RGUHS J Pharm Sci_2_2_09.pdf

Hepatoprotective activity of Methanolic extract of
Vigna mungo (Linn.) Hepper in Ethanol-induced
Hepatotoxicity in Rats
M. Nitin 1 , S. Ifthekar 2 *, M. Mumtaz 3
Department of Pharmacology, H.K.E Society’s MTR Institute of Pharmaceutical Sciences, Sedam road, Gulbarga – 585105,
Karnataka, India
ABSTRACT
Acute administration of ethanol causes liver damage, the present study was undertaken to investigate the
hepatoprotective activity of methanolic extract of seeds of Vigna mungo (Linn) Hepper (MEVM) against ethanolinduced
hepatic damage in adult albino rats of either sex. The hepatoprotective effect was evaluated on the
basis of liver function parameters such as increased (liver weight and volume), elevated serum enzyme levels
(glutathione pyruvate transaminase, oxaloacetate transaminase, alkaline phosphatase and total bilirubin) and
increased (thiopentone induced sleeping time). The MEVM treated rats produced statistically significant protection.
The results were also supported by histological studies on liver cells.
Keywords: Ethanol toxicity, Hepatoprotective activity, Methanolic extract, Vigna mungo.
INTRODUCTION
At the crossroads between the digestive tract
and the rest of the body resides the largest
solid organ of the body: the liver. Because
of its interposition, the liver has a dual
blood supply. Nutrient-rich blood arrives
through the portal vein and oxygen-rich
blood through the hepatic artery. Together
these channels import a large variety of
endobiotics and xenobiotics, ranging from
nutrients to toxic substances derived from
the digestive system. The main function
of the liver, therefore, is to maintain the
body’s metabolic homeostasis, and also
to protect the hazards of harmful drugs
and chemicals. This includes the efficient
uptake of amino acids, carbohydrates, lipids
and vitamins and their subsequent storage,
metabolic conversion, and release in to
blood and bile; synthesis of serum proteins;
hepatic biotransformation of circulating
compounds, a process which converts
hydrophobic substances into water-soluble
derivatives that can be secreted into bile or
urine, as well as phagocytosis of foreign
macromolecules and particles such as
bacteria. Fibrosis or scarring of the liver
occurs after damage to liver tissue. Most
chronic liver diseases eventually result in
excess scarring leading to liver cirrhosis. 1 In
spite of tremendous scientific advancement
in the field of hepatology in recent years;
liver problems are on the rise. Jaundice and
hepatitis are two major liver disorders, with
a high toll of death rate.
Liver disease is the most common medical
complication of alcohol abuse; it is estimated
that 15–30% of chronic heavy drinkers
eventually develop severe liver disease.
Clinically significant alcoholic liver disease
may be insidious in onset and progress without
evidence of overt nutritional abnormalities.
Research Article
Received Date : 14-03-2012
Revised Date : 20-05-2012
Accepted Date : 30-05-2012
DOI: 10.5530/rjps.2012.2.9
Address for
correspondence
Dr. Nitin Mahurkar
Professor and HOD,
Department of Pharmacology
H. K. E. Society’s MTR Institute of
Pharmaceutical Sciences,
Sedam road, Gulbarga – 585105,
Karnataka, India.
E-mail: ifthekar85@gmail.com
Mobile: 09036334393.
www.rjps.in
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Nitin M. et al.: Hepatoprotective activity of Methanolic extract of Vigna mungo (Linn.) Hepper in Ethanol-induced Hepatotoxicity in Rats
Alcoholic fatty liver, a reversible condition, may progress
to alcoholic hepatitis and finally to cirrhosis and
liver failure. In the USA, chronic alcohol abuse is the
leading cause of liver cirrhosis and of the need for liver
transplantation. The risk of developing liver disease is
related both to the average amount of daily consumption
and to the duration of alcohol abuse. Women appear
to be more susceptible to alcohol hepatotoxicity than
men. Another factor that increases the risk of severe
liver disease is concurrent infection with hepatitis B or
C virus. 2
There are only a few synthetic drugs available for the
treatment of hepatic disorders. In allopathic system of
treatment, corticosteroids and immunosuppressants
are commonly used, but these drugs have adverse
effects such as immunosuppression and bone marrow
depression and success rate of treating liver diseases
is low. Silymarin, a flavonoid ligand mixture extracted
from the Silybum marianum (milk thistle) is a popular
remedy for hepatic diseases, however there are several
herbs/herbal formulations claimed to possess beneficial
activity in treating hepatic disorders. 3
Vigna mungo (Linn) Hepper commonly known as Black
gram (Mash, Urid), belongs to family Fabaceae. Vigna mungo
is a diffuse annual herb; stem 30–60 cm long, clothed
with brownish silky hairs. Leaves 3 foliolate; leaflets
5–10 cm long, flowers yellow in axillary racemes, pods
subcylindric; 3.8–6.3 cm long. It is extensively cultivated
all over the India. It has been used for various medicinal
purposes in Ayurvedic and Unani systems of medicine.
