Hepatoprotective Activity of Gloriosa superba on
Liver Damage Caused by CCL4 in Rats
Pradeep Kumar Samal
SLT Institute of
Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur Chhattisgarh
*Corresponding Author E-mail: samalpharmacology@rediffmail.com
ABSTRACT:
The objective of this study was to
investigate the hepatoprotective activity of
alcoholic extract of Gloriosa superba
leaves against carbon tetrachloride (CCl4) induced hepatotoxicity. The plant material were dried in shade then
powdered and extracted with alcohol. Preliminary phytochemical
tests were done. Alcoholic extract showed presence of phenolic
compound and flavanoids. The hepatoprotective
activity of the alcoholic extract was assessed in CCl4 induced hepatotoxic rats. Alteration in the levels of biochemical
markers of hepatic damage like SGOT, SGPT, ALP, Billirubin
and Protein were tested in both CCl4 treated and untreated groups.
CCl4 (1ml) has enhanced the SGOT, SGPT, ALP and Total Billirubin
where decrease in total protein level in liver. Treatment of alcoholic extract
of Gloriosa superba
(200mg/kg) has brought back the altered levels of biochemical markers to the
near normal levels in the dose dependent manner. Our findings suggested that Gloriosa superba
alcoholic leaf extract possessed hepatoprotective
activity. Moreover, it prevented CCl4 induced prolongation in pentobarbital
sleeping time confirming hepatoprotectivity and
validates the traditional use of this plant against liver damage.
KEYWORDS: Gloriosa superba , Hepatoprotective,
Silymarin, CCl4, alcohol
1.
INTRODUCTION:
Liver
is the key organ of metabolism and excretion is constantly endowed with the
task of detoxification of xenobiotics, environmental
pollutants and chemotherapeutic agents. Thus, disorders associated with this
organ are numerous and varied1. Liver disease has become a global concern
worldwide. Liver is often abused by environmental toxins, poor eating habits,
alcohol and over-the-counter drug use, that damage and weaken the liver leading
to important public health problems like hepatitis, cirrhosis and alcoholic
liver diseases2. The conventional drugs used in the treatment of
liver diseases viz., corticoasteroids, antiviral and
immunosuppressant agents are sometimes inadequate and may lead to serious adverse effects. In India, numerous medicinal
plants and their formulations are used for liver disorders in traditional systems
of medicine. Some of these plants are evaluated for their hepatoprotective
actions against hepatotoxins.
However, the readily available hepatoprotective herbal drugs are not sufficiently active
to effectively combat severe liver disorders. In view of lack of synthetic
agents for the treatment of hepatic disorder, there is a growing focus to
evaluate traditional herbal medicines for hepatoprotective
activity.3 Therefore; there is a need to develop satisfactory hepatoprotective drugs. Gloriosa superba (Colchicacea)
is a large, evergreen shurb growing up to 1.5 4 m
tall. Leaves are 10-20 cm long oblanceolate to obovate, entire, tip narrow with short stalks. Its bark is
used in Arthritis, Rheumatism where as its leaf is used in Fabrifuge
generally healing, stomach troubles.
2. MATERIALS
AND METHODS: -
2.1 Plant
Materials:
The leaves of Gloriosa superba (Colchicacea) were collected from Thakur Chedilal Barristor Agriculture
College and Research Centre, Bilaspur, India, in the month of Oct
2012, and air dried at room temperature after wash with tape water. The Plant identification was
done by Dr. H. B. Singh Chief Scientist Head of the Raw Materials Herbarium and
Museum, NISCAIR, New Delhi (NISCAIR/ RHMD/Consult/-2012-13/2164/170).
Table No. 1 Effect of the Gloriosa superba leaves extracts on pentobarbital Induced
sleeping time in CCl4 induced hepatotoxic
rats.
