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IJPRD, 2013; Vol 5(04): June-2013 (071 – 076) International Standard Serial Number 0974 – 9446
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ANTIHEPATOTOXIC EFFECT OF“HOLOPETELEA INTEGRIFOLIA (ROXB)”. LEAVES AGAINST PARACETAMOL
INDUCED HEPATIC DAMAGE IN ALBINO RATS
Khushboo Saxena* 1 ,
R.Irchhaiya 1 , Vihangesh Dixit 1
1 Institute Of Pharmacy; Budelkhand University;Jhansi(U.P)
ABSTRACT
The objective of this study was to investigate the hepatoprotective
activity of ethanolic extract of leaves of Holopetlea
integrifolia(Roxb).againt paracetamol induce hepatic damage. There
are lack of reliable hepatoprotective drugs in modern medicine to
prevent and treat paracetamol induce liver damage. The ethanolic
extract showed presence of flavonoid and steroids. The plant material
was dried in shade, powdered and extracted with ethanol. The
hepatoprotective activity of the ethanolic extract was assessed in
paracetamol induced hepatotoxic rats. Biochemical parameters like
SGOT, SGPT, ALP and Bilirubin were tested in drug induced and
untreated groups of rats. Treatment of ethanolic extract of Holopetlea
integrifolia(Roxb).leaves has brought back the altered level of
biochemical parameter to the normal level in the dose dependent
manner and also compared with silymarin used as standard drug.
KEYWORDS : Hepatoprotective, ethanol, leaves.
paracetamol,silymarin.
Correspondence to Author
KHUSHBOO SAXENA
Institute Of Pharmacy; Budelkhand
University;Jhansi(U.P)
Email: Khushbusaxena1987@gmail.com
INTRODUCTION
The liver is one of the most important vital organs
in the human body. Liver plays a significant role not
only in metabolism and detoxification of
exogenous toxins and therapeutic agents, but also
in the bio regulation of fats, carbohydrates, amino
acids, proteins, blood coagulation and
immunomodulation. Many toxins target the liver
and cause hepatotoxic effects that can be observed
through some biochemical parameters.
Impairment of the liver generally occurs from
excessive exposure to xenobiotics, alcohol,
chemotherapeutic agents, virus and protozoan
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infections. Depending upon the severity of toxicant
insult, hepatic cell injury can lead from acute to
chronic hepatitis, which if left untreated can result
in cirrhosis or malignant lesions. Liver damage is
associated with cellular necrosis, increase in tissue
lipid peroxidation and depletion in the tissue GSH
levels. In addition serum levels of many
biochemical markers like SGOT, SGPT, triglycerides,
cholesterol, bilirubin, alkaline phosphatase are
elevated [1] . Liver diseases are the most serious
ailment and are mainly caused by toxic chemicals
(Excess consumption of alcohol, high doses of
paracetamol, carbon tetrachloride,
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International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446
chemotherapeutic agents, peroxidised oil,
etc).Plant drugs are known to play a vital role in the
management of liver diseases [2,3] . Holoptelea
integrifolia, the versatile medicinal plant is the
unique source of various types of compounds
having diverse chemical structure a very little work
has been done on the biological activity and
plausible medicinal application of the compounds
and hence extensive investigation is needed to
exploit their therapeutic utilities to combat
diseases. Phytochemical investigation shows the
presence of chemical constituent such as
terpenoids, alkaloids, glycoside, carbohydrates,
steroids, sterols, saponins, tannins, protein, and
flavonoids [5] . The isolated principle are Beta
amyrin, Beta sitosterols, octacosanol, holopettelin-
A, holopetelin-B, hederagenin, hexacosanol, Beta-
D-glucose,fridelin, epifriedelin, 2-amino
napthaqiunone, 1,4-napthalenedione are
considered as responsible for various activity [7] . The
traditional uses, reported
biological/pharmacological activity, isolated
compounds and therapeutic application of
holoptelea integifolia which might be useful for
scientific and researchers to find out new entities
responsible for therapeutic activity.
MATERIAL AND METHOD:
Drugs and Chemicals:
All the chemicals were analytical grade.
