumour necrosis factor α; SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum



187

6.6 Hepatoprotective Activity of Flax Oil and Fish Oil against Ethanol Induced

Hepatotoxicity


In the present study, flax oil intervention in ethanol treated rats produced

significant normalization of liver function. Phenolic components in flaxseed have

been shown to be effective in restoration of increased activities of liver function

enzymes (Kasote et al., 2012). Ismail et al. (2009) have reported normalization of

serum SGPT and SGOT levels after flax oil treatment in carbon tetrachloride

intoxicated rats. Shakir and Madhusudhan (2007) reported that rats treated with

flaxseed chutney showed decrease in the levels of liver markers such as SGOT,

SGPT, ALP and bilirubin in serum and liver homogenate against carbon tetrachloride

induced hepatotoxicity. In present study also, treatment of rats with flax oil (500

mg/kg) significantly reduced levels of SGOT, SGPT, ALP and bilirubin as compared

to ethanol treated group.

Raw flaxseeds as well as dietary flaxseeds as baked products, are known to

have hypolipidemic, hypoglycemic and hypocholesterolemic effects (Cunnane et al.,

1995; Shakir and Madhusudhan, 2007). Several animal and human intervention

studies indicate that flaxseed oil has beneficial effects on serum lipid profile

(Mahmud et al., 2004; Vijaimohan et al., 2006; Riediger et al., 2008; Bassett et al.,

2009; Newairy and Abdou, 2009; Kaithwas and Mujumdar, 2012). Administration of

15% flaxseed chutney or flaxseed oil to rats showed improvements of the lipid profile

(Increased level of HDL, decreased level of total cholesterol and LDL) in serum and

liver homogenates (Shakir and Madhusudhan, 2007). Ismail et al. (2009)

demonstrated that supplementation of flaxseed oil resulted in significant reduction in

serum total cholesterol, LDL and VLDL in rats treated with carbon tetrachloride.

High cholesterol diet-induced hypercholesterolemic rats exhibited lower serum total

cholesterol, triacylglycerols, LDL, VLDL, phospholipids, and increase in HDL after

flaxseed oil intervention (Hussein et al., 2014). In the present study also, flax oil

showed normolipidemic effects with decreased levels of total cholesterol, LDL,

triglycerides, VLDL and increased HDL in serum as well as liver homogenates.

188

Hepatoprotective and anti-oxidant activity of flaxseed has been demonstrated

through restoration of antioxidant enzymes in the liver of flaxseed pre-treated animals

challenged with carbon tetrachloride (Rajesha et al., 2006). Treatment of the hull

fraction of flaxseed also resulted in a significant increase in hepatic anti-oxidant

enzymes such as SOD, CAT, peroxidase as compared to carbon tetrachloride treated

group which is attributed to secoisolariciresinol diglucoside (SDG) content of the

flaxseed hull (Rajesha et al., 2010). Treatment of rats with flax lignans showed

decrease in TBARS and increase in the activities of glutathione S-transferase, SOD,

glutathione reductase and CAT as compared to lead acetate treated rats (Newairy and

Abdou, 2009). Several studies reported reduced levels of lipid peroxides (MDA) in

liver tissue in flaxseed oil treated animals (Lee and Prasad, 2003; Abdel-Moneim et

al., 2011). The present study also reports significant increase in activity of antioxidant

enzymes (SOD, CAT), increase in reduced glutathione and reduced levels of lipid

peroxides in flax oil treated animals as compared to ethanol treated group.


Several studies on liver damage due to ethanol induced hepatotoxicity have

already been discussed. In the present study, animals treated with flax oil showed near

normal hepatic architecture. There are several reports which indicate almost normal

liver architecture with minor histological anomalies after treatment with omega-3

fatty acids like flax oil or fish oil (Ismail et al., 2009; Meganathan et al., 2011; Kasote

et al., 2012; Khanchandani et al., 2014).



