Content uploaded by Dileep Chandra Sreerama
Author content
All content in this area was uploaded by Dileep Chandra Sreerama
Content may be subject to copyright.
Venkata Kiran Kumar Y. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 1(1), 2012, 80 - 86.
Available online: www.ijrpns.com 80
ISSN: XXXX - XXXX
HEPATOPROTECTIVE ACTIVITY OF ETHANOLIC EXTRACT OF EUGENIA
JAMBOLANA LEAF ON LIVER DAMAGE CAUSED BY PARACETAMOL IN RATS
Y. Venkata Kiran Kumar*1, G. H. Srinivasa Rao1, M. Santhiswaroop1, S. Dileep Chandra2
*1Department of Pharmacognosy, Saastra College of Pharmaceutical Education & Research,
Nellore, Andhra Pradesh, India.
2Department of Pharmacognosy, Natural Remedies Pvt.Ltd, Bangalore, Karnataka, India.
INTRODUCTION
Medicinal plants are the oldest type of healthcare
known, and have been used throughout history by
many cultures. Many of the modern medicines are
produced indirectly from medicinal plants, for
example aspirin1. Plants are directly used as
medicines by a majority of cultures around the
world, for example Chinese medicine and Indian
ABSTRACT
Ethanolic extract of Eugenia jambolana leaves were investigated for their Hepatoprotective potential against
liver injury induced by paracetamol in rats. The liver damage was induced in male albino rats (200 - 220gm)
by administering paracetamol (2 gm/kg) for 7 days and extent of damage was studied by assessing the
biochemical parameters such as Total bilirubin, Direct bilirubin, Indirect bilirubin, Serum glutamate pyruvate
transaminase (SGPT), Serum glutamate oxaloacetate transaminase (SGOT), Alkaline phosphatase (ALP),
Total protein (TP), Albumin and Globulin. Histopathological changes of liver sample were compared with
respective control. Treatment of rats with ethanolic extract (200mg/kg) for 7 days resulted in significant
hepatoprotection for total protein (6.6 g/dl) only, while at 400 mg/kg, the restoration was significant for
enzyme markers, viz. SGOT, SGPT, ALP (22.5 U/L, 18.5 U/L, 58.5 U/L ) in paracetamol-treated rats. The
lethal dose (LD50) of the plant was greater than 3000 mg/kg. The ethanolic extracts of Eugenia jambolana
leaves have protected liver from paracetamol damage against induced liver injury.
KEYWORDS:
Hepatoprotective, Paracetamol, Serum glutamate oxaloacetate transaminase, alkaline phosphatase.
International Journal of Research
in
Pharmaceutical and Nano Sciences
Journal homepage: www.ijrpns.com
Author for Correspondence:
Y.Venkata Kiran Kumar,
Saastra College of Pharmaceutical Education &
Research, Nellore, Andhra Pradesh, India.
E-mail: kirankumaryellu@gmail.com
Venkata Kiran Kumar Y. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 1(1), 2012, 80 - 86.
Available online: www.ijrpns.com 81
medicine. Studying medicinal plants helps to
understand plant toxicity and protect human and
animals from natural poisons. Many food crops
have medicinal effects, for example garlic.
Medicinal plants are resources of new drugs. It is
estimated there are more than 250, 000 flower plant
species 2.
The liver is an essential body organ that forms an
important barrier between the gastrointestinal blood,
which contains large amounts of toxins and
antigens, and the body. Around 60% of the liver is
formed by liver cells, hepatocytes, which are
radially grouped into thick unicellular layers around
the terminal hepatic veins, forming the smaller
anatomic units of the liver, the classic lobes. The
liver produces a large amount of hormones,
enzymes, and performs several functions essential
to life. It is also the organ responsible for cleansing
of toxins from the bloodstream, by turning them
into removable substances. Detoxification is
essential as toxins may damage both liver cells
membranes and the cell membranes of other organs.
Glutathione, the main detoxifying and antioxidant
agent of the liver, is essential for enhancing the
removal of toxins and prevents cell damage. The
term liver disease refers to many diseases and
disorders that may cause impaired liver function can
make the liver a decrease of its functions. The
dysfunction may be primary, but the liver is often
secondarily affected by disorders of other organ
systems, since it is involved in many metabolic and
detoxifying processes. This is especially due to
toxics agents produced by the diseases of the
gastrointestinal tract 3. Usually, more than 75% or
three quarters of liver tissue needs to be affected
before decrease in function occurs. The Eugenia
jambolana (Family: Myrtaceae) is well known for
its medicinal properties in indigenous medicine in
India4.