The seeds are sweet, laxative, aphrodisiac, tonic,
appetizer, diuretic, galactagogue and styptic; useful in
piles, asthma, scabies, leucoderma, gonorrhea, pains,
epistaxis, paralysis, rheumatism and affections of the
nervous system, liver and cough. It is also prescribed for
dropsy and cephalalgia. 4 Vigna mungo L. (Fabaceae) has
been reported to be used in a variety of disease conditions
of liver in Indian traditional system of medicine. 5
Vigna mungo has been reported for its hepatoprotective
activity against acetaminophen and CCl 4 model, 6
for antioxidant activity, 7 and for its diuretic activity. 8
However, no other models were used for screening
of its hepatoprotective activity. Therefore the present
investigation was undertaken to evaluate the hepatoprotective
activity of Vigna mungo using ethanol model.
MATERIALS AND METHODS
Drugs and chemicals
Silymarin was obtained as a gift sample from Micro
Labs. Bangalore. The kits for the biochemical estimation
were purchased from Pathozyme diagnostics Kagal,
Dist. Kolhapur, India. The ethanol, solvents and other
chemicals were procured from reputed manufacturers.
Preparation of seed extract
The seeds of Vigna mungo (Linn.) Hepper (500 g) were
purchased from the local market of Gulbarga, Karnataka;
were authenticated at Pharmacognosy department of
HKES’s MTR Institute of Pharmaceutical Sciences,
Gulbarga. The seeds were powdered and defatted with
petroleum ether and then subjected to extraction with
methanol (95%) using Soxhlet apparatus for 18 h. the
extract was concentrated on water bath to obtain thick
pasty mass. The percentage yield was 2.6% w/w.
Phytochemical screening
Preliminary phytochemical screening of Methanolic
extract of seeds of Vigna mungo was carried out as
described by Khandelwal. 9
Experimental animals
The albino rats of either sex (200–250 g) were used for
this study. The rats were housed in polypropylene cages
and maintained under standard conditions (12 h. light
and dark cycles, at 25 ± 5°C and 35 – 60% humidity)
standard pelletised feed and tap water were provided
ad libitum. The animals were acclimatized to laboratory
conditions for 48 hour prior to the experimental protocol
to minimize any nonspecific stress. The Institutional
Animal Ethics Committee of H.K.E.S’s College of
pharmacy, Gulbarga, India, approved the experimental
protocol in accordance with the guidelines provided by
Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA) with registration
no. 142/1999 CPCSEA 5th July 1999.
Acute toxicity studies
According to earlier reports the dose of Vigna mungo
seed extract 500 mg/kg body weight p.o. was used as
hepatoprotective. 6 As Vigna mungo seeds are used as food
and can be consumed in daily life. The dose selection
was based upon literature review and our studies in
the laboratory. Solanki YB and Jain SM have reported
safety levels of drug and used 500 mg/kg. our laboratory
reports also revealed concurrent values. Hence the above
dose was selected.
Hepatoprotective activity
The albino rats of either sex (200 – 250 g) were selected
and divided into four groups of six animals each.
The treatment protocol is summarized as shown below. 10
Group 1- Normal control: 2% suspension of gum acacia
(1ml/kg) p.o. once daily for 25 days.
Group 2- Ethanol control: Ethanol (3.76 g/kg) twice
daily, p.o. for 25 days.
Group 3- Standard: Silymarin (100 mg/kg) p.o. followed
by ethanol (3.76 g/kg) p.o. after a gap of 30 min for 25 days.
