English abstract
Snake envenomation is one of the most neglected tropical diseases, which result in high mobidity and mortality. Snake venom is the most complicated among all poisons. It casues many systemic and local complications e.g. local haemorrhage and tissue necrosis, which cannot be reversed with anti-snake venoms. The objectives of this study were to evaluate the capacity of Pithecellobium dulce (P. dulce) leaf extract to neutralise local haemorrhage induced by the venoms of Bothrops jararaca, Crotalus atrox and Echis carinatus and isolating the active components responsible for the anti-haemorrhagic activity. P. dulce leaves were extracted with three different solvents, resulted in a total of nine extracts. The results showed that the increasing doses of crude ethanolic extract, injected subcutaneously, have the best neutralising efficacy of local haemorrhage induced by either individual or pooled venoms of the three snake species. The in vivo assay of the anti-lethality (LD50) of the crude extract tested with B. jararaca venom (1.1mg/kg) showed that, the activity of the extract was significant at doses of 2mg/kg and 8mg/kg where the majority of animals survived. However, higher doses were accompanied with high toxicity as 50-75% of BALB/C mice, were killed. Consequently, the active components of the extract were separated and identified using high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionisation mass spectrometry (MALDI-TOF MS), respectively. The HPLC resulted in 173 fractions in which only three (Frc11, Frc13 and Fr14) were found to have antihaemorrhagic acitivities. The active fractions showed disparity in neutralising snake venoms individually, as Frcll was the only one that showed the best neutralising capacity among the three fractions. However, using the pooled fractions, a complete neutralising achievement against individual and pooled venoms was observed. This was confirmed in the anti-gelatinase activity assay, where pooled fractions inhibited the gelatinase activity of snake venom metalloproteinase. A phytochemical characterisation revealed that the isolated fractions showed the presence of flavonoids and tannins, which was confirmed by MALDI-TOF MS. In parallel to the anti-haemorrhagic capacity of the isolated active fractions, their anti-lethal activity was assessed against pooled venoms using LD100. The results were found to be statistically insignificant as all mice died within less than an hour including the positive controls (commercialised antivenoms). However, the active fractions showed an ability to elongate the animal survival time, especially Frc13 with an average survival period of 37 minutes in contrast with one of the positive controls (Indian antivenom) where the longest survival time was scored with 48 minutes. In conclusion, as the crude extract was found to be highly toxic to mice, therefore, isolating the active fractions was essential in this study. The active fractions were found to be mainly consisting of secondary metabolites such as polyphenols, diterpenoids and tannins, which are linked to their ability of inhibiting the enzymatic activity of the snake venoms.
