INVESTIGATIONS ON HEPATOPROTECTIVE POTENTIAL OF

i

INVESTIGATIONS ON HEPATOPROTECTIVE POTENTIAL

OF CENTRATHERUM ANTHELMINTICUM IN

PARACETAMOL AND CARBON TETRACHLORIDE (CCl4)-

INDUCED

LIVER INJURY

Sumera Rais Abbasi

Department of Biochemistry

University of Karachi

Karachi-75270,

Pakistan

ii

2017




INDUCED

LIVER INJURY

Sumera Rais Abbasi



Karachi-75270,

iii

Pakistan

2017




INDUCED

LIVER INJURY

Thesis submitted for the fulfillment of the degree of

DOCTOR OF PHILOSOPHY

By

Sumera Rais Abbasi



Karachi-75270,

Pakistan

2017

iv

DECLARATION

I hereby undertake that this research entitled “Investigations on Hepatoprotective

Potential of Centratherum Anthelminticum in Paracetamol and Carbon

Tetrachloride (CCl4)-Induced Liver Injury” is my original research work of synopsis

approved from the Board of Advanced Studies and Research, University of Karachi and

no part of it falls under plagiarism. None of the part of this work has been previously

submitted by any other person or approved for the award of any degree or certificate by

the university or the academy, except where due acknowledgment is necessary.

______________________

SUMERA RAIS ABBASI

Enrolment No. SCI / BCH / KU-41002 / 2014

Department of Biochemistry,

University of Karachi.

Date: __________________

v

CERTIFICATE

I hereby certified that Ms. Sumera Rais Abbasi, (Enrolment No. SCI / BCH / KU-

41002 / 2014) PhD student of Department of Biochemistry, University of Karachi, has

successfully completed her research work entitled, “Investigations on Hepatoprotective

Potential of Centratherum Anthelminticum in Paracetamol and Carbon Tetrachloride

(CCl4)-Induced Liver Injury” under my supervision. The entire work is her own and to

the best of my knowledge, it contains no formerly published text nor any part of this thesis

has been approved for the award of any degree or certificate by the university, except due

acknowledgment is necessary.

_________________________

Dr. SHAMIM A. QURESHI

Professor and Research Supervisor



Dated: __________________________

vi

vii

DEDICATION

Every challenging work required self-efforts as well as help of seniors those who were

very close to our hearts.

I dedicated this thesis to

My Great Parents Mr. Rais Ahmed and Mrs. Nasira Rais

Whose love, overwhelming, reinforcement, and Pray of day and night make me capable

to get this achievement.

My loving Husband, Ahmed Siraj and sweet Daughter, Mishal Siraj

Whose sacrifices, which were realized by over loss of precious time together, were for me.

My younger brothers Fahad Rais and Osama Rais, Their motivation in every step of my life.

And

My deepest thankfulness and warmest affection to my supervisor Dr. Shamim A.

Qureshi who has been constant source of knowledge and unwavering support.

Without none of them my success would not be possible.

viii

Acknowledgment

First Praise be to, ALLAH, his majesty for uncountable blessings, and giving me

opportunity, strength for my research and for all that.

I am deeply grateful to my supervisor, Dr. Shamim A. Qureshi, Professor,

Department of Biochemistry, University of Karachi, for the leadership, guidance, and

constructive comments and patience over the years throughout the study. She also

groomed me professionally. Thank you so much for pushing me, to look my work in

diverse ways and to open my mind .Your backing was essential for my success. I deeply

say that may ALLAH grandly praise her.

My warm appreciation is for Dr. Viqar Sultana, Chairperson, Department of

Biochemistry, University of Karachi for providing me facilities for research.

I deeply indebted thankful to my friend and senior lab fellow Dr. Tooba Lateef,

Assistant Professor, Department of Biochemistry, University of Karachi, for her, info,

support, and comprehensive advice on different aspects of my research.

I also grateful to my friend and fellow Mussarat Jehan, for his kind endless help,

generous advice and cooperation during the study. A special thanks also goes to my all

lab fellows especially Mr. M. Asad khan for guidance and supporting me.

In the end I want to show indebtedness to my family: my Parents, brothers Mr.

Fahad Rais, Osama Rais, my in-laws and friends for their unconditional love and support

during the entire period.

Finally, I am thankful to my Husband and Daughter my shining armor, for their

love, sacrifice, and for having both of you in my life.

Ms. Sumera Rais Abbasi

ix

Table of Contents

S. No Title Page No.

Acknowledgement

List of Figures

List of Tables

Summary i-iv

1 Chapter 1: Introduction 1-26

2 Chapter 2: Materials and Methods 27-50

3 Chapter 3: Results 51-85

4 Chapter 3: Discussion 86-94

5 Conclusion 95

6 Future Extension of the Present Research Work 96

7 References 97-114

8 Appendices 115-116

x

List of Figures

S. No Title Pg No.

INTRODUCTION

Figure A Liver Structure 4

Figure B Liver Functions 5

Figure C Mechanism of Action of Silymarin in Hepatotoxic Models 15

Figure D Mechanism of Paracetamol (PCM)-Induced Hepatotoxicity 18

Figure E Mechanism of Carbon tetrachloride (CCl4)-Induced Hepatotoxicity 19

Figure F Outline of the Present Work 20

MATERIALS AND METHODS

Figure 1 Flow Chart for the Extraction of ESEt and HSF 32

Figure 2 Animal Grouping of PCM-Induced Hepatotoxic (PIH) Rats Model 33

Figure 3 Animal Grouping of CCl4-Induced Hepatotoxic (CIH) Rats Model 34

RESULTS

Figure 4 Outcome of ESEt on Body Weight Change in PCM-Induced

Hepatotoxic Rats 53

Figure 5 Outcome of ESEt on Percent Protection in Body Weights of PCM-

Induced Hepatotoxic Rats 54

Figure 6 Outcome of ESEt on Liver Associated Enzymes in PCM-Induced

Hepatotoxic Rats 56

Figure 7 Outcome of ESEt on Percent Protection by ALT, AST and ALP in

PCM-Induced Hepatotoxic Rats 57

Figure 8 Outcome of ESEt on Total Protein (TP) and Albumin (ALB) in

PCM-Induced Hepatotoxic Rats 60

Figure 9 Outcome of ESEt on Percent Gain in Protein Profile of PCM-


Figure 10 Outcome of ESEt on Hemoglobin in PCM-Induced Hepatotoxic

Rats

62

xi

Figure 11 Outcome of ESEt on AST/PLT Ratio Index (APRI) in PCM-


Figure 12 Outcome of ESEt on Percent Inhibition of CAT and SOD in PCM-


Figure 13 Outcome of ESEt on Percent Inhibition of GSH and LPO in PCM-


Figure 14 Liver Histology of PCM-Induced Hepatotoxic Model 68

Figure 15 Outcome of ESEt and HSF on Percent Body Weights Change in

CCl4-Induced Hepatotoxic Rats 70

Figure 16 Outcome of ESEt and HSF on Percent Protection of Body Weights

in CCl4-Induced Hepatotoxic Rats 71

Figure 17 Outcome of ESEt and HSF on Liver Associated Enzymes in CCl4-


Figure 18 Outcome of ESEt and HSF on Protection of ALT, AST and ALP

in CCl4-Induced Hepatotoxic Rats 74

Figure 19 Outcome of ESEt and HSF on Total Protein (TP) and Albumin

(ALB) levels in CCl4-Induced Hepatotoxic Rats 77

Figure 20 Outcome of ESEt and HSF on Gain of TP and ALB levels in CCl4-


Figure 21 Outcome of ESEt and HSF on Uric acid in CCl4-Induced

Hepatotoxic Rats 79

Figure 22 Outcome of ESEt and HSF on Inhibition of CAT and SOD in


Figure 23 Outcome of ESEt and HSF on Inhibition of GSH and LPO in


Figure 24 Liver Histology of CCl4-Induced Hepatotoxic Model 85

xii

List of Tables

S. No Title Page No.

Table A Pharmacological Activities and Isolated Compounds Reported

from the Seeds of Centratherum anthelminticum 22-24

Table 1 Outcome of ESEt on Liver Weights (LW) of PCM-Induced

Hepatotoxic Rats 55

Table 2 Outcome of ESEt on GGT, TB and UA in PCM-Induced

Hepatotoxic Rats 59

Table 3 Outcome of ESEt on Hematological Parameters in PCM-

Induced Hepatotoxic Model 64

Table 4 Outcome of ESEt and HSF on Liver Weights (LW) and Liver

Index in CCl4-Induced Hepatotoxic Rats 72

Table 5 Outcome of ESEt and HSF on Total Bilirubin (Direct &

Indirect) and GGT in CCl4-Induced Hepatotoxic Rats 76

Table 6 Outcome of ESEt and HSF on Lipid Profile in CCl4-Induced

Hepatotoxic Rats 84

i

Summary

A vast range of liver diseases are increasing day by day that also enhancing the

rate of hospitalization and death in the world. Beside genetic, acquired causes including

viruses, chemicals, alcohol, toxins, pollutants and drugs are uplifting this scale severely

especially in developing or low income countries. Pakistan is also a big victim of this

health hazard. Liver is one of the vital organs of the body, equipped with enzymes that

are actively involved in all metabolism related to the synthesis, storage, detoxification

and excretion of substances. However, it is at high risk of dysfunction and inflammation

when it is exposed with the acquired factors of liver problems especially chemicals used

in professional environment and daily use of high doses of analgesics. These reversible

liver problems may turn into irreversible damages like fibrosis and cirrhosis if neglected.

Interestingly, there are few medicines available for the treatment of liver problems, and

majority of them are plant origin, though these are in clinical practice but not found as

effective as they expected in the regeneration of liver cells. Therefore, researchers are

finding new medicinal plant having improved hepatoprotective activity with the aim for

development of new medicine in this regard.

Centratherum anthelminticum (Wild) Kuntz (family Asteraceae), its seeds are

commonly called as kali zeri or black cumin. These seeds are not only well famous for

their culinary uses in Pakistan and neighbor countries but also for number of medicinal

purposes especially anticancer, antidiabetic, and antihyperlipidemic. However, its

antihepatotoxic activity was not reported. Therefore, in the present work, the

hepatoprotective action of organic solvent extract of C.anthelminticum seeds in carbon

tetrachloride (CCl4) and paracetamol (PCM)-induced hepatotoxic rats models was

investigated and reported.

In PCM-induced hepatotoxic model (PIH), except normal control group (distilled

water 1 ml; group I), all other experimental rats were made hepatotoxic by administering

PCM (1 gm/kg/day) orally and divided into PIH control (distilled water 1 ml; group II),

positive control group (silyamrin 100 mg/kg; group III) and three test groups (IV, V &

VI) treated with 200, 400 and 600 mg/kg of ESEt of C. anthelminticum separately for

ii

consecutive 9 days. After that, rats of all groups (6 rats/ group) were decapitated for the

evaluation of hematological, biochemical and antioxidant parameters. Whereas physical

parameter including percent body weight change (PBWC) was calculated by assessing

the body weights of each rat of each group on first and last day of each treatment. In

addition, weights of liver tissues (LW) of each group of rats were measured and

histopathological studies of liver tissues have also been done. The results stated that the

dosage of ESEt (200, 400 & 600 mg/kg) found efficient in decreasing the percent loss in

body weights of rats in all tests in compared to group intoxicated with only PCM. Plus

the weights of liver tissues of test groups were also found almost as same as observed in

normal control group. Similarly, the same doses of extract was found normalizing the

levels of liver-specific biomakers such as aminotransferases (both alanine; ALT &

aspartate; AST), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP),

total bilirubin (TBR) especially indirect one (IDBR), total protein (TP) especially

albumin (ALB) and uric acid (UA) in their own test groups when compared to PCM

hepatotoxic control group.

Hematological parameters including hemoglobin (Hb), red & white blood cells

(RBC & WBC), hematocrit (HCT) and platelets (PLT) were also found better in all three

extract treated test groups. In addition, aspartate aminotransferase: platelate ratio index

(APRI) was also calculated and found lower than 0.5 in test groups that showed the liver

protective property of extract. Similarly, percent inhibition (PI) of antioxidant parameters

including superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and

lipid peroxidation (LPO) was calculated in liver homogenates of all rats of all groups and

found that all ESEt treated test groups have low PI of SOD, CAT & GSH and high PI of

LPO when compared with PCM control group. The betterment in all physical,

biochemical, hematological and antioxidant parameters showed by ESEt in test groups

was strongly evident by observing decrease in necrosis and inflammation in histological

slides of liver tissues of same test groups as compared to liver tissue slides of hepatotoxic

group.

Before conducting CCl4-induced hepatotoxic model, hexane soluble fraction

(HSF) of ESEt was prepared and separated. Then rats were divided into two main groups

iii

normal control (distilled water 1 ml; group I) and CCl4-induced hepatotoxic group which

sub-divided into hepatotoxic control (distilled water 1 ml/kg; group II), positive control

(silyamrin100 mg/kg; group III), and three test groups (IV& V) administered with ESEt

in doses of 600 & 800 mg/kg and group VI with HSF 600 mg/kg for consecutive 5 days.

CCl4 (3ml/kg; in 1:1 dilution with olive oil) was injected intra-peritoneally in groups II to

VI on 3rd and 5th day of experiment after 1 hour of their allocated treatments. After 24

hours of last injection of CCl4, rats of all groups were decapitated to collect serum and

liver tissues. Methodology includes the determination of physical [PBWC, LW & (liver

index; LI)], biochemical [ALT, AST, ALP, GGT, TBR, DBR (direct bilirubin), IDBR,

TP, ALB, UA, TG (triglycerides), TC (total cholesterol), VLDL-c, LDL-c & HDL-c

(very low, low & high-density lipoprotein cholesterols)] and PI of antioxidant [CAT,

SOD, GSH & LPO] parameters.

The doses (600 and 800mg) of ESEt and HSF (600 mg) were significantly

decreased the PBWC, LW and LI in their respective test groups which was also

accompanied with much decreased levels of ALT, AST, ALP, GGT, TBR, IDBR, UA

and increased levels of TP and ALB in these test groups when compared to CCl4

intoxicated group that displayed completely vice versa situation of all these parameters.

Similarly, ESEt and HSF were also found beneficial in decreasing the levels of bad lipids

(TG, TC, VLDL-c & LDL-c) in test groups. Moreover, antioxidant status of test groups

was found improved by observing decreased in PI of CAT, SOD, GSH and increased in

PI of LPO as compared to high level of oxidative stress was found in hepatotoxic group

by observing high PI of CAT, SOD, GSH and less PI of LPO.

The liver protective effect of ESEt and its HSF was clearly identified by

observing gradually improved structure of liver tissues in rats belonged to test groups

administered with doses (600 & 800 mg) of ESEt as compared to hepatotoxic group

whose liver tissue slide displayed harmful effects of CCl4 including fatty accumulation,

ruptured and inflamed hepatocytes around abnormally enlarged central vein. Whereas,

the most amazing finding of present study is the recovery of complete normal structure of

liver tissue dissected out from rat of test group treated with HSF (600 mg).

iv

Therefore, the present study concludes that ESEt and its HSF are strong

hepatoprotective and liver regenerative agents not only by normalizing the liver function

test parameters, lipid profile and uplifting the antioxidant parameters but also capable of

reversing the adverse anatomical changes induced by PCM and CCl4 in liver tissues by

completely healing up the same tissue upto the normal structure. The liver protective

property of ESEt and HSF may be resides in polyphenol, flavonoid and steroidal contents

of ESEt which were already reported in our previous studies and fatty acids,

hydrocarbons or waxes which could be possibly present in HSF of ESEt of C.

anthelminticum

1

Centratherum Anthelminticum

Whole Plant Seeds

2

Investigations on Hepatoprotective Potential of Centratherum

Anthelminticum in Paracetamol and Carbon Tetrachloride

(CCl4)-Induced Liver Injury

1. Introduction

The liver is the indispensable organ and performs all important functions in the

human body. Below the diaphragm, liver lies in the abdominal cavity. The liver

circulatory system is different from other organs. About 70% of blood enters in liver

through the portal vein which brings venous blood from where all nutrients, xenobiotic

and drugs are absorbed. On the other hand, hepatic artery brings 25% of the arterial blood

(oxygenated) from the pulmonary system to the liver (Dolley et al., 2011).