Group |
Treatment |
Dose |
Mean Sleeping
time (min) |
I |
Solvent control |
5ml/kg p.o |
145±4 |
II |
Vehicle+CCl4 |
1 ml/kg p.o(2 ml/kg, s.c.) |
229±8*** |
III |
Silymarin+CCl4 |
50mg/kg p.o(2 ml/kg, s.c.) |
177±5*** |
IV |
AEGS+CCl4 |
100mg/kg p.o(2 ml/kg, s.c.) |
199±4*** |
V |
AEGS+CCl4 |
200mg/kg p.o(2 ml/kg, s.c.) |
189±3*** |
Values expressed as mean ± SEM,
from six observations, ***p<0.001 when compared with normal control group.
Using one-way ANOVA followed by Tukey Kramers post
hoc test.
2.2 Drugs and Chemicals:
Analytical
grades Chemical were used in this study. Silymarin
(Micro labs, Bangalore) was purchased from local market. Chemical like ethanol
(CDH, Mumbai), anaesthetic ether (CDH, Mumbai), CCl4
(Ranbaxy, Delhi) and other phytochemical reagents
were obtained from Institute. For estimation of biochemical parameter;
biochemical kits like SGOT, SGPT, ALP, albumin, total protein, direct bilirubin and total bilirubin
were obtained from Span Diagnostics ltd. Surat, India
were procured from Matushri Trading Company, Bilaspur.
2.3 Animals:
Each
experiment had separate set of animals and care was taken to ensure that
animals used for one response were not employed elsewhere. Animals were
habituated to laboratory conditions for 48 hours prior to experimental protocol
to minimize if any of non-specific stress. The approval of the Institutional
Animal Ethical Committee (IAEC) of SLT Institute of Pharmaceutical Sciences, Bilaspur (Chhattisgarh) was taken prior to the experiments (Reference No.
25/IAEC/Pharmacy/2013 dated 15/06/13). All the protocols and the
experiments were conducted in strict compliance according to ethical principles
and guidelines provided by Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA).
2.4
Preparation of plant Extracts:
About
300 g of the leaves powder of Gloriosa superba was extracted with 1.2 L of alcohol using
Soxhlet apparatus for 72 hrs at 40-50°C. The extract was concentrated to Ό of
its original volume by distillation as it was adapted to recover the solvent,
which could be used again for extraction.4
2.5 Acute toxicity study (AOT):
Acute
toxicity study was performed according to the procedure OECD guideline no. 425.5 AOT was performed on Swiss albino mice and
the animal were kept fasting for overnight providing water ad libitum, after which the alcoholic extract of Gloriosa superba (AEGS) was
administered orally 2000 mg/kg and observed the mortality of animals.
2.6 Preliminary
Phytochemical Analysis:
The extracts
obtained were subjected to various chemical tests to detect the chemical
constituents present in extracts of Gloriosa superba .6-9
2.7 Assessment of liver function:
The
alcoholic extract of Gloriosa superba was evaluated
for their hepatoprotective activity by using CCl4
induced acute hepatotoxicity model. Either sex of
Wistar rats, weighing (180-220) was divided into 5 groups consisting of 6
animals in each group. Group 1 received distilled water (6 ml/kg, p.o.) for 7 days. Group 2 were treated with vehicle (0.5%
of Tween 40, 1 ml/kg, p.o.)
for 7 days. Group 3 received silymarin (50 mg/kg, p.o.) for 7 days. Group 4, 5 pretreated with alcoholic
extract of Gloriosa superba 100 mg and 200
mg/kg body weight respectively for 7 days. Food was withdrawn 16 hrs before
administration to enhance the acute liver toxicity. Group 2, 3, 4 and 5 were treated
with CCl4 was administered (2 ml/kg, s.c.)
diluted in olive oil (1:1) was administered on 7th day after 1 hrs
of extracts treatment and sacrificed 24 hours after administration of CCl4.5
Animals were anesthetized using anesthetic ether and blood sample were
collected by cardiac puncture method and serum was used for estimation of AST,
ALT, ALP, albumin, total protein, total and direct bilirubin.
Immediately after the collection of blood, their livers were separated. The
liver was washed by normal saline were preserved in 10% formalin for histopathological studies.
2.8 Statistical analysis:
The experimental results were expressed as the Mean
± SEM for six animals in each group. The biochemical parameters were analysed statistically using one-way ANOVA followed by Tukey Kramers post hoc test. P value of < 0.05 was considered as statistically significant.
