Effect of Methanolic Extract of Vernonia amygdalina (Common Bitter Leaf) on Lipid Peroxidation and Antioxidant Enzymes in Rats Exposed to Cycasin

RESULTS

Colonic lipid peroxidation, represented by the level of maondialdehyde, was reported to be significantly elevated (p<0.05) in rats maintained on diet containing cycads compared with the control (Fig. 1). Prolonged administration of the extract of Vernonia amygdalina (VA) seemed to reversed this increase as there was no significant difference (p>0.05) in the colonic malondialdehyde levels of rats exposed to VA for a minimum of 5 weeks compared with the control (Fig. 1).

Exposure of the experimental animals to 5% cycads without VA intervention appeared to significantly reduce (p<0.05) the activity of colonic Superoxide Dismutase (SOD) compared with the controls (Fig. 2). Administration of VA appeared to have been effective in restoring the activity of colonic SOD to levels that are not significantly different (p>0.05) from the controls (Fig. 2).

Fig. 1:	Concentration of malondialdehyde (MDA) expressed as μmole MDA g-1 colonic tissue (n = 10). ND is normal diet; C is 5% Cycas revoluta; VA is Vernonia amygdalina (200 mg kg-1 b.wt.)

Fig. 2:	Activity of superoxide dismutase (SOD) expressed as Units/g colonic tissue (n = 10). ND is normal diet; C is 5% Cycas revoluta; VA is Vernonia amygdalina (200 mg kg-1 b.wt.). (1 Unit of enzyme activity is the amount of enzyme that is required to cause 50% inhibition of the auto-oxidation of adrenaline to adrenochrome)

Fig. 3:	Activity of catalase expressed as μmoles/min/mg colonic tissue (n = 10). ND is normal diet; C is 5% Cycas revoluta; VA is Vernonia amygdalina (200 mg kg-1 b.wt.)

Priming of the animals with VA for 5 weeks before exposure to cycads seemed to have elicited the most statistically significant increase (p<0.05) in the activity of colonic SOD compared with the controls (Fig. 2).

Treatment of the rats with cycads and extracts of VA did not result in any significant change (p>0.05) in the activity of colonic catalase (Fig. 3).

DISCUSSION

It has been strongly suggested that colorectal carcinogenesis is associated with intense oxidative stress and the progression of the cancer is certain in the face of increased imbalance in the oxidative-antioxidative disorder (Skrzydlewska et al., 2005). The use of synthetic chemotherapeutic drugs has a major limitation of toxicity considering the damage that is done to several organs, especially the liver in the course of detoxifying the drugs. An attractive approach to mitigate this side effect involves the increasing application of natural plant products which are currently being investigated in several laboratories for their chemotherapeutic potentials. In this regard, the presence of beneficial phytochemicals in both the aqueous and organic extracts of Vernonia amygdalina (VA) has been reported to be effective in the management of some biochemical alterations in leukemic and breast cancers (Izevbigie, 2003).

Lipid peroxidation is mainly caused by the action of reactive oxygen species on polyunsaturated fatty acids which are integral structural components of biological membranes. In addition, the products of lipid peroxidation have been shown to interfere with the functional capabilities of cells considering their propensity to cause DNA cross-linkage, DNA breakage and DNA adducts (Mattagajasingh et al., 2008). Cycas revoluta and other members of the Cycas family have been shown to contain cycasin which has the ability to cause the formation of agents like azoxymethane which have free radical activity (Rosenberg et al., 2009). This may account for the elevated amount of malondialdehyde-an index of lipid peroxidation in the experimental animals exposed to cycads only compared with the controls.

Among the measures that are employed by cells to counteract the deleterious effects of lipid peroxidation is the alteration of the activity of antioxidant enzymes such as catalase, peroxidases and superoxide dismutase (Bhor et al., 2004). Non-enzymatic antioxidants such as flavoinoids and other secondary plant metabolites may also be administered to animal cells to augment the activities of the antioxidant enzymes (Eriyamremu et al., 2008). This may partly explain the observed amelioration of the effects of lipid peroxidation following oral exposure to the methanolic extract of VA (Fig. 1). This result is in consonance with the findings of similar works where carbon tetrachloride-induced hepatic lipid peroxidation was ameliorated by the methanolic extract of VA (Adesanoye and Farombi, 2010; Arhoghro et al., 2009). As VA contains high amounts of luteolin, luteolin 7-O-β-glucoside, luteolin 7-O-β-glucuronoside and phenolic antioxidants (Igile et al., 1994), it may in part account for the ability of VA to counteract lipid peroxidation.

Superoxide dismutase is a metalloenzyme that scavenges superoxide anions and exists as isoforms classified on the basis of their relative requirement for manganese, copper and zinc to maintain structural and functional integrity (Sasaki et al., 2000). The observed restoration of the activity of superoxide dismutase to values within the normal range by the VA extract may be as a result of the established presence of flavonoids as well as micronutrients like zinc, manganese and copper in VA (Fig. 2) (Eyong et al. 2011). Factors that could impair the availability of these micronutrients such as dietary restrictions or chelators present in diets have long been reported to adversely affect the activity of the different isozymes of SOD (Harris, 1992).

Although the scope of this work did not include estimation of the activity of the different isozymes of SOD, it might not be out of place to speculate that the observed overall reduction of SOD activity in the absence of VA extract, as shown in Fig. 2, might be related to the presence of antinutrients in the cycad plant. This finding is consistent with the work of Eriyamremu et al. (2007) who, among other things, investigated the effect of different diet formulations containing cycads on the activity of SOD. Further studies will be required to ascertain the SOD isozymes that are most susceptible to the effects of cycads as well as their relative interaction with identified micronutrients in VA.

Another antioxidant enzyme that is known to be recruited by cells to check oxidative damage is catalase which catalyses the decomposition of hydrogen peroxide produced during the harmful oxidation process (Shinde et al., 2012). This study did not record any change in catalase activity suggesting that this enzyme may not be among the first line of enzymatic antioxidant defence during the first weeks of exposure to cycasin (Fig. 3). This is in agreement with the earlier report from a similar work by Eriyamremu et al. (2007). The results presented in this study strongly suggest that the methanolic extract of Vernonia amygdalina could mitigate cycasin-induced oxidative damage in colonic tissues.