INVESTIGATIONS ON HEPATOPROTECTIVE POTENTIAL OF ...
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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
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2017
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,
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Pakistan
2017
INVESTIGATIONS ON HEPATOPROTECTIVE POTENTIAL
OF CENTRATHERUM ANTHELMINTICUM IN
PARACETAMOL AND CARBON TETRACHLORIDE (CCl4)-
INDUCED
LIVER INJURY
Thesis submitted for the fulfillment of the degree of
DOCTOR OF PHILOSOPHY
By
Sumera Rais Abbasi
Department of Biochemistry
University of Karachi
Karachi-75270,
Pakistan
2017
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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: __________________
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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
Department of Biochemistry
University of Karachi
Dated: __________________________
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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.
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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
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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
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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-
Induced Hepatotoxic Rats 61
Figure 10 Outcome of ESEt on Hemoglobin in PCM-Induced Hepatotoxic
Rats
62
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Figure 11 Outcome of ESEt on AST/PLT Ratio Index (APRI) in PCM-
Induced Hepatotoxic Rats 63
Figure 12 Outcome of ESEt on Percent Inhibition of CAT and SOD in PCM-
Induced Hepatotoxic Rats 66
Figure 13 Outcome of ESEt on Percent Inhibition of GSH and LPO in PCM-
Induced Hepatotoxic Rats 67
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-
Induced Hepatotoxic Rats 73
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-
Induced Hepatotoxic Rats 78
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
CCl4-Induced Hepatotoxic Rats 82
Figure 23 Outcome of ESEt and HSF on Inhibition of GSH and LPO in
CCl4-Induced Hepatotoxic Rats 83
Figure 24 Liver Histology of CCl4-Induced Hepatotoxic Model 85
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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
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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
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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
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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).
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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
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Centratherum Anthelminticum
Whole Plant Seeds
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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
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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).
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Figure A: Liver Structure
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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
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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).
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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).
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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.
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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).
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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
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
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
23
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
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
Reagent blank Standard Sample
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
Reagent blank Standard Sample
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
Reagents Test Control
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
Reagents Test Control
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
Reagents Test Control
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
Each bar stands for mean (n=6).
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
Each bar stands for mean (n=6).
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
Each bar stands for mean (n=6).
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
Each bar stands for mean (n=6).
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
Each bar stands for mean (n=6).
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
Each bar stands for mean (n=6).
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).
81
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
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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