Biomedical studies on the protection of mucuna pruriens seed extract against the toxic actions of snake venoms

Abstract

Seed of Mucuna pruriens (Velvet beans) has been prescribed by traditional medicine practitioners in Nigeria as anti-snake bite remedy. Recent studies indicated that pretreatment with Mucuna pruriens seed extract (MPE) in mice did confer protection against the lethal effects of Echis carinatus and cobra venom. In the present studies, the protective effects of MPE against the lethalities and pharmacological actions of several snake venoms were investigated and the mechanism of the protective action was explored. Survival studies using rats showed that the MPE pretreatment did confer effective protection against lethality of Naja naja sputatrix (NNS) venom, and moderate to weak protection against the lethalities of Calloselasma rhodostoma (CR), Bungarus candidus, Ophiophagus hannah and Vipera russelli russelli venoms. Indirect ELISA and immunoblotting studies showed that there were extensive cross-reactions between anti-Mucuna pruriens (anti-MPE) IgG and various snake venoms, suggesting the involvement of immunological neutralization in the protective effect of MPE pretreatment against snake venom poisoning. The ELISA results were confirmed by Western blotting studies. In vitro neutralization experiments using mice showed that anti-MPE antibodies effectively neutralized the lethalities of Naja naja sputatrix, Naja kaouthia and Naja nivea venom, but were not very effective against other venoms tested, presumably because the antibodies were not of sufficiently high titres. Both immunoblotting and in vitro neutralization experiments showed that the anti-MPE antibodies cross-reacted with the two major toxins of NNS venom: phospholipase A2 and neurotoxin, but was not reactive against the venom cardiotoxin. The results indicated that anti-MPE antibodies could be used in the antiserum therapy of Asiatic cobra (Naja) bites, and that the protective mechanism of MPE pretreatment against Asiatic cobra venom poisoning involves immunological neutralization of cobra venom phospholipase A2 and neurotoxin by antibodies elicited by MPE pretreatment. x Pharmacological experiments using anesthetized rats showed that the MPE pretreatment significantly attenuated the depression effects of the NNS and CR venoms on the blood pressure, heart rate, respiratory rate and nerve-evoked muscle twitch tension. The protective effect against the cardiovascular, respiratory and neuromuscular depressant effects of NNS venom is probably due mainly to the ability of anti-MPE antibodies to neutralize phospholipases A2 and neurotoxins of the venom. However, experiments using isolated spontaneous beating rat atrial preparation showed that the MPE pretreatment also prevented the reduction in atrial contractility and rate induced by the cobra venom, indicating that there are mechanisms other than that of immunological interactions, by which MPE can exert its protective effect against NNS venom. MPE pretreatment may have a direct effect on rat heart rendering the heart more resistant to venom-induced cardiovascular depressant effect. As a whole, pharmacological investigations indicated that the protective action of MPE pretreatment against NNS venom involves protection against the toxic action of the venom in heart and, to a lesser extent, the neuromuscular depressant effect. Histological studies on rat organs confirmed results from the pharmacological studies: injection of NNS venom in untreated rats caused disruption in the striations of the heart muscle but the histological damages were prevented by MPE pretreatment. The effects of MPE pretreatment on gene expression alterations in rat heart induced by injection of NNS venom were also examined using microarray analysis and real time-PCR. Microarray analysis demonstrated that NNS venom injection resulted in the down-regulation of a vast number of genes encompassing a wide variety of biochemical pathways that affect the metabolism and structure of rat heart resulting in its dysfunction. Principal component analysis of the microarray data showed that MPE pretreatment greatly altered the gene expression changes in rat heart induced by the NNS venom, presumably mainly via the immunological neutralization of the cobra phospholipases A2 and neurotoxins. However, examination of the microarray analysis and real time-PCR of gene expression in rat heart as a result of MPE pretreatment showed that MPE may exert a direct protective effect too. Close to 50 genes were significantly up-regulated. In addition to the expected up-regulation of genes involved in immune and inflammatory responses, certain genes involved in energy supply and maintenance of homeostasis of the heart were also significantly up-regulated. Thus, gene expression studies together with pharmacological studies using isolated spontaneously beating rat atrial preparation do support the possibility that the protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on heart. However, further experiments are necessary to prove this hypothesis.