The entire liver is covered with connective tissue called Glisson’s capsule. This

capsule provides support, network and route for the bile ducts, lymphatic and afferent

blood vessels to enter and leave the liver. The liver divides into many small lobules

which are separated by means of fibrous partition called interlobular septum (Kuntz,

2006). These lobules are hexagonal in shape, having central vein, plus within each lobule

hepatocytes are arranged in irregular single cell thick plates covered with microvillae.

These plates are separated by narrow channels called sinusoids, its endothelial cells lacks

the basal membrane and have large openings for the release of metabolites like newly

synthesized plasma proteins. The hepatocytes plates and endothelium separated by the

sub-endothelium space, known as space of Disse. At each corner of lobule, specific area

present known as triad or portal area (Jacobs et al., 2010) as represented in Figure A

(Mescher, 2009).

About 60-80% liver is populated with hepatocytes while remaining 20-40%

comprises kupffer’s, stallate, biliary epithelial, sinusoidal, endothelial, non-parenchymal

cells, lymphocytes and bile canaliculi. The kupffer’s cells (local macrophages) are

phagocytic in nature and located in sinusoidal endothelium (Hall, 2015). Between

hepatocytes, the stellate cells are reside within perisinusoidal space of Disse and performs

https://www.ncbi.nlm.nih.gov/pubmed/?term=Jacobs%20F%5BAuthor%5D&cauthor=true&cauthor_uid=19948827

https://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22Anthony+Mescher%22

3

many function like vitamin A storage, cytokines secretions, hepatic extracellular matrix

synthesis and even take main part in the progression of fibrosis, when liver damages

(LeCluyse et al., 2012). The biliary epithelial cells of the liver also known as

cholangiocytes present in the portal triads of the bile duct. They maintain the bile

composition by varying water and solute content (Katsuda, 2013). Endothelial cells are

the non-parenchymal of the liver. The circulatory intra-hepatic vessels are covered with

these cells and provide huge area for nutrient absorption plus serve as selective barrier

between exchange of molecules and pathogens (Tortora & Derrickson, 2008).

Lymphocytes especially natural killer cells provide defense against pathogens (Taub,

2004). Functions of liver cells are also summarized in Figure B.

1.1. Prevalence

The rate of liver diseases over the years are gradually increasing globally. Liver

diseases have been ranked as the fifth most common cause of death in UK while among

all digestive disorders it becomes the second leading cause of mortality according to

National Statistics in the US (Sarin & Maiwall, 2016). A study described that an estimate

of about one million deaths in 2010 due to liver cirrhosis worldwide which was around

2% of all deaths and expected further increase in this due to liver cancer and acute

hepatitis (Byass, 2014). The most affected one is the male gender. Similarly, liver,

stomach and throat cancers are common in Asia. Therefore, liver problem and deaths are

alarming issue to be addressed immediately at national and international levels both like

only in China nearly above half of the newly diagnosed world liver cancer cases are

reported (Pourhoseingholi, 2015). On the other hand, the major cause of liver disease in

Pakistan is Hepatitis C virus (HCV) (Umar & Bilal, 2012).

Drug-induced liver problem/injury (DILI) is also contributing increase in

hospitalization and estimated annual incidence rate about 13.9-24.0 per 100,000

inhabitants. It is one of the leading causes of acute liver failure in the US (Suk & Kim,

2012). NAFLD (Nonalcoholic fatty liver diseases) prevalence is increasing worldwide

that affects approximately 15-40% of general population with only 30% comes from

South Asia regions even 14% have appeared in Pakistan (Parkash & Hamid, 2013).

https://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22Gerard+J.+Tortora%22&source=gbs_metadata_r&cad=5

https://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22Bryan+H.+Derrickson%22&source=gbs_metadata_r&cad=5

https://www.ncbi.nlm.nih.gov/pubmed/?term=Pourhoseingholi%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=25584172

https://www.ncbi.nlm.nih.gov/pubmed/?term=Suk%20KT%5BAuthor%5D&cauthor=true&cauthor_uid=23091804

https://www.ncbi.nlm.nih.gov/pubmed/?term=Kim%20DJ%5BAuthor%5D&cauthor=true&cauthor_uid=23091804

4

Figure A: Liver Structure

5

Figure B: Liver Functions

Liver Functions (more than 500 vital functions)

Synthesis of

albumin,

globulin, clotting factors,

angiotensinogen,

insulin like

growth factor

(IGF-1),

transport &

binding proteins

Detoxify

xenobiotics,

antibiotics and

other endogenously

synthesized

toxic

metabotites,

& ammonia

Excretion of

Dye (BSP and rose Bengal)

Conjugated

bilirubin,

calcium and

chemically

altered

steroid/thyriod

hormones

secreted in bile

Kupffer’s cells

present in

sinusoids

perform

phagocytosis

Storage of fat

soluble

vitamin, folic acid,

glycogen,

copper & iron

Carbohydrate, lipid, amino

acid,

cholesterol,

mineral,

vitamin,

nucleic acid &

ammonia

formation &

interconversion

of sugars

Met

aboli

c

funct

ions

Synth

etic

funct

ions

Sto

rage

funct

ions

Det

oxif

icat

ion

funct

ions

Excr

etory

funct

ions

Sec

reta

ry

funct

ions

Imm

unolo

gic

al

funct

ions

6

1.2. Pathological Conditions Caused by Drug and Chemical-induced

Liver Injury

During detoxification, liver subject with toxic substances like carbon

tetrachloride, acetaminophen, thioacetamide, antibiotics and several other carcinogens

that are degraded or may induce hepatocytes damage (Ramadori et al., 2008). Most of the

substances transformed into water soluble components and excreted out of the body but

continuous inhalation or intake of these substances produce problems by generating more

and more free radicals and reactive metabolites that encounter with DNA, membrane

proteins, lipids and alter their functions plus reduce the half life of cells (Gu &

Manautaou, 2012).

The diagnosis of DILI is challenging in many cases because there is no specific

maker (Khoury et al., 2015). However, drug-induced liver damage commonly results in

number of acute and chronic conditions include hepatitis, cholestasis, steatosis, cirrhosis

and even hepatic failure (Vinken et al., 2013).

1.2.1. Hepatitis

Inflammation of liver is referred as hepatitis. Various agents like viruses, alcohol,

drug, toxins and autoimmune conditions results in hepatitis (Seifter et al., 2005). Drug-

induced hepatitis are mainly acute and chronic but cytolytic hepatitis, cholestatic hepatitis

and mixed hepatitis belong to the important category. Several clinical and biological

pictures show drug-induced hepatitis, mostly similar to those of viral hepatitis. The

prognosis is good and their evolution favorable. Cytolytic hepatitis, a wider hepatocytic

necrosis has more severe prognosis. Fulminant acute hepatitis is the most severe form,

characterized by a substantial necrosis of the hepatic parenchyma. Chronic hepatitis is the

result of prolonged administration of some drugs with toxic action. Clinical and

biological indicators are not specific but progression towards cirrhosis is possible (Leise

et al., 2014).

https://www.ncbi.nlm.nih.gov/pubmed/?term=Khoury%20T%5BAuthor%5D&cauthor=true&cauthor_uid=26356634

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1.2.2. Cholestasis

The cholestasis refers to the stoppage of bile to release into the small intestine or

stagnant bile. In cholestatic damage an elevation of alkaline phosphatase (ALP) greater

than twice of its original value and the ratio of serum ALT/ ALP of less than 2 reported

(Giannini et al., 2005). Drugs may also induce intra- and extra-hepatic cholestatic

diseases where elevation in ALP is an only clinical sign. In addition, cholestatic injury

could be the result of mixed hepatocellular pure intra-hepatic due to the loss of

canalicular bile flow or an “obstructive” drug induced cholangiopathy which is the initial

site of injury that damage bile duct epithelium at many levels (Padda et al., 2011).

1.2.3. Steatosis

Steatosis, due to NALD, is a deposition of fat within hepatocytes which appeared

as vacuoles in a microvesicular or macrovesicular form. Micro-vesicular steatosis

represented by multiple small, fat vesicles distributed in the hepatocyte, while

macrovesicular steatosis as a large droplet of fat within the cytoplasm, that pushes the

nucleus to the edge of the cell and it is typical characteristic of nonalcoholic fatty liver

disease. However, both forms of hepatic steatosis are reported as a result of several drugs

toxicity, though differ in triggering events, each of these lead to excessive fat deposition,

increased formation of reactive oxygen species (ROS), mitochondrial dysfunction, and

stress in endoplasmic reticulum (ER) which induces inflammation, cell death and

ultimately leads to fibrosis, cirrhosis and other complications such as hepatocellular

carcinoma, thus accelerating the risk of morbidity and mortality (Ratziu et al., 2005).

The risk factors of liver problem without alcohol consumption include obesity,

dyslipidemia, and type 2 diabetes mellitus. This type of liver problem has 2 main

phenotypes first showed the presence of steatosis without inflammation and in second

steatosis accompanied with inflammation and ballooning injury of hepatocyte that

progresses to cirrhosis (Arab et al., 2017).

https://www.ncbi.nlm.nih.gov/pubmed/?term=Padda%20MS%5BAuthor%5D&cauthor=true&cauthor_uid=21480339

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1.2.4. Cirrhosis

It described as increased deposition of collagen in matrix outside the cell. Mostly

chronic liver injury leads to cirrhosis, hepatocellular carcinoma, liver failure, and in

severe cases it needs liver transplantation. Steatosis and inflammation without

administration of alcohol are the major cause of liver fibrosis. Strikingly, the estimated

mortality from cirrhosis rank 14th and 10th in the world and in developed countries, and

expected to reach 12th position as a leading cause of death in 2020 (Schuppan & Afdhal,

2008). The worst problems led by cirrhosis include ascites, renal failure, hepatic

encephalopathy, and variceal bleeding. Reports described that compensated cirrhosis

almost free of major complications for many years while decompensated cirrhosis

reduces the life span of patients and requires liver transplantation (Bataller & Brenner,

2005). Last stage of liver cirrhosis is primary sclerosing cholangitis (PSC), destruction of

intrahepatic bile ducts and prolonged obstruction in extrahepatic biliary tracts (Ahmed,

2011).

1.3. Treatments

According to World Health Organization (WHO), the aims to provide global

control and safety towards liver diseases include encouraging secure injection methods

necessary for minimizing the worldwide HBV-related illness and mortality, unfolding

knowledge of liver problems induced by viruses, drugs and chemicals among physicians,

law makers and general public by advising that hepatitis B vaccine should be included in

routine vaccination services plus rooms should be properly ventilated in mills and

industries to minimize inhalation of toxic chemicals (Thun et al., 2010). Liver has a

unique property to regenerate, the rate of hepatocytes proliferation accelerates as a result

of toxic and infectious injury. However, liver transplant is the only cure of last stage

cirrhosis.

9

1.3.1. Liver Transplantation

The use of liver transplant is now increasing for the treatment of irreversible liver

problems or end stage liver cirrhosis. But the vital part of this procedure is the selection

of patient and the availability of donor. The left and right lobes of liver can be

transplanted into separate recipients. Liver grafting is also another hope of liver

regeneration, though size of graft is associated with the degree of liver function available

in recipient. In the future, bioengineered organs may overcome the problem of shortage

of donors’ livers (Nicolas et al., 2016). The recent survival rates after transplantation is

about 79% in 1 year and 72% in 5 years so outcomes will enhance with time. In

paracetamol-related acute liver failure, the survival without liver transplantation was

observed more apparently as compared to other drug-induced cases (O’Grady., 2014).

1.3.2. Stem Cells

Use of stem cells is an alternate method to restore liver function. Different sources

of stem cell are using for the therapy of liver diseases. For example liver-derived stem

cell obtained from adult (adult liver stem cell also called oval cells) and fetuses derived

stem cells (livers obtained from fetal known as hepatoblasts). Both are bipotent so they

can be developed into hepatocytes or cholangiocytes. Oval cells participate in tissue

restoration when its liver regenerative ability becomes weaken, whereas cells obtained

from fetal liver used to rejuvenate liver in experimental animals trials. Stem cells derived

from bone-marrow include hematopoietic and mesenchymal stem cells (MSCs). Of

which, MSCs are more potent in liver regeneration as compare to hematopoietc ones,

these are easily accessible, rapidly expanded in culture, and also have

immunomodulatory or immunosuppressive properties. Other stem cells (Annex) also

obtained from human placental tissue, umbilical & blood cords and amniotic fluid, these

are pluripotent. The last and the best stem cells are embryonic stem cells (ESCs) which

can easily differentiated into hepatocyte-like cells, colonized into liver and replaced the

damage areas of this tissue (Nicolas et al., 2016).

10

Other therapies are supporting liver by detoxifying the circulating toxic

substances, calming down the medical conditions of patients till they are waiting for

perfect donor or helping liver to regenerate itself. Among such, extracorporeal liver-assist

devices (ELAD) are non-biologic dialysis like systems used for invivo detoxification and

bio-artificial devices used to implant hepatic cells of porcine or human in order to

recover detoxification and synthetic functions (Bernal & Wendon, 2013).

1.3.3. Allopathic Medicines

Hepa-Merz (syrup) contains L-Orinithine L-Aspartate (300 mg), Nicotinamide

(24 mg) and Riboflavin sodium phosphate. This medicine help to improve the

detoxifying ability of liver via its L-Ornithine which is an important intermediate of urea

cycle thereby converting ammonia into water soluble urea and get it excreted out of body

through kidneys. The physicians normally prescribed this medicine for acute & chronic

hepatitis, cirrhosis, fatty liver with hyperammonemia (Vela et al., 2011).

1.3.4. Homeopathic Medicines

This is natural form of therapy used from many years by millions of people in the

world to treat number of clinical conditions. The objective of this therapy is to use

substance in small quantity will cure the same symptom which it could cause if it is taken

in large quantity. Highly recommended homeopathic medicines for liver problems are

Chelidonium, Carduus marianus and Natrum sulphuricum. Chelidonium is a great

remedy for liver infections, hepatitis, gallstones and jaundice (Biswas, 2002), Carduus

marianus has shown remarkable results in liver cirrhosis while Natrum sulphuricum is

the most valuable among homeopathic medicines for liver problems like jaundice,

hepatitis and other bilious complaints (Chakraborty, 2005)

1.3.5. Home Remedies

Vegetable juices and detox recipes are a great way to cleanse the liver, reduce

inflammation and help in digestion, immunity, metabolism and the storage of nutrients

(Page & Abernathy, 2017). Add turmeric into diet and used to reduce swelling and treat

the digestive system, cilantro and ginger are both great for detoxifying the liver and

https://www.ncbi.nlm.nih.gov/pubmed/?term=Blanco%20Vela%20CI%5BAuthor%5D&cauthor=true&cauthor_uid=22228883

11

supporting the immune and digestive systems (Cobot, 2014). Chelation therapy and

heavy metals detoxification with organic juices are highly recommended for hepatitis

patients (Oyakhire, 2010).

