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.
The Tibetan antelope (chiru, Pantholops hodgsoni), a heavily poached species and symbol of the QinghaiTibetan Plateau (QTP), is noted worldwide for its special calving migration. This species originated in the early Quaternary and it is interesting to know how the following climatic oscillations affected its demographic dynamics in the climate-sensitive QTP. In this study, we analyzed the mitochondrial D-loop region from 312 individuals sampled in all of the six major populations. We found high rates of gene flow and little genetic differentiation between populations, suggesting that the calving migration may have homogenized the genetic pool of this species. Both mismatch distribution analyses and coalescent simulations suggested that this species experienced a demographic expansion approximately 600-200 Kyr following the retreat of the large glaciers developed in the QTP at 800-600 Kyr, rather than at the end of the last glacial age, as previously suggested, based on a limited sample size. In addition, we found evidence of a chiru population decrease probably related to the human settings at the QTP during the middle Holocene.
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