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International Journal of Integrative sciences, Innovation and Technology (A Peer Review E-3 Journal of Science Innovation Technology)
Section A – Basic Sciences; Section B –Applied and Technological Sciences; Section C – Allied Sciences
Available online at www.ijiit.net
Research Article
HEPATOPROTECTIVE EFFECT OF Β-SITOSTEROL ON LIPID PEROXIDATION
AND ANTIOXIDANT STATUS IN ETHANOL-INDUCED HEPATOTOXIC RATS
S. M. REXLIN SUJILA1, M. RAJADURAI*, S. M. REXLIN SHAIRIBHA
1
1Faculty of Science, Centre for Biosciences, Department of Biochemistry, Muthayammal College of Arts & Science,
Rasipuram-637 408, Namakkal.
*Sri Venkateshwaraa College of Arts & Science, Dharmapuri- 636 809, Tamil Nadu, India.
email: [email protected]
ABSTRACT Liver is the key organ regulating homeostasis in the body. It plays a key role in food processing and in the process of
detoxification. The present study was designed to investigate the effect of β-sitosterol on lipid peroxidative products
such as thiobarbituric acid reactive substance (TBARS) and lipid hydroperoxide and antioxidant status in ethanol-
induced hepatotoxic rats. Ethanol induction (3 g/kg) caused toxicity to liver that lead to various physiological and
biochemical alterations. Albino Wistar rats were divided in to 5 groups, β-sitosterol was suspended in carboxy
methyl cellulose and administered to rats orally at the dosage of 20 and 40 mg/kg/day for 35 days. Hepatotoxic rats
had elevated levels of TBARS and hydroperoxide and decreased antioxidant activities (both enzymatic and non-
enzymatic) when compared with normal control rats. Treatment with β-sitosterol shows the decreased level of lipid
peroxides and increased levels of antioxidants in ethanol-induced hepatotoxic rats. These results demonstrated that
β-sitosterol protected liver damage and it may have beneficial properties in the prevention of liver toxicity.
KEYWORDS: Hepatotoxic, lipid peroxidation, antioxidant, ethanol, β-sitosterol
INTRODUCTION Liver is one of the largest organs in human body and
the chief site for intense metabolism and excretion
which play an important role in the maintenance,
performance and regulating homeostasis of the body.
It is involved with almost all the biochemical
pathways to growth, fight against disease, nutrient
supply, energy provision and reproduction (Nasir, et
al., 2013). The major functions of the liver are
carbohydrate, protein and fat metabolism,
detoxification, secretion of bile and storage of
vitamin. Thus, to maintain a healthy liver is a crucial
factor for overall health and well being. Liver is
continuously and variedly exposed to environmental
toxins and abused by poor drug habits and alcohol
which can eventually lead to various liver ailment
like hepatitis, cirrhosis and alcoholic liver disease
(Noorani, et al., 2010).
Alcohol-induced liver injury is one of the world’s
major health problems and excessive alcohol
consumption caused several fetal diseases, which are
evidenced by considerable experimental and clinical
studies (Castilla, et al., 2004). Alcohol was absorbed
through the mucous membrane of the oesophagus and
stomach, and it metabolized entirely in the liver
(Zhang, et al., 2008). When alcohol was metabolized
in liver, it gives rise to oxidative stress by the
generation of excess amounts of reactive oxygen
species (ROS), and further affected antioxidant
defence system (Ozaras, et al., 2003). 80-90% of
alcohol breakdown in liver metabolism in the cells
leads to ROS production (Pronko, et al., 2002).
Phytochemicals are a large group of plant-derived
compounds hypothesized to be responsible for much
of the disease protection conferred from diets high in
fruits, vegetables, beans, cereals and plant-based
beverages such as tea and wine. Phytochemicals are
classified in to alkaloids, glycosides, flavonoids,
phenolics, saponins, tannins, terpenes,
anthraquinones and steroids (Arts, and Hollman,
2005). Plant steroids (or steroid glycosides) also
referred to as ‘cardiac glycosides’ are one of the most
naturally occurring plant phytoconstituents that have
found therapeutic applications as cardiac drugs (Firn,
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2010). Ergosterol, stigmasterol and β-sitosterol are
three important sterols in fungal and plant samples. β-
sitosterol (24-ethyl-5-cholestene-3-ol), a well-known
plant sterol (Fig 1), has been reported to reduce
serum cholesterol levels and to prevent
cardiovascular events mainly by inhibition of
cholesterol absorption in the intestines (Pouteau, et
al., 2003). β-sitosterol is also known to regulate key
molecules involved in inflammation, the immune
response, anti-cancer defenses and apoptosis. In
addition, the plasma β-sitosterol concentration was
found to be significantly reduced in type 2 diabetes
patients, suggesting a possible role of β-sitosterol in
lowering the blood glucose level (Awad, and Fink,
2000). The aim of the present study is to investigate
the hepatoprotective effect of β-sitosterol on lipid
peroxidation and antioxidant status in ethanol-
induced hepatotoxic rats.
