Assessment of Hepatoprotective Activity of Strobilanthes
asperrimus in Thioacetamide
Induced Hepatoxic Rats
Pradeep Kumar Samal*
SLT Institute of Pharmaceutical Sciences, Guru
Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh.
ABSTRACT:
The objective of
this study was to investigate the hepatoprotective
activity of ethanolic
extract of Strobilanthes asperrimus leaves
against Thioacetamide induced acute hepatotoxicity. The plant materials were dried in shade
then powdered and extracted with alcohol. Preliminary phytochemical
tests were done. Ethanolic extract showed presence of
phenolic compound and flavanoids.
The hepatoprotective activity of the ethanolic extract was assessed in Thioacetamide
induced hepatotoxic rats. Alteration in the levels of
biochemical markers of hepatic damage like SGOT, SGPT, ALP, Billirubin
and Protein were tested in both Thioacetamide treated
and untreated groups. Thioacetamide (100mg/kg s.c) has enhanced the SGOT, SGPT, ALP and Total Billirubin where decrease in total protein level in liver.
Treatment of ethanolic extract of Strobilanthes asperrimus (500mg/kg) has brought back
the altered levels of biochemical markers to the near normal levels in the dose
dependent manner. Our findings suggested that Strobilanthes asperrimus ethanolic
leaves extract possessed hepatoprotective activity.
Moreover, it prevented Thioacetamide induced
prolongation in pentobarbital sleeping time confirming hepatoprotectivity
and validates the traditional use of this plant against liver damage.
KEYWORDS: Strobilanthes asperrimu, Hepatoprotective, Silymarin, Thioacetamide , ethanol
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 varied. 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 diseases. The
conventional drugs used in the treatment of liver diseases viz., corticoasteroids, antiviral and immunosuppressant agents
are so EESA 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. Therefore;
there is a need to develop satisfactory hepatoprotective
drugs.
2. MATERIALS AND METHODS:
2. 1 Plant Materials: - Fresh leaves of Strobilanthes asperrimus (Acanthaceae) were collected from Thakur Chedilal
Barristor Agriculture College and Research Centre, Bilaspur, India, in the month of September 2011, 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 & Museum, NISCAIR, New
Delhi (Ref:- NISCAIR/RHMD/Consult/-2011-12/1830/130).
2.2
Drugs, Chemicals and
Biochemical kits: - Drug silymarin
(Micro labs, Bangalore) was purchased from local Medicine store (Jagat Medical Hall, Bilaspur). Thioacetamide
(Lova Laboratories Pvt. Ltd., Mumbai) were purchased through
local dealer (Prateek scientific & Stationery, Bilaspur). Chemicals like Anesthetic ether (CDH, Mumbai)
and Methanol (S.D. Fine-Chem Ltd., Mumbai) and
reagents (L R grade) used for phytochemical analysis
were provided by university. 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 School of
pharmaceutical sciences, S.O.A. University, Bhubaneswar was taken prior to the experiments
(1171/c/08/CPCSEA). 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 Strobilanthes asperrimus was extracted with 1.2 L of
alcohol using Soxhlet apparatus for 72 hrs at 40-50°C. The extract was
concentrated to Ľ of its original volume by distillation as it was adapted to
recover the solvent, which could be used again for extraction.
2.5
Acute toxicity study (AOT): - Acute toxicity study was performed according
to the procedure OECD guideline no. 425.
AOT was performed on Swiss albino mice and the animal were kept fasting
for overnight providing water ad libitum, after which
the ethanolic extract of Strobilanthes asperrimus (EESA) was administered orally 5000 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 Strobilanthes asperrimus .
2.7
Assessment of liver
function: -
Either sex adult albino rats
(Wistar strain) weighing between 175-230 g body
weights were divided into 5 groups, each group consisting of 6 rats. Group 1
received distilled water (5 ml/kg, p.o.) for 7 days.