1.3.6. Herbal Medicines

Heptocam and Sarpilin-B contains silymarin and vitamins, are opening up a new

promising and therapeutic action on the liver cells (Muriel & Rivera-Espinoza, 2004).

Silymarin, the active principle of heptocam, is isolated from the milk thistle (Silybum

marianum; family Compositae) plant. The fruits extract of this plant contains Silybum, a

complex polyphenolic mixture that actually contains seven flavonolignans molecules

including silibin A & B, isosilibin A & B, silichristin, isosilichristin, silidianin and the

effective antioxidant taxifolin, the flavonoid (Hellerband, 2016). Silymarin is also

available individually with different names like silliver from different pharmaceuticals.

Around 33% of patients of chronic HCV and cirrhosis are reported to use this medicine

(Fried, 2012).

Silymarin alone or in combination of vitamin (B-complex) acts as a

hepatoprotective agent by performing number of activities like i. Showing free radical

scavenging activity by uplifting the status of superoxide dismutase and increasing the

cellular amount of glutathione (GSH) thereby preventing lipid peroxidation (Surai, 2015),

ii. Regulating the ability of membrane permeability and increasing the stability of

membrane during xenobiotic destruction (Karimi, 2011), iii. Disturbing the packing of

acyl chains and repairing the liver microsomes and mitochondrial membrane fluidity

(Abou Seif, 2016), iv. Regulating nuclear expression ability by steroidal-like effect

followed via regeneration of tissue (study reported the structural similarity of steroid

hormones to silymarin) (Karimi, 2011), v. Reducing the conversion of inactive hepatic

stellate cells into active myofibroblasts that leads to the cirrhosis by the deposition of

collagen fibres (Fraschini et al., 2002), vi. Showing inhibitory effect on inflammatory

cytokines (anti-inflammatory effect), thereby reducing the hepatic inflammation and

tissue damage, vii. Inhibiting the leukotrienes formation from polyunsaturated fatty acids

http://www.sciencedirect.com/science/article/pii/S2314853516300282

12

by inhibiting the action of lipoxygenase (Surai, 2015). The hepatoprotective action of

silymarin is summarized in Figure C.

Heptocam (Silymarin with vitamins) delay the entry of exogenous and

endogenous toxins into the hepatocytes thereby further damage is prevented. Silymarin

stimulates the phagocytic and bactericidal functions of the sinusoidal cells, hence

protecting the liver against deleterious agents. It also stimulates Kupffer’s cells and

increases the synthesis of ribosomal RNA and proteins. Vitamins act by replacing the

bio-substrates, hence mobilizing the liver cell metabolism. The vitamin B-complex forms

a functional unit in different intermediate metabolism. Experiments have shown

heptocam (silymarin with vitamin) exert a protective effect on the liver through their

enzymatic influence on protein and carbohydrates metabolism. They accelerate the repair

of damaged liver parenchyma and promote organ detoxifying action. This can be

prescribed in liver problem like hepatitis, jaundice, fatty liver, chronic liver cirrhosis,

hepatotoxicity due to alcohol or drugs (North-Lewis, 2008).

Liv-52, contains Capparis spinosa, Cichoriumin intybus, Solanum nigrum,

Terminalia arjuna, Cassia occidentalis, Achille amillefolium, Tamarix gallica, (Freedom

Press Topanga, 2014). This medicine restores the functional efficacy of the liver by

shielding the hepatic cells and accelerating their regeneration. Its anti-peroxidative

activity stop the loss of integrity of the cell membrane and speed up the recovery period

of cytochrome P-450 thereby restoring the liver function in infective hepatitis. This

medicine hastens the removal of toxic metabolite (acetaldehyde) of alcohol-induced liver

injury. It also inhibit fatty deposition in liver by reducing the lipotropic activity in chronic

alcoholism. In conditions prior the cirrhosis, this herbal medicine ceased the spread of

disease to prevent more liver damage (Huseini et al., 2005). Physicians prescribed it in

virus and alcohol-induced hepatitis.

Hepanox capsules contain silymarin and vitamins A, C and E. It is

hepatoprotective and antioxidant. Lovenox contains active principles such as curcumin

2%, silymarin 80% and dandelion extract (Taraxacum officinale). It improves liver

functions. Another silymarin preparation named Legalon improves liver function and

13

protects against liver damage. Lipocholine tablets are actually lipotropic agent

containing active constituent extract of artichoke (Cynara scolymus) and a group of

vitamin B (Abou Seif, 2016).

1.3.7. Medicinal Plants

Interestingly, herbal medicines (either contains single plant or combination of many

plants) are playing main role in the treatment of liver problems. In recent years, several

plant constituents such as phenol, glycoside, alkaloids, and terpenes have been discovered

with hepatoprotective activity. Researchers are still looking forward to find out medicinal

plant having better hepatoprotective activity as the existing herbal or allopathic medicines

are not as effective as they are expected. In this regard, about 101 plants and 160

phytoconstituents have been investigated and isolated to have protective effect on liver

(Arpita et al., 2011) like alcoholic fruits extract (250mg/kg) of Coccinia grandis

(Cucurbitaceae) Linn was found beneficial in reducing the harmful effects of CCl4 in

rats (Kalpana & Gopinathan, 2016), the alkaloid (colchicine) isolated from Colchicum

autumnale (Colchicaceae) was found hepatoprotective by reporting its ability to bind

with microtubuler protein in many hepatotoxins [paracetamol (PCM), carbon

tetrachloride (CCl4) and D-galactosamine]-induced animal models (Hamzawy, 2015),

chloroform extract of Eucalyptus maculate Hook. and its phenolic isolates were reported

for antioxidant and hepatoprotective properties in rats and mice (Ahmad & Sharafatullah,

2008).

Similarly, aerial parts of Indigo feratinctoria (Fabaceae) is fractionated by

petroleum ether, isolated a bioactive, Indigtone categorized as trans-tetracos-15- enoic

acid (TCA) and displayed dose dependent hepatoprotective activity in PCM and CCl4

induced liver damage in mice and rats (Jannu, 2012). Methanolic extract of Lepidium

sativum (Brassicaceae) also found beneficial in same regard (Al-Asmari et al., 2015).

The seeds of Nigella sativa L, (Ranunculaceae) and Urtica dioica L. (Urticaceae) are

also famous in the healing of advanced cancer and showing antioxidant activity plus

decreasing the level of fatty degeneration and liver enzymes in the CCl4-induced rats

(Kanter et al., 2005). Orthosiphon stamineus (Lamiaceae) extract also proved to have

14

hepatoprotective role in PCM administered liver damage in rats (Alshawsh et al., 2011).

The water extract of Veronica amygdalina (Compositae) leaves have hepatoprotective

and antioxidant effects against PCM-induced hepatotoxicity in mice (Johnson et al.,

2015).

15

Figure C: Mechanism of Action of Silymarin on Hepatotoxic Models

Silymarin

Protective and

regenerative

effect on cells

Membrane

stabilizing

effect

Nuclear effect Xenobiotic

metabolism by

microsomes

GSH sparing

activity

Free radical

Scavenger activity

Activation of

cytochrome

P-450

Synthesis of

Ribosomal

RNA

Translation

increased

Treatm

ent

Key

effect C

ellular effect

Molecu

lar

effect

16

1.4. Models Used in Present Study

1.4.1. Paracetamol (PCM)-Induced Hepatotoxic Model

Paracetamol (Acetaminophen, N-acetyl-p-aminophenol; NAPAP) commonly used

to relief pain and fever. It is safe for human consumption especially in recommended

doses. It is similar to aspirin and ibuprofen in analgesic and antipyretic properties (Verbic

et al., 2016). Whereas in higher doses it can be fatal as it produces a centrilobular hepatic

necrosis. The PCM hepatotoxicity was first reported in humans in 1960s (Prescott, 2000)

plus rats, mice and hamsters were also found very sensitive with high doses of this drug

(Bidhan et al., 2009). The hepatotoxic mechanism of PCM includes a complex series of

actions like i. A cytochrome P450 enzyme metabolized PCM into reactive intermediate

N-acetyl-p-benzoquinone imine (NAPBQI), excess formation of NAPBQI bypasses the

steps of glucuronidation and sulfation thereby inducing depletion of glutathione (GSH)

and build-up itself, ii. Loss of GSH results in increased formation of ROS species, iii.

Changes in calcium homeostasis encourage the transition in mitochondrial permeability,

iv. Loss of mitochondrial membrane potential to synthesize ATP, results in the depletion

of ATP. All these events induce necrosis that also associated with number of

inflammatory mediators such as certain cytokines and chemokines that can more

aggravate the toxicity (Hinson et al., 2010).

The clinical and biochemical changes induced by PCM hepatotoxicity include an

increase in alanine & aspartate aminotransferases (ALT & AST) and total bilirubin in

serum, plus increases the prothrombin time. Some patients with hepatotoxicity can also

develop nephrotoxicity (Yoon et al., 2016).

1.4.2. Carbon Tetrachloride (CCl4)-Induced Hepatotoxic Model

CCl4 used as a solvent for cleaning or degreasing agent, fumigant for grain,

synthesis of refrigeration fluid and aerosol cans propellants in the industries. CCl4

accumulate in the atmosphere and groundwater. Major ways with its exposure include

inhalation of fumes and ingestion of contaminated water (El-kabalawy & Bakheet, 2008).

17

CCl4 used to induce liver injury as a typical hepatotoxicant by converting itself through

cytochrome P-450 enzyme into its highly reactive metabolic product viz., trichloromethyl

which damages hepatocytes by initiating lipid peroxidation. Free radical activates the

kupffer cells that release cytokines and help in the progression of damage. Single/double

oral, intraperitoneal, or subcutaneous doses of CCl4 induced acute liver injury by

accelerating fibrosis (Hewawasam et al., 2016).

`The halogenated alkanes like carbon tetrachloride (CCl4) are generally used as

model compound to induce liver injury (Bahashwan et al., 2015). Ingestion of CCl4

activate cytochrome CYP2E1, CYP2B1 or CYP2B2, and maybe CYP3A, trichloromethyl

(CCl3*) radical is formed which develop toxicity such as fatty degeneration/ deposition,

fibrosis, carcinogenicity and hepatocellular apoptosis. The CCl3* radical binds to cellular

macromolecules (proteins, lipids) and induce essential cellular processes like fatty

degeneration, whereas the CCl3* reacts with DNA then become hepatic cancer initiator.

On the other hand, a highly reactive species trichloromethylperoxy (CCl3OO*) radical is

produced when CCl3* reacts with oxygen. The lipid peroxidation initiates the chain

reaction (Huang et al., 2012). This leads to the alteration in mitochondrial permeability,

plasma membranes, and endoplasmic reticulum, resulting in cell damage with subsequent

loss of cellular calcium and calcium homeostasis. CCl3 also activate tumour necrosis

factor (TNF) α, nitric oxide (NO), and transforming growth factors (TGF)-α and -ß

processes, accelerate fibrosis/necrosis. The TGFs directs to the fibrosis and cells headed

toward apoptosis (Weber et al., 2003). The consequence of the CCl4 results is the

reducing effect on cytochromes, successive active trichloromethyl radical formation and

increased expression of NFкB, TNFα and TGF –α and –β, so withdrawing effects of CCl4

have been proven by using antagonist of cytochrome P450, free radical scavengers and

antioxidants. CCl4 induced hepatotoxicity ameliorates by some plants because of their

antioxidant activities (Bahashwan et al., 2015).


18

Figure D: Mechanism of PCM-Induced Hepatotoxicity

NAPBQI

(Toxic)

PCM

Glucouronidation (Non-Toxic)

45-55%

Sulfation (Non-Toxic)

35-45%

Renal excretion

5-10%

CYP-P450

95%

Conjugation

GSH-derived conjugates

Glutathione

conjugation Elimination &

Detoxification

Reactive metabolite

Covalent binding

Oxygen species

Oxidative stress (decrease GSH)

NAPBQI-GSH adduct

NAPBQI-Protein adduct

Lipid peroxidation

SH group on cellular Ca+2 ATPase

GSH-depletion

Oxidative stress

Protein Nitration (ONOO-)

Cell Death/Necrosis

Decrease membrane

permeability

Massive Mitochondrial

damage

Decreased ATP

Increased in calcium

Activated degradative

enzyme by stimulation of calcium

(toxic doses)

19

Figure E: Mechanism of CCl4-Induced Hepatotoxicty

CCl4

CCl3*

Bind to the tissue components

CCl3OO*

ROS (Increase oxidative stress)

Decrease CAT & SOD

(Increase ER stress)

Activates TNF-alpha

Decrease

COX 2 and NO

Lipid peroxidation

Decrease

GPx & GSH

Increase MDA

Mitochondrial

Dysfunction

Liver damage

+O2

PUFA

pp

P

uFAkjk

j+O2

20

1.5. Plant of Present Study

1.5.1. Centratherum anthelminticum (Wild) Kuntz

(English: bitter/black cumin; Hindi: Somraj; Urdu: kali zeri)

Centratherum anthelminticum (Syn: Vernonia anthelmintica Roxb. and Conyza

anthelmintica (L.) Wild) belongs to family Asteraceae. This plant is an erect, leafy

annual, robust 3-5 ft high, leaves petioled long, pale violet florets, achenes that contains

single seeded fruit (seed) which is greenish-brown in color. It is commonly use all over

India and Pakistan for culinary purpose and it also distributed in Southeast Asia countries

(Lateef & Qureshi, 2013). The different parts of kali zeri have been used traditionally as

medicine such as its aerial part reported for anthelmintic, laxative, mild hypotensive,

and smooth muscles relaxant. Purgative flowers are used to treat asthma, kidney trouble

and inflammatory swellings. Roots are useful in curing diarrhoea, stomachache, ulcers

and cough. Fruits are used for diuretics and seeds having hot sharp taste, reported as

astringent, anthelmintic, antiseptic, antiviral and used to treat intestinal colic, flatulence,

hiccups, sores, fever, white leprosy and other skin diseases. Seeds powder also used

externally to treat leg paralysis (Negi et al., 2014). However, its hepatoprotective

potential was not reported before conducting this work.

1.5.2. Phytochemical Profile of C. anthelminticum

Previous studies described that nearly 120 bioactive compounds were isolated

from this plant including proteins, carobohydrates, lipids, alkaloids, phenols, tannins,

flavanoids, saponins, sterols, and resins, etc (Bhatia et al., 2008). Many potential

compounds isolated from seeds of this plant and examined for biological activities are

flavonoids (2´, 3, 4, 4´-tetrahydroxychalcone, 5, 6, 7, 4´ tetrahydroxyflavone and butin,

kaempferol, etc), nine steroids (stigmastane-type) called vernoanthelcin A to I, two

steroidal glycosides (vernoantheloside A & B and vernoanthelsterone A) involved in the

production of estrogen from human ovarian granulosa-like cells (Srivastava et al., 2014),

other steroids (vernodalidimers A and B) also reported with anti-cytotoxic potential. The

various other compounds were also reported from seeds includes, naphthalene derivative

21

(centratheram naphthylpentol and hexol), phenolic acid (caffeic acid, 3-O-caffeoylquinie

acid), triterpenoid saponins (3-O-[β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-

(1→2)-α-Larabinopyranosyl]-28-O-[β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-

(1→3)-β-D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid), Fatty acids (vernolic

acid trivernolin, palmitic acid etc), sesquiterpene lactone (vernodalol, vernodalidimers A

and B, vernodalin), carohydrates (lactose, raffinose) and ethyl 4-isothiocyanatobutyrate

etc (Paydar et al., 2013).

1.5.3. Pharmacological Activity of C. anthelminticum

The pharmacological activities of seeds extracts of C. anthelminticum are

summarized Table A.

