Research Article
Protective Effect of Erythrina senegalensis (DC) Leaf Extract on Carbon Tetrachloride-induced Liver Injury in Rats

H.Y. Wakawa and M. Hauwa

Asian Journal of Biological Sciences, 2013, 6(4), 234-238.

Abstract

The aqueous leaf extract of Erythrina senegalensis (ES) was evaluated for its protective activity against carbon tetrachloride (CCl₄)-induced liver injury. A 100, 200 and 400 mg kg^-1 b.wt. of the ES leaf extract was administered to different groups of rats for 7 days prior to the CCl₄ administration. A significant (p<0.05) decrease was observed in both the groups pretreated with 200 and 400 mg kg^-1 b.wt. of the leaf extract on the levels of the enzymes and non enzyme markers of tissue damage, lipid peroxidation and relative organ weights and this is shown to be dose dependant when compared to rats that were given CCl₄ only. These results showed that ES possess hepatoprotective principle (s) that was (were) able to prevent the toxicity of CCl₄ against the liver of rats.

ASCI-ID: 62-237

Timbrell, 1991). CCl₄ is a chemical model commonly used for animal experiment to induce reactive oxygen formation and depletion of glutathione, this may reduce antioxidant enzymes as well as the substrate to induce oxidative stress (Dahiru et al., 2010). The liver damage is associated with membrane lipid peroxidation and cell necrosis (Williams and Burk, 1990; Obidah et al., 2010) which change enzyme activity and finally induce hepatic injury. ES (DC) is a tropical plant of the family fabaceae. The plant has shown to exhibited medicinal potentials in many separate studies (Linuwa et al., 1994; Togola et al., 2005; Mann et al., 2008) and the leaf is also used as vegetable. Some diseases that has been reported by traditional healers to have been treated by the plant include amenorrhea, malaria, jaundice, ulcer, diarrhea, gastrointestinal disorder sterility and wound and body pain (such as chest, back and abdominal pains) (Togola et al., 2005).

This study was designed to evaluate the effect of pretreatment with the leaf extract of ES on CCl₄-induced liver injury in rats.

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MATERIALS AND METHODS

Plant: ES leaf was collected from uncultivated from farmland in Girei Local Govt. Area of Adamawa state and authenticated in plant sciences Department of Modibbo Adama University of technology yola and given a voucher specimen number WH/ESS015/05. The leaf was dried at room temperature.

Preparation of aqueous extract: Freshly plucked leafs of ES were dried at room temperature and ground to powdered form with laboratory mortar and pestle. The powder was sieved and 150 mg was weighed and mixed with 400 mL of distilled water and allow to stand for 6-12 h with continues shaking at time interval. The mixture was then filtered with Whatman filter paper No. 4. The filtrate was then evaporated using rotary evaporator at room temperature (>50°C).

Animals: Thirty male rats weighing 110-150 g were purchased from the animal house of Biochemistry Department of University of Jos, plateau state Nigeria. The rats were then house in cages at room temperature under 12/12 light/dark and were fed with pelleted standard laboratory feed (Vital feed, Grand cereal and oil Mills Jos) and water ad libitum. They were allowed to stand for a period of 7 days to acclimatized before the commencement of the study.

Experimental design: The rats were divided into 5 groups (1-5) o f 6 rats each and were given the extract as follows:

Groups 3, 4 and 5 were pretreated with aqueous leaf extract of ES for 7 days prior to CCl₄ administration. The CCl₄ was dissolved in olive oil and administered intraperitonealy (1:1) 2 mL kg^-1 b.wt. to induce liver injury.

Collection of samples (serum and liver): Rats from all the different groups were sacrificed 48 h after CCl₄ administration and blood samples were collected via ocular vein and allowed to stand for 7-10 min, it was then centrifuged at 300 rpm for 15 min to obtain serum. This was separated for the estimation of transaminases, alkaline phosphatase and bilirubin.

Liver of the rats were quickly excised, weighed and used for the determination of lipid peroxidation. The hepatic lipid peroxidation was determine as thiobarbituric acid reactive substance (TBARS) and expressed as the amount of malondialdehyde (MDA) (Uchiyama and Mihara, 1978).

Statistical analysis: The data generated was subjected to statistical analysis and the result expressed as Mean+SEM. Student t-test was used to determine the statistical difference between 2 mean values at 95% level of confidence (p<0.05).

RESULTS

The result of the pretreatment with the aqueous leaf extract of ES on transaminases, alkaline phosphatase and Total Bilirubin (TB) levels in CCl₄ induced liver injury is shown in Table 1.

The result above demonstrate a trend of increased levels of ALT, AST and ALP significantly (p<0.05) in group 2 (group that was administered CCl₄ alone) as compared to the control group (group 1). There was however an observed significant (p<0.05) decreased in the groups pretreated with 200 and 400 mg kg^-1 b.wt. of ES leaf extract for the period 7 days prior to the CCl₄ administered on the levels of the marker enzymes as compared to group ii.