Group 2 were treated with vehicle (2 % tween 80, 1
ml/kg, p.o.) for 7 days. Group 3 received silymarin (50 mg/kg, p.o.) for 7
days. Group 4 & 5 were pretreated with ethanolic
extract of Strobilanthes asperrimus leaves 250 and 500 mg/kg respectively for 7
days. Food was withdrawn 16 hrs before Thioacetamide
administration to enhance the acute liver
damage in animals Group II, III, IV and V. Rats of above groups were
treated with a single dose of Thioacetamide (100
mg/kg s. c.) as 2 % w/v solution in
double distilled water after 1 hrs of last treatment.
On
the 8th day, the rats were anesthetized with light ether anesthesia
and blood sample were collected by cardiac puncher. The collected blood samples
were allowed to clot for 45min at room temperature. Serum was separated by
centrifugation at 4000 rpm for 20 min. Serum was used for estimation of SGOT, SGPT,
ALP, albumin, total protein, direct and total bilirubin.
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
Kramer’s 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 ethanolic extract of Strobilanthes asperrimus leaves (EESA) was screened in albino mice for their
acute oral toxicity. No mortality was recorded till 5000 mg/kg body weight. Hence the extract was found to be safe up to
the dose levels of 5000 mg/kg. So 1/10th
and 1/20th of these dose i.e. 250 & 500 mg/kg body weight of
EESA 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: 3.1 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 Thioacetamide induced toxic
control group (group-2) of animals was found to 229 ± 8 min. When sleeping time
of toxic control group of animals was compared with test groups, the higher
dose of EESA (500mg/kg body wt) (Group-V) was highly significant 182±2 min (P<0.001)
which is very nearer to that of silymarine induced
standard (Group-3) (177±5). The effects of EESA 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 Thioacetamide induced liver damage in rats are summarized
in Table - 3.2 and 3.3. Administration of Thioacetamide
(100 mg/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 Thioacetamide 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 EESA (Group IV & 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 EESA (500 mg/kg p.o.) was found to
be significantly greater than EESA (250 mg/kg p.o.).
4.
DISCUSSION:
Toxicity
experienced by the liver during thioacetamide
poisoning results from the production of a metabolite, thioacetamide
S-oxide which is a direct hepatotoxin (Neal and Halpert, 1982).1 Thioacetamide
induces centrilobular necrosis within 3 h of
administration. It has also been observed that thioacetamide
causes specific changes in the nucleolus and increased synthesis of guanine and
cytosine-rich RNA, with concomitant decrease in ribosomal RNA in the cytoplasm
(Zimmerman, 1976).2 It is quite likely that the extracts under study
antagonize the effect of thioacetamide by acting,
either as membrane stabilizer, thereby preventing the distortion of the cellular
ionic environment associated with thioacetamide
intoxication, or by preventing interaction of thioacetamide
with the transcriptional machinery of the cells.
Furthermore, protective mechanism not specific to thioacetamide may be responsible for hepatoprotective
activity of the methanolic extract of the leaves of
Strobilanthes asperrimus.
Thus, the stimulation of hepatic regeneration known to cause the liver to
become more resistant to damage by toxins (Lesch et
al., 1970)3 could explain the hepatoprotective
effect of the extracts. Likewise, activation of the functions of reticuloendothelial system (Gruen
et al., 1974)4 or inhibition of protein biosynthesis (Castro et al.,
1977)5 are some of the mechanisms which can reduce the hepatotoxicity of thioacetamide (Iwu et al., 19906; Dwivedi
et al., 19917).
Table No. – 3.1 Effect of the Strobilanthes asperrimus leaves extracts on pentobarbital Induced
sleeping time in Thioacetamide induced hepatotoxic
rats.
|
Group |
Treatment |
Dose |
Mean Sleeping time (min) |
|
I |
Solvent control |
5ml/kg p.o |
145±4 |
|
II |
Vehicle + Thioacetamide |
1 ml/kg p.o (2 ml/kg, s.c.) |
229±8*** |
|
III |
Silymarin + Thioacetamide |
50mg/kg p.o (2 ml/kg, s.c.) |
177±5*** |
|
IV |
EESA + Thioacetamide |
100mg/kg p.o (2 ml/kg, s.c.) |
197±4*** |
|
V |
EESA + Thioacetamide |
200mg/kg p.o (2 ml/kg, s.c.) |
180±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
Kramer’s post hoc test.