22

Table A: Pharmacological Activities and Isolated Compounds Reported from the Seeds of C. anthelminticum

S # Biological Activity Extracts Active Components References

1 Antibacterial and Antiviral Activity - Palmitic,stearic, oleic, and linoleic acid Patel et al., 2012

Paydar et al., 2012

2 Antimicrobial

Activity

Anti-bacillus spp.,

Activity

Aqueous Methanol Acetone

Extract - Ani & Naidu., 2008

Ethanol Extract - Patel et al., 2012

- 24 μ-hydroperoxy-24-vinyllathosterol Hua et al., 2012b

Anti-Enterobacteriaceae

activity

Benzene:Acetone Extract

(24α/R)-Stigmasta-7-en-3-one and (24α/R)-Stigmasta-7, 9(11)-dien-3-

one,(24α/S)-Stigmasta-5, 22- dien-3β-ol and (24α/S)-Stigmasta-7, 22-dien-3β-ol

Mehta et al., 2005

Ethanol Extract - Patel et al., 2012

Anti-Staphylococcus aureus activity

Aqueous Methanol Acetone

- Ani & Naidu., 2008

Methanol and Acetone

Triterpenoidsaponins, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl]-28-O-[β-D-

xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid and 3-O-[β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl]-28-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-hederagenin

Mehta et al., 2010

- 24ξ-hydroperoxy-24-vinyllathosterol Hua et al., 2012b

Anti-Pseudomonas aeruginosa activity

Ethanolic Extract - Patel et al., 2012

3 Antifungal Activity

Methanolic Extract

Centratherumnaphthylpentol and centratherumnaphthylhexol Singh et al., 2012

(24α/R)-stigmasta-7-en-3-one and (24α/R)-stigmasta-7, 9(11)-dien-3-

one Mehta et al., 2005

3-O-[β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-

arabinopyranosyl]-28-O-[α-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid and 3-O-[β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-

(1→2)-α-L-arabinopyranosyl]-28-O-[β-D-glucopyranosyl-(1→3)-β-D- glucopyranosyl]-hederagenin

Mehta et al., 2010

Various Extracts - Ani & Naidu., 2008

http://scialert.net/fulltext/?doi=ijp.2013.211.226#15066_tr

http://scialert.net/fulltext/?doi=ijp.2013.211.226#12914_op

http://scialert.net/fulltext/?doi=ijp.2013.211.226#1136372_ja


http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=Staphylococcus+aureus

http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=Staphylococcus+aureus

http://scialert.net/fulltext/?doi=ijp.2013.211.226#15066_tr



http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=Pseudomonas+aeruginosa

http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=Pseudomonas+aeruginosa


http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=ethanolic+extract




http://scialert.net/fulltext/1136292_ja

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Anti-candida species

Petroleum Ether Oil of seeds Gopalkrishna et al., 2016

4 Analgesic and Antipyretic Activities Petroleum Ether and Alcohol Extracts

- Purnima et al., 2009

5

Anti-Filarial Activity

Macro-Filaricidal Activity

Aqueous and Alcoholic Extract

Methanol Extract

-

-

Singhal et al., 1992

Nisha et al., 2007

6 Antioxidant Activity

Aqueous Methanol AcetoneExtract

Phenolic compounds Ani and Naidu, 2011

Choloroform Fraction - Arya et al., 2012a

7 Anti-InflammatoryActivity Petroleum Ether and Alcoholic Extracts

- Ashok et al., 2010

8 Anti-Arthritic Activity Ethanolic Extract - Otari et al,. 2010

9 Anti-Diabetic Activity

Aqueous Methanol-Acetone Extract

- Ani & Naidu, 2008

Crude Methanolic Fraction - Arya et al., 2013 Arya et al., 2012b

Ethanolic Extract - Mudassir & Qureshi, 2015

Aqueous Extract - Bhatia et al., 2008

Shah et al., 2008

10 Anti-HypergylcemicActivity - Polyphenolic constituents Ani and Naidu, 2008

11 Anti-Hyperlipidemic Activity Ethanolic Extract - Lateef & Qureshi, 2013

12 Anti-Diuretic & Anti-Urolithiatic Activity

Aqueous Extracts

Alcohol and Chloroform Extracts

-

-

Shenoy et al., 2009

Koti and Purnima., 2008

Kumar et al., 2010


http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=phenolic+compounds










24

13 Anti-Nephrolithiatic Activity 70% Methanolic Extract - Galani & Panchal., 2014

14 Anti-Cancer Activity

Anti-Tumor Activity

Chloroform Fraction - Arya et al., 2012ab

- vernodalin and 12,13-dihydroxyoleic acid Looi et al., 2013

Anti-Cytotoxic Activity

- vernodalidimers A and B Liu et al., 2010

Anti-Proliferative Activity

- - Lambertini et al., 2004

15 Anti-Helmintic Activity Alcohol, Methanol and Aqueous Extract

- Iqbal et al., 2006

16 WoundHealing Activity Aqueous Methanolic Extract ointment

- Sahoo et al., 2012

17 Inhibition of Aromatase - -

Bhatnagar et al., 2001

- 24μ-hydroperoxy-24-vinyllathosterol Hua et al., 2012b

18 Larvicidal Activity Petroleum Ether Extract of Fruits and Leaf

- Srivastava et al., 2008

19 Melanogenesis Activity - 2',3,4,4',-tetrahydroxychalcone, 5,6,7,4',-tetrahydroxyflavone and Butin. Tian et al., 2004

Ethanol Extract of Fruit - Zhou et al., 2012












25

1.6. Objective of the Present Study

There was not any scientific data available related to the hepatoprotective effect

of seeds extract of Centratherum anthelminticum. Therefore, it became the idea of

present study to evaluate the hepatoprotective ability of organic solvent extracts of seeds

of this plant in carbon tetrachloride (CCl4) and paracetamol (PCM)-induced liver injury.

The outline of the present work is given in Figure F.

26

Histopathalogical Studies

Preparation of Ethanolic Seeds Extract of C.anthelminticum Seeds of Plant

Grinded

Soaked in Ethanol(1L) Overnight Filtered

Concentrated by Rotary Vacuum Evaporator

Hepato-Protective Activity

ESEt

Hepato-Protective activity of HSF

Fractionated with n-Hexane

Defatted Sample

PCM-Induced Hepatotoxic Model CCl4-Induced Hepatotoxic Model

Biochemical Analysis Liver-Specific parameters

Lipid Profile

Hematological Analysis

Complete Blood Profile

Antioxidant Analysis

Antioxidant Protein & Enzymes

Statistical Analysis

Hexane Soluble Fraction (HSF)

Physical Analysis Body and organ weights

Figure F: Outline of Present Work

27

2. Materials & Methods

2.1. Animals

Healthy female albino Wister rats (180-220 g) were bought from breeding house

of Dow University of Health Sciences (DUHS), Karachi, Pakistan. The animals were

placed in conventional animal house of the University of Karachi according to

internationally accepted guidelines for animal handling, provided with standard

laboratory diet and free access to water ad libitum.

2.2. Chemicals

The analytical grade chemicals include acetic acid (C2H4O2), acetic anhydride

(C4H6O3), disodium hydrogen phosphate (Na2HPO4), dimethyl sulfoxide (DMSO; C2H6OS),

ethyenediamine tetra acetic acid (EDTA; C10H16N2O8), epinephrine (C9H13NO3), ethanol

(C2H6O), glacial acetic acid (C2H4O2), hydrochloric acid (HCl), hydrogen peroxide (H2O2),

perchloric acid (HClO4), picric acid (C6H3N3O7), potassium dichromate (K2Cr2O7),

potassium ferricyanide (C6N6FeK3), sodium bicarbonate (NaHCO3), sodium carbonate

(Na2CO3), sodium chloride (NaCl), sodium citrate (Na3C6H5O7), sodium dihydrogen

phosphate (NaH2PO4), sodium hydroxide (NaOH), sulphuric acid (H2SO4), thiobarbituric

acid (TBA; C4H4N2O2S), trichloro acetic acid (TCA; C2HCl3O2) used in experimental work

were purchased from BDH (United Kingdom), Merck (United Kingdom), Sigma-Aldrich

(United States) and Fisher Scientific, United kingdom (UK).

2.3. Dimethyl Sulphoxide

DMSO (0.05%) was used as dissolving medium for preparing the doses of

ethanolic seeds extract of Centratherum anthelminticum.

28

2.4. Hepatotoxic Inducers

Paracetamol (4-acetamidophenol) formula C8H9NO2 molecular weight 151.16

g/mol was orally administered in a dose of 1 gm/kg/day for 9 days consecutively in

paracetamol-induced hepatotoxic animal model and carbon tetrachloride formula CCl4

molecular weight 153.82 g/mol was given intra-peritoneal (i.p) in a dose of 3ml/kg (with

olive oil in 1:1 ratio) on 3rd and 5th day in CCl4-induced hepatotoxic animal model. These

were purchased from BDH Laboratories, England.

2.5. Instruments

Beckman coulter AU- 480, centrifuge machine (M-800 centrifugal machine), digital

weighing balance (Sartorius secrua®) for weighing chemicals, kitchen scale weighing

machine (1800) for rat’s body weight, micropipettes (adjustable and fixed, 10-1000µl) of

Eppendrof, Germany, rotary vacuum evaporator (Eylea-18), hematological analyzer (Sysmex

X-80), tissue homogenizer (Janke & Kunkel IKA-Labortechnik Ultra-Turrax T25), UV-

Visible spectrophotometer (Jenway 6305 Spectrophotometer), digital water bath.

2.6. Positive Control

Hepatoprotective medicine including Silymarin (Silliver 200mg/kg) was

purchased from Abbott laboratories (Pvt) Ltd, Pakistan and used as positive control in

present study.

2.7. Experimental Plant Material

Seeds of Centratherum anthelminticum (L.) Kuntze plant purchased from

Hamdard Dawakhana, Saddar, Karachi, identified and authenticated by taxonomist in

Botany Department, University of Karachi, Karachi-75270, Pakistan and kept with a

voucher No. KU/BCH/SAQ/05 in Department of Biochemistry of the same university,

stored in clean airtight bottle at room temperature.

29

2.8. Preparation of Seeds Extracts

Extraction of seeds of C. anthelminticum with the help of ethanol is mentioned in

Figure 1.

2.9. Percent Yield of Seeds Extracts

The percent yield of ESEt and HSF was determined by using the following

formula,

Percent yield ( ) =

Where,

Y= ESEt or HSF (gm).

A= Seed powder or ESEt (gm).

Results expressed in percent (gm / 100 gm of starting material)

2.10. Animal Models

2.10.1. Paracetamol (PCM)-Induced Hepatotoxic Rats Model

In PCM-induced hepatotoxic (PIH) rats model, experimental rats (n=6) were

categorized into 2 broad groups viz., normal control (group I) treated with distilled water

(1 ml/kg) and PIH rats administered with PCM in a dose of 1gm/kg/day for 9 days orally.

These PIH rats were further divided into 5 groups according to the treatments (Figure 2).

Each treatment was given orally once in a day for consecutively 9 days. After 24 hour of

last dose of PCM, body weights of all rats were recorded, rats were sacrificed to collect

blood and serum was separated whereas body tissues including liver, kidney, heart and

pancreas were dissected out carefully.

2.10.2. Carbon Tetrachloride (CCl4)-Induced Hepatotoxic Rats Model

In CCl4-induced hepatotoxic (CIH) model was mainly divided into two groups

including normal control group treated with distilled water (1 ml/kg) and CIH group

30

administered with CCl4 (i.p) in a dose of 3 ml/kg diluted with olive oil in 1:1 ratio on 3rd

and 5th day of animal trial which was further divided into different groups according to

the treatment (Figure 3). Each group consist of 6 rats and treatment was given orally once

in a day for 5 consecutive days. After 24 hour of last dose of CCl4, body weights of all

rats were recorded and sacrificed them to collect blood, serum and liver tissue.

2.11. Determination of Physical Parameters

i. Determination of Percent Body Weight Change

The percent gain/loss in body weights was determined with the help of the

following formula (Azmi & Quershi., 2013).

Body weight change (%) =

ii. Determination of Wet Organ Weight

Wet liver organ weight organ was measured by a kitchen scale.

2.12. Determination of Hematological Parameters

Hematological parameters including heamoglobin (Hb), red blood cells (RBC),

white blood cells (WBC), haematocrit (HCT), platelets (PLT), were determined by an

automatic hematology analyzer.

2.13. Estimation of Biochemical Parameters

Biochemical parameters including alanine amino transfrese (ALT), aspartate

aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBR), direct

bilirubin (DBR), indiect bilirubin (IDBR), γ-glutamyl transferase (GGT) were done by

Beckman Coulter AU 480 Inc. While triglycerides (TG), total cholesterol (TC), high

density lipoprotein-cholesterol (HDL-c), total protein (TP), albumin (ALB), and uric acid

(UA) were estimated from commercially available enzymatic kits (Randox, UK). The

low density lipoprotein-cholesterol (LDL-c), very low density lipoprotein-cholesterol

(VLDL-c) and percent protection were calculated by standard formulae. In liver

(Final weight – Initial weight)

Initial weight x 100

31

homogenate, catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH)

and lipid peroxidation (LPO) were estimated by manual methods (Ellman, 1959; Misra

& Fridovich, 1972; Pari & Latha, 2004).

32

Figure 1: Flow Chart for the Extraction of ESEt and HSF

Seeds of Centratherum anthelminticum (40 gm)

+ 1 L of Ethanol (95%)

Soaked overnight at room temperature

Filtered through Whatman’s filter paper No.1 twice

Concentrated at 40°C by using rotary vacuum

evaporator

Dark brown residue was obtained, termed as ESEt

Hexane insoluble fraction Hexane soluble fraction (HSF),

oil in nature

Defatted with n-hexane

33

Note: ESEt =Ethanolic seed extract of C.anthelminticum

Figure 2: Animal Grouping of PCM-Induced Hepatotoxic (PIH) Rats Model

34

Note: ESEt =Ethanolic seed extract of C.anthelminticum

HSF = Hexane soluble fraction of ESEt

Figure 3: Animal Grouping of CCl4-Induced Hepatotoxic (CIH) Rats Model

35

2.13.1. Principles of Methods Used to Determine Biochemical Parameters through

Beckman Coulter AU 480 Inc

i. Alanine Aminotransfersae (ALT)

Principle

The alanine aminotransferase (ALT) transfers the amino group of the alanine to α-

oxoglutrate (ketoglutarate) and formed pyruvate and glutamate. The pyruvate reduced to

lactate in the presence of lactate dehydrogenase (LD) by utilizing NADH + H+ and

producing NAD+.

ii. Aspartate Aminotransferase (AST)

Principle

Aspartate aminotransferase catalyzed the transamination of aspartate and α-

oxoglutarate (α-ketoglutarate) and formed L-glutamate and oxaloacetate. The

oxaloacetate was reduced L-malate by malate dehydrogenase (MDH) and NADH was

rapidly converted to NAD+.

36

iii. Alkaline Phosphatase (ALP)

Principle

ALP hydrolyzes the p-nitrophenyl phosphate a colorless and synthetic substrate to

p-nitrophenol a yellow-colored product, and inorganic phosphate.

iv. Gamma-Glutamyl Transferase (GGT)

Principle

This method was the modification of the szasz procedure (Szasz & Klin 1974),

GGT catalyzes the transfer of the gamma-glutamyl group from the substrate, gamma-

glutamy-3-carboxy-4-nitroanilide, to glycylglycine, formed 5-amino-2-nitrobenzooate.

