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Hepatoprotective Effects of Ethanol Extract of Caesalpiniabonduc Against Carbon Tetrachloride Induced Hepatotoxicity in Albino Rats

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Hepatoprotective Effects of Ethanol Extract of Caesalpiniabonduc Against Carbon Tetrachloride Induced Hepatotoxicity in Albino Rats June. JASEM ISSN 1119-8362 Full-text Available Online at J. Appl. Sci. Environ. Manage. 2016 All rights reserved www.ajol.info and Vol. 20 (2) 396 – 400 www.bioline.org.br/ja Hepatoprotective Effects of Ethanol Extract of Caesalpiniabonduc against Carbon Tetrachloride Induced Hepatotoxicity in Albino Rats. *1UBHENIN, AE; 2IGBE, I., 3ADAMUDE FA; 4FALODUN ,A. 1,3 Department Biochemistry, Faculty of Basic Health Science, Federal University, Lafia. Nasarawa, Nigeria. 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin,City, Nigeria. 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Benin, Benin,City, Nigeria. *Author for correspondence: +2348133893080, [email protected] ABSTRACT: The present study was carried out to evaluate the acute toxicity, hepatoprotective and in -vivo antioxidant activities of ethanolic extract of Caesalpinia bonduc leaf on carbon tetrachloride (CCl 4) induced liver damage using Swiss albino rats. The ethanolic extract of the plant of C. bonduc were suspended in 5 % tragacanth and then administered orally at doses of 250 and 500 mg/kg body weight for fourteen days before intraperitoneally injection of Carbon tetrachloride (CCl 4) at dose of 2 mL/kg body weight. The plant extracts at 250 and 500mg/kg b.wt showed a remarkable hepatoprotective and invivo antioxidant activities against carbon tetrachloride CCl 4 – induced hepatotoxity judged from the serum marker enzymes .The CCl 4 induced significant increase in aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phophatase (ALP), total bilirubin, and malondyaldehyde (MDA) with a reduction of total protein, catalase, and glutathione peroxidase. Treatment of rats with different doses of plant extract (250 and 500 mg/kg b.wt.) significantly (P< 0.001) altered serum maker enzymes and antioxidant levels to near normal levels. The study suggests that C. Bonduc specifically chloroform and ethyl acetate fraction may be good sources of natural antioxidant and hepatoprotective substance. © JASEM http://dx.doi.org/10.4314/jasem.v20i2.22 KEYWORDS:Ceasalpiniabonduc , hepatoprotective, intraperitoneally , antioxidant Introduction brickles. Vine stem, diameter up to 5cm recorded, Liver is one the most complex organ in the body and usually grows as a vine but also flowers and fruits as a it plays a key role in the metabolism, secretion, shrub. The leaves are with large, leafy branched, basal detoxification and excretion of xenobiotics(Alcamo appendanges, 30-60cm long. The plant grows all over 2004; Rowen et al., 2009).The main causes of liver tropical part of the world and the whole plant of injury are viral infection, xenobiotics, toxic Caesalpinia bonduc contain all major chemical chemicals, excessive drug therapy, environmental constituents such as alkaloid, steroidal saponin, fatty pollutants,chronic alcohol ingestion and acid, hydrocarbon, phytosterols, isoflavones, amino chemotherapeutic drugs (King and Perry,2001; acids and phenolics. Sharma, et al., 1991; Subramonium and Pushpangadan 1991). Due to inadequacy of liver protective agents, Patil et al . reported antidiabetic activity of bark and researchers and traditional medicine practitioners have root of Caesalpinia bonduc ( Patil et al ., 2011). The focused on herbal based remedies for various liver seed extract were screened for adaptogenic activity disorders. There are no safe hepatoprotective drug using cold stree model and surium endurance model. available for the treatment of liver disorders (Karan et It was also found that extracts proved efficient in al., 1999; Chaterrjee, 2000). Carbon tetrachloride controlling the hyperlipidaemic condition due to stress (CCl4)-induced hepatotoxicity model is widely used (Kannur et al ., 2006). It was found that the extract for the study of hepatoprotective effects of drugs and showed significant anthelmintic activity (Wadkar et plant extracts (Rubinstein, 1962; Suja et al., 2002). al ., 2010).However, literature review reveals that Caesalpiniabonduc has not been scientifically Caesalpinia bonduc (Linn.) Roxb popularly called fever investigated for its hepatoprotective properties. nut belonging to Caesalpiniaceae family is used to treat Therefore the plant is tested for its potential fevers and roasted seeds are used to treat diabetes hepatoprotective in experimental animal models. (Chevallier, 1996). It is a prickly and extensive climber, very thorny shrubs, branches finely grew- MATERIALS AND METHODS downy, armed with hooked and straight hand yellow Collection and identification of plant material : Fresh Correspondence author Email:, [email protected] Hepatoprotective Effects of Ethanol Extract of Caesalpiniabonduc 397 leaves of Caesalpiniabonduc were collected in June b.wt. ip) on day 14. 