An Efficient Strategy Based on Liquid–Liquid Extraction with Three-Phase Solvent System and High Speed Counter-Current Chromatography for Rapid Enrichment and Separation of Epimers of Minor Bufadienolide from Toad Meat

This study presents an efficient strategy based on liquid–liquid extraction with three-phase solvent system and high speed counter-current chromatography for rapid enrichment and separation of epimers of minor bufadienolide from toad meat. The reflux extraction conditions were optimized by response surface methodology first, and a novel three-phase solvent system composed of n-hexane/methyl acetate/acetonitrile/water (3:6:5:5, v/v) was developed for liquid–liquid extraction of the crude extract. This integrative extraction process could enrich minor bufadienolide from complex matrix efficiently and minimize the loss of minor targets induced by repeated extraction with different kinds of organic solvents occurring in the classical liquid two-phase extraction. As a result, four epimers of minor bufadienolide were greatly enriched in the middle phase and total content of these epimers of minor bufadienolide was increased from 3.25% to 46.23%. Then, the enriched four epimers were separated by HSCCC with a two-phase solvent system composed of chloroform/methanol/water (4:2:2, v/v) successfully. Furthermore, we tested Na+,K+-ATPase (NKA) inhibitory effect of the four epimers. 3β-Isomers of bufadienolide showed stronger (>8-fold) inhibitory activity than 3α-isomers. The characterization of minor bufadienolide in toad meat and their significant difference of inhibitory effect on NKA would promote the further quantitative analysis and safety evaluation of toad meat as a food source.