The change in absorbance at 480 nm is due to the formation of 2-amino-2-nitrobenzoate

and is directly proportional to the GGT activity in the sample.

v. Total Bilirubin (TB)

Principle

Total bilirubin is composed of direct (conjugated) and indirect (unconjugated)

bilirubin in serum. A bilirubin reacts with stabilized diazonium salt, 3, 5-

dichlorophenyldiazonium tetrafluoroborate (DPD), to form azobilirubin. Caffeine and a

surfactant was used as reaction accelerators.

37

vi. Direct Bilirubin (DB)

Principle

Direct bilirubin (conjugated) reacts with a diazonium salt, 3, 5-dichloroaniline

in an acid medium and formed an azobilirubin (Van den Bergh, 1916).

2.13.2. Methods Used to Determine Biochemical Parameters through Randox Kits

i. Total Protein (TP) by Biuret Method

Principle

In an alkaline medium, cupric ions interact with protein peptide bond and formed

coloured complex (Tietz, 1995).

Reagents

R1: Biuret reagent (sodium hydroxide 100 mmol/l, Na-K-tartrate 15 mmol/l,

potassium iodide 6 mmol/l, cupric sulphate 6 mmol/l)

R2: Blank reagent (sodium hydroxide 100 mmol/l, Na-K-tartrate 16 mmol/l) The

content of R2 was diluted with 400 ml of double distilled water.

Standard: Total protein 5.95g/dl

38

Procedure

Reagent blank Standard Sample

Distilled water 0.02 ml - -

Standard - 0.02 ml -

Serum - - 0.02 ml

R1 1.0 ml 1.0 ml 1.0 ml

Mixed and incubated for 30 min at 25ºC. Absorbance of the sample (A sample) was

measured against the reagent blank at 530nm.

Calculation

Total protein conc. (g/dl) = (5.95g/dl)

ii. Albumin (ALB) by Bromocersol Green Method

Principle

The serum albumin was estimated by its quantitative binding to the indicator 3,

3’, 5, 5’-tetrabromo-m cresol sulphonephthalein (bromocersol green BCG). The albumin-

BCG-Complex absorb maximally at 578 nm. The absorbance is directly proportional to

the concentration of the albumin present in sample (Grant, 1987).

Reagents

R1: BCG concentrate (succinate buffer 75 mmol/l; pH 4.2, bromocersol green

0.15 mmol/l, Brij 35)

One bottle of R1 was diluted with 87 ml of distilled water.

Standard: Albumin 4.60g/dl

39

Procedure


Distilled H2O 0.01ml - -

Standard - 0.01ml -

Sample - - 0.01ml

R1(BCG Reagent) 3.00 ml 3.00 ml 3.00 ml

Mixed and incubated for 5 minutes at 25ºC. Absorbance of the sample ( Asample) and

standard ( Astandard) was measured against the reagent blank at 630nm.

Calculation

Albumin conc. (g/dl) = (4.60g/dl)

iii. Uric acid (UA) by Enzymatic Colorimetric Method

Principle

Reagents

R1a: Buffer (Hepes buffer 50 mmol/l pH 7.0, 3, 5-Dichloro-2-hydroxy

benzenesulfonic acid 4 mmol/)

R1b: Enzyme reagent (4-aminophenazone 0.25 mmol/l, peroxidase ≥1000Ul,

uricase ≥ 200Ul)

One vial of R1b was reconstituted with 15 ml of R1a.

Standard; Uric acid 10.18 mg/dl

40

Procedure

Reagent blank (µl) Sample(µl) Standard(µl)

Sample - 20 -

Standard - - 20

Reagent(R1) 1000 1000 1000

Mixed and incubated for 5minutes at 37ºC. Absorbance of sample (Asample) and Standard

was measured against reagent blank at 520 nm.

Calculation

Uric acid conc. = (10.18mg/dl)

iv. Total Cholesterol (TC) by Enzymatic End Point Method (Triender P, 1969)

Principle

Reagents

R1: Buffer Reagent : (4-aminoantipyrine 0.30 mmol/l (pH 6.8), phenol 6 mmol/l,

peroxidase ≥ 0.5 U/ml, cholesterol esterase ≥ 0.15 U/ml, cholesterol oxidase ≥ 0.1

mmol/ml, pipes buffer 80 mmo/ml

Standards : Cholesterol 200mg/dl

41

Procedure

Reagent blank Standards Sample

Distilled water 10 µl - -

Standard - 10 µl -

Serum - - 10µl

Reagent (R1) 1000 µl 1000 µl 1000 µl

Mixed and incubated for 5min at 37ºC. Absorbance of the sample (Asample) and standard

(Astandard) was read against reagent blank at 500 nm.

Calculation

Cholesterol (mg/dl) =

v. Triglycerides (TG) by GPO-PAP Method (Tietz N.W, 1990)

Principle

42

Reagents

R1a: Buffer (pipes buffer 40 mmol/l pH7.6, 4-cholro-phenol 5.5 mmol/l,

Magnesium-ions 17.5 mmol/l)

R1b: Enzyme reagent (4-aminophenazone 0.5 mmol/l, ATP 1.0 mmol/l, Glycerol-

kinase ≥ 0.4 U/l, Lipases ≥ 150 U/l, Glycerol-3-phosphate oxidase ≥ 1.5U/l,

Peroxidase ≥ 0.5U/l)

One vial of R1b was reconstituted with 15 ml of R1a before used.

Standard: Triglycerides 194 mg/dl

Procedure


Standard - 10 µl -

Sample - - 10 µl

Reagent (R1) 1000 µl 1000 µl 1000 µl

Mixed and incubated at 37ºC for 5minutes. Absorbance of the sample (Asample) and

standard (Astandard) was measured against the reagent blank at 500 nm within 60min.

Calculation

Triglycerides (mg/dl) =

2.13.3. Estimation of Biochemical Parameters through Formulae

i. Low & Very Low-Density Lipoproteins-Cholesterols (LDL-c & VLDL-c)

LDL-c & VLDL-c were determined by using Friedwald formulae (Friedewald,

1972), as

43

LDL-c (mg/dl) = TC – (TG/5) –HDL-c

VLDL-c (mg/dl) = TG/5

ii. Indirect Bilirubin (IDBR)

IDBR was calculated according to the

IDBR (mg/dl) = Total Bilirubin - Direct Bilirubin

iii. Aspartate transaminase and Platelet Ratio Index (APRI)

APRI was calculated according to the formula (McGoogan et al., 2010)

APRI=AST/Platelet count x 100

iv. Percent Protection

Percent protection was calculated according to the formula (Quershi et al.,

2016)

Where, At = Rats treated with test dose + CCl4 or Silymarin +CCl4

Ax = Rats treated with CCl4

Ao = Rats treated with Distilled water

v. Liver Index

Liver index was calculated according to the formula (Hai et al., 2011)

Liver Index = (liver weight / body weight) × 100.

44

2.14. Determination of Antioxidant Parameters

i. Percent Inhibition of Catatlase (CAT)

Principle

CAT is chiefly present in liver, kidney and erythrocytes. It is responsible to

decompose hydrogen peroxide (H2O2) into oxygen and water, which is produced due to

dismutation of superoxide radical.

CAT

2H2O2 2H2O+ O2

The presence of CAT in liver homogenate was determined by observing the

decomposition of substrate H2O2 at 620 nm in the presence of color developer reagent for

three minutes consecutively. Finally results expressed as percent inhibition of CAT (Pari

& Latha, 2004).

Reagents

Phosphate Buffer (pH 7.0) 0.01M: 1.735g disodium hydrogen phosphate and

sodium dihydrogen phosphate were dissolved in 1 liter of distilled water.

Liver homogenate (0.5%): 0.5g liver tissue was homogenized in 100 ml of

distilled water.

Hydrogen peroxide (2M H2O2): 6.8 ml of H2O2 was dissolved in 93.2 ml of

distilled water.

Potassium dichromate (5%): 5gm of potassium dichromate was dissolved in

100ml of distilled water.

Dichromate-acetic acid reagent (color reagent): 5% potassium dichromate and

glacial acetic acid (1:3) were freshly mixed before used.

45

Procedure

Reagents Test Control

Phosphate buffer (pH 7.0) 1.0 ml 1.0 ml

Distilled water - 0.1 ml

Liver Homogenate 0.1 ml -

H2O2 0.4 ml 0.4 ml

Dichromate-acetic acid 2.0 ml 2.0 ml

After 1 minute absorbance was start reading at 620 nm till the next 3 minutes against the

control.

Calculation

ii. Percent Inhibition of Superoxide Dismutase

Principle

Superoxide dismutase (SOD) involved in the scavenging system against reactive

oxygen species (ROS). SOD at high reaction rate decomposes superoxide anion into

oxygen and hydrogen peroxide.

The percent inhibition of SOD was estimated through method described by Misra

& Fridovich, 1972.

46

Reagents

Ethylenediamine tetraacetic acid (EDTA; 0.6 mM): 0.175 g of EDTA was

dissolved in 1000 ml of distilled water

Carbonate-bicarbonate buffer (0.1 M; pH 10.2): Solution A (0.2 M): 21.2 gm of

sodium carbonate was dissolved in 1 litre of distilled water whereas Solution B

(0.2 M): 16.8 gm of sodium bicarbonate was dissolved in 1 liter of distilled water.

Then 33 ml of solution A and 17 ml of solution B were mixed and made up the

volume to 200 ml with distilled water.

Epinephrine (1.8 mM): 0.329 gm of epinephrine was dissolved in 1000 ml of

distilled water.

Ethanol: 95%

Chloroform: Ice chilled

Liver homogenate (0.5%): 0.5 g of liver tissues was homogenized in 100 ml of

distilled water.

Procedure


Homogenate 0.1 ml -

Ethanol 0.75 ml 0.75 ml

Chloroform (Chilled) 0.15 ml 0.15 ml

Centrifuged at 3000 rpm for 10 minutes

Supernatant 0.5 ml -

EDTA 0.5 ml 0.5 ml

Buffer 1.0 ml 1.0 ml

Epinephrine 0.5 ml 0.5 ml

The increased in the absorbance was measured for 3 minutes at 480 nm.

47

Calculation

Rate of Absorbance of Test (R) =

iii. Reduced Glutathione (GSH)

Principle

Reduced glutathione (GSH) is tripeptide (gamma-glutamylcystenylglycine)

peroxidase enzyme family and has a free thiol group. Reduced GSH converts into

oxidized glutathione (GSSG) and involved in the reduction of lipid hydroperoxides to

their corresponding alcohols, and/or reduction of hydrogen peroxide into water. The total

amount (GSH+GSSG) of glutathione react with Ellman’s reagent [5, 5’-dithiobis-2-

nitrobenzoic acid (DTNB)] to give yellow chromogenic compound [5-thiol-2-

nitrobenzoic acid (TNB)] that can be quantified at 412 nm (Ellman, 1959). The reaction

is as follows.

Reagents

Liver homogenate (1%): 1g of liver tissue was homogenized with 100 ml of distilled

water.

Sodium citrate (1%): 1 g of sodium citrate was dissolved in 100 ml of distilled water.

Ellman’s reagent: 9.9 ml of DTNB was dissolved in 50 ml of 1% sodium citrate.

Trichloroacetic acid (TCA 5%): 5 g of (TCA) was dissolved in 100 ml of distilled

water.

48

Procedure


Homogenate 0.5 ml -

Distilled water - 0.5 ml

TCA (5%) 2 ml 2 ml

Centrifuged at 3000 rpm for 15 minutes

Supernatant 1ml 1 ml

Ellman’s reagent 0.5 ml 0.5 ml

Phosphate buffer (0.2M, pH-8.0) 3.0 ml 3.0 ml

The increased in the absorbance was measured for 3 minutes at 412 nm

Calculation

The percent Inhibition of GSH was calculated using

iv. Lipid peroxidation (Thio Barbituric Acid Reactive Substances (TBARS)

Principle

Lipid peroxidation (LPO) was used in cells as an indicator of oxidative stress.

LPO decomposes and form a series of complex compound like reactive carbonyl

compounds. LPO decomposes polyunsaturated fatty acid and generate a malondialdehyde

(MDA). The free MDA reacts with thiobarbituric acid (TBA) to form a MDA-TBA

adduct, which can be easily quantified colorimetrically at 535 nm (Alam et al., 2011).

49

Reagents

Liver homogenate (10%): 10 g of liver tissue was homogenized in 100 ml of

distilled water.

Thiobarbituric acid (TBA; 0.37%): 0.37 g of TBA was dissolved in 100 ml of

distilled water.

15% Trichloroacetic acid (TCA; 15%): 15 g of TCA was dissolved in 100 ml of

distilled water.

Hydrochloric acid (HCl; 0.25N): 48 ml of HCl was dissolved in 1000 ml of

distilled water.

TBA-TCA-HCl Reagent: TBA, TCA and HCl with 1:1:1 ratio.

Procedure


Liver homogenate 0.1ml -

Distilled water - 0.1ml

TBA-TCA-HCl 2ml 2ml

All tubes were kept on boiling water bath for 30 minutes then cooled and read the amount

of malondialdehyde formed in sample using reagent blank at 535 nm.

Calculation

The percent inhibition of LPO was calculated using

50

2.15. Histopathological Examination of Liver Tissues

Dissected out liver tissues from each group were immersed in 10% formaldehyde

solution separately and sent to Dr. Essa’s diagnostic laboratory, Abul Hasan Isfahani

Road, Karachi Pakistan for conducting histological studies. Where 2 - 4 µm thickness of

liver sections were cut from each sample, dehydrated by a series of ethanol solutions,

embedded in paraffin separately and stained in haematoxylin. The stained tissues were

observed through an Olympus microscope (BX-51) and photographed by Olympus DP-

72.

2.16. Statistical Analysis

Results of the present study are expressed as mean ± SD (standard deviation). All

data were analyzed by means of one-way ANOVA followed by LSD (least significant

difference) test at p < 0.05 through statistical package for social sciences (SPSS version

16). The differences of means of each parameter of test groups were considered

significant at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 when compared with means of

hepatotoxic control groups.

51

3. Results

3.1. Total Amount of Seeds Extracts Obtained

The total obtained amount of ESEt of C. anthelminticum was 12.6gm/100gm of

dried seeds powder. Wheares the HSF of same extract was obtained as 16.8gm/100gm of

ESEt.

3.2. Outcome of ESEt on PCM-Induced Hepatotoxic Rats Model

3.2.1. On Physical Parameters

Percent Body Weight Change

There was marked reduction observed in percent body weight up to -8.62 and -

10.23 respectively in PCM-induced hepatotoxic and positive control groups (II & III) as

compared to control rats (group I). Whereas all three doses of ESEt (200, 400 & 600

mg/kg) gradually and significantly (p<0.0001) improved the body weights when matched

to PCM intoxicated group II (Figure 4) and showing percent protection from 91 to 163%

in the same physical parameter. However, silymarin (100 mg/kg) in positive control

(group III) protected body weight reduction only 49% as compared to group II (Figure 5).

Liver Weights

The liver weight of PCM control group was found 9.1 ± 0.92g. Whereas the same

parameter becomes significantly decreased in groups III-VI administered with silymarin

(100 mg/kg), ESEt 200, 400 & 600 mg/kg respectively by showing percent decrease from

-13.1 to -21.4% (Table 1).

3.2.2. Outcome of ESEt on Liver Associated Enzymes Activities

The levels of liver associated enzymes (ALT, AST & ALP) increased in PCM-

induced hepatotoxic control group II. Whereas silymarin (100mg/kg) treated group III

and ESEt (200 - 600 mg/kg) treated test groups IV- VI displayed significant decreased in

levels of ALT below 50 U/l even the highest dose of ESEt 600mg/kg induced 97%

52

protection in the same ALT level irrespective to PCM control group II (Figure 6 & 7).