MATERIALS AND METHODS
Preparation of ethanolic extracts of Eugenia
jambolana leaves
The leaves was separated from plant and it was
washed with absolute ethanol to avoid the microbial
growth, the leaves were dried at open air under the
shade, cut in to small pieces and powdered
mechanically, then 50 gm of powder Eugenia
jambolana leaves was extracted with 250ml ethanol
in a soxhlet apparatus for 72 hrs. The extract
obtained was concentrated by recovery of ethanol.
The concentrated product was used as ethanolic
extract of leaves of Eugenia jambolana 5, 6.
Experimental animals
Male Wistar Albino rats, weighed 200 ± 20 g were
utilized for this study. They were housed in
polypropylene cages under standard laboratory
conditions (12-h light/ 12-h dark cycle, 21 ± 2 ºC,
and relative humidity 55-70 %).
Experimental design
The rats were divided into six groups (A to F), each
group consisting of six rats.
Group A - Served as normal control and received
subcutaneous administration of 5% tween 80 at a
dose of 5 ml/kg on alternate days, for one week.
Group B - Rats were subcutaneously injected with
paracetamol (2g/kg) in a suspension of 5 % tween
80 at a dose of 5 ml/kg body weight, on alternate
days, for a week and were considered as diseased
control.
Group C - Rats were subcutaneously injected with
paracetamol (2g/kg) in a suspension of 5 % tween
80 at a dose of 5 ml/kg body weight and Silymarin
at a dose of 50 mg/kg body weight was treated
orally for a week.
Group D - Rats were subcutaneously injected with
paracetamol (2g/kg) in a suspension of 5 % tween
80 at a dose of 5 ml/kg body weight and extract of
Eugenia jambolana leaves at the dose of 100 mg/kg
was treated orally for one week.
Group E - Rats were subcutaneously injected with
paracetamol (2g/kg) in a suspension of 5 % tween
80 at a dose of 5 ml/kg body weight and extract of
Eugenia jambolana leaves at the dose of 200 mg/kg
was treated orally for one week.
Group F - Rats were subcutaneously injected with
paracetamol (2g/kg) in a suspension of 5 % tween
80 at a dose of 5 ml/kg body weight and extract of
Eugenia jambolana Leaves at the dose of 400
mg/kg was treated orally for one week.
Venkata Kiran Kumar Y. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 1(1), 2012, 80 - 86.
Available online: www.ijrpns.com 82
Acute toxicity study as per OECD guideline 425
In the assessment and evaluation of the toxic
characters of the substance, determination of acute
oral toxicity is usually an initial step. It provides
information of health hazards likely to arise from a
short-term exposure by the oral route. Acute oral
toxicity is the adverse effects occurring within a
short time of oral administration of a single dose of a
substance or multiple doses given within 24h. Data
from an acute study may serve as a basis for
classification and labeling. LD oral, is a statistically
derived single dose of a substance that can be
expected to cause death in 50% of animals when
administered by the oral route. The LD value 50
expressed in terms of test substance per unit weight
of test animal (mg/kg). If there is no reversal before
reaching the selected upper (2000 or 5000 mg/kg)
limit dose, then a specific number of animals are
dosed at the limit dose 7.
RESULTS
Phytochemical investigation on a tropical tree
Eugenia jambolana leaves
The preliminary phytochemical screening like
Saponins, Tannins, Amino acids, Proteins,
Glycosides, Cardiac glycosides, Alkaloids,
Carbohydrates and Flavonoids was done with the
ethanolic extract of Eugenia jambolana leaves
according to the procedure. In the above chemical
test the ethanolic extract of Eugenia jambolana
gives positive results for Saponins, Tannins, Amino
acids, Proteins, Cardiac glycosides, Alkaloids,
Carbohydrates and Flavonoids except glycosides.
The results of preliminary test of ethanolic extract
of Eugenia jambolana leaves were shown in Table
No.1.
Hepatoprotective activity of Eugenia jambolana
leaves
The results of paracetamol - induced hepatotoxicity
are shown in Table No.2. Animals treated with a
four subcutaneous dose of paracetamol developed
significant liver damage in inducer control, as
evident from a significant increase in the serum
activities of Total Bilirubin, Direct bilirubin,
Indirect bilirubin, SGOT, SGPT, Albumin,
Globulin, and Alkaline phosphate concentrations
and a decrease in the total protein compared with
normal control rats, indicating acute hepatocellular
damage and biliary obstruction. The restoration
level of elevated serum enzyme markers and
increase in protein level was observed in silymarin-
treated animals. The elevated levels of serum
markers were reduced in the animal groups treated
with ethanolic extracts Eugenia jambolana leaves.