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Nitin M. et al.: Hepatoprotective activity of Methanolic extract of Vigna mungo (Linn.) Hepper in Ethanol-induced Hepatotoxicity in Rats
Group 4- Methanolic extract of seeds of Vigna mungo
(500 mg/kg) p.o. followed by ethanol (3.76 g/kg) p.o. after
a gap of 30 min for 25 days.
The study was carried out for 25 days. On 26th day
thiopentone sodium (40 mg/kg, i.p.) was injected and
the sleeping time was recorded. After complete recovery,
the blood samples were collected from all animals by
retro orbital puncture method. Serum was separated
by centrifugation at 2500 rpm for 15 min. and analyzed
for various biochemical parameters such as serum
glutathione pyruvate transaminase (SGPT), oxaloacetate
transaminase (SGOT), alkaline phosphatase (ALP),
and total bilirubin (BIT). Immediately after collection
of blood the animals were euthanized with an over
dosage of ether and sacrificed by cervical dislocation.
The livers were removed, washed in saline and the wet
weight and volume was determined then transferred
into 10% formalin for its histopathological studies. 11
Histopathological studies
Histopathological study of livers was performed in histopathology
Laboratory by consultant histopathologist.
Haematoxylin-eosin was used for staining in histopathology
studies.
Statistical analysis
The data obtained in the experiment was expressed in
terms of mean ± SEM. Statistical significance of data
was assessed by one way analysis of variance (ANOVA)
followed by a comparison between different groups
using “Tukey-Kramer” multiple comparison test. A
value of P

Nitin M. et al.: Hepatoprotective activity of Methanolic extract of Vigna mungo (Linn.) Hepper in Ethanol-induced Hepatotoxicity in Rats
a. Group 1 (Normal control): Showing normal histology
of rat liver.
c. Group 3 (Standard): C&INF- mild Congestion and
inflammation.
b. Group 2 (Ethanol control): CVC-Central vein
congestion, INF-Inflammation.
d. Group 4 (MEVM): RH-Showing regenerating hepatocytes.
Figure 1: Effect of methanolic extract of seeds of Vigna mungo (MEVM) on histopathological examination of rat liver in ethanol-induced
hepatotoxicity.
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Nitin M. et al.: Hepatoprotective activity of Methanolic extract of Vigna mungo (Linn.) Hepper in Ethanol-induced Hepatotoxicity in Rats
induced by ethanol. The hepatoprotective effect offered
by methanolic extract of seeds of Vigna mungo was
found to be significantly greater than silymarin treated
group (Table 2).
Hepatocytes of the normal control group showed a
normal histology of the liver. In the ethanol treated
group the liver showed loss of lobular architecture,
extensive central vein dilation, sinusoidal congestion
and inflammation. Silymarin-and MEVM-pretreated
groups showed mild central vein dilation, architecture
of the liver was maintained, and hepatocytes showed
regeneration. (Figure 1)
DISCUSSION
Alcoholic beverages are used universally and alcohol
is the world’s most widely used psychoactive drug,
but chronic, excessive alcohol consumption leads to
permanent organ damage or death. Alcohol is rapidly
oxidized in the liver tissue to acetaldehyde and acetate
by alcohol dehydrogenase (ADH) and acetaldehyde
dehydrogenase (ALDH), respectively. 12 Recent study
indicates that oxidative stress is involved in the
pathogenesis of liver diseases including drug induced
hepatic damage, alcohol hepatitis and viral hepatitis
or ischaemic liver injury. 13 Increased formation of
lipoperoxides, conjugated dienes and malondialdehyde
and reduced levels of antioxidants like vitamin E and
glutathione in the tissues have been demonstrated in
experimental animals administered with ethanol as well
as in alcoholic human subjects. The increased level of
SGPT, SGOT, ALP, and total bilirubin is conventional
indicator of liver injury. 14 Oxidative stress is one major
factor in etiology of ethanol injury, mainly by Kupffer
cell derived reactive oxygen species (ROS), ethanol
activates Kupffer cells primarily through the action of a
substance called endotoxin, which is released by certain
gram-negative bacteria present in the intestine, which
generates ROS and pro inflammatory cytokines (TNF
alpha, IL 1) and both of them can lead to liver damage. 15
The hepatic injury leads to elevation of serum levels of
SGPT (ALT), SGOT (AST), ALP, and BIT in rats and
are used as markers for assessing toxicant effect and also
hepatoprotective agents. During hepatic damage these
enzymes present in the liver cells leak into the serum,
resulting in increased concentrations. 16
In the present study ethanol administration for 25 days
resulted in morphological changes such as enlargement
of liver, scratches, dark brown coloration and increased
volume. Barbiturates are a class of xenobiotics that
are extensively metabolized in the liver. Deranged liver
function leads to delay in the clearance of barbiturates,
resulting in a longer duration of hypnotic effect. 17 In the
present study, administration of thiopentone sodium to
rats treated with ethanol resulted in an increased duration
of thiopentone-induced sleeping time. Whereas the
Vigna mungo seed extract (MEVM) pretreated animals
showed similar morphology of livers compared to that of
normal control animals that were healthy in appearance
and significantly decreased the weight and volume, and
also showed decreased thiopentone-induced sleeping
time, an indirect evidence of their hepatoprotective
effect.
