Isolation, purification and characterization of the toxins secreted by the paratoid glands of Bufo biporcatus Gravenhorst

Paratoid secretions of Bufo biporcatus were collected, precipitated by 95% ethanol and subjected to solvent partitioning yielding three fractions: diethylether, ethyl acetate and aqueous. Each fractions was concentrated and analyzed via thin layer chromatography [TLC]. Bufadienolides (bufotoxins), visualized after spraying with 20% Sb Cl3 were detected in the highest concentration in the ether extract. The aqueous extract did not show significant amounts of the bufadienolide. HPLC [high pressure liquid chromatography] analysis of the ether fraction yielded three peaks. Subsequent spiking with resibufogenin as standard resulted in an increased peak area for the major component in the fraction. This indicated that the peak could be resibufogenin. TLC of the ether extract yielded three spots where the major component was subjected to HPLC. IR [infrared] analysis indicated the presence of all functional groups in the structure of the standard which further suggested that the bufodienolide could be resibufogenin or its derivative. Another major compound detected in the paratoid secretion was an aromatic amine. This was detected through TLC with n-butanol: acetic acid-H2O (4:1:5) as mobile phase. The major spot localized by the Ehrlich's reagent was evaluated through IR analysis and probably corresponds to epinephrine. The isolation and identification of compounds from the skins of Bufo marinus were also done for comparative studies following the procedure mentioned for Bufo biporcatus which showed parallel results. Acute toxicity of the crude extract and the ether fractions (1:1 w/v) of the paratoid secretions of Bufo biporcatus and Bufo marinus were evaluated. The crude and the ether extracts of the paratoid secretions of B. marinus were more effective in killing the laboratory animals as compared to that of B. biporcatus. There were extensive damages to the kidney, lungs and liver based on gross and microscopic examinations, which were more pronounced in animals treated with B. marinus. There seemed to be no abnormalities on the heart and spleen sections, which indicated no apparent toxicity on these organs. Although the aqueous extracts from B. biporcatus did not result to mortality, histopathological examination revealed damage to kidneys, e.g. tubular necrosis. This was observed to a lesser degree with the aqueous extract from B. marinus and the crude extract of B. biporcatus. This could have been due to the differential secretion of the toxic elements in the toads on the possibility of producing additional toxic compounds with different properties in each food species