MATERIALS AND METHODS
Experimental Animals
All the experiments were done with male albino
Wistar rats weighing 150-200 g, obtained from the
Venkateswara Enterprises, Bangalore. They were
housed in polypropylene cages (47x34x20 cm) lined
with husk, renewed every 24 hours under 12:12 hour
light/dark cycle at around 22˚ ± 2˚C and had free
access to water and food. The rats were fed on a
standard pellet diet (Pranav Agro Industries Ltd.,
Maharashtra, India). The pellet diet consisted of
22.02% crude protein, 4.25% crude oil, 3.02% crude
fibre, 7.5% ash, 1.38% sand silica, 0.8% calcium,
0.6% phosphorus, 2.46% glucose, 1.8% vitamins and
56.17% nitrogen free extract (carbohydrates). The
diet provides metabolisable energy of 3,600 kcal. The
experiment was approved by Institutional Animal
Ethics Committee (IAEC) of Muthayammal College
of Arts & Science, Rasipuram, Tamilnadu, India,
(Clearance No: 1416/Po/a/11CPSCEA & 7 March
2011) and carried out in according with the
guidelines of the Committee for the Purpose of
Control and Supervision of Experiments on Animals
(CPCSEA), New Delhi, India.
Drugs and Chemicals
β-Sitosterol was purchased from Sigma Chemical
Company, St. Louis, MO, USA. Ethanol was
purchased from Changshu Yangyuan Chemicals Pvt.
Ltd., China. Thiobarbituric acid (TBA), 1, 1’ ,3 , 3’
tetramethoxy propane, butylated hydroxy toluene
(BHT), xylenol orange, dithionitro bis benzoic acid
(DTNB), ascorbic acid, 2, 2’ dipyridyl, p-phenylene
diamine and sodium azide were obtained from
Himedia laboratory, Mumbai, India. All other
chemicals were obtained under analytical grade.
Experimental Induction of hepatotoxicity Ethanol (3 gm/kg) was dissolved in water and
injected intragastrically, for a period of 35 days
(Rajat, and Gill, 1999). β-sitosterol (20 and 40
mg/kg) was suspended in carboxy methyl cellulose
(CMC) and administered to rats orally using an
intragastric tube daily for a period of 35 days.
Experimental Design
In this experiment, a total of 30 rats (18 toxicity
surviving rats, 12 control rats) were used. The rats
were divided into 5 groups of 6 rats in each.
Group 1: Normal control rats
Group 2: Normal rats treated with β-
sitosterol (40 mg/kg)
Group 3: Ethanol control rats
Group 4: β-sitosterol (20 mg/kg) + Ethanol
Group 5: β-sitosterol (40 mg/kg) + Ethanol
After the last treatment, all the rats were sacrificed by
cervical decapitation. Plasma was separated from
blood after centrifugation. The tissues (liver and
kidney) were excised immediately from the rats,
washed off blood with ice-cold physiological saline.
A known weight of the liver and kidney were
homogenized in appropriate buffer solution. The
homogenate was centrifuged and the supernatant was
used for the estimation of various biochemical
parameters.
Biochemical estimations
The level of plasma TBARS was estimated by the
method Yagi (1987), and the tissue TBARS was
estimated by the method of Fraga et al. (1988). The
levels of lipid hydroperoxides (HP) were estimated
by the method of Jiang et al. (1992). The activity of
SOD, catalase and GPx was assayed according to the
procedures of Kakkar et al. (1984), Sinha (1972) and
Rotruck et al. (1973) respectively. The level of
ceruloplasmin was estimated by the method of Ravin
(1961). The level of GSH was estimated by the
method of Ellman (1959). The level of vitamin C
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and E was estimated by the methods of Omaye et al.
