Combination of pressurized liquid extraction with countercurrent chromatography for systematical isolation of ginsenosides from Panax ginseng

• A novel three-stage PLE coupled with HSCCC was successfully established.
• The solvent systems of PLE/HSCCC were calculated by mathematical model.
• We elaborate the mechanism of three-stage PLE/HSCCC in detail.
• More than nine compounds were separated within 450 min.

Automation of pressurized liquid extraction (PLE) coupled with high-speed counter-current chromatography based on mathematical calculation was firstly developed and applied to the systematic extraction and online isolation of chemical constituents with a wide range of polarity from Panax ginseng. The experiments were designed under the guidance of optimized mathematical model, the K values of the target compounds and the peak resolutions were employed as the research indicators to calculate and optimize the solvent systems and the flow rates of the mobile phases. At the first stage, the upper phase of the solvent system of ethyl acetate–n-butanol–water (1.1:1.0:2.1, v:v:v) was used as both the PLE solvent and HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase of the solvent system to separate the polar common compounds. At the second stage, the upper phase of the solvent system of ethyl acetate–n-butanol–methanol–water (3.1:0.6:1.0:2.6, v:v:v:v) was used as both the PLE solvent and HSCCC stationary phase (extracted at 110 °C), and the target compounds were eluted with the corresponding lower phase of the solvent system to separate the moderate polar compounds. Finally, the upper phase of the solvent system of n-hexane–n-butanol–methanol–water (4.2:1.0:1.2:4.5, v:v:v:v) was used as both the PLE solvent and HSCCC stationary phase (extracted at 125 °C), and the target compounds were eluted with the corresponding lower phase of the solvent system to separate the low polar compounds. Nine target compounds with purities above 93.35% and two non-target compounds with purities above 73.65% were successfully semi-preparative separated in 460 min.

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