Summary HEPATOPROTECTIVE ACTIVITY OF CHRYSOPHYLLUM ALBIDUM AGAINST CARBON TETRACHLORIDE INDUCED HEPATIC DAMAGE IN RATS

The liver is the central organ in the metabolism and detoxification of drugs and toxins. Consequently, drugs affect the liver more frequently than any other organ and place the liver at great risk for toxic damage (Bussieres and Habra, 1995). After absorption by the intestines, drugs reach the liver via the portal system. In the hepatocytes, these chemicals undergo complex metabolic processes to be converted into hydrophilic substances, readily soluble in the blood stream and easily eliminated thereafter (Lee, 2003). Drugs or their metabolites can cause toxic effect on the liver. Many of the intermediate metabolites have a short half-life, some estimated to be less than a minute, which makes detecting them a challenging task (Park et al., 2005). This chemical-driven liver damage is referred to as hepatotoxicity. The use of herbal medicine can be traced back to 2100 BC in ancient China at the time of the Xia dynasty and during the Vedic period in India. The first written reports are timed to 600 BC with Charaka Samhita in India and to 400 BC with the early notes of the Eastern Zhou dynasty in China (Dhiman and Chawla, 2005). The study of African medicinal plants Table 1. Effect of ethanolic extract of C. albidum on CCl4 induced hepatotoxic rats. Parameters GROUP A (Negative Control) GROUP B (Positive Control) GROUP C (500 mg/kg) GROUP D (1000 mg/kg) GROUP E (1500 mg/kg) AST (U/L) 28.00 ± 4.36 62.80 ± 11.04† 41.75 ± 5.27 (75.2%) 28.70 ± 2.22a (121.8%) 26.75 ± 4.21a (128.8%) ALT (U/L) 107.86 ± 4.79 123.25 ± 3.75† 104.83 ± 5.88a (17.1%) 91.71 ± 3.10a,b (16.2%) 88.83 ± 3.36a,b (17.6%) ALP (U/L) 228.19 ± 11.09 487.57 ± 19.00† 386.27 ± 28.60a,b (44.4%) 354.90 ± 11.87a,b (58.1%) 317.86 ± 31.93a,b (74.4%) Total Bilirubin (µmol/L) 14.31 ± 3.18 26.41 ± 3.13† 26.18 ± 3.87b (1.6%) 16.51 ± 3.43a (69.2%) 11.15 ± 1.68a (106.6%) Albumin (g/dL) 3.65 ± 0.21 3.55 ± 0.07 3.98 ± 0.22 3.62 ± 0.34 3.60 ± 0.25 Total protein (g/dL) 7.41 ± 0.56 6.96 ± 0.20 8.25 ± 1.39 7.43 ± 0.50 7.51 ± 0.81 Values represent mean ± SE of 8 replicates. a p 0.05 versus positive control; b p 0.05 versus negative control; †p 0.05 positive control versus negative control. Values in parenthesis are percentage decrease of parameters analyzed (p 0.05) after pretreatment *Corresponding author email: has not in the past been taken as seriously, or documented as fully, as Indian and Chinese traditional medicines (Adebayo, 2010). Over 5000 plants are known to be used for medicinal purposes in Africa, but only a few have been described or studied (Adebayo et al., 2010). Chrysophyllum albidum belongs to the Sapotaceae family and native to the Central, Eastern and Western Africa (Amusa et al., 2003). The plant is specifically distributed in Nigeria, Uganda, Niger, Cameroun and Cote d’ Ivoire (Adewusi, 1997). It is often called the white star apple and distributed throughout the southern part of Nigeria (Idowu et al., 2006). The fruit is popularly called “agbalumo” and “udara” in South Western and Eastern Nigeria respectively. From our previous investigation, we observed that the leaf extract of C. albidum significantly reduced the levels of liver function parameters (Adebayo et al., 2010). Many folk remedies from plant origin are tested for their potential hepatoprotective effect on liver damage in experimental animal model. Carbon tetrachloride (CCl4) induced hepatotoxicity model is widely used for the study of hepatoprotective effects of drugs and plant extracts (Rubinstein, 1962; Suja et al., 2002). CCl4 is biotransformed by the cytochrome P450 with the extract and CCl4 with respect to the control groups. system to produce the trichloromethyl free radicals, which in turn covalently binds to cell membranes