Rajadurai et al eISSN 2278-1145 International Journal of ... · Rajadurai et al eISSN 2278-1145 1 Int. J. Int sci. Inn. Tech. Sec. B, Aug. 2014, Vol.3, Iss 4, pg 01-11 International

Rajadurai et al eISSN 2278-1145

1

Int. J. Int sci. Inn. Tech. Sec. B, Aug. 2014, Vol.3, Iss 4, pg 01-11

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

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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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6


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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8



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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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Rajadurai et al eISSN 2278-1145 International Journal of ... · Rajadurai et al eISSN 2278-1145 1 Int. J. Int sci. Inn. Tech. Sec. B, Aug. 2014, Vol.3, Iss 4, pg 01-11 International