International Journal of Applied Research in Natural Products Vol. 3 (3), pp. 19-26, Sep-Oct 2010 Directory of Open Access Journals ©2010-2011. IJARNP-HS Publications
Original Article Hepatoprotective Activity of Bombax ceiba Linn against Isoniazid and Rifampicin-induced Toxicity in Experimental Rats Ravi V*, Patel SS, Verma NK, Dutta D, Saleem TSM Cardiovascular Research Unit, Himalayan Pharmacy Institute, Majhitar, Sikkim-737136, India. Abstract: Hepatoprotective activity of methanolic extract of flowers of Bombax ceiba L. (MEBC) was investigated against hepatotoxicity produced by administering a combination of two anti-tubercular drugs Isoniazid and Rifampicin for 10 and 21 days by intraperitoneal route in rats. MEBC were administered at three graded dose i.e. 150, 300 and 450 mg/kg i.p. 45 min prior to anti-tubercular challenge for 10 and 21 days. MEBC was evident in the all doses as there was a significant decrease in AST, ALT, ALP, and Total Bilirubin levels, but increased the level of total protein in comparison to control. MEBC significantly decreased the level of TBARS and elevated the level of GSH at all doses as compared to control. Histology of the liver section of the animals treated with MEBC improved the hepatotoxicity caused by antitubercular drugs. The results obtained from the analysis of biochemical parameters and histopathological studies, enabled to conclude that the MEBC were not able to revert completely the hepatic injury induced by INH + RIF, but it could limit the effect of INH + RIF to the extent of necrosis. Industrial relevance Herbal medicines are getting more importance in the treatment of liver disease because the modern medicine does not find curative treatments. A large proportion of the Indian population for their physical and psychological health needs depend on traditional system of medicine. Medicinal plants have become the focus of intense study in term of conservation as to whether their traditional uses are supported by actual pharmacological effects or merely based on folklore. Herbal medicine is free from side effects and less costly when compared to synthetic drugs. The present study will help the industry to produce herbal drugs with fewer side effects, which are affordable and more effective in the treatment of hepatotoxicity. Key words: Bombax ceiba; Hepatotoxicity; Isoniazid; Oxidative stress; Rifampicin
Introduction Tuberculosis is one of the fatal communicative diseases and is spread easily amongst people. Over one-third of the world's population is estimated to be infected with Mycobacterium tuberculosis and over 2 million people a year will die of the disease (Shishoo et al., 2001). Multi-drug resistant strains of M. tuberculosis have emerged and a coinfection with AIDS was found out. This turned out that the WHO declared tuberculosis as ‘Global health emergency’ (Anon, 1997). The administration of INH and RIF, the most common medication prescribed against tuberculosis, produces many metabolic and morphological aberrations in liver due to the fact that liver is the main detoxifying site for these antitubercular drugs. These antitubercular drugs induce hepatotoxicity by a multiple step ______________________ *Corresponding Author: E-mail:
[email protected] Tel: 91-3592-246462 Available online http://www.doaj.org/doaj?func=openurl&issn=19406223&genre=journal
Hepatoprotective Activity of Bombax ceiba L. mechanism. The exact mechanism responsible for liver injury caused by these drugs is not clear. Isoniazid is acetylated and then hydrolyzed, resulting in isonicotinic acid and monoacetylhydrazine; the later compound can be activated to a toxic species by cytochrome P-450 (Thomos et al., 1981). In vitro studies indicated that metabolic oxidation of acetylhydrazine leads to a reactive acylating species that binds covalently to microsomal protein. It is postulated that acetylhydrazine and hydrazine act as acetylating agents by binding covalently with liver cell macromolecules, causing hepatocyte injury (Noda et al., 1983). RIF, a powerful inducer of mixed-function oxidase, increases the hepatotoxicity of INH by enhancing the production of toxic metabolites from acetylhydrazine (Ellard et al., 1976, Kalra et al., 2007). Rats show a similar genetically determined acetyltransferase activity as in humans and are more sensitive to INH-induced hepatotoxicity due to a high amidase activity, which results in release of large amount of acetylhydrazine, which induces hepatotoxicity. Antitubercular drugs mediated oxidative damage is generally attributed to the formation of free radicals, which act as stimulator of lipid peroxidation and source for destruction and damage to the cell membrane (Georgieva et al., 2004). Alterations of various cellular defense mechanisms consisting of enzymatic and non-enzymatic components [reduced glutathione (GSH)] have been reported in INH and RIF-induced hepatoxicity (Tasduq et al., 2005). Bombax ceiba (Bombacaceae) is an important medicinal plant of tropical and subtropical India. Its medicinal usage has been reported in the traditional systems of medicine such as Ayurveda, Siddha and Unani. Bombax ceiba described as a cotton tree and has been used extensively for treatment of some diseases like inflammation (Buckingham, 1992), algesia, hepatotoxicity (Saleem et al., 2003), and