An effective method based on medium-pressure liquid chromatography and recycling high-speed counter-current chromatography for enrichment and separation of three minor components with similar polarity from Dracocephalum tanguticum.

The separation of minor compounds, especially those with similar polarities from a complex sample, remains challenging. In the proposed study, an effective method based on medium-pressure liquid chromatography and recycling high-speed counter-current chromatography was developed for the enrichment and separation of three minor components from Dracocephalum tanguticum. The crude extract was directly introduced to medium-pressure liquid chromatography for the enrichment of the three minor components. Based on high-performance liquid chromatography analysis, the total content of these three compounds increased from 0.48% in the crude extract to 85.3% in the medium-pressure liquid chromatography fraction. In addition, high-speed counter-current chromatography was employed to separate the enriched compounds using the solvent system hexane/ethyl acetate/methanol/water (1.18:8.82:1.18:8.82, v/v/v/v). As a result, compound 3 and a mixture of compounds 1 and 2 were obtained. In order to improve the resolution of compounds 1 and 2 while saving separation time, a recycling and heart-cut mode was used. Finally, compounds 1 and 2 were obtained after five cycles. These compounds were identified as 3-phenylethyl β-d-glucopyranoside (1), tazettoside E (2), and cirsiliol-4'-glucoside (3). Compounds 1 and 2 were primarily separated from D. tanguticum. Moreover, the developed method provided a reference for the separation of minor components from the complex sample.