3. RESULTS:
Preliminary phytochemical
studies with extract revealed the phytoconstituents
like cardiac glycoside, carbohydrates, phytosterols, saponins, phenolics and tannins.
Different doses of alcoholic extract of Gloriosa superba
leaves (AEGS) was screened in albino mice for their acute oral toxicity.
No mortality was recorded till 2000 mg/kg body weight. Hence
the extract was found to be safe up to the dose levels of 2000 mg/kg. So 1/10th and 1/20th
of these dose i.e. 200 and 100 mg/kg body weight of AEGS for oral dose was
select as therapeutic dose for pharmacological activity screening.
Effect of plant extract on pentobarbital sleeping time was studied in rats and
the results are shown in Table: 1 Pentobarbital at a dose of 75 mg/kg. i.p. in normal control( group-1) caused sleep in rats for a
period of 145 ± 4 min (mean ± SEM. n = 6). Whereas the sleeping time in the CCl4
induced toxic control group (group-2) of animals was found to 229 ± 8 min.
Table No. 2 Effect of the Gloriosa superba leaves extracts on serum enzyme in CCl4
induced hepatic damage in rats.
Group |
Treatment |
SGOT(IU/L) |
SGPT(IU/L) |
ALP(IU/L) |
I |
Solvent control |
96.38±4.02 |
48.28±3.63 |
107.45±8.98 |
II |
Vehicle+CCl4 |
436.16±7.45 |
196.48±2.88 |
283.10±3.12 |
III |
Silymarin+CCl4 |
218.40±2.77*** |
69.46±2.75*** |
186.23±9.85*** |
IV |
AEGS+CCl4 |
389.27±9.56*** |
116.14±4.89 *** |
231.12±11.26 |
V |
AEGS+CCl4 |
283.10±3.52*** |
94.41±3.781 *** |
205.16±8.46*** |
Values
expressed as mean ± SEM, from six observations, **p<0.01, ***p<0.001 when
compared with CCl4 control group. Using one-way ANOVA followed by Tukey Kramers post hoc test.
Table No. 3 Effect of the Gloriosa superba leaves extracts on serum biochemical in
CCl4 induced hepatic damage in rats.
Group |
Treatment |
Dose |
Albumin (mg/dl) |
Total
protein(mg/dl) |
Direct
bilirubin (mg/dl) |
Total
bilirubin (mg/dl) |
I |
Solvent
control |
5ml/kg
p.o |
4.67±0.29 |
14.78±0.54 |
0.28±0.01 |
0.39±0.01 |
II |
Vehicle+CCL4 |
1
ml/kg p.o100mg/kg s.c |
2.47±0.0.27 |
7.089±0.15 |
0.81±0.06 |
1.09±0.05 |
III |
Silymarin+CCL4 |
50mg/kg
p.o100mg/kg s.c |
3.94±0.18** |
13.92±0.41*** |
0.391±0.026*** |
0.59±0.01*** |
IV |
AEGS+CCL4 |
250mg/kg
p.o100mg/kg s.c |
3.59±0.18* |
11.86±0.57
*** |
0.5±0.01* |
0.75±0.02*** |
V |
AEGS+CCL4 |
500mg/kg
p.o100mg/kg s.c |
4.04±0.062*** |
13.21±0.24*** |
0.42±0.031** |
0.6±0.01*** |
Values
expressed as mean ± SEM, from six observations, **p<0.01, ***p<0.001 when
compared with CCl4 control group. Using one-way ANOVA followed by Tukey Kramers post hoc test.