In the present study, PPARγ level was significantly increased in fish oil

treated animals and SREBP-1 was significantly decreased in flax oil and fish oil as

compared to ethanol treated animals. Omega-3 fatty acids are known to have their

effects on modulation of gene expression in cultured hepatocytes as well as in the

animal livers. The mRNA and protein expression of SREBP-1c was found to be

suppressed after treatment of the cells or animals with PUFAs (Sekiya et al., 2000;

Hannah et al., 2001; Yoshikawa et al., 2002; Nakatani et al., 2003). Fish oil has been

reported to increase the expression of PPARγ mRNA in mice liver (Yamazaki et al.,

189

2007). Fish oil also has effects on reduction of SREBP-1c and increase in the PPARα

expression in ethanol induced fatty liver (Wada et al., 2008).

Nanji et al. (2004) have reported that the expression of FABP decreases with

increase in the extent of fatty liver in ethanol intoxicated rats. They detected lowest

expression of L-FABP in the animals which developed severe fatty liver. Intestinal

expression of L-FABP was found to be upregulated in mice treated with sunflower oil

which was shown to be due to linoleic acid in sunflower oil (Poirier et al., 1997). In

the present study also, near normal liver function and histology was associated with

flax oil treatment which showed increase in the level of FABP1 expression as

compared to ethanol treated animals.


Fish oil supplementation in healthy human volunteers lead to decreased

production of inflammatory cytokines (like TNF-α, IL-1 and IL-6) by mononuclear

cells (Caughey et al., 1996; Baumann et al., 1999; Trebble et al., 2003). Baumann et

al. (1999) have exclusively demonstrated that only omega-3 and not omega-6 and

omega-9 fatty acids, can significantly alter the expression levels of the genes involved

in inflammation and atherosclerosis. Treatment of mice with omega-3 fatty acids

reduced serum alanine aminotransferase (SGPT) levels and decreased inflammatory

response with decreased plasma TNF-α levels and this treatment also led to reduced

hepatic expression of TNF-α, IL-1β, IFN-γ and IL-6 as compared to

lipopolysaccharide/D-galactosamine-induced hepatitis model (Schmocker et al.,

2007). In the present study, expression of NF-κβ and TNF-α was significantly

decreased in flax oil treated animals as compared to alcohol treated animals.


modulation studies in animals receiving interventions of polyunsatured fatty acids

(flax oil and fish oil), hepatoprotective activity of flax oil was found to be better than

that of fish oil, in ethanol induced hepatotoxicity.

190

Fig. 31. A Model for Probable Molecular Mechanism of Action of Flax Oil and Fish Oil Interventions. A: Effect of Flax Oil, B: Effect of Fish Oil

A B

PPARγ: Peroxisome proliferator-activated receptor γ; SREBP: Sterol regulatory element binding protein; NF-κβ: Nuclear factor kappa β; Acyl-CoA: Acetyl

coenzyme A; FAS: Fatty acid synthase; TNF-α: Tumour necrosis factor α; SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum glutamic pyruvic

transaminase; ALP: Alkaline phosphatase; BIL: Total bilirubin; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density

lipoprotein; TG: Triglycerides: CH: Total cholesterol.

191

6.7 Protective and Corrective Effects of Combination of Neem-giloe Satwa and

Fish Oil against Acetaminophen Induced Hepatotoxicity


Hepatoprotective effects of Neem-giloe satwa and fish oil were separately

evaluated against acetaminophen induced hepatotoxicity. The studies revealed that the

comparative hepatoprotective activity of Neem-giloe satwa and fish oil was higher

than other two satwa (T. cordifolia and T. sinensis) and flax oil, respectively. The

present study was carried out to evaluate protective and corrective effects of

combination of Neem-giloe satwa and fish oil against acetaminophen induced

hepatotoxicity.