Paracetamol was obtained from Amol Pharma
Jaipur (Rajasthan).and silymarin from kypton
pharma.
Plant collection and authentication
The leaves of Holoptelea integrifolia was collected
from Jhansi (U.P), and Identified and authenticated
by National vrkshayurveda research institute
Jhansi. The accession no. is NVRI/05551/2011. The
leaves was dried in shade, and finally grounded in
powdered form in and electronic grinder and
stored in cellophane bags at 4 o C until use.
Preparation of Extract:
The air dried and coarsely powdered leaves (350 g)
were first extracted with 1ltr petroleum ether
about 40-80 o C to remove all fatty acids and again it
is extracted with ethanol (95%) in a soxhlet
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apparatus for 70 hr .The extract were concentrated
to dryness under reduced pressure and controlled
temperature (30-50 o C).The yield value of both the
leaves extract is recorded.
Animals:
Healthy Wistar-albino rats weighing about (180-
250gm) of either sex were obtained from animal
house, Institute of Pharmacy, Bundelkhand
University, Jhansi. The animals were housed in
specific standard laboratory conditions. The
conditions were kept in a temperature-controlled
environment (25±1 0 C) and with a regular 12h
light/12h dark cycle. All animals were fed with
commercial diet & water ad libitum, during the
experiment. All protocols of the study was
approved by the Institutional Animal Ethical
Committee with reference no.
BU/Pharm/IAEC/11/022.The IAEC is approved by
Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA)
with registration no. 716/02/a/CPCSEA Dose and
Treatments: Rats were divided into different
groups (n=6). Silymarin syrup (0.5ml/100gm),
Paracetamol (2gm/kg) & HIEE (200 and 400 mg/kg)
were administered orally. The control group
received 1.5% Tween 80 in distilled water as
vehicle (10ml/kg B.W.).
Experimental Design: The general principle
involved in the evaluation of Hepatoprotective
activity is to induce liver toxicity or infection with
the help of hepatotoxin in the liver of experimental
animals. The magnitude of the protective activity is
measured in-vivo by estimating the following
parameters:
Biochemical Parameters: These are the most
reliable parameter in the in-vivo study & include
the estimation of different enzyme like SGOT or
Aspartate transferase & Alanine transferase or
SGPT & Serum alkaline phosphate (SALP). It also
includes estimation serum bilirubin (SBLN) &
estimation of Hydroxyproline fat & protein content
of Livers.
Groups of Animals: The animals were divided into
five groups of six animals each.
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International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
• Group І: served as control & received 1.5%
Tween 80 in distilled water as vehicle (10ml/kg
• Group IV: received HIEE (200mg/kg/day)
simultaneously 7 days followed by Hepatotoxin.
B.W.) for 7 days.
• Group V: received HIEE (400mg/kg/day)
• Group II: received vehicle for 7 days followed
by Hepatotoxin (2gm/kg B.W.).
simultaneously Hepatotoxin. [11]
7 days followed by
• GroupIII: received silymarin (0.5ml/100gm B.W.
per day) simultaneously 7 days followed by
Hepatotoxin.
Paracetamol was administered to animals of
Groups II, III, IV & V in single dose (2gm/day B.W.)
as suspension of 1.5% tween 80.
Table1:Effect of Holopetlea integrifolia. Extract on Biochemical Parameters in Rats Subjected to Paracetamol
Induced Hepatotoxicity.
Enzyme group
SGOT
SGPT
SALP
Bilirubin
Control group 25.30±0.089 27.5524±0.085 89.217±1.5 0.78±0.06
PCM treated group 67.28±1.25 42.350±0.150 2414.25±26.03 1.875±0.006
Standard group 56.20±0.103 34.383±0.160 1456.20±0.839 1.317±0.006
HIEE(200mg/kg B.W.) 44.283±1.22 30.283±0.054 424.100±0.052 1.11±1.696
HIEE(400mg/kg B.W). 29.183.40±0.060 21.383±0.145 116.217±0.122 1.157±0.011
Statistical Analysis The results were expressed as
mean ± SEM of six animals from each group. The
statistical analysis were carried out by one way
analysis of variance (ANOVA) P values < 0.05 were
solution for histopathological study. The pieces of
liver were processed and
embedded in paraffin wax sections were made
about 4-6μm in thickness. They were stained with
considered significant.