2011 from Igueben in Igueben Local Government Area of Edo State. It was identified and authenticated After 24 hours of the last treatment, all the animals were by Ugbogu O.A. and Shasanya O.S. of the Forest anaesthesized with chloroform and blood was collected Research Institute of Nigeria (FRIN), Ibadan. A via cardiac puncture. The blood was put into a plain Specimen number 109493 was given and the plant sample tubes and sera was obtain from it by allowing it specimen was deposited at the FRIN Herbarium. to stand for 2hrs at room temperature before centrifuging for at 2000rpm to separate the serum. The serum was Processing & Extraction of crude powdered used for estimation of various biochemical parameters. sample : The fresh leaves were air dried and the dried material was grounded into powder using an electric Assessment of liver function : Serum Aspartate blender (pyeUnicam, Cambridge, England). The andAlanineTransferase(AST and ALT) The activities powdered plant material was subjected to cold of these enzymes were estimated by the method of extraction by soaking 800g of dry powder in 5L of Reitman and Frankel (1957). 0.2 ml of serum was distilled ethanol for three days (72hours) at room added to 1 ml of phosphate buffer containing temperature with occasional shaking. The ethanol substrate, mixed and incubated for 30 min for ALT and extract was filtered using a Buchner funnel and 60 min for AST at 37°C, Then 1 ml of Whatman No.1 filter paper.Dried ethanol extracts dinitrophenylhydrazine was added and incubated for were obtained after removing the solvent by 20 min at room temperature and 10 ml of 0.4% sodium evaporation under reduced pressure using Rotary hydroxide was added, mixed well and after five evaporator.The extract was stored in an air-tight minutes read at 550 nm against sample blank .The container and kept in the refrigerator at 4°C until use value of AST and ALT are calculated from a series of standards curve. Animals : Swiss albino mice weighing 25-30 g and Wistar albino rats weighing 200-250 g were procured from the Alkaline Phosphatase : Serum alkaline phosphatase Animal House, Department of Pharmacology, Faculty activity was measured following the method of of Pharmacy, University of Ibadan. Food and water King and Armstrong (1934),usingdisodium phenyl were provided ad libitum. Animals were exposed to phosphate as substrate.Thecolour developed was read at controlled environmental of temperature (28± 2°C), 510 nm. Activities are expressed as KAU/L. relative humidity (50± 5%) and 12 hour light or dark. The handling procedures were conducted in Total Bilirubin : Serum total bilirubin was estimated accordance with the Faculty of Pharmacy, University following the method of King and Coxon (1950).In of Benin Ethical committee on experimental animals. brief, 1 ml of serum was mixed with 0.5 ml of The animals were also allowed two weeks under these diazoreagent, followed by 0.5 ml (NH 4)2SO4 .The conditions to acclimatize before the commencement volume was made up to 10 ml with 85% ethanol.The of the experiments. contents were mixed well and allowed to stand for 30 min for even distribution of the precipitate.The Preparation of the hepatotoxin: Carbon tetrachloride precipitate was filtered and measured using (CCl 4) was given with olive oil (1:1 v/v) at dose of 2 mL/kg colorimeter body weight intraperitoneally. Protein Content: Protein content of serum was Hepatoprotective Activity : Rats were divided into five determined by biuret method described Kingsley (1942). groups, each group consisting of six animals. To 0.02 ml of sample, 1ml of solution 1 was Group I: (Control) received 5 % tragacanth (1mL/kg b.w. added and mixed gently. The mixture was incubated for orally) daily for 14 days and olive oil (1mL/kg b.wt. ip) on 30mins at 30 oC, The absorbance of the sample and of the day 14. standard was read at 546 nm against the blank. Group II: (CCl 4 induced) received 5% tragacanth (1mL/kg b.wt. orally) daily for 14 days and CCl 4: olive oil Determination of malondialdehyde (MDA) (1mL/kg b.wt. ip) on day 14. Group III: (Standard) concentration.: The level of malondialdehyde was received silymarin (50 mg/kg b.w orally) for 14 days determined by modified procedure described by Guidet and CCl 4: olive oil (1ml/kg b.wt. ip) on day 14. Group and Shah (Guidet and Shah, 1989).Briefly, 150 μl of IV:received ethanol leaf extract of C. bonduc (250 serum sample was mixed in a test tube with 1 ml of mg/kg orally) for 14 days CCl 4 : olive oil (1ml/kg b.wt. 17.5 %TCA.1ml of 0.6 % TBA was added, and the ip) on day 14. mixture was mixed well by vortexing, incubated Group V: received ethanol leaf extract of C. bonduc (500 in boiling water bath for 15 min, and then mg/kg b.w orally) for 14 days and CCl 4 :olive oil (1ml/kg allowed to cool. *1UBHENIN, AE; IGBE I 2, FALODUN A 3 Hepatoprotective Effects of Ethanol Extract of Caesalpiniabonduc 398 1ml of 70 % TCA was added, with mixing, the containing 3ml of H 2O and 0.01M phosphate buffer mixture was left to stand at room temperature for 20 (pH 7.0) and the OD change was measured at 240nm, minutes. The mixture was centrifuged at 2000 the time taken for decrease in the absorbance from g for 15 minutes, and 1ml the supernatant was 0.45 to 0.4 is noted as 4T.The activity of the enzyme taken out for measuring the absorbance at 532 nm.
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