hypertension, as well as for antiangiogenic and antioxidant activities (Vieira et al., 2009). The effect of Bombax ceiba on INH and RIF induced liver injury is still unclear; hence the present work includes the hepatoprotective activity of methanolic extract of flowers of Bombax ceiba (MEBC) against INH and RIF-induced liver damage in rats. Materials and methods Chemicals and drugs: Isoniazid and Rifampicin were purchased from Panacea Biotech Limited (New Delhi, India). Bilirubin, Total Protein, Alkaline phosphatase (ALP), Alanine transaminases (ALT), and Aspartate transaminases (AST) were assayed by using kits from Ranbaxy diagnostic (New Delhi, India). GSH and TBARS were assayed by using chemicals from M/s Sigma Chemical Company (St. Louis, MO, USA). Animals: Wistar strain male albino rats, having weight range of 120–150 g, were used for the experiment. The animals were housed in polypropylene cages under hygienic condition and maintained at 28±2 °C temperature. The animals were allowed to have food and water ad libitum. The experiment was conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India and approved by the Institutional Animal Ethical Committee (HPI/08/60/IAEC/0052). Preparation of Plant Extract: Flowers of Bombax ceiba were collected from the Majhitar, in the month of January and identified by Pharmacognocist, Himalayan Pharmacy Institute, East Sikkim, India. The shade dried flowers were powdered to get a course granule. About 750 g of dried powder was extracted with methanol using Soxhlet apparatus. The resulted dark – brown extract was concentrated up to 100 ml. The concentrated crude extract was powdered and used for the study. Experimental design. Acute toxicity study: The acute toxicity study of MEBC was performed (Turner, 1965; Verappan et al., 2007). The dead animals obtained from primary screening studies, and LD50 value was determined. Induction of experimental hepatotoxicity: INH and RIF solution were prepared in sterile distilled water. Rats were treated with INH+RIF at the dose of (100 mg/kg b.w., i.p.) to the experimental animals for 10 and 21 days. (Jiang et al., 2004; Saleem et al., 2008). In order to study the effect of MEBC in rat, 150 mg/kg b.w., 300 mg/kg b.w. and 450 mg/kg b.w. were administered by intraperitoneal route. Silymarin (2.5 mg/kg b.w., p.o.) was used as a standard drug in this study (Parthasarathy et al., 2007). Rats were divided into six groups. Each group contains 6 animals and treatments were followed as per treatment protocol given below. Treatment protocol: G1 – Rats were treated with normal saline G2 – Rats were treated with INH + RIF G3 – Rats were treated with INH + RIF + MEBC (150 mg/kg) G4 – Rats were treated with INH + RIF + MEBC (300 mg/kg) G5 – Rats were treated with INH + RIF + MEBC (450 mg/kg) G6 – Rats were treated with INH + RIF + Silymarin (2.5 mg/kg) Rats were treated as per the treatment protocol for 10 and 21 days. Biochemical estimation: Rats were sacrificed one hour after administration of last dose on 10th and 21st day under anesthesia (pentobarbitone sodium, 45 mg/kg, i.p.). The blood was collected by retro-orbital bleeding. Blood 20
samples were centrifuged for 10 minutes at 3000 rpm to separate the serum. ALP, ALT, AST, Total protein and Bilirubin levels were estimated using standard kits (Rajesh et al., 2005). Liver were excised. One gram of the tissue was taken and homogenized (Homogenizer REMI RQM-122, Remi Instrument, India) with 1ml of 0.3M phosphate buffer, pH 8.4. This was then centrifuged at 4°C at 5000 rpm for 10min., thereafter the supernatant was collected and stored in freeze for the estimation of TBARS (Thiobarbituric acid reactive substances) and GSH (reduced glutathione) (Okhawa et al., 1979; Ellman, 1959). Histopathological studies: The liver was excised quickly and fixed in 10% buffered-formaldehyde at room temperature. After dehydration using graded ethanol, pieces of tissues were embedded in paraffin, cut in fine (5 µm) sections and mounted on glass slides. Sections were then deparaffinized with xylene, counterstained with hematoxylin and eosin and viewed under a light microscope at X400. Statistics: The results were expressed as Mean ± SEM. Statistical analysis was carried out by using One-way ANOVA followed by Newman-Keul’s multiple tests. Results The dead animals obtained from the acute experiments usually presented with their cardiac arrest. Apart from this characteristic observation, no other significant observation deviant from the normal was seen in these dead animals. The LD50 value of MEBC is 3147.81 mg/kg. Three fold rises in biochemical parameters in toxic control group (G2) is indication for liver injury by INH + RIF treatment. Increased biochemical parameters (ALP, AST, ALT, and TB) significantly reduced with co-administration of MEBC at three different doses (150, 300 & 450 mg/kg b.w.) in G3, G4, and G5 and silymarin treated group (G6). The results were present in Table 1 and 2 for 10 and 21 days treatment period respectively. Similarly it decreased the level of total protein after INH + RIF treatment, and increased significantly with co-administration of MEBC and silymarin treatment group (G6). Increased liver TBARS level in G2 group is indication for increased oxidative stress by treatment of INH + RIF. Increased liver TBARS, significantly (p