Histopathological Examination: Small pieces of
liver tissue were collected in 10% formaldehyde
hematoxylin and photographed.
Photograph:1,2 Liver of rat treated with vehicle, Liver of rat treated with paracetamol.
Photograph :3 Liver of rat treated with syrup silymarin.
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International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
Photograph: 4 Liver of rat treated with HIEE (200mg/kg bodyweight)
Photograph 5: Liver of rat treated with HIEE (400mg/kg bodyweight)
RESULT AND DISCUSSION:
The present work was to carried out with the
objective of ethanolic extract of leaves of
marker of the extent & type of hepatocellular
damage [6] . The mode of action of paracetamol on
the liver is by covalent binding of its metabolite, n-
Holopetlea integrifolia(Roxb.). Phytochemical acetyl-p-benzoquinone-amine amine to sulfhydryl group
investigation, evaluation of the Hepatoprotective of protein resulting in cell necrosis and lipid
activity (in-vivo) against PCM induced peroxidation [7] . Due to liver injury caused by PCM
Hepatotoxicity model.Liver is a versatile organ in overdose, the transport function of the
the body concerned with regulation of internal
chemical environment. Therefore damage to the
liver inflicted by a hepatotoxic agent is of grave
consequence. In the present study, the ethanolic
extract of Holopetlea integrifolia(Roxb.). was
observed to exhibit hepatoprotective effect as
demonstrated by a significant decrease in SGOT,
SGPT and SALP concentration, and preventing liver
hepatocytes gets disturbed resulting in the leakage
of plasma membrane [8] thus causing an increase in
serum enzyme levels. Assay of the activities of
these serum marker enzymes and HIEE helps to
assess the liver function [9] .
Herbal Silymarin is a unique, all natural, complex
multi-ingredient formula. It helps in protecting the
liver from harmful toxins & regulates levels of
histopathological changes in rats induced with PCM enzymes and optimizes assimilation. Herbal
hepatotoxicity. Paracetamol is a well known
antipyretic & analgesic agent, which is safe in
therapeutic doses but can produce fatal necrosis in
experimental animals & humans & is employed as
an experimental hepatotoxic agent. A sign of
hepatic injury is the leaking of cellular enzymes into
the plasma due to the disturbances caused in the
transport functions of hepatocytes. The estimation
Silymarin has been found to be associated with an
increase in serum albumin & restores the
functional efficiency of the liver by promoting the
hepatocellular regeneration. Silymarin is believed
to be the first multi-herb remedy granted
regulatory approval as a drug, Silymarin, the well
known hepatoprotective product. Silymarin is a
favoured drug for different liver diseases because
of enzymes in the serum is a useful quantitative
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International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446
of its oral effectiveness, good safety profile,
avaialvility in India and importantly affordable [10].
In this experiment, it is observed that the level of
hepatic biochemical markers i.e. SGOT, SGPT, SALP
& Bilirubin is increased due to PCM in comparison
to the control group. This clearly indicates that
there is significant hepatic damage due to the
paracetamol. The toxic effect of PCM was
controlled in animals treated with ethanolic extract
of Holopetlea integrifolia. 400 mg/kg/day by way of
restoration of the markers levels in the liver with
comparison to positive control Silymarin.
Hepatoprotective effect of HIEE was further
confirmed by histopathological studies of the liver,
which basically supported the results from the
serum assay. HIEE administration resulted in
bringing about an almost normal histological
architecture of the liver. The preliminary
phytochemical screening of Holopetlea integrifolia
leaves shows the presence of flavonoids and
Steroids compounds as major active principle in
ethanolic extract of H.integrifolia. leaves. Many of
flavonoids and steroids(Beta- steroid) compounds
have been reported for hepatoprotective activity.
ACKNOWLEDGEMENT
The authors are grateful to Dr.R,Irchhaiya Head of
the department of pharmacy and Mr.Vihangesh
Dixit, Assistant professor, Bundelkhand university
Jhansi(U.P).
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