Table No. – 3.2 Effect of the Strobilanthes asperrimus leaves extracts on
serum enzyme in Thioacetamide 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 + Thioacetamide |
436.16±7.45 |
196.48±2.88 |
283.10±3.12 |
|
III |
Silymarin + Thioacetamide |
218.40±2.77*** |
69.46±2.75*** |
186.23±9.85*** |
|
IV |
EESA + Thioacetamide |
303.41±4.65*** |
112.78±4.78*** |
237.45±3.56** |
|
V |
EESA + Thioacetamide |
227.56±4.62*** |
86.23±3.511*** |
192.35±13.32*** |
Values expressed
as mean ± SEM, from six observations, **p<0.01, ***p<0.001 when compared
with Thioacetamide
control group. Using one-way ANOVA followed by Tukey
Kramer’s post hoc test.
Table No. -3.3 Effect of the Strobilanthes asperrimus leaves extracts on serum biochemical
parameters in Thioacetamide induced hepatic damage in rats.
|
Group |
Treatment |
Albumin (mg/dl) |
Total protein (mg/dl) |
Direct bilirubin (mg/dl) |
Total bilirubin (mg/dl) |
|
I |
Solvent control |
4.67±0.29 |
14.78±0.54 |
0.28±0.01 |
0.39±0.01 |
|
II |
Vehicle + Thioacetamide |
2.47±0.0.27 |
7.089±0.15 |
0.81±0.06 |
1.09±0.05 |
|
III |
Silymarin + Thioacetamide |
3.94±0.18** |
13.92±0.41*** |
0.391±0.026*** |
0.59±0.01*** |
|
IV |
EESA + Thioacetamide |
2.49±0.35 |
10.21±0.75** |
0.62±0.020*** |
0.85±0.02*** |
|
V |
EESA + Thioacetamide |
3.68±0.19* |
12.13±0.45 *** |
0.58±0.072*** |
0.65±0.01*** |
Values expressed
as mean ± SEM, from six observations, *p<0.05, **p<0.01 and ***p<0.001
when compared with Thioacetamide control group. Using one-way ANOVA followed
by Tukey Kramer’s post hoc test.
In the present
study, thioacetamide 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 EESA the sleeping time was decreased
as compared to thioacetamide treated group and nearly
restored back to the initial sleeping time.
In living systems, liver is considered to be
highly sensitive to toxic agents. The study of different enzyme activities such
as SGOT, SGPT, SALP, albumin, total protein, direct bilirubin
and total bilirubin have been found to be of great
value in the assessment of clinical and experimental liver damage.8
In the present investigation it was observed that the animals treated with thioacetamide resulted in significant hepatic damage as
shown by the elevated levels of serum markers. These changes in the marker
levels will reflect in hepatic structural integrity. The rise in the SGOT is
usually accompanied by an elevation in the levels of SGPT, which play a vital
role in the conversion of amino acids to keto acids.9
The pretreatment with CETI and EESA, both at
the dose of 250mg/kg and 500mg/kg, significantly attenuated the elevated levels
of the serum markers. The normalization of serum markers by CETI and EESA
suggests that they are able to condition the hepatocytes
so as to protect the membrane integrity against Thiocetamide
induced leakage of marker enzymes into the circulation. The above changes can
be considered as an expression of the functional improvement of hepatocytes, which may be caused by an accelerated
regeneration of parenchyma cells. Serum ALP and bilirubin
levels, on the other hand are related to hepatic cell damage. Increase in serum
level of ALP is due to increased synthesis in presence of increasing billiary pressure.10 Effective control of bilirubin level and alkaline phosphatase
activity points towards an early improvement in the secretory
mechanism of the hepatic cell.
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 Thioacetamide
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 Thioacetamide was observed,
indicating considerable hepatocellular injury. Our
results indicated that Strobilanthes asperrimus (500
mg/kg) administration significantly alleviated the increased serum enzyme
activity induced by Thioacetamide, indicating
improvement of the functional status of the liver. The recovery towards
normalization of serum enzymes and liver histological architecture caused by Strobilanthes asperrimus 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 Strobilanthes asperrimus in
Thioacetamide 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 Strobilanthes asperrimus 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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