An Efficient Strategy to Separate the Main Ginsenosides From Steamed Panax quinquefolius by a Combination of Consecutive and Online‐Storage Inner‐Recycling High‐Speed Counter‐Current Chromatography

This study aimed to isolate and purify compounds from seven-steamed and seven-dried Panax quinquefolius L. by employing a novel separation strategy combining macroporous resin column chromatography, high-speed countercurrent chromatography (HSCCC), and an on-line storage inner-recycling countercurrent chromatography (OS-IRCCC) mode for the first time, which yielded 12 compounds, including 11 ginsenosides and one methyl naphthalene derivative. Steaming processing of P. quinquefolius triggers significant chemical transformations, characterized by the progressive degradation of major ginsenosides and the concomitant formation and marked accumulation of rare ginsenosides. After preliminary fractionation of the crude extract using a D101 macroporous resin column, three fractions (Fractions III, V, and VI) were selected for further targeted separation. Fractions III and VI were separated using the solvent system of dichloromethane/methanol/H₂O/isopropyl alcohol with different ratios of 6:5:4:2 and 6.2:4.8:4:2, respectively. For fraction V, traditional HSCCC combined with OS-IRCCC technology was adopted, using methyl tert-butyl ether/n-butanol/acetonitrile/H₂O (4:2:3:7, v/v). Ultimately, 12 compounds were obtained, including ginsenoside Rd (1, 6.2 mg), ginsenoside Re (2, 7.5 mg), ginsenoside Rg₁ (3, 2.3 mg), 20(R)-ginsenoside Rh₁ (4, 8.3 mg), ginsenoside Rb₁ (5, 3.7 mg), rubilactone (6, 6.4 mg), ginsenoside Re₈ (7, 6.3 mg), 20(S)-ginsenoside Rg₂ (8, 12.1 mg), 20(R)-ginsenoside Rg₂ (9, 5.7 mg), damulin B (10, 2.7 mg), ginsenoside Rb₃ (11, 4.3 mg), and ginsenoside Rg₃ (12, 5.1 mg). This research highlights the efficacy of the novel separation technique for isolating and purifying valuable compounds from seven-steamed and seven-dried P. quinquefolius, providing a robust methodological framework for the targeted purification of complex natural products.