(1979) and Baker et al. (1980) respectively.
Statistical analysis
Statistical analysis was performed by one way
analysis of variance (ANOVA) followed by
Duncan’s Multiple Range Test (DMRT) using
Statistical Package for the Social Sciences (SPSS)
software package version 16.00. P values <0.05 were
considered significant.
RESULTS
Effect of β-sitosterol on lipid peroxidation in
plasma and tissues The levels of lipid peroxidative products (TBARS
and hydroperoxides) in plasma and tissue of normal
and ethanol-induced rats were presented in Figs 2, 3,
4 & 5. Ethanol-induced rats show the elevated levels
of TBARS and hydroperoxides in plasma and tissue.
Treatment with β-sitosterol in ethanol-induced rats
showed reversal of these parameters to near normal
levels.
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Effect of β-sitosterol on antioxidants in serum and
tissues The activities of enzymatic antioxidants such as
superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GPx) in the liver and kidney
of normal and ethanol-induced rats are shown in Figs
6, 7 & 8. Ethanol-induced rats showed significant
reduction in the activity of SOD, CAT and GPx. Oral
administration of β-sitosterol exerted a significant
effect on the antioxidants in ethanol-induced rats.
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The levels of non-enzymatic antioxidants such as
reduced glutathione (GSH), ceruloplasmin, vitamin C
and vitamin E in the plasma, liver and kidney of
normal and ethanol-induced rats are shown in Figs 9,
10, 11, 12, 13 & 14. The level of ceruloplasmin,
GSH, vitamin C and E was found to be significantly
reduced in serum, liver and kidney of hepatotoxic
rats. Oral administration of β-sitosterol significantly
increased the levels of ceruloplasmin, GSH, vitamin
C and E in ethanol-induced rats.
For all the parameters, β-sitosterol treatment at 40
mg/kg to ethanol-induced hepatotoxic rats showed
better effect than β-sitosterol 20 mg/kg. β-sitosterol
treatment (40 mg/kg) to normal rats didn’t show any
significant effect.
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DISCUSSION Liver is an important organ actively involved in
several metabolic functions mainly in detoxification
of toxicants (Meyer, and Kulkarni, 2001). Chronic
alcohol provokes successive hepatic changes,
consisting of alcoholic fibrosis, alcoholic hepatitis
and cirrhosis (Kai, 1995). Increasing evidence
support the hypothesis that ethanol-induced tissue
damage may be a consequence of oxidative stress and
nutritional deficiencies (Anbu, and Anuradha, 2012).
In liver, ethanol is metabolized to highly toxic
metabolite acetaldehyde that interacts with the
cellular macromolecules (lipids and proteins) and
leads to the damage of membrane lipids besides
altering the enzyme activities (Negre, et al., 1999).
Alcohol dehydrogenase (ADH) leads to the formation
of acetaldehyde from ethanol with the simultaneous
reduction of NADP to NADH which in turn elevates
xanthine oxidase activity and leads to the enhanced
production of superoxide (Lieber, 2005).
Increased lipid peroxidation associated with chronic
ethanol administration has often been used as an
indicator of oxidative stress in both animal models
and human clinical trials (Nordmann, 1994). Ethanol-
induced rats showed increased plasma and tissue
levels of lipid peroxidation markers such as TBARS
and lipid hydroperoxides. The increased peroxidation
can result in changes in cellular metabolism of the
hepatic and extrahepatic tissues. Increased
accumulation of lipid peroxidation products in cells
can result in cellular dehydration, whole cell
deformity and cell death (Winrow, et al., 1993).
β-sitosterol (20 and 40 mg/kg) co-administered rats
for a period of 35 days showed significantly lowered
levels of these lipid peroxidative markers compared
to ethanol-induced rats. Decreased lipid peroxidation
with β-sitosterol administration suggests a decreased
impact of reactive oxygen species (ROS) on lipid
membranes, and therefore increased protection
against ethanol-induced liver injury. Thus the
inhibition of lipid peroxidation by β-sitosterol may be
one of the mechanisms by which β-sitosterol exerts
its protection against ethanol-mediated tissue injury.