Table 1:	Effects of pretreatment with ES aqueous leaf extract against CCl4 induced liver damage on enzyme and non enzyme markers of liver damage
Results are Mean±SEM. (n = 6), *Significantly higher than control group (p<0.05), **Significantly lower than group given CCl4 only

Table 2:	Effects of pretreatment with ES aqueous leaf extract against CCl4 induced liver damage in liver lipid peroxidation
Results are Mean±SEM. (n = 6), *Significantly higher than control group (p<0.05), **Significantly lower than group given CCl4 only

Table 3:	Effects of pretreatment with ES aqueous leaf extract against CCl4 induced liver damage on relative organ weight
Results are Mean±SEM. (n = 6), **Significantly lower than group given CCl4 only

Also the decrease in the pretreated groups was observer to be dose dependent as group 4 (400 mg kg^-1 b.wt.) indices of the tissue damage was observed to be decreased significantly (p<0.05) than group 3 and 4 (100 and 200 mg kg^-1 b.wt., respectively).

There is a significant (p<0.05) increase in bilirubin concentration in group administered CCl₄ alone as compared to control (group i). However pretreatment with 200 and 400 mg kg^-1 b.wt. of ES leaf extract prior to CCl₄ administration results in significant (p<0.05) decrease in the level of bilirubin concentration as observed in groups iv and v as compared to group administered CCl₄ alone.

Table 2 shows the effects of pretreatment of with ES leaf extract on lipid peroxidation. The levels of MDA in the group treated with CCl₄ alone increased significantly (p<0.05) when compared to the control group, however the is a significant (p<0.05) decreased as observed in the pretreated groups and it dose dependent manner.

From Table 3, the result shows that pretreatment with the aqueous leaf extract resulted in a significant (p<0.05) increase in the relative organ weight when compared to normal. The Mean final body weight is not however affected by either the CCl₄ or the extract administration.

DISCUSSION

Hepatotoxicity was observed in rats treated with CCl₄ alone, this is an experimental model widely used for hepatoprotective drug screening, as shown by the increased serum levels of the transaminases, this may reflect the cytosolic release of liver marker enzymes into serum that results from the necrotic and degenerative response of hepatocytes (Chawla, 1999) following the CCl₄ administration. The marker enzymes assay plays a significant role in diagnosis of diseases, investigation and assessment of drugs or plant extract for safety and toxicity (Adeoyo and Oyedepo, 2004).

CCl₄ undergoes a biotransformation by hepatic microsomal cytochrome P₄₅₀ to produce a metabolite trichloromethyl free radicals, this hepatotoxic metabolite can react with protein and lipid in the membrane of the cells or organelle leading to necroses of hepatocytes and as a result of the injury, the altered permeability of the membrane causes the enzymes from the cells to be released into the circulation (Drotman and Lawhorn, 1978). The observed significant increase in the serum levels of ALP activity and total bilirubin concentration further demonstrate hepatoxicity in the rats.

Pretreatment with leaf extract of E. senegalensis protect the organ against CCl₄ induced hepatotoxicity as demonstrated by significant decrease in the levels of the liver marker enzymes and other biochemical indices.

The magnitude of hepatic damage is usually assessed by measuring the level of release of cytosolic transaminases (AST, A LT) in circulation (Perez Gutierrez and Solis, 2009). Thus, the observed increase in the levels of these transaminases in group 2 could be attributed to the damage caused by CCl₄ on the structural integrity of the liver, as reported by other researchers (Dahiru et al., 2005, 2010; Galati et al., 2005; Perez Gutierrez and Solis, 2009). Since they are located in the cytoplasm and are released into circulation after damage.

Increase in bilirubin concentration is an index of jaundice, a condition of liver injury, possibly due to increase production or decrease uptake by the liver, decreased conjugation and decreased secretion from the liver or blockage of bile duct (Bun et al., 2006). This was observed in group 2 after CCl₄ administration as an indication of hepatoxicity.

There was an observed significant elevation in the levels of MDA in the CCl₄-treated group. The elevated levels could be seen to be elicited by the administration of a toxicant, CCl₄ which is known to increase lipid peroxidation. However pretreatment with E. senegalensis leaf extract indicated an effective protection of the animals against CCl₄ induced liver lipid peroxidation. Consumption of the extract may be seen to decrease liver organ susceptibility to lipid peroxidation and invariably oxidative stress.

Also pretreatment with the leaf extract protect the liver against change in relative organ weight which normally reflects the pathological state of the organ. The relative weights were observed to consistent with the serum levels of the marker enzymes and MDA.

These findings further demonstrated that from the result presented above and earlier reports by other researchers that CCl₄ causes mononuclear cell infiltration, necrosis and degeneration of hepatocytes (Hung et al., 2006; Liu et al., 2006), the leaf extract of E. senegalensis have the capability of protecting the liver against CCl₄ induced hepatoxicity as well as cellular degeneration and fatty liver development. Thus the leaf extract could be seen to block or minimize to a greater extend adipogenesis.

CONCLUSION

From the results obtained in this study, it is clear that the leaf extract of E. senegalensis has a potent hepatoprotective agent against CCl₄ induced liver damage in rats.

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