Herbal medicines and nutraceutical supplements have been shown to have

preventive role in several diseases. Several medicinal herbs with hepatoprotective

properties have been reported in literature, e.g. Allium sativum (Garlic) (Ajayi et al.,

2009), Silybum marianum (Milk Thistle) (Freitag et al., 2015), Glycyrhizza glabra

(Licorice Root) (Sharma and Agrawal, 2014), Curcuma longa (Turmeric root) (Bae et

al., 2006) and Neem-guduchi (Chavan et al., 2013a; Nagarkar et al., 2013), Tinospora

cordifolia (Bishayi et al., 2002). In Asian countries, several studies revealed that

many dietary food items and supplements possess hepatoprotective activity (Shukla

and Kumar, 2013). There are a number of different food items and supplements e.g.

hepatoprotective spices like Turmeric (Luper, 1999), Coriander (Samojlik et al.,

2010), Garlic (Ajai et al., 2009), Red chili (Kim et al., 2005); hepatoprotective fruits

like Grapes (Carbo et al., 1999), Custard apple (Chavan et al., 2011), Apple (Miura et

al., 2007), Pomegranate (Toklu et al., 2007); hepatoprotective vegetables and grains

like Carrot (Bishayee et al., 1995), Sweet corn (Guo et al., 2009), Soy (Hu et al.,

2004), hepatoprotective drinks like Green tea (Luper, 1999), Coffee (Wang et al.,

2009); hepatoprotective omega-3 fatty acids like flax oil (Shakir and Madhusudhan,

2007; Ismail et al., 2009; Kasote et al., 2012) and fish oil (Meganathan et al., 2011;

Asaad and Aziz, 2012; Khanchandani et al., 2014). Food ingredients contain several

phytochemicals beneficial in liver injuries and possess potential to prevent or reverse

different kinds of liver injuries. There are several studies reported on either herbal or

nutritional supplements (Omega-3 fatty acid) against different hepatotoxicants

192

(Bishayi et al., 2002; Khanchandani et al., 2014; Sharma and Agrawal, 2014; Freitag

et al., 2015). A recent report shows that treatment of rats with a combination of

Glycyrrhizin and omega-3 fatty acid (fish oil) leads to significant decrease in SGOT

and SGPT activities as compared to thioacetamide treated rats (Abo El-Magd et al.,

2015). In the current study, protective and corrective treatment of rats with a

combination of Neem-giloe satwa and fish oil showed significant decrease in the

levels of SGOT, SGPT, ALP and bilirubin as compared to acetaminophen treated

groups. In a single-blind, placebo-controlled, crossover study of combinatorial

treatment of fish oil and garlic showed decrease in cholesterol, triglyceride, and LDL

levels, as well as increase in HDL as compared to placebo treated group (Morcos,

1997). In present study also, protective and corrective treatment of combination of

Neem-giloe satwa and fish oil showed improvement in the lipid profile (decrease in

cholesterol, LDL, VLDL and triglyceride and increase in HDL) in serum and liver

homogenates. Total protein of liver homogenate was significantly improved in

corrective effect of the combination as compared to acetaminophen treated group.

A combination of Glycyrrhizin and fish oil to thioacetamide intoxicated rats

exhibited strong antioxidant activity with significant increase in hepatic MDA levels

(Abo El-Magd et al., 2015). In the present study, combination of Neem-giloe satwa

and fish oil was also found to have antioxidant effects and it decreased the extent of

lipid peroxidation and improved the levels of SOD, reduced glutathione and CAT as

compared to acetaminophen treated group.


In the present study, sections of liver from the animals treated with a single

dose of acetaminophen on 8th

day showed swelling and degenerative changes in

hepatocytes with compressed sinusoids. The protective treatment of combination of

Neem-giloe satwa and fish oil showed improvement in hepatic architecture which still

showed some hepatocytes with degenerative changes. The corrective treatment of

combination of Neem-giloe satwa and fish oil showed near normal liver histology as

compared to that in the animals treated with acetaminophen which exhibited

congestion of central vein and swollen hepatocytes with ballooning degeneration.

193



Thorough literature search indicated lack of references on expression studies

of genes from lipid metabolism, in acetaminophen induced hepatotoxicity and

combination of herbal and nutraceutical interventions.

In the present study, the animals with the intervention of combination of satwa

and fish oil, exhibited significant up-regulation of PPARγ and FABP1 while

expression of SREBP-1 was down-regulated.