Similarly, AST levels in silymarin treated group III and extract treated groups (IV, V &

IV) were significantly reduced below 200 U/l (p<0.01 & p<0.05) while matched with

PCM control group II that showed elevated level of same parameter (Figure 6). The

highest dose of ESEt produced 99% protection in the same AST level in test group IV as

compared to group II (Figure 7). In the same way, the ALP levels in test groups IV, V

and VI were significantly (p<0.05) decreased when matched with PCM control group II

and showed 64% protection in the same enzyme level by 600mg/kg of ESEt in group VI

(Figure 6 & 7).

Another bile duct specific enzyme GGT was also found decreased (p<0.0001)

from -52 to -68.4% in test groups IV, V and VI treated with 200, 400 & 600 mg/kg of

ESEt as compared with PCM-induced hepatotoxic group II. Whereas the level of same

enzyme found increased in positive control group (Table 2).

53

Each bar stands for mean ± S.D (n=6). d = p<0.0001, matched with PCM control group II.

Figure 4: Outcome of ESEt on Body Weights Change in PCM-Induced Hepatotoxic Rats

54

Each bar stands for mean (n=6).

Figure 5: Outcome of ESEt on Percent Protection in Body Weights of PCM-Induced Hepatotoxic Rats

55

Table 1: Outcome of ESEt on Liver Weights (LW) in PCM-Induced Hepatotoxic Rats

Each value stands for mean ± SD (n=6). a, c & d = p<0.05, p<0.001 & p<0.0001 respectively, matched with PCM control

group II.

S. No Groups Treatment LW (g)

1 Group I: Normal Control D. water 1ml/kg 6.9±0.78

2 Group II: PCM Control PCM 1gm/kg + D. water 1ml/kg 9.1±0.92

3 Group III: Positive Control PCM 1gm/kg + Silymarin 100mg /kg 7.8±0.8d

(-14.2%)

4 Group IV: Test group PCM + ESEt 200mg 7.60±0.22d

(-16.4%)

5 Group V: Test group PCM+ ESEt 400mg 7.15±0.43d

(-21.4%)

6 Group VI: Test group PCM + ESEt 600mg 7.9±0.30d

(-13.1%)

56

Each bar stands for mean ± SD (n=6). a, b & d = p<0.05, p<0.01 & p<0.0001 respectively, matched with PCM

control group II.

Figure 6: Outcome of ESEt on Liver Associated Enzymes in PCM-Induced Hepatotoxic Rats

57


Figure 7: Outcome of ESEt on Percent Protection of ALT, AST and ALP in PCM-Induced Hepatotoxic Rats

58

3.2.3. On Biochemical Parameters

Total Bilirubin

A significant (p<0.0001) decrease -50% was observed (p<0.0001) in total

bilirubin concentration (mg/dl) in test groups V to VI when matched with PCM control

hepatotoxic group II (Table 2).

Total Protein (TP) and Albumin (ALB)

The TP and ALB levels were found prominently (p<0.01 &p<0.05) increased in

positive control group III and test groups IV to IV by showing gain from 18-25 and 18-

29.7% respectively when matched with PCM control group II where decreased levels of

same parameters were found (Figure 8 & 9).

Uric Acid

The highest dose of ESEt 600mg/kg in group VI induced marked decreased

(p<0.0001) in uric acid level when compared with the PCM control group II. However,

the percent reduction in the levels of uric acid in all test groups VI-IV and positive

control group III was found from -4.1 to -13.1% (Table 2).

3.2.4. On Hematological Parameters

Hb, RBC, WBC, HCT and PLT levels were sufficiently decreased in PCM-

induced hepatotoxic control group II. Whereas ESEt (200 - 600 mg/kg) was observed

beneficial in increasing the levels of all these parameters in their respective test groups

IV-VI where they almost became upto the levels which were found in normal control

group I (Figure 10 & Table 3). Similarly, the APRI was also found reduced in silymarin

treated group III and all three test groups (IV-VI) when matched with PCM control group

II (Figure 11).

59

Table 2: Outcome of ESEt on GGT, TB and UA in PCM-Induced Hepatotoxic Rats

GGT= Gammaglutamyl transpeptidase, TB= Total bilirubin, UA = uric acid. Each value stands for mean ± SD (n=6).

Values with - / + signs in parenthesis represent reduction / gain in parameter. d = p<0.0001, matched with PCM control group II.

S. No Groups Treatment GGT (U/l) TB (mg/dl) UA (mg/dl)

1 Group I D. water 1ml/kg 10 ± 0 0.10±0.00 8.51 ± 0.21

2 Group II PCM 1gm/kg + D. water

1ml/kg 6.33 ± 0.81 0.2 ± 0.00 10.18 ± 0.26

3 Group III PCM 1gm/kg + Silymarin

100mg /kg 8.83 ± 1.32d (34.7%) 0.19 ± 0.00 (-5%) 9.76 ± 0.37 (-4.1%)

4 Group IV PCM + ESEt 200mg 2.5 ± 0.54d (-60.5%) 0.10 ± 0.00 d (-50%) 9.74 ± 0.42 (-4.3%)

5 Group V PCM+ ESEt 400mg 2 ± 0.63d (-68.4%) 0.1 ± 0.01d (-50%) 9.64 ± 0.72 (-5.3%)

6 Group VI PCM + ESEt 600mg 3 ± 0.63 d (-52.6%) 0.1 ± 0.00d (-50%) 8.84 ± 0.85d (-13.1)

60

Each bar stands for mean ± SD (n=6). a,b & d = p<0.05 , p<0.01 & p<0.0001 respectively when matched with PCM

hepatotoxic group II

Figure 8: Outcome of ESEt on Total Protein (TP) and Albumin (ALB) in PCM-Induced Hepatotoxic Rats

61


Figure 9: Outcome of ESEt on Percent Gain in Protein Profile of PCM-Induced Hepatotoxic Rats

62

Each bar stands for mean ± S. D (n=6). d = p<0.0001, matched to PCM hepatotoxic control group II.

Figure 10: Outcome of ESEt on Hemoglobin in PCM-Induced Hepatotoxic Rats

63


Figure 11: Outcome of ESEt on AST/PLT Ratio Index (APRI) in PCM-Induced Hepatotoxic Rats

64

Table 3: Outcome of ESEt on Hematological Parameters in PCM-Induced Hepatotoxic Model

Each value stands for mean ± S. D (n=6). a, b= p<0.05, p<0.01 respectively, matched to PCM hepatotoxic control group II.

S.No Groups RBC (1012/L) WBC (109/L) HCT (%)

1 Group I 5.64 ± 0.42 10.93 ± 4.17 35.46 ± 2.88

2 Group II 4.88 ± 0.87 8.21 ± 2.7 26.71 ± 3.71

3 Group III 4.41 ± 0.34 4.91 ± 0.31 a 29.03 ± 2.97

4 Group IV 5.16 ± 0.97 8.66 ± 4.16 32.58 ± 6.12

5 Group V 6.03 ± 0.34 b 9.63 ± 1.13 33.35 ± 3.45d

6 Group VI 5.66 ± 0.26 5.38 ± 2.17 31.36 ± 0.63c

65

3.2.5. On Percent Inihibition (PI) of Antioxidant Parameter

PI of CAT and SOD Enzymes

The PI of CAT efficiency was significantly suppressed from 24 to 29.6 %

(p<0.0001) in test groups administered with doses of ESEt 200,400 & 600 mg/kg and

group III treated with silymarin in dose 100mg/kg also showed almost same significant

result when compared with PCM hepatotoxic control group II which exhibited 43.6 %

inhibition of same enzyme (Figure 12). Similarly, the same three doses of ESEt and

silymarin in test and positive control groups showed good reduction in percent inhibition

of SOD ranging from 26 to 30.8%(p<0.01) in comparison with PCM hepatotoxic control

group which exhibited about 44% inhibition of same SOD activity (Figure 12).

PI of GSH and LPO

All groups including positive control (III) and test groups (IV,V & IV) showed

low (p<0.05 &p<0.01) PI of reduced glutathione (GSH) activity ranging from 42.4 to

38.5% as compared with PCM control group II which showed 52.6% inhibition in the

same (Figure 13). On contrary, PI of lipid peroxidation (LPO) was highly prominent

(p<0.0001) from 36 to 41.5% in silymarin treated group III and all ESEt treated test

groups (IV, V& VI) in comparison to the PCM intoxicated group II which showed very

low inhibition of LPO about 13% (Figure 13).

3.2.6. On Histopathology of Hepatic Tissue

Histopathalogical observation of liver tissue indicated that paracetamol (PCM)

induced cellular degenerative alterations including necrotic and inflamed cells around

enlarged central vein in PCM hepatotoxic control group II (slide A). Whereas slides B &

C showed that treatment with different doses of ESEt (200 & 400mg/kg) reduced the

degree of these adverse signs in liver tissues of their respective groups (IV &V). All these

findings proved the hepatoprotective characteristic of ESEt (Figure 14).

66

Each bar stands for mean ± S. D (n=6). b & d = p<0.01 & p<0.0001 respectively, matched to PCM hepatotoxic control

group II.

Figure 12: Outcome of ESEt on PI of CAT and SOD in PCM-Induced Hepatotoxic Rats

67

Each bar stands for mean ± SD (n=6). a,b & d = p<0.05,p<0.01 & p<0.0001 respectively, matched to PCM hepatotoxic

group II.

Figure 13: Outcome of ESEt on PI of GSH and LPO in PCM-Induced Hepatotoxic Rats

68

Figure 14: Liver Histology of PCM-induced Hepatotoxic Model. A= PCM intoxicated

control group that displayed necrosis and inflammation around enlarged center vein.

These adverse signs are gradually decreased with improving the structure of liver tissues

in test groups administered with ESEt @ 200 & 400 mg/kg (B & C).

Dilated central vein

Inflammation

A B

C

69

3.3. Outcome of ESEt and Hexane Soluble Fraction (HSF) on CCl4-

Induced Hepatotoxic (CIH) Rats Model

3.3.1. On Physical Parameters

Percent Body Weight Change

There was marked decreased observed in percent body weight up to -12.18 and -

8.96 in CIH control and positive control group (II & III) respectively when matched with

normal control rats (group I). However, orally administered doses of ESEt (600 &

800mg/kg) in test groups IV & V significantly decreased the percent reduction (p<0.01&

p<0.0001) in body weight by showing protection from 54 to 80%. Similarly, HSF of

ESSt in a dose 600 mg/kg (p<0.01) also improved the body weight up to 54.6% when

matched with CIH control group II (Figure15 &16).

Liver Weights and Liver Index

The weights (g) of livers in CIH control group II were evidently enlarged

(p<0.0001) when matched with silymarin treated III (silymarin 100mg), tests IV & V

(ESEt 600 & 800mg) and VI (HSF 600mg) where their respective treatments were

observed successful in bringing the weights of livers normal (Table 4). Similarly, the

liver index was also improved (p<0.0001) in all these positive and test groups by showing

reduction from -22 to -37% in liver weight of these groups (Table 4).

3.3.2. Outcome on Liver Associated Enzymes Activities

Liver associated enzymes viz., ALT, AST and ALP (U/l) were prominently

increased in CIH group II. However, all these enzymes were amazingly decreased

(p<0.01 and p<0.0001) in positive control and test groups including III, IV, V and VI

which administered with silymarin (100 mg/kg), ESEt (600 and 800 mg/kg) and HSF

(600mg/kg) respectively and displayed prominent protection particularly in ALT from 95

to 98 %, AST from 65 to 81% and ALP from 37 to 100% (Figure 17 & 18). While GGT

(U/l) monitored low in all groups when matched with normal control group I (Table 5).

70

Each bar stands for mean ± S.D (n=6). b & d = p<0.01 & p<0.0001, matched with group II.

Figure 15: Outcome of ESEt and HSF on Body Weights Change in CCl4-Induced Hepatotoxic Rats

71


Figure 16: Outcome of ESEt and HSF on Percent Protection of Body Weights in CCl4-Induced Hepatotoxic Rats

72

Table 4: Outcome of ESEt and HSF on Liver Weights (LW) and Index in CCl4-Induced Hepatotoxic Rats

Each value stands for mean ± SD (n=6). d = p<0.0001 , matched to hepatotoxic control group II.

ESEt = Ethanolic seeds extract. HSF= Hexane soluble fraction of ESEt.

Groups LW (g) Liver Index

I: Normal Control 7.5 ± 1.3 4.58±0.91

II: Hepatotoxic Control 15.0 ± 2.1 9.25±1.04

III: Positive Control 12.1 ± 1.2 d

(-21.9%) 6.87±.75 d

IV: ESEt 600mg 11.30±1.43 d

(-26.1%) 6.82±0.42 d

V: ESEt 800mg 10.60±1.07 d

(-30.7%) 5.54±0.39 d

VI: HSF 600mg 9.63±0.52 d

(-37.0%) 5.84±0.11 d

73

Each bar stands for mean ± S.D (n=6). b &d = p<0.01 & p<0.0001 respectively, matched with group II.

Figure 17: Outcome of ESEt and HSF on Liver Associated Enzymes in CCl4-Induced Hepatotoxic Rats

74


Figure 18: Outcome of ESEt and HSF on Protection of ALT, AST & ALP in CCl4-Induced Hepatotoxic Rats

75

3.3.3. Outcome of ESEt and HSF on Biochemical Parameters

Total Bilirubin (Direct & Indirect)

A noteworthy decreased (p<0.05) was monitored in total bilirubin levels in

silymarin, ESEt and HSF treated groups including III, IV, V and VI respectively by

showing decrease in the level of same parameter from -30 to -56% as compared to CIH

group II. This decrease was actually due to the -73% reduction (p<0.05) found in indirect

bilirubin (mg/dl) in all these groups (Table 5).

Total Protein (TP) and Albumin (ALB)

The levels (g/dl) of TP and ALB were observed low in CIH group II but the levels

of both these parameters were outstandingly increased (p<0.0001) in silymarin treated

and extracts treated groups (III, IV, V and VI) by showing increase from 37 to 59% in TP

and 41 to 56% in ALB levels in all these treated groups (Figure 19 & 20).

Uric Acid

Silymarin (100mg), ESEt (600 & 800mg) and HSF (600mg) induced decreased

(p<0.0001) in uric acid levels in groups from III- VI when matched to CIH control group

II (Figure 21).

76

Table 5: Outcome of ESEt and HSF on Total Bilirubin (Direct & Indirect) and GGT in CCl4-Induced Hepatotoxic Rats

Each value stands for mean ± S. D (n=6). a =p<0.05, b=p<0.01 and d =p<0.0001, matched to CCl4-Induced hepatotoxic control group II.

ESEt = Ethanolic seeds extract. HSF = Hexane soluble fraction of ESEt.

S.No Groups TBR (mg/dl) DBR (mg/dl) IDBR (mg/dl) GGT(U/L)

1 I. Normal Control 0.23 ± 0.3 0.10 ± 0.01 0.0 ± 0.0 10±0

2 II. Hepatotoxic Control 0.43 ± 0.22 0.2 ± 0.16 0.38 ± 0.21 8.1±6.3

3 III.Positive Control 0.19 ± 0.01a

(-55.8%) 0.10 ± 0.01 a

0.10 ± 0.008 a

(-73.6%) 0.71 ± 0.47 d

4 IV. ESEt 600mg 0.20 ± 0.01a

(-53.4%) 0.10 ± 0.009 a

0.10 ± 0.007 a

(-73.6%) 1.36 ± 0.32 d

5 V. ESEt 800mg 0.26 ± 0.01

(-39.5%) 0.19 ± 0.009

0.10 ± 0.009 a

(-73.6%) 3.05 ± 0.08 b

6 VI. HSF600mg 0.30 ± 0.01

(-30.2%) 0.10 ± 0.01

0.10 ± 0.008 a

(-73.6%) 3.66 ± 0.08 a

77

Each bar stands for mean ± S.D (n=6). d =p<0.0001, matched to hepatotoxic control group II.

