Hepatoprotective Effect of Methanolic Leaves Extracts of Tylophora asthmatica Against Paracetamol-Induced Liver Damage in Rats

INTRODUCTION

Liver is one of the most important organs in the biotransformation of food, drugs, endogenous and exogenous substances. Profuse supply of blood and the presence of many redox systems (e.g., cytochromes and various enzymes) enable liver to convert these substances into different kinds of inactive, active or even toxic metabolites. The burden of metabolism and exposure to dangerous chemicals make liver vulnerable to a variety of disorders, such as acute or chronic inflammation, toxin-/drug-induced hepatitis, cirrhosis and hepatitis after viral infection (Sherlock and Dooley, 2002). Drugs can cause toxic effects which can mimic almost every naturally occurring liver disease. Normally, the toxins and drugs are absorbed from the intestinal tract gain access first to the liver resulting in a variety of liver ailments. Thus liver diseases remain one of the serious heath problems (Lin et al., 2002).

Oxidative stress is one out of several etiological and pathophysiological factors implicated in various diseases. Exposure of biological systems to xenobiotics, ionizing radiations and certain normal metabolic processes in the body leads to overproduction of Reactive Oxygen Species (ROS) that overwhelms the antioxidant armory. Recent studies have demonstrated that overproduction of ROS can further aggravate the oxidative stress and the result is a unifying mechanism of injury that occurs in many developments of clinical disease processes, such as heart disease, diabetes, liver injury, cancer, aging, etc. (Giordano, 2005; Rolo and Palmeira, 2006; Jaeschke, 2000; Klaunig and Kamendulis, 2004; Bokov et al., 2004). Maintaining the balance between ROS and antioxidant enzymes [especially superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] is therefore crucial and could serve as a major mechanism in preventing damage by oxidative stress. This balance has been suggested to play an important role in drug toxicity, such as from paracetamol (Jaeschke et al., 2003).

Paracetamol is an antipyretic analgesic drug that is available over-the-counter and an overuse of paracetamol can cause overproduction of ROS during formation of N-acetyl-p-benzoquinoneimine (NAPQI) by cytochrome P450 (Dahlin et al., 1984). This mechanism has been suggested to participate in the development of oxidative stress and injury in paracetamol-induced hepatotoxicity (James et al., 2003).

Modern medicines have little to offer for alleviation of hepatic diseases and it is chiefly the plant based preparations which are employed for their treatment of liver disorders. But there are not much drugs available for the treatment of liver disorders (Karan et al., 1999; Chaterrjee, 2000). Therefore, many folk remedies from plant origin are evaluated for its possible antioxidant and hepatoprotective effects against different chemical-induced liver damage in experimental animals. Paracetamol-induced hepatotoxicity model is frequently used for the investigation of hepatoprotective effects of drugs and plant extracts. The changes associated with paracetamol-induced liver damage are similar to that of acute viral hepatitis (James et al., 2003).

Tylophora asthmatica, a wild indigenous plant, belongs to the family Asclepidaceae and is commonly called as Indian ipecac. It is an endangered plant species, endemic to the state of Tamilnadu in India. The powdered leaves, stems and root contains 0.2-0.3% alkaloids, of these, tylophorine, tylophorinine and tylophorinidine are important alkaloids. Various studies have confirmed the anti-inflammatory activity (Gopalakrishnan et al., 1980), direct stimulant of adrenal cortex (Udupa et al., 1991), anti-asthmatic (Shivpuri et al., 1972) and the treatment of bronchitis, rheumatism and dermatitis (Nadkarni, 1976). The extract from the leaves and stems of T. asthmatica have been reported to possess anti-feedent, insecticidal and anti-tumour properties (Verma et al., 1986).

Review of the literature revealed that this rare medicinal plant remained unexplored for many of its claimed pharmacological activities. The aim of the present study was to evaluate the hepatoprotective and antioxidant properties of the methanolic leaves extracts of Tylophora asthmatica using rat model.

MATERIALS AND METHODS

Collection and Processing of Plant Material
The fresh leaves of T. asthmatica were collected during the month of February-2006 in the Banks of Cauvery River, Tiruchirappalli, South India. It was botanically identified and authenticated. A voucher specimen (TAL-12) has been kept in our laboratory for future reference. The leaves were shade dried, powdered, sieved through 40 mesh and stored in a tightly closed container for further use.

Preparation of the Plant Extract
The powdered plant material (500 g) was extracted with petroleum ether (60-80°C) using soxhlet apparatus to remove lipids. It was filtered and the filtrate was discarded. The residue was extracted with methanol by soxhlet apparatus. The extract was completely dried in vauco, stored in refrigerator at 4°C and protected from sunlight until the time for extract administration. The yield of methanolic dried extract was 8.63% (w/w).

Animals Used
Adult healthy male Wistar strain rats weighing 180 to 200 g were procured from Fredrick Institute of Plant Protection and Toxicology, Padappai, Chennai, India. They were maintained under uniform laboratory conditions in standard steel cages and provided with food (Hindustan Lever Ltd., Bangalore) and water ad libitum. The animals were maintained under laboratory conditions for 2 weeks to acclimatize before performing the experiment.

Experimental Protocol
Paracetamol (Acetaminophen; Sigma Chemical Company, USA) was suspended in saline solution in boiling water and was administered intraperitoneally (i.p.) after cooling (37°C) at a dose of 1 g kg^-1 body weight. This dose is known to cause liver damage in rats (Akintonwa et al., 1990). The experimental animals were divided into four groups, each group comprising six animals.

Biochemical Investigations
At the end of the experimental period (21 days), the animals were killed by cervical dislocation. Blood was collected immediately and the sera were separated by centrifugation. Liver was excised, weighed and homogenized in Tris-HCl buffer (0.1M, pH 7.4). Serum and liver homogenate were used for the estimation of proteins (Lowry et al., 1951) and serum was used for the estimation of aminotransferases (King, 1965a), lactate dehydrogenase (King, 1965b), alkaline phosphatase (King, 1965c) and bilirubin (Malloy and Evelyn, 1937) levels. Estimation of lipid peroxidation (Ohkawa et al., 1979), superoxide dismutase (Misra and Fridovich, 1972), catalase (Sinha, 1972), glutathione peroxidase (Rotruck et al., 1973) and reduced glutathione (GSH) (Maron et al., 1979) were carried out in the liver homogenate.

Statistical Analysis
All data were evaluated with SPSS/10 software. Hypothesis testing methods included one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test. p-values of <0.05 were considered to indicate statistical significance. All the results were expressed as mean±SD for six animals in each group.