A recent report by Abo El-Magd et al. (2015) has reported increase in the

expression of NF-κβ in hepatic tissues of thioacetamide treated rats which was

reduced after treatment with a combination of glycyrrhizin and fish oil (Abo El-Magd

et al., 2015). In the present study also, protective and corrective treatment of

combination of interventions showed significant down-regulation of NF-κβ and TNF-

α.

194

Fig. 32. A Model for Probable Molecular Mechanism of Action of Neem-giloe and Fish oil. A: Protective Effect of Combination of Neem-giloe

Satwa, B: Corrective Effect of Combination of Neem-giloe Satwa

A B


β; Acyl-CoA: Acetyl coenzyme A; FAS: Fatty acid synthase; TNFα: Tumour necrosis factor α; SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum



195

6.8 Prophylactic Effect of Combination of Tinospora sinensis Satwa and Flax Oil

against Ethanol Induced Hepatotoxicity


Hepatoprotective effects of T. sinensis satwa and flax oil were separately

evaluated against ethanol induced hepatotoxicity. The studies revealed that the

comparative hepatoprotective activity of T. sinensis satwa and flax oil was higher than

other two satwa (T. cordifolia and Neem-giloe) and fish oil, respectively. The present

study was carried out to study the prophylactic effects of combination of T. sinensis

satwa and flax oil against ethanol induced hepatotoxicity. In the present study,

combination of T. sinensis satwa and flax oil showed significant reduction of SGOT,

SGPT, ALP and bilirubin as compared to ethanol treated group. The treatment also

normalized serum and hepatic lipid profile and improved hepatic antioxidant status. A

report by Wahba and Ibrahim (2013) showed improvement in the serum levels of liver

function markers, lipid profile as well as increase in tissue antioxidant enzymes after

treatment with flax oil in combination with vitamin E in potassium bromate treated

rats.


Effects of ethanol treatment and interventions on the hepatic architecture are

described in the results section. There are no previous reports describing hepatic

architecture in ethanol intoxicated rats treated with combination of herbal and

nutraceutical interventions.



PPARγ level was significantly up regulated and SREBP-1 level was found

normalized in treatment with combination of T. sinensis satwa and flax oil. In

prophylactic effect, FABP1 was down regulated in ethanol treated group while it was

up regulated in treatment with combination of T. sinensis satwa and flax oil.

There are no previous reports on the gene expression studies of combination

of interventions in ethanol induced hepatotoxicity. In the present studies, PPARγ was

196

significantly up-regulated while NF-κβ was shown to be significantly down-regulated

in intervention group.


In the present study, NF-κβ and TNF-α expressions were up regulated in

ethanol treated animals. NF-κβ gene expression was found significantly decreased in

the animals treated with a combination of T. sinensis satwa and flax oil. TNF-α

expression was significantly decreased in healthy control and no significant change

was observed due to treatment of combination of T. sinensis satwa and flax oil.

Treatment of animals with combinations of Neem-giloe and fish oil and T.

sinensis and flax oil in acetaminophen and ethanol induced hepatotoxicity

respectively, showed comparatively better results than intervention of any one of

them. So combination of herbal and nutritional intervention has shown beneficial

effects on liver disease.

197

Fig. 33. A Model for Probable Mechanism of Action of T. sinensis Satwa and Flax oil

A

PPARγ: Peroxisome proliferator-activated receptor γ; SREBP: Sterol regulatory element

binding protein; NF-κβ: Nuclear factor kappa β; Acyl-CoA: Acetyl coenzyme A; FAS: Fatty

acid synthase; TNF-α: Tumour necrosis factor α; SGOT: Serum glutamic oxaloacetic

transaminase; SGPT: Serum glutamic pyruvic transaminase; ALP: Alkaline phosphatase;

BIL: Total bilirubin; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL:

Very low density lipoprotein; TG: Triglycerides: CH: Total cholesterol.