Figure 19: Outcome of ESEt and HSF on Total Protein (TP) and Albumin (ALB) levels in CCl4-Induced Hepatotoxic Rats

78


Figure 20: Outcome of ESEt and HSF on Gain of TP and ALB levels in CCl4-Induced Hepatotoxic Rats

79

Each value stands for mean ± SD (n=6). d =p<0.0001, matched to hepatotoxic control group II.

Figure 21: Outcome of ESEt and HSF on Uric acid in CCl4-Induced Hepatotoxic Rats

80

3.3.4. Outcome of ESEt and HSF on Lipid Profile

Administration of CCl4 increased TC, TG, VLDL-c and LDL-c in hepatoxic

control group II whereas concentration of all these parameters was found decreased

appreciably (p<0.01,p<0.001 & p<0.01) in positive control (group III) and test groups

(IV, V and VI) administered with silymarin, ESEt (600 & 800mg) and HSF (600mg).

Where percent reduction in TC was found from -25.3 to -44.5%, TG from -8.63 to -

16.2%, VLDL-c from -8.24 to -18.4% and LDL-c from -80.8 to -93.5% when matched

with CIH control group II (Table 6).

3.3.5. Outcome of ESEt and HSF on Percent Inhibition (PI) of Antioxidant Markers

PI of CAT and SOD

The PI in CAT efficiency was considerably decreased (p<0.05 &p<0.0001) from

12 to 41.3% in all ESEt & HSF treated test groups (IV, V & VI) and silymarin treated

positive group (III) whereas only CCl4 treated group II that showed 46% inhibition in

same CAT activity. Similarly, PI of SOD was found from 16.9 to 39.4% (p<0.0001) in all

treated groups (test and positive control) in comparison with hepatotoxic control group II

that exhibited 50% inhibition in SOD activity (Figure 22).

PI of GSH and LPO

All groups III, IV,V &VI showed low (p<0.0001, p<0.001&p<0.05) PI in GSH

activity ranging from 26 to 34.4 % when matched with CIH control group II which

indicated 65 % inhibition in same GSH level. On the other hand, PI of LPO significantly

(p<0.0001) increased from 1.8 to 30% in all test groups (p<0.0001) in comparison with

CIH rats which showed only 8.1% inhibition in LPO (Figure 23).

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3.3.6. Outcome of ESEt & HSF on Histopathaological Examination of Hepatic

Tissues

Histopathological examination was done by fixing liver tissues on slides and

stained with hematoxylin and eosin. Where slide A represents the liver deterioration

together with fatty accumulation (ballooning) and inflamed cells (Inflammation) present

around abnormally enlarged center vein in lobules of CCl4-induced hepatotoxic control

group. However, all these toxic signs were gradually disappeared in liver tissues (slide C,

D& E) dissected out from groups administered with ESEt (600 & 800 mg/kg) and HSF

(600mg/kg) respectively (Figure 24). The results showed that treatment with two doses

(600 & 800 mg/kg) of ESEt greatly improved liver structure or anatomy (slide C & D).

Whereas, the HSF (600mg/kg) was found successful in bringing the liver structure back

to normal (slide E). Silymarin 100 mg was not found effective in this respect (slide B).

82

Each bar stands for mean ± S. D (n=6). a & d = p<0.05 & p<0.0001 respectively, matched with group II.

Figure 22: Outcome of ESEt and HSF on Inhibition of CAT and SOD in CCl4-Induced Hepatotoxic Rats

83

Each bar stands for mean ± S. D (n=6). a,b & d = p<0.05, p<0.01 & p<0.0001 respectively, matched with group II.

Figure 23: Outcome of ESEt and HSF on Inhibition of GSH and LPO in CCl4-Induced Hepatotoxic Rats

84

Table 6: Outcome of ESEt and HSF on Lipid Profile in CCl4-Induced Hepatotoxic Rats

Each value stands for mean ± S. D (n=6). b & d = p<0.01 & p<0.0001, matched with hepatotoxic control II.

ESEt= Ethanolic seeds extract. HSF = Hexane soluble fraction of ESEt.

S.No Groups Treatment TC

(mg/dl)

TG

(mg/dl) VLDL-c (mg/dl)

LDL-c (mg/dl)

1 I: Control Distilled water

1ml/kg 129.59 ± 10.81 148.23 ± 3.59 27.97 ± 4.16 37.02 ± 8.19

2 II: Hepatotoxic

Control CCl4 3ml/kg 242.6 ± 29.4 182.9 ± 5.37 36.4 ± 1.20 73.1 ± 29.29

3 III: Positive Control CCl4+Silymarin

100mg /kg

180.1 ± 9.40 d

(-25.3%)

156.9 ± 9.02 d

(-14.2%)

31.3 ± 1.81 b

(-14.0%)

14.0 ± 10.01 b

(-80.8%)

4 IV: Test groups CCl4 +ESEt

600mg/kg

171.0 ± 10.61 d

(-29.5%)

153.1 ± 11.92 d

(-16.2%)

30.6 ± 2.37 b

(-15.9%)

13.2 ± 3.45 b

(-81.9%)

5 V: Test groups CCl4 + ESEt

800mg/kg

134.5 ± 35.71 d

(-44.5%)

167.1 ± 2.31 b

(-8.63%)

33.4 ± 0.45

(-8.24%)

7 ± 1.99 b

(-90.42%)

6 VI: Test groups CCl4 +HSF

600mg/kg

154.9 ± 10.33 d

(-36.1%)

148.9 ± 15.91 d

(-15.3%)

29.7 ± 3.17 d

(-18.4%)

4.7 ± 1.35 d

(-93.5%)

85

Figure 24: Liver Histology of CCl4-Induced Hepatotoxic Model. A= CCl4 treated

group displayed fatty accumulation (ballooning), necrotic and inflamed cells around

abnormally enlarged center vein. These harmful signs are gradually decreased in test

groups administered with ESEt 600 and 800 mg/kg (C & D) and completely disappeared

in test group treated with HSF @ 600 mg/kg (E). However, ballooned and inflamed cells

were present in liver of silymarin (100 mg/kg) treated group (B).

B

C D

E

Dilated central vein

Necrosis & Ballooning

Inflammation

A

B

C

B

D

C

B

86

4. Discussion

The prevalence of liver diseases increases day by day globally. The major causes include

viruses and toxicants like environmental pollutants, occupational chemicals, drugs or xenobiotics,

which normally metabolize by liver to detoxify and get rid of the body. However during this,

some of these toxicants directly or few converts into more reactive compounds which can damage

the integrity of hepatocytes and that if ignore may leads to severe necrosis, inflammation, fibrosis

and eventually cirrhosis. Therefore, liver diseases are in top-twelve causes of death in the world

(Sarin & Maiwall 2016). Now-a-days, high and prolong use of analgesics like paracetamol

(PCM) and work related chemicals like carbon tetrachloride (CCl4) contributing a lot in this

regard and increasing the risk of hospitalization (McLachalan et al., 2011). This is the basic

theme of present work to overcome or reverse the harmful effects of PCM and CCl4 in liver by

using ethanolic seeds extract of Centratherum anthelminticum and its hexane soluble fraction.

4.1. Effect of ESEt of C. Anthelmenticum in PCM-Induced

Hepatotoxic Model

Paracetamol (PCM) is widely used analgesic and antipyretic drug which cheaply and

freely available in market without prescription in Pakistan, if its therapeutic dose use, no side

effects and no interaction with other drugs can be observed but when consume in toxic doses,

PCM develops a potent hepatotoxic substances that can induce fatal hepatic necrosis in animals

and humans (Hinson et al., 2010). Studies report that PCM in higher doses can induce acute

centrilobular hepatic necrosis which may leads to failure of liver function and death of

experimental animals and human in severe cases (Rehman & Hodgson., 2000). In the United

Kingdom (UK) and the United States (US), PCM is one of the main causes of acute liver

failure. However, other study in Asian populations reported a lower rate of the same health

problem (Marzilawati et al., 2012).

Normally, PCM is metabolized by drug metabolizing enzymes into water soluble

compounds and excreted in the urine. Whereas chronic high doses of PCM metabolize

oxidatively by hepatic CYP450 enzyme to N-acetyl-p-benzoquinone imine (NAPBQI), a

injurious compound usually detoxify via a reduced glutathione (GSH) which reported for both its

oxidant scavenger and redox-regulation capabilities thus prevent cell injury. High consumption of

PCM causes depletion of GSH and accumulation of NAPBQI that interacts covalently with the

87

sulfhydryl groups of cysteine in cellular proteins and make protein-NAPBQI complex. As a result

of which hydrogen peroxide (H2O2), superoxide anion (SO-) and hydroxyl (OH−) radicals

generate and alter the cellular membrane by diminishing the ATP levels, changing Ca++

homeostasis, lipid peroxidation and thereby inducing hepatic necrosis (Mehmood et al., 2014;

Rabiul et al., 2011). A study reports that excess NAPBQI causes initial hepatic damage and later

tumor necrosis factor-alpha (TNF-α), an inflammatory mediator contribute in tissue necrosis

(Mayyuren et al., 2010).

The PCM-induced hepatotoxic model is frequently used to evaluate the

hepatoprotective activity of medicinal plant extracts/compounds. PCM in a dose of 1 g/kg

was orally administered in experimental rats daily for successive 9 days in the present

investigation with the objective to induce hepatic dysfunction. Loss of appetite and body

weight is the prompt symptom of acute liver problem. The same was found in present

study by observing severe percent reduction in body weights of PCM-induced

hepatotoxic control rats. However, this was effectively appeared vice versa in three

separate PCM-induced hepatotoxic test groups which were treated with ESEt in doses of

200, 400 & 600 mg/kg respectively. Therefore, ESEt was found beneficial in protecting

the body weights of rats in test groups either by reducing the PCM induced oxidative

stress cell damage or lipid peroxidation thereby reducing tissue protein degradation (Li et

al., 2015). Another important sign of hepatoxicity is the elevation of liver related

enzymes viz., ALT, AST, GGT & ALP and bile pigment (bilirubin). ALT is a more

sensitive and better index of acute liver injury than AST (Botros & Sikaris, 2013).

However, increase in ALT and AST levels together provide more clear picture of liver

injury and if these are accompanied with increase in GGT and ALP levels, tells that bile

duct is also affected (Giannini et al., 2005). The same was observed in present study

where PCM administered hepato-toxic control group displayed severe high serum AST,

ALT, and ALP levels. On contrary, the level of these enzymes become decreased in all

three ESEt treated test groups even the highest dose 600 mg of same extract produced 95,

71 and 64% protection in ALT, AST and ALP levels in its test group whereas 97, 99 and

56 % protection was observed in these three liver-specific enzymes by silymarin in

positive control group. This finding indicates that ESEt is hepatoprotective in nature.

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Liver is the site where indirect bilirubin (IDBR) converts into direct bilirubin

(DBR) by conjugating with two molecules of diglucuronide to make water insoluble

IDBR into water soluble DBR. This conjugation reaction is only takes place in liver cells

(Bijlani & Manjunatha, 2010). Damage to liver cells will inhibit this process and elevate

the levels of total bilirubin especially IDBR in serum. Interestingly, the hepatoprotective

outcome of extract of C. anthelminticum was strengthened in present study by observing

the decreased levels of total bilirubin especially IDBR in all rats of three test groups

administered with ESEt (200-600mg) when compared with hepatotoxic control rats

which showed elevated levels of IDBR and total bilirubin. This indicates that conjugation

reaction resumed by inhibiting the hepatic necrosis through oral administration of ESEt in

test groups that’s why percent reduction in total bilirubin level was found as -50% in all

test groups whereas only -5% decreased in the same parameter was found in silyamrin

treated positive control group.

The hepatic parenchymal cells are responsible for synthesizing the most of the

serum proteins (albumin, α and β globulins, fibrinogen, coagulation factors, etc).

Decrease in total protein level is an indicator of liver damage especially contributed by

decrease in albumin level which 100% synthesized by liver cells. Study states that liver

injury causes disturbance and disassociation of polyribosomes on endoplasmic reticulum

thereby reducing the protein biosynthesis that was also showed in PCM-induced

hepatotoxic group. Whereas silymarin (100mg) and ESEt (200, 400 & 600 mg) treated

groups showed the restoring the normal levels of total protein especially by raising the

levels of albumin in their respective positive and tests groups by producing percent gain

from 19-25 % in total protein and 18.5-30% in albumin levels.This liver function

restoring property of ESEt was also evident by measuring reduced and/or normal liver

weights in all three test groups which were almost similar to the liver weights found in

normal control group. However, slightly increased liver weights were found in PCM

hepatotoxic control group which may be attributed to the accumulation of collagen and

extracellular matrix protein in liver tissue (Mehmood et al., 2014). Besides this, other

study indicated that impaired liver growth and organs function may be due to blocking in

secretion of hepatic triglyceride into plasma (Aniya et al., 2005).

89

The uric acid is the excretory yield of purine metabolism, its increase level in serum

reflects the cell damage including the liver and kidney cells. The cell defensive result of ESEt of

C.anthelminticum was also supported by monitoring the decrease in serum uric acid levels in all

three test groups and showing nice percent reduction in uric acid level especially the highest dose

(600mg) of ESEt was found more effective (-13%) by comparing with PCM hepatotoxic group.

Pharmacologically 70% methanolic seeds extract of same plant has also been accounted for

providing protection against nephrotoxicity (Galani & Panchal, 2014).

Liver plays a principal role in hematopoiesis and biosynthesis of coagulation

factors and similarly many hematological abnormalities are found associated with a wide

range of liver disease (Madkour & Abdel-Daim, 2013). Hence in current study, a

significant reduction in hematological parameters (Hb, RBC, and HCT levels) was found

in hepatotoxic rats by comparing with control. This found compatible with the studies

that showed that the chronic doses of PCM induced mature RBCs’ destruction, reduction

in erythropoiesis rate by inhibiting the release of erythropoietin from the kidney. As a

result of which, the oxygen-carrying ability of blood become declined and fewer amount

of oxygen delivered to tissues thereby lowering the Hb concentration and producing

slight anemic condition in animals and human. However, this unusual situation was

completely reverse in all three test groups treated with ESEt from low to high doses

whereas no significant decrease was found in WBCs level in test groups. Toxic

paracetamol metabolites (NAPBQI) also reported to alter platelet function and existence

which causes the thermbocytopenia (Miyakawa et al., 2015) that also affects aspartate

transaminase and platelet ratio index (APRI). APRI is a useful indicator to predict

significant inflammation/ fibrosis as well as cirrhosis. Study showed that when APRI >

1.5, it represents the moderate to severe fibrosis (Shaheen & Meyers, 2007). The almost

same was observed in PCM intoxicated rats that showed high APRI no doubt not above

1.5 but as compared to test rats of groups IV to VI treated with ESEt (200-600mg) which

displayed APRI much less than intoxicated rats. This finding also supports the

hepatoprotective potential of ESEt in normalizing the liver function.

