MATERIALS AND METHODS

Animals: Sprague Dawley rats, of both sexes, weighing between 150 and 180 g were used for the experiment. They were housed in standard rat plastic cages under laboratory conditions with 12:12 h light/dark cycle at 25°C±2. The animals were allowed to acclimatize for two weeks (Eghoghosoa et al., 2011).

Treatment: Animals were randomized into three groups; the negative control, positive control and the extract-treated groups. The negative control received only 2 mL of distilled water daily. The positive control received 2 mL of 10% alcohol, while the extract-treated group received 1 mL of the aqueous extract of Aloe vera and 2 mL of 10% alcohol. Treatments lasted for 4 weeks.

Extraction of aqueous extract of Aloe vera: Aqueous extract of Aloe vera was obtained as previously described (Saka et al., 2011). Briefly, fresh stems of Aloe vera were washed thoroughly to get rid of all forms of debris. The leaves were then sliced longitudinally to cut open the inner part of the leaves. The gel in the leaves was scrapped into a clean bowl and blended to obtain a finer and liquefied form of the gel, the aloe juice. The juice was refrigerated below 4°C for preservation (Ige et al., 2011).

Blood sample collection: After the experimental period, blood sample was collected from the animals by jugular puncture. Rats were anaesthetized in chloroform vapour. The neck was quickly cleared of fur and skin to expose the jugular veins. The veins after being slightly displaced to avoid contamination with interstitial fluid were sharply cut with a sterile scalpel (Oladiji et al., 2007) and blood was collected into lithium-heparinized bottles. Sera were obtained by centrifuging at 3000 rpm for 15 min (Akhigbe et al., 2008) and liver function test was done.

Assessment of liver function and histopathological examination: Alkaline phosphatase, aspartate aminotransaminase, alanine aminotransaminase and total bilirubin were determined using standard laboratory kit according to manufacturer’s instruction.

The excised livers were used in normal saline and fixed immediately in 10% formalin solution. A paraffin embedding technique was carried out and sections were taken at 5 mM thickness, stained with hematoxylin and eosin and examined microscopically for histopathological changes (Preece, 1972).

Statistical analysis: Data are presented as Mean±standard error of mean (n = 5). Statistical analyses used one-way Analysis of Variance (ANOVA) to account for the different treatments and were complemented with unpaired t-test. Differences were considered statistically significant at p<0.05.

RESULTS

Liver functions are summarized in Fig. 1-4. Alanine aminotransaminase (ALT) activities were comparable in all groups, though there was a marginal insignificant raise in alcohol-treated group compared to other groups.

Fig. 1:	Effect of aloe vera aqueous extract on serum alkiline phosphatase in alcohol-induced hepatic damage; bars carrying different letters are significantly different (p<0.05)

Fig. 2:	Effect of aloe vera aqueous extract on serum alanine aminotransaminase in alcohol-induced hepatic damage; bars carrying different letters are significantly different (p<0.05)

Alkaline Phosphatase (ALP), Aspartate Aminotransaminase (ASP) and Total Serum Bilirubin (TSB) were significantly increased in alcohol-treated group. These rises were significantly abrogated by Aloe vera Extract (AvE).

The histopathological examination of the negative control group livers showed a normal arrangement of the hepatocytes, with clearly visible nuclei, central vein and portal triad. Areas of congestion of sinusoids, cloudy swelling, congestion of central vein, centrilobular fatty change and necrosis of hepatocytes were seen in animals treated with alcohol. There was marked reduction in sinusoidal congestion, cloudy swelling and fatty change, with areas of regeneration in animals treated with AvE (Fig. 5).

Fig. 3:	Effect of aloe vera aqueous extract on serum aspartate aminotransaminase in alcohol-induced hepatic damage; bars carrying different letters are significantly different (p<0.05)

Fig. 4:	Effect of aloe vera aqueous extract on serum bilirubin in alcohol-induced hepatic damage; bars carrying different letters are significantly different (p<0.05)

Fig. 5:	Effect of aloe vera aqueous extract on serum bilirubin in alcohol-induced hepatic damage bars carrying different letters are significantly different (p<0.05)

DISCUSSION

Polysaccharides, carbohydrates, proteins, amino acids, vitamins, lipids, inorganic compounds, chromones, anthraquinones/anthrones, triterpenoid, flavonoids and some enzymes and hormones are the constituent phytochemicals in Aloe vera (Hu et al., 2003; Hamman, 2008; Sharrif Moghaddasi and Verma, 2011).

Hepatic damage is characterized by a rise in serum enzymes viz ALT, AST and ALP. Generally, ALT concentrations are consistently higher than AST levels and this is expected as body cells generate more ALT than AST (Mayne, 1996). Most ALT is found in the mitochondria, unlike AST which is entirely cytosolic. Thus, AST is a more sensitive marker of hepatocellular damage than ALT (Al-Mamary et al., 2002).

The administration of alcohol to the animals resulted in significant rise in serum ALP, AST and TSB. Administration of AvE ameliorated the toxic effect of alcohol. Compared with the alcohol-treated animals, AvE caused a significant decrease in ALP, AST and TSB levels. However, ALT levels were similar in all groups. The observed increase in the concentrations of ALP and AST is indicative of loss of the functional integrity of the hepatic cell membranes and cellular leakage of these enzymes into the bloodstream (Lahon and Das, 2011). Histological study showed normal cytoarchitecture of the control rats, however, the alcohol-treated animals exhibited areas of hepatic necrosis. The animals treated with AvE revealed appreciable protection of hepatic tissue from alcohol. Thus, AvE showed hepatotherapeutic activity. This agrees with previous studies (Rajasekaran et al., 2006; Saritha and Anilakumar, 2010) that reported that Aloe vera maintains normal levels of aminotransaminases, ALP, TSB and hepatic cytoarchitecture integrity.

The hepatoprotective activity of AvE might be associated with its anti-oxidant and anti-inflammatory potentials against alcohol-induced hepatic damage. AvE preserved the metabolizing enzymes of the liver through an antioxidant activity (Chandan et al., 2007), via an inhibitory action on the arachidonic acid pathway through cyclooxygenase (Vazquez et al., 1996). It also significantly reduced leukocyte adhesion and tumour necrosis factor α (Prabjone et al., 2006). Thus, AvE reduced prostaglandin synthesis and leucocytes infiltration (Cross and Roberts, 2004) with consequent enhancement of hepatic functions.

The hepatotherapeutic effect of AvE may be attributed to the anti-oxidant properties of its bioactive principles via a membrane-stabilizing mechanism. This study thus lends pharmacological support to the folkloric, ethnomedical use of the plant in the management of alcohol-induced hepatic damage.