International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

______________________________________________________________________Research Paper

Effect of Calotropis procera Linn. against paracetamol induced

hepatotoxicity in rats
E.Manivannan*, S. Rajaram, R. Kothai1, B. Arul1 and B. Jayakar1
Department of Pharmacology, Vinayaka Missions Kirupanandha Variyar Medical College,
Salem-636 308, Tamilnadu, India
1. Vinayaka Missions College of Pharmacy, Salem.
__________________________________________________________________________________________
ABSTRACT
Calotropis procera Linn. showed remarkable hepatoprotective activity against paracetamol-induced
hepatotoxicity as judged from biochemical parameters such as serum aspartate amino transferase (AST),
alanine amino transferase (ALT), alkaline phosphatase (ALP), total bilirubin, total protein and gamma
glutamate transpeptidase (GGTP) and levels of lipid peroxides in liver, which was comparable to the control
and activity exhibited by the reference standard Silymarin.
Key words: Chloroform extract; Calotropis procera; paracetamol-induced; anti-hepatotoxic.
INTRODUCTION
Calotropis procera is a small, erect and
compact shrub, covered with cottony tomentum, up
to 5.4 m in height, found growing wild throughout
India in comparatively drier and warmer areas up
to an altitude of 1050 m (1). It is commonly known
as arka and it is used for the treatment of different
ailments in ayurvedic and unani systems of
medicines (2). It is used as an analgesic (3),
antipyretic (4), anthelmintic (5), antitussive (6),
contraceptive (7) and antidiarrhoeal (8). A review
of literature afforded no information on the
hepatoprotective aspects of this plant against
paracetamol induced hepatotoxicity in rats. So the
present study is therefore, an attempt to assess the
efficacy of this indigenous herb for its
hepatoprotective activity against paracetamol
induced toxicity model in rats.
MATERIALS AND METHODS
Plant material
The aerial parts were collected from the foothill
of Yercaud, Salem, in June 2008 and cleaned to
remove the debris. The plant was identified and
authenticated by a botanist Dr. A. Marimuthu,
Department of Botany, Government Arts College,
Attur. A voucher specimen (CPM-1) has been kept
in our college museum for future reference. The
plant parts were dried at room temperature for 10
days and coarsely powdered with the help of a
hand-grinding mill and the powder was passed
through sieve No. 60.
_______________________________________
*Address for correspondence:
E-mail: emanivannan@yahoo.co.in

Vol. 2 (2) Apr Jun 2011

Preparation of the extract:

The powder of Calotropis procera was
extracted separately by continuous hot extraction
process using Soxhlet apparatus with different
solvents in increasing order of polarity from
petroleum ether, chloroform, acetone, alcohol, to
finally chloroform:water. After extraction, the
extracts were concentrated under reduced pressure
in tared vessel. The marc of crude drug powder was
then once again subjected to successive extraction
with other solvents and the extractive values were
calculated with reference to the air-dried drug. The
dried extracts were subjected to various chemical
tests to detect the presence of different
phytoconstituents.
Test animals
Wister rats of either sex and of approximately
the same age, weighing about 150-175 g were used
for the present study. They were housed in
polypropylene cages and fed with standard chow
diet and water ad libitum. The animals were
exposed to alternate cycle of 12 h of darkness and
light each. Before each test, the animals were
fasted for atleast 12 h. Male mice weighing about
20-25 g each were used for acute toxicity studies.
The experimental protocols were subjected to the
scrutinization of the Institutional Animal Ethics
Committee and were cleared by the same.
Acute toxicity studies
The animals were divided into control and test
groups containing six animals each. The control
group received the vehicle (1 % acacia), while the

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International Journal of Research in Pharmaceutical and Biomedical Sciences