SUMMARY AND Conclusions

Awards

Prophylactic effect of combination of herbal and nutritional interventions against

alcohol induced hepatotoxicity in rats

Hepatoprotective Effect Of Polyunsaturated Fatty Acids And Satwa From Three

Tinospora Species Exhibits Differential Hepatoprotective Activity Against Repeated

Alcohol Dosing In Rats

Hepatoprotective Effect Of Polyunsaturated Fatty Acids Against Repeated Subacute


Comparative Hepatoprotective Potential Of T.cordifolia, T.sinensis and Neem-Giloe

(Tinospora Cordifolia Growing On Azadirachta Indica (Neem)) Against

Paracetamol Intoxication In Rats

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Liver diseases have serious adverse effects on health. Allopathic medicines

prescribed for liver diseases have several side effects and hence modern medicine is

still short of reliable and effective drugs for protection and repair of liver. There are

many herbs being used to treat various liver disorders. In India, traditional medicine

system and ethno-medical practices rely on several medicinal plants and their

formulations to treat liver disorders.

The objective of the present study was to assess hepatoprotective activity of

ayurvedic formulation and nutritional supplements that can potentially be used as

hepatoprotective agent having less/no side effects. Based on the literature review,

medicinal values and availability, three different forms of Tinospora (satwa of stem)

and omega-3 fatty acids (Flax oil and fish oil) were selected to evaluate their

hepatoprotective potential.

The current section summarizes organoleptic characteristics and nutritional

analysis of satwa forms three Tinospora forms and also hepatoprotective activity of

satwa from three forms of Tinospora as well as that of flax oil and fish oil against

acetaminophen and alcohol induced hepatotoxicity.

Methodology

The different organoleptic characteristics and nutritional parameters like

protein, carbohydrates, lipid, starch, crude fiber, and ash were analyzed from satwa,

from three different forms of Tinospora. The hepatoprotective activity of satwa from

three different forms of Tinospora and omega-3 fatty acids (Flax oil and fish oil) was

studied by employing acetaminophen and alcohol induced hepatotoxicity in animal

models. The biochemical parameters like serum SGPT, SGOT, ALP, bilirubin, total

cholesterol, triglycerides, HDL cholesterol and LDL cholesterol, Hepatic antioxidant

parameters like catalase, SOD, MDA, GSH, total protein and functional parameters

like histopathology of liver, were studied. Further study on modulation of expression

of the genes involved in lipid metabolism and inflammation was also carried out. As

per the current study, maximum yield of satwa was obtained in T. sinensis and Neem-

giloe. T. sinensis and Neem-giloe are rich sources of nutritional contents. The contents

of lipid, ash, and carbohydrates are found to be greater in Neem-giloe than

199

T. cordifolia and T. sinensis. Protein, starch and crude fiber content is found higher in

T. sinensis as compared with T. cordifolia and Neem-giloe.

Results of this study reported significant increase in serum levels of liver

function tests (SGOT, SGPT, ALP, and bilirubin) and lipid profile (Total cholesterol,

LDL cholesterol, VLDL, Triglycerides and decrease in HDL cholesterol levels) in the

groups treated with acetaminophen and alcohol indicating induction of hepatotoxicity.

It further indicated abnormal integrity of hepatocytes, resulting into liver damage.

This resulted into significant elevation of lipid peroxidation with simultaneous decline

in GSH, CAT, SOD and total protein levels in liver tissue.

Among the three satwa, Neem-giloe satwa and T. sinensis satwa exhibited

higher hepatoprotective/hepatoregenerative activity in acetaminophen and alcohol

induced liver toxicity. The intervention of fish oil and flax oil proved beneficial in

acetaminophen and alcohol induced liver damage. The combination of Neem-giloe

satwa and fish oil in acetaminophen induced liver toxicity and combination of T.

sinensis satwa and flax oil for alcohol induced liver damage indicated improvement

over their individual activities. These treatments showed significant decrease in serum

levels of SGOT, SGPT, ALP, bilirubin and lipid profile (Total cholesterol, LDL

cholesterol, VLDL, Triglycerides and increase in HDL cholesterol levels) and also

exhibited reduction in lipid peroxidation and significant increase in levels of GSH,

CAT, SOD and total protein as compared with acetaminophen and alcohol treated

groups respectively. Histopathological observations showed that the above treatments

also improved hepatic architecture when compared with acetaminophen and alcohol

treated groups.

