Comparison of toad venoms from different Bufo species by HPLC and LC-DAD-MS/MS

Abstract

Ethnopharmacological relevance Toad venom, called Chansu in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers for a long time in clinic. Aim of the study The aim of the study is to investigate the chemical differences among a variety of toad venoms from different geographic locations and related Bufo species. Materials and methods Ten batches of commercial toad venom collected from different regions in China, one batch of fresh toad venom obtained from Bufo bufo gargarizans, and six batches of related Bufo species were analyzed by HPLC and LC-DAD-MS/MS. Individual components were identified by comparison of retention times, UV spectra, and mass spectra with authentic compounds, standard addition, as well as summarized MS fragmentation rules. Based on the profile of identified constituents and the content of cinobufagin and resibufogenin, the chemical differences observed among different samples are discussed. Results Overall, 43 compounds were identified in the methanolic extracts of the different samples of toad venom. Besides of suberoyl arginine, several free bufadienolides, bufadienolide sulfates, and suberoyl esters of bufadienolides were found. The total amounts of cinobufagin and resibufogenin, which are the only two control markers according to the current Chinese Pharmacopoeia, varied widely from 0.7% to 10.9% in the commercial Chansu samples collected in the different locations in China. Low levels of resibufogenin, but no cinobufagin was observed in the samples from Bufo melanosticus and Bufo marinus, and even neither of both compounds was found in the sample from Bufo viridis. Conclusions The chemical profiles of the different commercial and collected toad venoms from related Bufo species differed significantly, not only in the absolute and relative contents, but also in the number and type of the constituents. The main reason for this variation are species-specific differences, but additional factors, such as the harvest and post-harvest processing, and adaption to environmental factors in different geographic locations, also seem to contribute.