20-11-2012, 02:16 PM
HEPATOPROTECTIVE ACTIVITY OF SOPHORA INTERRUPTA AND HOLOPTELEA INTEGREFOLIA AGAINST CARBONTETRACHLORIDE INDUCED HEPATOTOXICITY IN RATS
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ABSTRACT
Carbon tetrachloride (CCl4) is a well-known hepatotoxin and exposure to this chemical is known to induce oxidative stress and causes liver injury by the formation of free radicals. The objective of this study was to investigate the hepatoprotective activity of methanolic extracts of Sophora interrupta and Holoptelea integrefolia against CCl4 induced hepatotoxicity. Animals were pretreated with the methanolic extracts of Sophora interrupta (400 mg/kg) and Holoptelea integrefolia (500 mg/kg) for one week and then challenged with CCl4 (1.5 ml/kg) in olive oil (1:1, v/v) on 7th day. Serum marker enzymes (ALP, AST, ALT and Total bilirubin) were estimated in all the study groups. Alteration in the levels of biochemical markers of hepatic damage like AST, ALT, ALP and Total bilirubin were tested in both CCl4 treated and extract treated groups. CCl4 has enhanced the AST, ALT and ALP in liver. Treatment of methanolic extracts of Sophora interrupta (400 mg/kg) and Holoptelea integrefolia (500 mg/kg) exhibited a significant protective effect by altering the serum levels of AST, ALT, ALP and Total bilirubin. These biochemical observations were supported by histopathological study of liver sections. From this preliminary study it has been concluded that among the two extracts tested, the methanolic extract of Sophora interrupta and Holoptelea integrefolia found to possess significant protective effect against CCl4 induced hepatotoxicity.
INTRODUCTION
Liver - a major metabolic organ affected by various chemicals and toxins daily and identification of a successful hepatoprotective agent will provide a useful tool for the treatment of hepatic diseases. In absence of reliable liver-protective drugs in modern medicine, a large number of medicinal preparations are recommended for the treatment of liver disorders and quite often claimed to offer significant relief1. Exposure to various organic compounds including a number of environmental pollutants and drugs can cause cellular damages through metabolic activation of those compounds to highly reactive substances such as reactive oxygen species (ROS). Carbon tetrachloride (CCl4) is a well-known hepatotoxin and exposure to this chemical is known to induce oxidative stress and causes liver injury by the formation of free redicals2.Sophora interrupta- Bedd commonly known as “ Pili Girgoli3 ” is a woody perennial shrub which grows endemically in seshachalam hill ranges, seshatheertham and kumaradhara theertham in Tirumala, India, belonging to the family “Leguminosae”. It contains Matrine, Oxymatrine type of Alkaloids4,5 , Flavonoids6,7 , Saponins and Polysaccharides8. It possess wide-reaching pharmacological actions, including anti-oxidant, anti-cancer, anti-asthmatic, anti-neoplastic, antimicrobial, anti-viral, antidote, anti-pyretic, cardio tonic, anti-inflammatory, diuretic and in the treatment of skin diseases like eczema, colitis and psoriasis.
MATERIALS AND METHODS
Plant Materials
The fresh leaves of Sophora interrupta and Holoptelea integrefolia were collected from Sri Venkateshwara University, Tirupati, Andhra Pradesh, India, in June 201014. The plant was authenificated by a Botanist, Dr. K. Madhava Chetty, Assistant professor, Department of Botany and voucher specimen was deposited in Sri Venkateshwara University, and a copy has been preserved for the future reference at the herbarium of the institute TRRCP. After authentification, the leaves were cleaned and shade dried and milled into coarse powder by a mechanical pulverizer.
Preparation of Extract
The coarse powder of plant material was defatted with petroleum ether (60-80°C) in a soxhlet extraction apparatus and marc was extracted with methanol (1000 ml). Overnight, at room temperature with constant stirring. The extract was filtered and the filtrate was concentrated at 30°C under reduced pressure in a rotary evaporator. Extract was dried in dessicator. The crude extract was suspended in 1% Tween-80 to required concentrations and used for the experiments.
Phytochemical screening
The methanolic extract obtained was subjected to preliminary phytochemical screening, to identify the chemical constituents15. The phytoconstituents in the extract was found to contain alkaloid, flavonides, glycosides, steroids and tannins.
Formulation: Suspensions were formulated of required concentrations 300 mg/kg and 500 mg/kg by using 1% Tween-80 and double distilled water. The formulated suspensions were compared for various evaluation parameters.
PHARMACOLOGICAL STUDIES
Animals
Male Wistar rats weighing between 150-200 gm were used for this study. The animals were obtained from NIN, Hyderabad, India. The animals were placed in polypropylene cages with paddy husk as bedding. Animals were housed at a temperature of 24±2°C and relative humidity of 30-70 %. A 12:12 light: day cycle was followed. All animals were allowed to free access to water and fed with standard commercial pelleted diet. All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics Committee (IAEC) and were in accordance with the guidelines of the CPCSEA (No. 1447/PO/a/11/CPCSEA).
Hepatoprotective Activity
Animals were divided into 5 groups of six rats each. Group- I and II served as normal and toxic control, and received only the vehicle (1% Tween-80; 1 ml/kg; p.o). Group- III animals were treated with standard silymarin at an oral dose of 100 mg/kg and group- IV and group- V received the Sophora interrupta and Holoptelea integrefolia extract at an oral dose of 400 mg/kg and 500 mg/kg respectively, as a fine suspension of 1% Tween-80. The treatment was continued for 7days, once daily. On the day of 7 for groups II - V, 30 min post-dose of extract administration animals received CCl4 at the dose of 1.5 ml/kg (1:1 v/v of CCl4 in olive oil) orally16, 17, 18.
Biochemical estimation
The animals were sacrificed 36h after administration of acute dose of CCl4. The blood was collected by retro orbital artery bleeding. Blood samples were centrifuged for 10 min at 3000 rpm to separate the serum. Alanine Transaminase (ALT), Aspartate Transaminase (AST), Alkaline Phosphatase (ALP) and Total Bilirubin (TB) levels were estimated from the serum by using standard kits19.