Chronic doses of PCM induced oxidative stress that suppresses the action of antioxidant

proteins and enzymes in body. Study reports that decrease in antioxidant enzymes activities is a

sensitive index of hepatocellular damage (Adam et al., 2016). In antioxidant guard system, SOD

90

is one of the most imperative enzymes. It hunts superoxide (O2−) anion and converts it to H2O2

hydrogen peroxide, which further hydrolyzes by CAT into water and oxygen, and thus diminishes

the free radical oxidative damage effect thereby protecting the body tissues from highly reactive

hydroxyl radicals. Similarly, GSH, a non-enzymatic biological antioxidant in liver, eliminates

ROS species (hydrogen peroxide) and maintains membrane protein thiol and integrity. Decrease

level of GSH is related to an enhance lipid peroxidation in PCM-treated rats (Kannan et al.,

2013). LPO is actually altering the membrane lipid due to ROS that induce destruction and cell

membrane damage, disturbance in membrane permeability, fluidity, enhancement in protein

degradation that leads to cell death (Singh et.,al 2015). The same was found in PCM intoxicated

rats that demonstrated increased percent hang-up of CAT, SOD, GSH while low for LPO.

Whereas ESEt treated test groups displayed low percent inhibition of CAT, SOD, GSH and high

percent inhibition of LPO which is another good sign of ESEt of C.anthelminticum by showing

its free radical scavenging property.

Literature states that high intake of PCM associated with centriloular necrosis in liver

depending upon spreading of damage and time. Cross section of liver tissues treated with PCM

reflects the destruction of architectural pattern, necrotic foci with intense inflammation by

eosinophilic invasion in the cytoplasm of hepatocytes that result in cell membrane irregularity.

Irregular cordon, intense erythrocyte aggregation, dilated sinusoids, congestion in the vascular

structures in central vein and portal region plus lipid droplets are also reported in other studies. In

the present study, not all adverse effects normally induced by PCM were observed in liver tissues

of PCM intoxicated rats but dilated central vein and inflammation due to mononuclear leucocytes

infiltration were appeared. However, these adverse features of PCM intake was gradually

subsides in test groups pretreated with ESEt in dose dependent manner by observing much better

or almost normal structure of liver tissues of test groups. This clearly confirms the betterment

observed in all liver-specific biochemical parameters in present study and proves the liver saving

ability of ESEt of C.anthelminticum. The hepatoprotective potential of ESEt might be resides in

its high phenolic content which has been known to contribute to the antioxidant activity (Yahya et

al., 2013). Moreover, it’s already reported steroid content with anti-inflammatory activity

(Hanafy et al., 2016) also play a significant role in this regard.

91

4.2. Effect of ESEt of C. Anthelmenticum in Carbon Tetrachloride

(CCl4)-Induced Hepatotoxic Model

CCl4 intoxicated model is another model used commonly in scientific research to

evaluate hepatoprotective effect of medicinal plant extracts or compounds (Malaguarnera

et al., 2012). CCl4 is a classic most potent hepatotoxic solvent used as cleaning agent in

leather industry, inhalation of its fumes severely damage body cells especially causes

acute hepatic damage (Bates et al., 2001). The CCl4 hepatotoxicity mediated by

production of its toxic radicals viz., trichloromethyl & peroxytrichloromethyl by

CYP3E1, binds with lipoprotein and leads to the lipid peroxidation thus alters the ions

exchange through cell membrane, results in enzyme escape in blood that leads

inflammation, cell degradation and death of cell (Dongare et al., 2013). It is well proven

that continuous exposure to CCl4, a pro-fibrogenic agent develop the hepatic fibrosis and

cell necrosis. Furthermore, in CCl4-induced advanced liver fibrosis, a gradually

deteriorating anisonucleosis (variation in the size of the hepatocyte nuclei) has been

reported related with hepatic oxidative stress (Uehara et al., 2014).

CCl4 hepatotoxicity is characterized by drastic increase of ALT, AST, ALP, total

bilirubin especially indirect bilirubin, uric acid and decrease in total protein especially

contributed by low levels of albumin plus accompanied with decrease intake of food. All

these happen due to severe hepatic necrosis, inflammation and steatosis (fatty deposits)

induced by CCl4. All these symptoms are evident one by one in CCl4 intoxicated rats like

severe percent reduction was found in body weights of rats which were only treated with

CCl4 (3ml/kg). This represents rupturing of body tissues proteins or cells. However, this

reduction was notably decreased in silymarin (100mg), ESEt (400, 600, 800mg) and

hexane soluble fraction (HSF; 600mg) of ESEt pre-treated test rats /groups by showing

protection in this physical parameter from 35 to 80%. The immediate indicators of liver

damage is the serum elevation of ALT & AST thrice than their upper limit, ALP and total

bilirubin especially the indirect one more than twice of their original values. Researches

divide liver damage into hepatocellular, characterized by increase in ALT and cholestatic,

that characterized by rise in ALP types (Giannini et al., 2005). The same was displayed

by CCl4 hepatotoxic group where serum elevation of ALT, AST and ALP was found

92

above quadrate higher than their values found in normal control group which was the

neat and clean resonance of change in cell membrane (Bishop et al., 2013). On the other

hand, the levels of all these hepatic markers become much lowered in all groups pre-

administered with silymarin, ESEt and HSF by showing 95% protection in ALT, from 65

to 81 % in AST and from 37 to 100% in ALP levels which is the good sign of

hepatoprotective action of ESEt and HSF by reversing the cellular intactness. While GGT

levels remain low in these test and positive control groups which normally represents the

intrahepatic cholestasis and low production of bile acid (Arias, 2009).

The promising involvement of ESEt and HSF in rejuvenating liver was also confirmed by

monitoring the usual levels of total bilirubin both direct and indirect, total protein, and albumin in

all test groups by comparing with CCl4-induced hepatotoxic control group where completely

opposite picture was observed about these parameters. Rise in serum total bilirubin particularly

the indirect one is the prompt reflection of liver dysfunction, impairment in conjugation, binding

and excretory abilities of hepatocytes (Boyer., 2013) . Studies show that CCl4 exposure leads to

damage in Golgi bodies which harmfully disturbs the protein packaging, release from the

hepatocytes and ofcourse the biosynthesis of albumin. Polysomes serve as protein synthesizing

factories bound to the endoplasmic reticulum losses the ability to synthesize albumin in liver due

to CCl4 induction (Amer et al., 2015). On contrary, betterment in all these hepatic abilities was

found in all test groups by showing increase from 37 to 59% in total protein, 41 to 56 % in

albumin levels and decrease from -37 to -56% in total bilirubin contributed by -73% decrease in

indirect bilirubin. Enlargement in liver weights and increment in liver index were found in CCl4

treated control rats. It might be due to acute extreme mitosis taking place in hepatocytes as it was

reported to happens normally whenever total protein levels declines or accumulation of

triglycerides, called hepatomegaly (Bishop et al., 2013) . While decrease or normal liver weights

and indices was found in all ESEt and HSF pre-treated test groups which confirms the

improvement of liver function. Interestingly, all liver regenerating signs of extract found in the

present study also compatible to the former in vitro study stated the inhibition of tumor necrosis

factor alpha (TNF-α) in human tumor cells by chloroform fraction of seeds of C. anthelminticum

(Arya et al., 2012a). Inhibition of cell degradation by ESEt and HSF was also supported by

monitoring decrease in uric acid levels in all tests by comparing with its high levels observed in

CCl4 intoxicated group. High uric acid level is directly proportional to body cell damage (Mbarki

et al., 2017).

93

Hyperlipidemia is inversely proportional to the degree of cirrhosis (Chrostek et al.,

2017). An elevation in the concentration of unsaturated fatty acid lipoperoxide and free peroxide

radical can alter the cholesterol profile (Khan et al., 2012). Similarly, CCl4 induced toxicity was

also associated with fat accumulation in the liver and overproduction of TG-enriched VLDL

particles results in serum elevated levels of VLDL and TG. Normally, 80%, cholesterol is

synthesized in liver. After intoxication with CCl4, movement of acetate increases into the liver

that in turn also accelerates the synthesis of cholesterol (Lien et al., 2017). In the present study, a

marked elevation in serum TG, TC, VLDL-c and LDL-c levels was found in CCl4-intoxicated

rats, in contrast with control rats. Pretreatment with ESEt and HSF resulted in decrease in lipid

profile parameters in test groups that could be due to inhibiting cholesterol & triglycerides

synthesis, interfering in lipoprotein production, increasing expression and function of hepatic

LDL receptor that causes an increase LDL-c removal from blood. Few of these possible

mechanism of lipid reducing action of ESEt of C.anthelminticum is approved by previous studies

on same extract which reported the inhibition of beta hydroxy beta methyl glutaryl Co A

reductase, the rate limiting enzyme of cholesterol biosynthesis and acceleration of TG degrading

enzyme lipase in high-fat induced hyperlipidemic rabbits and fructose-induced type 2 diabetic rat

models (Mudassir & Quershi, 2015; Lateef & Quershi, 2014).

Once again oxidative stress reducing activity of ESEt of C.anthelminticum observed in

CCl4 induced hepatotoxic model as same as it was found in PCM-induced hepatotoxic model in

the first phase of present study. It has been reported that CCl4 after passing through liver

cytochrome P450 oxidase generate oxidative stress by producing two trichloromethyl radicals

which severely accelerate lipid peroxidation (LPO) and alter the cell membrane permeability and

mitochondrial function. Study described that LPO stimulated with the removal of a hydrogen

atom from the double bond of unsaturated fatty acids and generate toxic radicals involved in

hepatic injury (Singh et al., 2014). In the same way, prominent oxidative stress induced by CCl4

was observed in hepatotoxic control group that displayed high LPO and low CAT, SOD and GSH

functional levels. However, ESEt and HSF of C. anthelminticum in their respective test groups

provided completely opposite picture by inhibiting LPO and accelerating the functions of CAT,

SOD and GSH by showing their low percent inhibition. It might possible that flavonoids,

polyphenols and steroids (Karuna et al., 2009; Quershi et al., 2016),which are already reported in

ESEt, on one hand responsible to scavenge free radicals either by inhibiting the enzymes involved

in their synthesis (xanthine oxidase, lipooxygenase,NADPH oxidase,aldose reductase) or by

breaking the reaction sequence of LPO.

94

All the bad effects induced by CCl4 including inflammation, necrosis, bollooning (fatty

deposites), dilated central vein, rupturing intralobular septum were found on their peak in liver

tissues dissected out from hepatotoxic groups. Whereas all these features were gradually

disappeared in liver tissues of test groups treated with ESEt 600 & 800mg and completely

disappeared in liver tissue of test group treated with HSF by surprisingly restoring the normal

structure of liver. Therefore, provide full evidence that ESEt of C.anthelminticum especially its

HSF is hepatoprotective in character.

In conclusion, hepatoprotective potential of ESEt and HSF of C.anthelminticum

investigated and proved for the first time in this present study.

95

5. Conclusion

The present study has proved that ethanolic seeds extract (ESEt) of C.anthelminticum has

hepatoprotective action against paracetamol (PCM) and carbon tetrachloride (CCl4)-induced

hepatotoxic rats models by minimizing the reduction in body weight loss and normalizing other

physical (liver weights, liver index) and biochemical parameters that not only reflect the altered

hepatic & biliary cellular integrity (ALT, GGT, ALP, AST, UA) but also functionality of liver

(TP, ALB, TBR, DBR, IDBR, TG, TC, VLDL-c, LDL-c & HDL-c). In addition, the same extract

was found beneficial in reducing oxidative stress (LPO) and upgrading the functions of

antioxidant proteins (GSH) and enzymes (SOD & CAT). Similarly, hematological parameters

(Hb, RBC, WBC, HCT, AST / APR index) were also improved in PCM induced hepatotoxic

model by ESEt. Interestingly, the hexane soluble fraction (HSF) of ESEt was found much better

in improving all these parameters in CCl4 induced hepatotoxic model plus showed best liver

regenerative ability in the same model.

The hepatoprotective, antioxidant and anti-inflammatory activities of ESEt may be due to

the presences of flavonoids, polyphenols and steroids which are already reported in the same

extract. Whereas fatty acids, hydrocarbons and waxes, which are possibly present in HSF can also

contribute in this regard and especially restoring the liver architecture successfully. Therefore,

seeds extract of C.anthelminticum could be used as a substitute of existing available medicine in

the treatment of liver problems. In addition, these extracts could serves as a source of active

principle that can be isolated and used in the formulation of future hepatoprotective medicine.

96

6. Future Extension of the Present Research Work

The present study first time proves that ethanolic seeds extract (ESEt) of

C.anthelminticum and its hexane soluble fraction (HSF) are antioxidant and hepatoprotective in

nature in paracetamol (PCM) and carbon tetrachloride (CCl4)-induced hepatotoxic rats model

plus show powerful liver regenerative property especially in CCl4-induced hepatotoxic model.

Therefore the following suggestions can be considered as future aspects of this research.

1. Further fractionation of ESEt should be done by using organic solvents of different

polarities and each fraction like HSF should be analyzed for hepatoprotective and

antioxidant effects in experimentally induced hepatotoxic models.

2. Each prepared organic solvent fraction of ESEt plus HSF used in this study should be

subjected to GC-MS and other spectrophometeric techniques to detect and isolate the

possible chemical compound (s) responsible for hepato-protective activity of this extract

that could be used in the preparation of medicine for the liver problem in future.

3. The evaluation of ESEt and its HSF should be done against tumor necrosis factor-α

(TNF-α) and IL-6 which are involved in degeneration and regeneration of liver tissues

respectively to confirm its importance in the future development of new hepatoprotective

medicine.

4. Hepatoprotective activity of seeds extracts of C.anthelminticum should be evaluated in

human volunteers having liver problems in order to confirm its use in herabal / alternative

/ integrated medicines.

97

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115

PUBLICATIONS

Publication from Thesis

Qureshi SA, Sumera Rais, Usmani R, Zaidi SS, Jehan M, Lateef T and Azmi

MB (2016). Centratherum anthelminticum seeds reverse the carbon

tetrachloride-induced hepatotoxicity in rats. African Journal of Pharmacy and

Pharmacology, 10(26): 533-539.

Publication Other than Thesis

Azmi MB, Qureshi SA, Sumera Rais, Sultana S. (2015). Methanolic root extract

of Rauwolfia serpentina lowers atherogenic dyslipidemia, arteriosclerosis and

glycosylation indices in type 1 diabetic mice. Journal of Applied Pharmaceutical

Science, 5(8): 61-67.

116

PRESENTATIONS

Poster Presentation from Thesis

Poster presentation on “ Centratherum anthelminticum (kali zeri) minimize the

risk of chemically-induced hepatotoxicty in rats” in International Conference on

“Translational Medicine from Discovery to Health Care,” organized by Ziauddin

University Pakistan,1-3rd Feb , 2016

Poster presentation on “ Centratherum antheminticum ameliorates liver function

in paracetamol-induced hepatotoxic rats” in symposium on World Diabtes Day

“Role of Ethanopharmcology in Metabolic Disorders” orgainzed by

Phytopharmacology and Biotechnology Research Laboratory, Department of

Biochemistry, University of Karachi,18th Nov,2014

Poster Presentation Other Than Thesis

E-paper presentation on “Improvement in antiatherogenic index by Centratherum

anthelminticum” in Golden Jubilee Symposium, organized by Jinnah Post

Graduate Medical Centre, Karachi,23rd -29th March, 2014

Poster presentation on “Centratherum athelminticum ameliorates

cardioprotective indices in hyperlipidemic rabbits” in 1st FUUAST Science

Symposium, organized by Department of Biochemistry, Federal Urdu University

of Arts, Science and Technology , Karachi, 20 th Feb, 2014

Documents

INVESTIGATIONS ON HEPATOPROTECTIVE POTENTIAL OF