Ethanol administration also produces significantly
increased serum marker enzymes such as SGPT, SGOT,
ALP and BIT in ethanol control group compared
to normal control group. Treatment with MEVM
significantly decreased the enzymes SGPT, SGOT, ALP
and BIT levels as compared to ethanol control group. All
the above parameters indicating their hepatoprotective
effect against ethanol-induced liver cell damage.
Histological changes such as loss of lobular architecture,
extensive central vein dilation, sinusoidal congestion and
inflammation were observed in ethanol control group.
The Vigna mungo seed extract (MEVM) pretreated animals
had significantly prevented these histological changes,
further indicating their hepatoprotective activity. All the
histological changes observed were in correlation with
the physical, biochemical and functional parameters of
the liver.
Ethanol is metabolized largely by sequential hepatic
oxidation, first to acetaldehyde and then to acetic acid. 18
Acetaldehyde is thought to have a number of adverse
effects like decreased transport and secretion of proteins
owing to tubulin polymerization, enhanced vitamin
metabolism and trace metals. Drugs like paracetamol
cause severe acute liver injury which is sometimes
fatal. 19,20,21 Antioxidants exhibit hepatoprotective activity
by blocking the conversion of ethanol to acetaldehyde. 22
Liver diseases are often associated with edema and ascites
in conjunction with elevated portal hydrostatic pressure
and reduced plasma oncotic pressure, the mechanisms
for retention of sodium by the kidney are complex, they
probably involve a combination of factors, including
diminished renal perfusion resulting from systemic
vascular alteration, diminished plasma volume as a result
of ascites formation and diminished oncotic pressure
due to hypoalbuminemia. In addition, there may be
primary sodium retention by the kidneys and diuretic
therapy is useful to overcome the ascites and edema. 2
From the previous studies it was found that Vigna mungo
has exhibited diuretic activity, 8 due to the presence of
strong antioxidants like ascorbic acid, total phenolic
compounds, tannins, flavonoids etc. The presence of
potent diuretic such as saponins in the extract may be
responsible for the hepatoprotective activity.
The earlier reports carried out with the extract of
Vigna mungo in acetaminophen and CCl 4 model have
66 RGUHS J Pharm Sci | Vol 2 | Issue 2 | Apr–Jun, 2012

Nitin M. et al.: Hepatoprotective activity of Methanolic extract of Vigna mungo (Linn.) Hepper in Ethanol-induced Hepatotoxicity in Rats
shown SGPT, SGOT, ALP and BIT values 99.33, 121.17,
59.067 and 1.84 IU/L. The present studies using ethanol
model have shown SGPT, SGOT, ALP, and BIT values
49.61, 74.51, 147.18, and 0.76 IU/L. levels respectively
indicating a greater level of protection in this model.
CONCLUSION
From the above studies it can be concluded that
methanolic extract of seeds of Vigna mungo (Linn.)
Hepper (MEVM) possesses a hepatoprotective activity
against ethanol-induced hepatotoxicity.
ACKNOWLEDGEMENT
The authors are thankful to authorities of H.K.E.S’s
MTR Institute of pharmaceutical Science, Gulbarga for
providing the necessary facilities to carryout the work.
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