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 ¼ 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.

 

6. REFERENCES:

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2.       Lesch, R., Reutter, W., Keppler, D. and Decker, K. (1970) Liver restitution after acute galactosamine hepatitis autoradiographic and biochemical studies in rats. Experimental and Molecular Pathology 12, 58-69.

3.       Gruen, M., Leihr, H., Gruen, W., Rasenack, U. and Branswig, D. (1974) Influence of liver-RES (reticulo-endothelial system) on toxic liver damage due to galactosamine. Acta

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7.       Dwivedi, Y., Rastogi, R., Sharma, S.K., Garg, N.K. and Dhawan, B.N. (1991) Picroliv affords protection against thioacetamide-induced hepatic damage in rats. Planta Medica 57, 25-28.

8.       Vaishwanar I, Kowale CN. Effect of two ayurvedic drugs Shilajeet and Eclinol on changes in liver and serum lipids produced by carbontetrachloride. Ind J Exp Biol 1976; 14: 58-61.

9.       Sallie R, Tredger JM, Willaiam. Drugs and the liver. Biopharm Drug Dispos 1999; 12: 251-259.

10.     Moss DW, Butterworth PJ. Enzymology and Medicine, London, Pitman Medical, 1974, p139.

11.     Liu, G.T., 1989. Pharmacological actions and clinical use of Fructus shizandrae. Journal of Chinese Medicine 102, 740–749.

12.     Rees, K.R., Spector, W.G., 1961. Reversible nature of liver cell damage due to carbon tetrachloride as demonstrated by the use of Phenergan. Nature (London) 189, 821–829.

13.     Morazzoni, P., Bombardelli, E., 1995. Silybium marianum (Carduus marianus). Fitoterapia 66, 3–42.

14.     Ramellini, G., Meldolesi, J., 1976. Liver protection by silymarin. In vitro effects on dissociated rat hepatocytes. Arzneimittel Forschung (Drug Research) 26, 69–73.

 

Received on 07.04.2013

Modified on 11.04.2013

Accepted on 14.04.2013

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 5(3): May-June 2013, 139-142