and organelles to elicit lipid peroxidation (Recknagel et al., 1989). The study is therefore aimed at investigating and validating the hepatoprotective properties of the leaf extract of C. albidum in CCl4 induced liver cell damaged rats. MATERIALS AND METHODS Plant material The leaves of C. albidum were obtained from the campus of Covenant University, Canaan land, Ota, Ogun State, Nigeria in November, 2009. The plant was authenticated at the Department of Pharmacognosy, University of Lagos, Lagos, Nigeria and a voucher specimen (PCGH 435) was deposited in the herbarium for reference purpose. Preparation of extracts The procedure described by Adebayo et al. (2010) was adopted. The leaves of C. albidum were collected and airdried in the laboratory for two weeks after which they were blended into fine powder. 400g were extracted with 95% ethanol. Evaporation of the extract in a rotatory evaporator (Buchi 461, Switzerland) at 40oC gave a yield of 98g. Experimental animals Male albino rats (40) of Wistar strain obtained from the University of Agriculture, Abeokuta, Ogun State, Nigeria weighing between 200-230g were used for the experiment. Animals were maintained in 12-h light: 12-h dark at a controlled temperature (25 ± 3oC), humidity (60 ± 5%) and kept in the animal house of the Department of Biological Sciences, Covenant University, Ogun State, Nigeria. The animals were allowed to acclimatize for six weeks. Feed and water were given ad libitum. All animals were treated in accordance with the recommendations of National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH, 1985). Experimental Design The model described by Chakraborti and Handa (1989) was employed with some modifications. The rats were divided into five groups of eight rats per group. The animals of group A served as normal control group and were given only vehicle (distilled water, 1 ml/kg b.w.) for 7 days. The animals of group B (positive control) were administered with vehicle on the first four days, and with the vehicle and CCl4 (50% solution of CCl4 in liquid paraffin, 2 ml/kg b.w.) on the fifth, sixth and seventh day. The animals of groups C, D and E were respectively administered with 500, 1000 and 1500mg/kg b.w. of ethanolic extract and distilled water for the first four days, and with distilled water, ethanolic extract and CCl4 on the last three days. Animals were subsequently anaesthetized and blood samples were collected for aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, albumin and total bilirubin, assays. Blood collection and preparation of sample At the end of the treatment period, the rats were anaesthetized in diethylether prior to dissection. The blood was then collected by cardiac puncture into lithium heparinized bottles. Plasma was obtained by centrifuging the blood at 10, 000 revolution per minute for 15 minutes into clean bottles and stored at –20oC until required for biochemical assays (Adebayo et al., 2006). The liver was also collected and fixed with 10% formaldehyde for histopathological examination. Adebayo et al. 1599 Analysis of biochemical parameters Commercial test kits obtained from Randox Laboratories, United Kingdom were used for all biochemical parameters measured. Standard methods were used to estimate aspartate amino transferase (AST), alanine aminotransferase (ALT) (Reitman and Frankel (1957), alkaline phosphatase (ALP) (Tietz et al., 1983), total protein (Weichselbaum, 1946), albumin (Doumas et al., 1971) and total bilirubin (Doumas et al., 1973). Histopathological analysis Small pieces of liver fixed in 10% buffered neutral formalin were processed for embedding in paraffin (Aliyu et al., 2007). Sections of 5-6µm thickness were stained with hematoxylin and eosin, examined for histopathological changes under a compound microscope. STATISTICAL ANALYSIS