EVALUATION OF ANTIOXIDANT AND HEPATOPROTECTIVE POTENTIAL OF FICUS BENGALENSIS

Abstract

Liver being a vital organ also plays a pivotal role in detoxification and metabolic control of many toxins which are further excreted out of the body (Lee and Senior, 2005). However, during such detoxification processes, liver itself faces a load of free radical that is generated from various sources like detoxification system, oxidative enzymes and immune system (Britton and Bacon, 1994; Lykkesfeldt et ai. 2007). Under normal physiological condition, hepatic aerobic metabolism results in a steady state production of pro-oxidants such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are balanced by a similar rate of their consumption by antioxidants. Imbalance in the pro-oxidant/antioxidant equilibrium in favor of pro-oxidant generates the oxidative stress phenomenon, a condition that may induce a number of pathophysiological events in the liver. Hepatotoxicity by oxidative stress maybe achieved through ROS, such as hydroxyl radical, superoxide radical anion and nitric oxide that causes cell membrane damage through lipid peroxidation (Halliwell and Chirico, 1993). It also modifies or damage biomolecules, like proteins, lipids, carbohydrates and DNA both in vitro and in vivo (Halliwell and Aruoma, 1991; Bandyopadhyay et ai, 1999; Manibusan et al., 2007; Halliwell, 2007; Loft et ai, 2008). Several attempts have been made to study carbon tetrachloride (CCI4) intoxicated rat liver toxicity model, because CCI4 is a potent hepatotoxin and it leads to hepatic oxidative stress toxicity and liver damage (Recknagel et al., 1989; Weber et al., 2003), which is reported to show great similarity with most of the chronic liver diseases (Cesaratto et al, 2004). Mechanisms like increase in fatty acid Q> oxidation as well as peroxisomal fatty acid oxidation, mitochondrial dysfunctioning and the biotransformation of CC14 by CYP2E1 enzyme to a more toxic free radical (Ingelman- Sundberg, 1988; Lieber, 1997) results in a consequent increase in intracellular oxidant load that ultimately leads to liver cell damage (Parola and Robino, 2001). Once hepatocellular function is impaired, accumulation of bile acid causes additional stress and toxicity (Jaeschke et al., 2002). Therefore, it is evident that ROS play an important role in pathological changes in the liver (Dianzani, 1987; Poli, 1993; Poli and Parola, 1997), particularly in case of liver cirrhosis (Natarajan et al., 2006). In view of above implications and importance of liver as an organ, there is need to protect it from oxidative stress toxicity by external supply of antioxidative agents, when endogenous protective mechanisms evolved to limit ROS and the damage caused by them is unable 1 to cope up with excessive free radical generation and subsequent oxidative stress damage (Sies, 1997; Serafini, 2000; Valko et ai, 2007). Therefore, antioxidants with free radical scavenging property could have much relevance as prophylactic and therapeutic agents in diseases in which oxidants or free radicals are implicated (Vitaglione et al., 2004; Meghana et al, 2007). A number of synthetic antioxidants such as BHA (Butylated hydroxy anisole) and BHT (Butylated hydroxy toluene) and tertiary butylhydroxyquinone (TBHQ) have also been developed that can assist in coping with oxidative stress. But some of their physical properties such as high volatility and instability at elevated temperatures, toxicity, higher manufacturing cost, strict legislation on the use of synthetic food additives and consumer preferences aroused the need to find alternatives to synthetic antioxidants (Rice-Evans, 1998; Abdalla et al., 1999). Besides, the conventional drugs used in the treatment of liver diseases are often inadequate. It is therefore necessary to search for alternative drugs for the treatment of liver diseases to replace the currently used drugs of doubtful efficacy and safety. Consequently, there is considerable interest in preventive medicine and food industry in the development of natural antioxidants. Among several sources of antioxidant and hepatoprotective agent, the importance of plants as a natural source have been well established due to its wide diversity and its ability to synthesize a wide array of phytochemicals as part of its defense strategy. Natural antioxidants from plant sources have better antioxidant activity and are safer to health, reliable and compatible with human diet without any side effects as caused by synthetic antioxidants. Conventional medicine is now pursuing the use of natural products such as herbs/ plant extracts to provide the support that the liver needs on a daily basis. For example, Liv.52, an Ayurvedic preparation (mixture of several herbal extracts) is a well prescribed liver tonic and provides protection to liver from the hepatotoxicity (Dhawan and Goel, 1994; Kataria and Singh, 1997). Presently, in spite of an increasing need for agents to protect the liver from damage, modern medicine lacks a reliable liver protective drug. Therefore, search for natural antioxidants showing hepatoprotective role in liver oxidative stress toxicity has been gaining momentum and considered to be thrust areas in biomedical sciences globally(Abalea et al, 1999; Seeffet al, 2001; Lee et al, 2008). Herbal medicines derived from plant extracts are being increasingly utilized to treat a wide variety of clinical diseases, though relatively little knowledge about their mode of action is available (Matthews et al, 1999). Recently, the number of cases with drug-induced liver injury has been increasing (Saad, 2006; Stickel et al, 2009), parallel to the growing number of drugs including health food and "natural" foods (Seeff, 2009). Further clarifying the role of oxidative stress in drug hepatotoxicity is needed for useful therapy of drug-induced liver injury, and many drugs and treatments now being investigated are directed toward preventing the damage from oxidative stress (Medina and Otero, 2005; Antoine, 2008). India due to its geographical location and climatic conditions is blessed with a widely diversified plant flora and are endowed with diversified classes of plant phytochemicals, which has been found to deliver preventive role in several oxidative stress involved human diseases including liver diseases (Dahanukar et al, 2000; Samarth et al, 2008). It is more likely that some of these maybe valuable sources of natural antioxidants. A large number of plants of dietary and medicinal importance in India have been evaluated for their antioxidant potential (Aqil et al, 2006; Kumar et al, 2008; Ali et al, 2008). However, a large number of these plants with diversified medical potential still remains unexplored; it is likely that some of these maybe valuable source of potent antioxidant and hepatoprotective agent. Therefore, there is need to explore the rich diverse flora to search new antioxidant and hepatoprotective agents and identify the active constituents. The present work was emphasized to search a potential source of antioxidant and identify its active constituents. This study was undertaken with the following objectives: 1. Screening of selected Indian medicinal plants for their antioxidant activity. 2. Extraction and bioactivity guided fractionation of active antioxidant constituents from selected plant using various biochemical leading techniques (liquid-liquid partitioning, Thin layer chromatography, column chromatography and suitable in vitro antioxidant assays). 3. Identification of components in active fraction using various analytical techniques like UV- Visible spectrophotometry, FTIR, ESI-MS and H'NMR. 4. Determination and evaluation of antioxidant activity and protective potential against oxidative damage to biomolecules including DNA, protein and lipid using various in vitro biochemical and molecular biology based assays. 5. Evaluation of hepatoprotective role against CCI4 intoxicated liver oxidative stress model employing biochemical, histological and molecular biology techniques.