Separation of six antioxidants from Hypsizygus marmoreus by high-speed countercurrent chromatography utilizing an approach based upon the polarity parameter model.

For a successful high-speed countercurrent chromatography (HSCCC) separation of multiple components, the suitable solvent system selection is a critical operation. Despite the difference in separation mechanism between HSCCC and HPLC, K values of compounds in the solvent system and the retention factor of compounds in HPLC were connected with polarity, and the polarity mainly depended on the structure and chemical properties of compounds. On the basis of the concept of "like dissolves like", the average polarity of the solvent system is equal to the average polarity of multiple components at the "sweet point" log K = 0. The result of theoretical deduction showed a negative linear correlation between the polarity of each component and the logarithm of the gradient range of the organic phase. Therefore, the average polarity of unknown multiple components could be obtained through the polarity parameter model established in the HPLC analysis. The suitable solvent system composed of n-hexane-ethyl acetate-methanol-water (8:3:8:1, v/v) was selected through the average polarity of multi-components combining K values of the enriched samples. In the context of bioassay, an efficient HSCCC separation procedure was established, and two groups of analogue benzaldehyde derivatives (four main antioxidants, namely, isodihydroauroglaucin, isoaspergin, isotetrahydro-auroglaucin, and flavoglaucin; two minor antioxidants, namely, 6'-oxo-chaetopyranin and chaetopyranin) were obtained from Hypsizygus marmoreus. The predicted polarity values of multi-components were sufficient to meet the HSCCC experimental requirements. The HPLC analysis of reference compounds and multi-components showed a significant consistency with different chromatographic columns. Therefore, the polarity parameter model established in the HPLC analysis was a simple, rapid, and helpful tool for looking an appropriate solvent system, which was a forwarding step for the HSCCC separation of multiple components.