Oxidative stress results from a disturbance in the
balance between generated oxidants and antioxidants
in favour of the oxidants. This is often caused by an
increase in the generation of ROS and a decrease in
the activity of antioxidant system (Postmal, et al.,
1996). According to Wu and Cederbaum (2003),
chronic alcohol consumption not only activates free
radical generation, but also alters the levels of both
enzymatic and non-enzymatic endogenous
antioxidant systems. This results in oxidative stress
with cascade of effects, thus, affecting both
functional and structural integrity of cell and
organelle membranes (De leve, et al., 1996).
Free radical scavenging enzymes such as superoxide
dismutase (SOD), catalase (CAT) and glutathione
peroxidase (GPx) are the first line of defense against
oxidative injury. The second line of defense consists
of the non-enzymatic scavengers, such as GSH,
ascorbic acid (vitamin C) and α-tocopherol (vitamin
E), which scavenge residual free radicals escaping
decomposition by the antioxidant enzymes.
Moreover, enzymatic antioxidants are inactivated by
the excessive levels of free radicals and hence the
presence of non-enzymatic antioxidants is
presumably essential for the removal of these radicals
(Allen, 1991).
Decrease in enzyme activity of SOD is a sensitive
index in hepatocellular damage and is the most
sensitive enzymatic index in live injury. It scavenges
the superoxide anion to form hydrogen peroxide and
thus diminishing the toxic effect caused by radicals.
Decrease in SOD production can be attributed to an
enhanced superoxide generation and utilization of
this enzyme during reactive metabolites
detoxification. High amounts of superoxide inhibit
catalase, which is another important antioxidant
enzyme (Flora, 2002). CAT decomposes hydrogen
peroxide and protects the tissues from highly reactive
hydroxyl radicals (Chance, and Greenstein, 1992).
Therefore reduction in the activity of CAT may result
in a number of deleterious effects due to the
assimilation of superoxide radical and hydrogen
peroxide. GPx works in tandem with CAT to
scavenge excess H2O2 as well as other free radicals in
response to oxidative stress. The equilibrium between
these enzymes is important for the effective removal
of oxidative stress in intracellular organelles. The
sulphydryl group is subjected to modification, which
is invariably affecting the activity of the enzyme.
This antioxidant defense system is significantly
altered by ethanol administration (Veerappan, et al.,
2004).
Ethanol-induced rats showed the decreased activities
of these enzymes; result in the accumulation of
highly reactive free radicals, leading to deleterious
effects such as loss of cell membrane integrity and
membrane function (Krishnakantha, and Lokesh,
1993). Treatment with β-sitosterol significantly
increases the hepatic and renal SOD, catalase and
GPx activity and thus reduces reactive free radical
induced oxidative damage to liver.
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Glutathione is one of the most abundant tripeptide,
non-enzymatic biological antioxidant present in the
liver. It removes free radical species such as
hydrogen peroxide, superoxide radicals and
maintains membrane protein thiols. Also it is a
substrate for glutathione peroxidase (Prakash, et al.,
2001), decreased level of GSH is associated with an
enhanced lipid peroxidation in ethanol-induced rats.
Vitamin C and E both are naturally occurring free
radical scavengers (Yu, 1994). Both vitamin C and E
are known to be decreased in liver diseases,
particularly in alcoholics (Bjorneboe, et al., 1987).
Under these conditions, thiol compounds, such as
GSH, might be involved in regenerating α-tocopherol
from its radical form (Wefers, and Sies, 1988). The
observed decrease in the levels of vitamin C and E
may be due to their increased utilization for
scavenging ethanol- and/or oxygen-derived radicals.
Administration of β-sitosterol in ethanol-induced rats
significantly increased the levels of these non-
enzymatic antioxidants. The ability of β-sitosterol
might be potentially useful in countering free radical-
mediated injuries involved in the development of
liver damage caused by alcohol abuse.
β-sitosterol acts as a competitive inhibitor of
cholesterol absorption. Hence administration of β-
sitosterol decreases the levels of cholesterol and other
lipids in ethanol-induced rats which is one of the
indirect effects of β-sitosterol on the lipid
peroxidative products as well as antioxidants.
Additionally β-sitosterol itself having strong
antioxidant action (Yoshida, and Niki, 2003) as well
as free radical scavenging activity (Backhouse, et al.,
2008). Thus the free radical scavenging antioxidant
and cholesterol lowering property are collectively
responsible for the hepatoprotective action in
ethanol-induced rats. However further clinical
evaluation is required for β-sitosterol to become a
drug for alcoholism.
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