Among the three graded doses of ethanolic extract,
200 mg/kg showed a significant hepatoprotection
for total protein (6.6 g/dl) only, while at 400 mg/kg,
the restoration was significant for enzyme markers,
viz. SGOT, SGPT, ALP (22.5 U/L, 18.5 U/L, 58.5
U/L ) and for the nonenzyme marker, viz. Bilirubin
in addition to total protein (0.4 mg/dl and 6.2 g/L,
respectively) exhibited significant restoration of
serum markers for all the parameters studied,
indicating its effectiveness.
Histopathological study of Eugenia jambolana
leaves
The histopathological studies of control,
paracetamol, silymarin and test sample-treated liver
sections are shown in Figure No.1. Histological
studies of control animals showed normal
hepatocyte whereas the liver section of paracetamol
-treated animals showed intense centrilobular,
necrosis, vacuolization and macro vesicular fatty
changes. The liver section of standard drug and
silymarin-treated animals showed normal
hepatocyte, which is comparable with the liver
section of normal animals. The animals treated with
different doses of the ethanolic showed recovering
of hepatocyte, especially in the 400 mg/kg bw-
treated group with minimal inflammation and near-
normal architecture possessing higher
hepatoprotective activity as compared with the
ethanolic extract treated at 100 mg/kg which shows
a moderate number of recovered hepatocyte with a
small amount of necrosis, vacuolization and macro
vesicular fatty changes. However, the liver sections
of the animals treated with ethanolic extract of
Eugenia jambolana leaves exhibited significant
liver protection against paracetamol, as evident by
the presence of normal hepatic cords, absence of
Venkata Kiran Kumar Y. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 1(1), 2012, 80 - 86.
Available online: www.ijrpns.com 83
necrosis and fatty infiltration, supplementing the
protective effect of ethanolic extract of Eugenia
jambolana leaves which is comparable to the
standard drug and control animals liver sections.
Acute toxicity study as per OECD guideline 425
Acute toxicity test at 3000mg/kg of leaf extracts of
Eugenia jambolana leaves produced no mortality
after 24 hours of observation. The median lethal
dosage (LD50) of the ethanolic leaves extract was
greater than 3000 mg/kg body weight. The extract
did not produce any grossly negative behavioral
changes such as excitement, restlessness,
respiratory distress, convulsions or coma. However,
a reduction in body weights of the rats was
observed. The reduction in weight may be due to
reduced fluid and water intake, which may be
secondary to feeling of fullness and loss of appetite
after administration of the extract. Despite the
above side effects, the very high value of the LD50
indicated that the extract of Eugenia jambolana
leaves is practically non-toxic. The effect of
Eugenia jambolana extracts at three dose levels
(100, 200 and 400 mg/kg) and paracetamol on
serum marker enzymes, total protein, albumin and
bilirubin in paracetamol-induced hepatic injury.
Hepatic injury induced by paracetamol caused
significant (p < 0.05) rise in SGOT, SGPT, ALP,
cholesterol and bilirubin, but decreases in levels of
total proteins and albumin were observed.
Administration of the plant extract and paracetamol
at three dose levels resulted in recovery as indicated
by the decrease in the hitherto increase of the serum
parameters, produced by paracetamol. The effect
was almost similar to that produced by silymarin
treatment.
DISCUSSION
The paracetamol induced liver damaged rats on
treatment with the ethanolic crude extract at the
dose 400 mg/ kg showed significant changes and
restored the biochemical parameters, thereby
indicating their protection against the injurious
effects of paracetamol, which may be due to the
inhibitory effects on cytochrome P450 resulting in
the hindrance of the formation of hepatotoxicity
free radicals8, 9. Thus, the histological study
supports the hepatoprotective activity of the
Eugenia jambolana from hepatic toxic effect of
paracetamol and silymarin. Our results demonstrate
a very good protective effect of E. jambolana leaves
extract against paracetamol induced liver injury,
which is probably due to the presence of antioxidant
potential of tannins, flavonoids and anthraquinones
present in the extract 10, 11.
Table No.1: Phytochemical screening results of Eugenia jambolana leaves
S.No PHYTOCONSTUTIENT RESULT
1 SAPONINS +
2 TANNINS +
3 AMINO ACIDS +
4 PROTEINS +
5 GLYCOSIDES -
6 CARDIAC GLYCOSIDES +
7 ALKALOIDS +
8 CARBOHYDRATES +
9 FLAVONOIDS +
PRESENT = (+), ABSENT = (-)
Venkata Kiran Kumar Y. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 1(1), 2012, 80 - 86.
