Hepatoprotective Activity of Tamarindus indica on Liver Damage Caused by Thioacetamide

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 Tamarindus indica seeds against thioacetamide induced hepatic damage in rats. The plant material were dried in shade then powdered and extracted with methanol. Preliminary phytochemical tests were done. Methanolic extract of Tamarindus indica seeds showed presence of phenolic compound and flavanoids. The degree of protection was measured by using biochemical parameters like serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), bilirubin (BRN), and total protein (TP). Alteration in the levels of biochemical markers of hepatic damage like SGOT, SGPT, ALP, Bilirubin and total protein were tested in both thioacetamide treated and untreaed groups. Thioacetamide has enhanced the SGOT, SGPT, ALP and bilirubin and decrease Total Protein level in liver. Treatment of methanolic extract of Tamarindus indica seeds (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 Tamarindus indica methanol seeds extract possessed hepatoprotective activity. Silymarin was used as reference standard.

KEYWORDS: Tamarindus indica, Hepatoprotective, Silymarin, Thioacetamide, Methanol.

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 methanolic 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.

Tamarindus indica (Fabaceae) is a large evergreen tree, usually about 20 m tall. Externally bark is used as an astringent. Orally it is used as a tonic, febrifuge and the ash obtained by heating the bark with salt in an earthen pot is mixed with water and taken orally for colic, indigestion, as a gargle for sore throat, and as a mouth wash for apthous sores, Hot water extract of dried bark is taken orally for paralysis and as a tonic. Fruit juice mixed with Calotropis gigantean latex is taken orally to relieve menstrual pains. Hot water extract of dried seeds is taken orally for inflammatory swellings and for urinary discharges. Leaf juice is taken orally to treat encephalitis. Four drops of leaf juice with three drops of latex from Caltropis gingantea are taken once a day for eight days. For rheumatic arthritis, leaf juice, latex of Calotropis gigantean, goat milk, and sesame oil is applied externally.

2. MATERIALS AND METHODS:

2.1 Plant Materials: -

The Tamarindus indica seeds were collected in September 2007 and was taxonomically identified and authenticated. The Plant material was authenticated by Dr. H.B. Singh, Head, Raw Material Herbarium & Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. (Ref. NISCAIR/RHMD/consult/-2010-11/1558/156 dated 27/10/10. After authentication, the plant materials were washed thoroughly with water and dried in shade for about five weeks. The dried materials were then ground to coarse powder by using by a mechanical grinder. This powder material was again dried in the shade at about 20°C for one week and store in air tight containers for further studies.

2.2 Drugs and Chemicals: -

Analytical grades Chemical were used in this study. Silymarin (Micro labs, Bangalore) was purchased from local market. Chemical like methanol (CDH, Mumbai), anaesthetic ether (CDH, Mumbai), Thioacetamide (Lova) 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. IAEC/Pharmacy/2012/36). 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 seeds powder of Tamarindus indica 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.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 methanolic extract of Tamarindus indica (METI) 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 Tamarindus indica .^6-9

2.7 Assessment of liver function: -

The methanolic extract of Tamarindus indica was evaluated for their hepatoprotective activity by using Thioacetamide 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 methanolic extract of Tamarindus indica 250 mg and 500 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 Thioacetamide was administered (100mg/kg s.c ) as 2 % w/v solution in double distilled water was administered on 7^th day after 1 hrs of extracts treatment and sacrificed 24 hours after administration of Thioacetamide.⁵ Animals were anesthetized using anesthetic ether and blood sample were collected by cardiac puncture method and serum was used for estimation of SGOT, SGPT, ALP, albumin, total protein, total and direct 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 methanolic extract of Tamarindus indica seeds (METI) 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 5000 mg/kg. So 1/10^th and 1/20^th of these dose i.e. 250 & 500 mg/kg body weight of METI for oral dose was select as therapeutic dose for pharmacological activity screening.

The effects of METI 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 Thioacetamidetreated 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 METI (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 METI (500 mg/kg p.o.) was found to be significantly greater than METI (250 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 methanolic extract of Tamarindus indica seeds were evaluated for the hepatoprotective activity using Thioacetamide induced hepatotoxicity in rat model and find out the therapeutically better efficacious extract. Thioacetamide is a well-known hepatotoxic agent and the preventive action of liver damage by Thioacetamide has been widely used as an indicator of liver protective activity of drugs in general.¹¹ Toxicity experienced by the liver during thioacetamide poisoning results from the production of a metabolite, thioacetamide S-oxide which is a direct hepatotoxin. 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).¹² 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.

Fig No. – 1 Effect of the Tamarindus indica seeds extracts on serum enzyme in Thioacetamide induced hepatic damage in rats.

Fig: 2 Effect of the Tamarindus indica seeds extracts on serum Albumin and total protein in Thiocetamide induced hepatic damage in rats.

Fig: 3 Effect of the Tamarindus indica seeds extracts on serum Direct billirubin and total billirubin in Thiocetamide induced hepatic damage in rats.

Furthermore, protective mechanism not specific to thioacetamide may be responsible for hepatoprotective activity of the chloroform and methanolic extract of the seeds of Tamarindus indica. Thus, the stimulation of hepatic regeneration known to cause the liver to become more resistant to damage by toxins (Lesch et al., 1970)¹³ could explain the hepatoprotective effect of the extracts. Likewise, activation of the functions of reticuloendothelial system (Gruen et al., 1974)¹⁴ or inhibition of protein biosynthesis (Castro et al., 1977)¹⁵ are some of the mechanisms which can reduce the hepatotoxicity of thioacetamide (Iwu et al., 1990 ¹⁶; Dwivedi et al., 1991¹⁷).

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.¹⁸ In the present investigation it was observed that the animals treated with thiocetamide 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.¹⁹

The pretreatment with METI, 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 METI 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.²⁰ Effective control of bilirubin level and alkaline phosphatase activity points towards an early improvement in the secretory mechanism of the hepatic cell.

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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