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.

中文翻译：

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基于液相萃取-三相溶剂系统和高速逆流色谱的快速策略，用于富集和分离蟾蜍肉中次生丁二烯内酯的差向异构体

这项研究提出了一种基于液相-液相萃取，三相溶剂系统和高速逆流色谱的有效策略，用于从蟾蜍肉中快速富集和分离次要丁二烯内酯的差向异构体。首先通过响应面法优化了回流萃取条件，并建立了由n组成的新型三相溶剂体系。开发了己烷/乙酸甲酯/乙腈/水（3：6：5：5，v / v）用于液-液萃取粗提物。这种综合萃取过程可以有效地从复杂基质中富集次要丁二烯内酯，并最大程度地减少了经典液相两相萃取中由于反复使用不同种类的有机溶剂反复萃取而引起的次要靶标的损失。结果，在中间相中大大增加了四个短丁二烯内酯的差向异构体，这些短丁二烯内酯的差向异构体的总含量从3.25％增加到46.23％。然后，通过HSCCC在由氯仿/甲醇/水（4：2：2，v / v）组成的两相溶剂系统中成功分离出富集的四种差向异构体。此外，我们测试了Na ⁺，K ⁺-ATPase（NKA）对四种差向异构体的抑制作用。布法地那利的3β-异构体显示出比3α-异构体更强（> 8倍）的抑制活性。蟾蜍肉中次生丁二烯内酯的特征及其对NKA抑制作用的显着差异将促进蟾蜍肉作为食物来源的进一步定量分析和安全性评估。