Available online: www.ijrpns.com 84
Table No.2: Effect of ethanolic extract of Eugenia jambolana leaves on paracetamol induced
hepatotoxicity in rats
Data represent means±SD (n=6), One Way ANOVA followed by Dunnett’s multiple comparison tests.*p<0.05,
**p<0.01, ***p<0.001 when compared with normal control group, #p<0.05, ##p<0.01 when compared with
diseased control.
Groups Total Bilirubin
(mg/dl) SGOT(U/L) SGPT(U/L) ALP(U/L) Totalprotein
(g/dl) Albumin
(g/dl) Globulin
(g/dl)
Normal Control 0.33±0.07 35.00±9.90 24.50±0.71 57.50±3.54 6.60±0.85 4.00±0.90 2.60±0.57
Paracetamol
induced
Diseased control 0.65±0.09** 75.50±2.12** 69.50±7.78** 112.50±10.61
*** 7.90±0.14** 5.90±0.80
** 2.90±0.14
Paracetamol
+Silymarin
( 50 mg/kg, p.o)
0.33±0.07## 45.00±8.49# 28.50±16.26# 65.00±21.21# 6.25±0.35# 3.90±0.14
# 2.25±0.35
#
Paracetamol +EJ
LEAF
(100 mg/kg, p.o)
0.55±0.07* 50.00±7.07*,# 40.50±2.12*# 76.00±5.66# 6.55±0.07# 3.80±0.28
# 2.75±0.35
Paracetamol +EJ
LEAF
(200 mg/kg, p.o)
0.40±0.09 42.00±2.83# 30.50±6.36# 59.50±7.78# 6.50±0.42# 4.10±0.14
# 2.40±0.28
Paracetamol +EJ
LEAF
(400 mg/kg, p.o)
0.35±0.06# 21.00±2.83*, # 17.00±2.83# 58.00±8.49# 6.10±0.14# 4.00±0.28
# 2.10±0.14
##
Venkata Kiran Kumar Y. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 1(1), 2012, 80 - 86.
Available online: www.ijrpns.com 85
Figure No.1: Histology changes in liver tissues (H&E Staining) with 10X magnification in rats
CONCLUSIONS
In this experimental model, the ethanolic extract
Eugenia jambolana leaves (100, 200, 400 mg/kg)
with reference standard Silymarin (4mg/kg)
significantly effective in abnormalities of enzyme
profile in experimental rats. The leaves have a
potential to be used in the treatment of
hepatoprotectivity. Data on the short and long term
adverse effects of Eugenia jambolana leaves
ingestion needs to be collected.
BIBLIOGRAPHY
1. Sharma D and Dubey S. Biotechnology and conservation of medicinal plants, Journal of Experimental
Sciences, 10, 2011, 2.
2. David Sadava, Craig Heller H, Gordon H. Orians, et al. The free encyclopedia, Life: The science of
biology, 4, 2010, 477.
3. http://www.bioiberica.com/Health_solutions/Digestive_Health/hepatic_disease/Pets.html.
4. Ernst E. Alternative medicine. "Complementary medicine: common misconceptions", J R Soc Med, 88,
1995, 244-247.
Venkata Kiran Kumar Y. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 1(1), 2012, 80 - 86.
Available online: www.ijrpns.com 86
5. Nirali Prakashan, Khandwal KR. Practical Pharmacognosy, Pune, 10th edition, 2003, 158.
6. Kokate CK, Vallabh Prakashan. Practical Pharmacognosy, 1999.
7. OCED 425 guidelines. OCED Guidelines for testing animals, 26, 2001, 1-26.
8. Gowri Shankar NL, Manavalan R, Venkappayya D, David Raj C. Hepatoprotective and antioxidant effects
of Commiphora berryi (Arn) Engl bark extract against CCl4-induced oxidative damage in rats, Food Chem
Toxicol, 46(9), 2008, 3182-3185.
9. Manjunatha BK, Mankani KL, Vidya SM, Krishna V, Manohara YN. Hepatoprotective activity of Butea
superba against carbon tetrachloride induced hepatic damage in rodents, Pharmacog Mag, 4, 2008, S41–
S45.
10. Jain A, Manish S, Lokesh D, Jain A, Rout SP, Gupta VB, et al. Antioxidant and hepatoprotective activity
of ethanolic and aqueous extracts of Momordica dioica Roxb. Leaves, J Ethnopharmacol, 115, 2008, 61–
66.
11. Surendra V, Prakash T, Uday RS, Divakar G, Snehal DF, Kotresha D. Hepatoprotective activity of aerial
parts of Cynodon dactylon against CCl4-induced in rats, Pharmacog Mag, 4, 2008, 195-201.