Expressions of FABP1 and PPARγ were down regulated while NF-κβ, TNF-α

and SREBP1 were upregulated in acetaminophen and alcohol treated groups.

Treatment with individual satwa and omega-3 fatty acids as well as their

combinations (as indicated above) lead to downregulation of the exression of NF-κβ,

TNF-α and SREBP1 and upregulation of FABP1 and PPARγ.

Herbal and nutraceutical intervations showed beneficial effects at biochemical,

histological and molecular levels in chemical induced hepatotoxicity. The

hepatoprotective effect of T. sinensis satwa, Neem-giloe satwa, fish oil and flax oil

200

may be due individual phytochemicals and nutritional constituents or their synergistic

action.

Conclusions

The present study clearly establishes the hepatoprotective/hepatoregenerative

effects of Neem-giloe and T. sinensis (Herbal interventions) and flax and fish oil

(Nutraceutical interventions) on hepatic disorders caused by repeated acetaminophen

and alcohol dosing in rats.

Neem-giloe satwa showed hepatoprotective effect against acetaminophen and

Tinospora sinensis satwa showed hepatoprotective effect against alcohol

induced hepatotoxicity.

Fish oil and flax oil showed hepatoprotective effect against acetaminophen

and alcohol induced hepatotoxicity respectively.

Combination of Neem-giloe and fish oil showed hepatoprotective effect

against acetaminophen and combination of T. sinensis and flax oil showed

hepatoprotective effect against alcohol induced hepatotoxicity.

The herbal medicines and omega-3 fatty acid supplements and their combination

counter the liver toxicity at the biochemical, histological and molecular levels by

normalizing oxidative stress and inflammation. Their action at cellular level could be

seen in the form of repair and regeneration of hepatocytes. The combination reported

in the present study is worth further investigation, especially in clinical cases of liver

damage due to long term alcohol abuse.

FUTURE Direction

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Future Direction

The hepatoprotective/hepatoregenerative effects of Neem-giloe satwa, T.

sinensis satwa and omega-3 fatty acids (Flax oil and fish oil) are worth further

investigations in humans through well-designed clinical trials to ascertain their

efficacy in clinical liver damage cases of varying severity.

CHAPTER 8

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PUBLICATIONs

ECES

Awards




Presentation

Awards


Presentation

273

Publications (Total no: 5)

Total Impact Factor (8.99)

[1] Chavan T, Khadke S, Harke S, Ghadge A, Karandikar M, Pandit V, Ranjekar

P, Kulkarni O, Kuvalekar A. Satwa from three Tinospora species exhibits

differential hepatoprotective activity against repeated acetaminophen dosing in

rats. Journal of Pharmacy Research. 2013; 123-128. (Impact Factor : 2.63)


P, Kulkarni O, Kuvalekar A. Hepatoprotective effect of polyunsaturated fatty

acids against repeated subacute acetaminophen dosing in rats. International

Journal of Pharma and Bio Science. 2013; 4(2): 286-295. (Impact Factor:

5.12)

[3] Chavan T, Mandhare A, Kulkarni O, Kuvalekar A. Nutritional evaluation of

satwa, an ayurvedic formulation of three Tinospora species from India

International Journal of Vedic Research Phytomedicine. 2014; 2(2): 53-58.

[4] Chavan T, Ghadge A, Karandikar M, Pandit V, Ranjekar P, Kulkarni O,

Kuvalekar A. Prophylactic effect of combination of herbal and nutritional

interventions against alcohol induced hepatotoxicity in rats. UGC Sponsored

Nation Conference on Innovative Ideas and Research in Life Science for

Sustainable Development: January 2015; ISBN: 978-81-925586-6-0,

Organized by: Nowrosjee Wadia College of Art and science, Pune. 80-89.


