Chemical profiling and cytotoxicity assay of bufadienolides in toad venom and toad skin.

ETHNOPHARMACOLOGICAL RELEVANCE

Toad venom and toad skin have been widely used for treating various cancers in China. Bufadienolides are regarded as the main anticancer components of toad venom, but the difference on composition and anticancer activities of bufadienolides between toad venom and toad skin remains unclear.

METHODS

Fractions enriched with free and conjugated bufadienolides were prepared from toad venom and toad skin. Bufadienolides in each fraction were comprehensively profiled by using a versatile UHPLC-TOF-MS method. Relative contents of major bufadienolides were determined by using three bufogenins and one bufotoxin as marker compounds with validated UHPLC-TOF-MS method. Furthermore, cytotoxicity of the fractions was examined by MTT assay.

RESULTS

Two fractions, i.e., bufogenin and bufotoxin fractions (TV-F and TV-C) were isolated from toad venom, and one bufotoxin fraction (TS-C) was isolated from toad skin. Totally 56 bufadienolides in these three fractions were identified, and 29 were quantified or semi-quantified. Bufotoxins were identified in both toad venom and toad skin, whereas bufogenins exist only in toad venom. Bufalin-3-conjugated bufotoxins are major components in toad venom, whereas cinobufotalin and cinobufagin-3-conjugated bufotoxins are main bufotoxins in toad skin. MTT assay revealed potent cytotoxicity of all the fractions in an order of TV-F>TV-C>TS-C.

CONCLUSIONS

Our study represents the most comprehensive investigation on the chemical profiles of toad venom and toad skin from both qualitative and quantitative aspects. Eight bufotoxins were identified in toad skin responsible for the cytotoxicity for the first time. Our research provides valuable chemical evidence for the appropriate processing method, quality control and rational exploration of toad skin and toad venom for the development of anticancer medicines.