test groups got graded doses of different extracts

orally and were observed for mortality till 48 h and
the LD50 was calculated.
Hepatoprotective study
For determining the hepatoprotective activity,
animals were divided into four groups containing 6
animals each. Group I served as normal control
and received orally 1 ml of propylene glycol daily
for 4 consecutive days and a single dose of 40 %
sucrose solution (1 ml/rat, p.o.,) on day three.
Group II was served as positive control and
received paracetamol which was suspended in 40
% w/v aqueous sucrose solution and administered
at a dose of 2 g/kg, p.o.(9). Group III and IV were
treated with chloroform extract of Calotropis
procera (100 and 200 mg/kg, p.o.) and reference
compound Silymarin (200 mg/kg, p.o.),
respectively for 4 consecutive days and a single
dose of paracetamol suspension (2 g/kg) on day 3.
After 48 h, blood samples were withdrawn from all
groups by cardiac puncture of nonanaesthetized
rats. The biochemical parameters such as ALT
(10), AST (11), ALP (12), total bilirubin (13), total
protein (14) and GGTP (15) were estimated as
reported earlier. A small portion of liver was cut
from the animals from each group and preserved in
neutral buffered formalin and was processed for
paraffin embedding, following the standard
microtechnique (16). 5 sections of the livers were
stained with alum haemotoxylin and eosin and
studied for degenerative and necrotic changes.
Statistical analysis
All values were expressed as meanSEM. The
data were statistically analyzed using one way
ANOVA followed by Newman Keuls multiple
range test and differences below P<0.05 are
considered as significant.
RESULTS AND DISCUSSION
The average percentage yield of chloroform
extract of C.procera was found to be 3.2 % w/w
and the LD50 was 993 mg/kg. The results of
biochemical parameters revealed to the elevation of
enzyme level in paracetamol-treated group
indicating that it induces damage to the liver. Liver
tissue rich in both transaminase increased in
patients with acute and chronic hepatic diseases.
AST, which is slightly elevated by cardiac necrosis,
is a more specific indicator of liver disease. A
significant reduction was observed in AST, ALT,
ALP, GGTP, total bilirubin and total protein levels
in the animals treated with chloroform extract of
C.procera (Table 1). The enzyme levels were
almost restored to the normal. So the animals
treated with chloroform extract of C. procera
exhibited statistically significant (P<0.001)
protection
against
paracetamol-induced

Vol. 2 (2) Apr Jun 2011

ISSN: 2229-3701

hepatotoxicity in rats, which is comparable to the

reference
compound
Silymarin.
The
histopathological studies support the biochemical
findings. Hepatotoxicity induced by Paracetamol
manifested itself by the 8th day with the liver
showing massive degeneration enveloping the not
so visible necrotic areas as compared to the normal.
The liver sections of rats treated with the
chloroform extract were similar to liver sections of
rats treated with Silymarin and showed micro
vesicular changes with mild congestion and
widening of the sinusoids. There was no evidence
of necrosis.
Paracetamol is a well known antipyretic and
analgesic, which produces hepatic necrosis in high
doses and of the most commonly used hepatotoxins
in the experimental study of liver disease.
Paracetamol damages liver by covalent binding of
its toxic metabolite N-acetyl-p-benzoquinone imine
to sulphydrl groups of proteins resulting in cell
necrosis and lipid peroxidation induced by decrease
in glutathione in the liver (17). This is evidenced
by an elevation in the serum marker enzymes
namely AST, ALT, ALP, GGTP, total bilirubin and
total protein. Estimation of serum transaminase
levels gives a fairly good idea about the functional
study of liver.
The efficacy of any hepatoprotective drug is
dependent on its capacity of either reducing the
harmful effect or maintaining the normal hepatic
physiology, which has been disturbed by a
hepatotoxin. The extract decreased paracetamolinduced elevated levels of the enzymes in group III,
indicate the production of structural integrity of
hepatocytic cell membrane or regeneration of
damaged liver cells.
Histopathological examination of the liver
section of the rats treated with toxicant showed
intense centrilobular necrosis and vascuolisation.
The rats treated with extracts alone with toxicant
showed sign of protection against these toxicants to
considerable extent as evident from formation of
normal hepatic cards and absence of necrosis and
vascuoles.
Decrease in serum bilirubin after treatment with
extract in liver damage indicated the effectiveness
of the extracts in normal functional status of the
liver.
CONCLUSION
The results of the present investigation indicate
that the chloroform extract of C.procera possesses
good
hepatoprotective
activity.
Further
investigations are required to characterize the
active hepatoprotective principle and its
mechanism of action.

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International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

Table 1. Effect of chloroform extract of C.procera on paracetamol-induced hepatotoxicity in rats

Treatment

Dose
mg/kg

SGOT
U/L

SGPT
U/L
42.17+1.88

Alkaline
Phosphate
(U/L)
180.33+6.53

Normal
Control
Toxic control
Silymarin
Drug
Extract of
C.procera
Extract of
C.procera

114.17+4.49

200

GGTP
U/L
112.674.41

Total
Bilirubin
mg/dl
0.48+0.003

220.83+3.51
117.83+2.20*

107.50+4.98
46.33+1.31*

268.17+5.47
190.83+1.55*

235.334.90
122.502.86*

0.92+0.005
0.62+0.009*

100

138.17+2.20*

54.17+2.11*

204.67+4.08*

140.172.11*

0.78+0.005*

200

127.33+1.31*

49.83+2.20*

198.17+2.20*

136.02.78*

0.72+0.009*

Data are expressed as Mean SEM, n = 6 in each group. *P<0.01 compared to control group.

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