When sleeping time of
toxic control group of animals was compared with test groups, the higher dose
of AEGS (200mg/kg body wt) (Group-V) was highly significant189±3 min (P<0.001)
which is very nearer to that of silymarine induced
standard (Group-3) (177±5). The
effects of AEGS on Serum glutamate oxaloacetate transaminase (SGOT), Serum glutamate pyruvate
transaminase (SGPT), Alkaline phosphatase
(ALP), Serum direct bilirubin(DBIL), Sreum total bilirubin(TBIL),
Serum albumin(ALB) and Serum total protein(TLP) levels in CCl4
induced liver damage in rats are summarized in Table - 2 and 3. Administration
of CCl4 (2 ml/kg, s.c.), after 24
hours of intoxication resulted a significant (P<0.05) elevation of hepatospecific serum enzymes markers like SGOT, SGPT and
ALP and serum biochemicals markers like DBIL and TBIL
in CCl4 treated groups, while seum biochemicals markers like albumin and total protein were
found to be decreased in comparison with the normal control group. On
administration of AEGS (Group IV and V) and Silymarin
at the dose of 50mg/kg (Group III) the level of these enzymes and biochemicals were found retrieving towards normalcy. The hepatoprotective effect offered by AEGS (200 mg/kg p.o.) was found to be significantly greater than AEGS (100
mg/kg p.o.).
4. DISCUSSION:
In
recent years, many studies have been undertaken with traditional medicines, in
an attempt to develop new drugs for hepatitis.10 In the present
study alcoholic extract of Gloriosa superba leaves
were evaluated for the hepatoprotective activity using
CCl4 induced hepatotoxicity in rat model
and find out the therapeutically better efficacious extract. CCl4 is
a well-known hepatotoxic agent and the preventive
action of liver damage by CCl4 has been widely used as an indicator
of liver protective activity of drugs in general.11 The changes
associated with CCl4-induced liver damage are similar to that of
acute viral hepatitis.12 CCl4 is biotransformed
by Cytochrome P-450 system to produce trichloromethyl free radicals. These free radicals may
again react with oxygen to form trichloromethyl peroxyl radicals, which may attack lipids on the membrane
of endoplasmic reticulum to elicit lipid peroxidation,
finally resulting in cell necrosis and consequent cell death.13 As
well known, phenolic antioxidants, such as flavonoids and tannins, are considered promising
therapeutic agents for free radical pathologies due to their scavenging ability
with ROS.14
In the present study, CCL4
was found to cause significant prolonged the pentobarbital induce sleeping
time. In spite of a large biological
variation in sleep time, it was observed that sleep time was reduced in the
treated group. In the group of animals administered with AEGS the sleeping time
was decreased as compared to CCL4 treated group and nearly restored
back to the initial sleeping time.
SGOT
is found in the liver, cardiac muscles, skeletal muscles, pancreas, lungs,
kidney, brain, etc., whereas SGPT concentrationis
highest in the liver and therefore, it appears to be a more sensitive test to hepatocellular damage than SGOT.15 Leakage of
large quantities of enzymes into the blood stream is often associated with
massive necrosis of the liver.16 CCl4 is known to cause
marked elevation in serum enzymes (SGOT and SGPT). In the present study, a
significant increase in the activities of SGOT and SGPT within 24 hr exposure
to CCl4 was observed, indicating considerable hepatocellular
injury. Our results indicated that Gloriosa superb
(200 mg/kg) administration significantly alleviated the increased serum enzyme
activity induced by CCl4, indicating improvement of the functional
status of the liver. The recovery towards normalization of serum enzymes and
liver histological architecture caused by Gloriosa superba was almost similar to that caused by silymarin,
in the present study. Similar results have been reported.17 Silymarin is a known hepatoprotective
compound. It is reported to have a protective effect on the plasma membrane of
hepatocytes.18
In
conclusion the possible hepatoprotective effect of Gloriosa superba in
CCl4 induced liver injuries may be due to: (1) inhibiting Cytochrome P- 450 activity, (2) preventing the process of
lipid peroxidation, (3) stabilizing the hepatocellular membrane and (4) enhancing protein and
glycoprotein biosynthesis. However the exact hepatoprotective
mechanism of Gloriosa superba is
still unknown. Further studies are warranted to isolate the active components.
5. ACKNOWLEDGEMENTS:
The authors wish to thank Prof. J.S. Dangi, Head of the Institute for facilities and Mr. Karteek Patra for technical
assistance.
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Received on 30.10.2013 Accepted on 22.11.2013
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J. Pharm. Res. 3(4): Oct. - Dec.2013; Page 194-197