Kuvalekar A. Mantri N. Hepatoprotective activity of stawa, an ayurvedic

formulation, from three forms of Tinospora against alcohol induced liver

injury in rats. Alternative Therapies in Health and Medicine. 2016

(Impact Factor: 1.24)

274

Publication (Communicated)

[1] Chavan T, Kuvalekar A. A review on drug induced hepatotoxicity and

alternative therapies. Asian Pacific Journal of Tropical Biomedicine

Under Preparation

[1] Chavan T, Ghadge A, Kuvalekar A. Mantri N. Modulation of expressions of

genes from lipid metabolism and inflammatory pathways in response to

intervention of satwa of three different Tinospora forms in acetaminophen and

alcohol induced hepatotoxicity in rats.

[2] Chavan T, Ghadge A, Karandikar M, Pandit V, Kuvalekar A.

Hepatoprotective effects and modulation of genes from lipid metabolism and

inflammatory pathway in response to intervention of polyunsaturated fatty

acids (flax oil and fish oil) in alcohol induced hepatotoxicity in rats

AWARDS

Awards











Protein synthesis


Presentation Award


Award

Awards











Protein synthesis


Presentation Award


Award

275

Awards

[1] SECOND PRIZE IN ORAL PRESENTATION Titled “Prophylactic

Effect of Combination of Herbal and Nutritional Interventions against

Alcohol Induced Hepatotoxicity in Rats” in National Conference on

“Innovative Ideas and Research in Life Sciences for Sustainable

Development” organized by Department of Zoology, Nowrosjee Wadia

College of Arts and Science, Pune on 16th

and 17th

January 2015.

[2] BEST POSTER PRESENTATION AWARD for paper titled

“Hepatoprotective Effect of Polyunsaturated Fatty Acids against

Repeated Subacute Alcohol Dosing in Rats” in National Conference on

“Recent Trends in Cell Biology and Biotechnology” organized by Department

of Zoology, Balwant College, Vita on 6th

and 7th

September, 2013.

1

INT

PRESENTATIONs

Awards











Protein synthesis


Presentation Award


Award

Awards











Protein synthesis


Presentation Award


Award

276

International Conference

Chavan T, Ghadge A, Karandikar M, Pandit V, Ranjekar P, Kulkarni O,

Kuvalekar A. “Hepatoprotective effect of polyunsaturated fatty acids and

satwa from three Tinospora species exhibits differential hepatoprotective

activity against repeated alcohol dosing in rats” at the “International

Conference on Biotechnology and Bioinformatics (ICBB-2014)”, organized

from 1-2 February, 2014 at Yashada Auditorium, Pune, India (Poster

Presentation)

National Conferences


Kuvalekar A. “Prophylactic effect of combination of herbal and nutritional

interventions against alcohol induced hepatotoxicity in rats” at the National

Conference on “Innovative ideas and research in life sciences for

sustainable development” organized by Department of Zoology, Nowrosjee

Wadia College of Arts and Science, Pune on 16th

and 17th

January 2015 (Oral

Presentation)


P, Kulkarni O, Kuvalekar A. “Hepatoprotective effect of polyunsaturated

fatty acids against repeated subacute alcohol dosing in rats” at the National

Conference on “Recent Trends in Cell Biology and Biotechnology”

organized by Department of Zoology, Balwant College, Vita on 6th

and 7th

September 2013 (Poster Presentation)

[3] Chavan T, Kulkarni R, Kasote D, Kulkarni O, Harsulkar A, Jagtap S,

“Comparative hepatoprotective potential of Tinospora cordifolia, Tinospora

sinensis and Neem-guduchi (Tinospora cordifolia growing on Azadirachta

indica (Neem)) against paracetamol intoxication in rats” at the “National

conference on Plant Biodiversity for sustainable development”, organized

by Department of Botany, University of Pune on 10th

to 12th

March 2011

(Oral Presentation).

tejaswi chavan ph.d. thesis.pdf

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