1. INTRODUCTION

Free radicals are highly reactive substances formed in the body's cells as a result of metabolic processes. A free radical is a chemical species with, which is capable of independent existence and possess one or more unpaired electrons that bestow it with immense reactivity, this reactivity is inversely related to their stability. Main sites of free radical generation are mitochondria, lysosomes, peroxisomes, nuclei, endoplasmic reticulum, plasma membranes and the cytosol. Free radicals, from both endogenous and exogenous sources are implicated in the etiologic of several degenerative diseases such as coronary artery diseases, stroke, rheumatoid arthritis, diabetes and cancer.¹ High consumption of fruits and vegetables is associated with low risk for these diseases, which is attributed to the antioxidant vitamins and other phytochemical.^2-4 There is a great deal of interest in edible plants that contain antioxidants and health promoting phytochemicals, in view of their health implications.

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.⁵ 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. It is cardinal to treat liver disorders since it directly affects the biochemistry of the cell through participating events like oxidative stress, redox change, etc.⁶ which induces lipid peroxidation. Peroxidation of membrane phospholipids not only alters the lipid mileu and structural as well as functional integrity of cell membranes, but also affects the activities of various membrane-bound enzymes including total ATPase, Mg2+ATPase, Ca2+ATPase and Na+K+ATPase.^7-8 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.⁹ Therefore; there is a need to develop satisfactory hepatoprotective drugs. Strobilanthes asperrimus (Acanthaceae) is a high-climbing liana or large shrub, with white blueish flower.is a large, evergreen shurb growing up to 1.5 – 4 m tall.

The leaves is used as a Hypoglycemic. It is also used in goiter, antitumor's, tuberculosis, bactericidal and also in fungicidal

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 and Chemicals: -

Analytical grades Chemical were used in this study. Silymarin (Micro labs, Bangalore) was purchased from local market. Chemical like ethanol (CDH, Mumbai), alcohol (CDH, Mumbai) anesthetic ether (CDH, Mumbai) and CCl₄ (Ranbaxy, Delhi) Thiobarbituric acid, Trichloro acetic acid, H₂O₂, Epinephrine, EDTA, Tric Hcl buffer, GSH, NADP and other phytochemical reagents were obtained from Institute.

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/2012/51). 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.¹⁰

2.5 Acute toxicity study (AOT): -

Acute oral toxicity study was performed according to the procedure OECD guideline no. 425.¹¹ 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 Strobilanthes asperrimus (EESA) 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 Strobilanthes asperrimus.^12-15

2.7 Assessment of antioxidant activity: -

The alcoholic extract of Strobilanthes asperrimus was evaluated for their antioxidant activity in 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 Strobilanthes asperrimus 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 7^th day after 1 hrs of extracts treatment. After the treatment animals were sacrificed by cervical decapitation under excessive anaesthesia on the 8^th day. Immediately after sacrifice, the liver was dissected out, washed in the ice-cold saline, and the homogenate was prepared in 0.1M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged and the supernatant liquid was used for the assay of marker enzymes, namely Glutathione peroxidise by Paglia, D.E., Valentine, W.N., (1967), superoxide dismutase by Saggu et al., (1989)., Misra and Fridovich, (1972), catalase by Chance and Maehly., (1955) and MDA by Esterbauer and Cheeseman (1990).

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 alcoholic extract of Strobilanthes asperrimus leaves (EESA) 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 1/10^th and 1/20^th of these dose i.e. 200 & 100 mg/kg body weight of EESA for oral dose was select as therapeutic dose for pharmacological activity screening.

The effects of EESA on rat liver MDA, SOD, GPx and CAT levels are shown in fig 1-4. The level of MDA was considerably increased in rats treated with CCl₄ as compared to the normal rats. Treatment with EESA (100 mg and 200 mg/kg/day) resulted in a significant decrease (P<0.05) in levels of MDA and brought them near to normal level. A significant decrease in the activities of enzymic antioxidants (SOD, GPx and CAT) were noted after administration of CCl₄. Upon administration of EESA (100 mg and 200 mg/kg/day), the activities of glutathione and enzymic antioxidants were significantly (P<0.05) reversed to near to normal (Fig.1 to 4). The effects of EESA (100 mg and 200 mg/kg/day) comparable with that of standard reference drug Silymarin. The EESA (at dose of 200 mg/kg body weight) show maximum effect as compared to EESA (at dose of 100 mg/kg body weight).

4. DISCUSSION: -

In biological systems, lipid peroxidation (oxidative degradation of polyunsaturated fatty acid in the cell membranes) generates a number of degradation products, such as malondialdehyde (MDA), and is found to be an important cause of cell membrane destruction and cell damage .¹⁶ CCl₄ is commonly used to induce hepatoxicity in animal models. Metabolic processes convert CCl₄ in to the trichloromethyl radical (CCl₃^-) which interacts with O₂ to yield the highly reactive trichloromethylperoxy radical (CCl₃O₂^-). Both radicals are capable of binding to protein and lipid or abstracting a hydrogen atom from unsaturated lipid, which induced lipid peroxidation and leads to changes in the endoplasmic reticulam, reduction in protein synthesis and elevation of serum transaminase enzyme levels.^17-18

Lipid peroxidation as measured by MDA formation increased in the liver tissue of rats treated with CCl₄.^19-20 MDA a secondary product of lipid peroxidation is a major reactive aldehyde, levels can lead to peroxidation of biological membrane. A major defense mechanism involves the antioxidant enzymes, GSH, CAT, and SOD.²¹ Decrease in SOD activity is a sensitive index of hepatocellular damage. SOD scanvenges the superoxide anion to form hydrogen peroxide, thus diminishing toxic effects caused the free radical. CAT is an enzymatic antioxidant widely distributed in all animals’ tissue, highest concentrations are found in erythrocytes and liver cell. CAT decomposed H₂O₂ and protects the tissue from highly reactive hydroxyl radicals.²²

Fig 1 Effect of Ethanolic extracts of S. asperrimus on CAT level in liver tissue

Fig 2 Effect of Ethanolic extracts of S. asperrimus on GPx level in liver tissue

Fig 5.8 Effect of Ethanolic extracts of S. asperrimus on MDA level in liver tissue

Fig 4 Effect of Ethanolic extracts of S. asperrimus on SOD level in liver tissue

CCl₄- treated rats showed significant decreases in GP_X, CAT, and SOD level compare with control. In contrast GSH, CAT, and SOD levels were significantly increased in EESA treated groups, suggesting enhanced antioxidant properties.²³

The qualitative analysis of Strobilanthes asperrimus leaves extract indicated the presence of flavonoids, polyphenols are natural antioxidants but have also been reported to significantly increase SOD, Glutathion and catalase and decrease MDA level.

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 15.04.2013 Accepted on 20.05.2013

Asian J. Pharm. Res. 3(2): April- June 2013; Page 71-74