Separation and Purification of Ginsenosides and Flavonoids in From the Leaves and Stems of Panax quinquefolium by High-Speed Countercurrent Chromatography and Online-Storage Inner-Recycling Countercurrent Chromatography

Abstract

Study aimed to isolate and purify compounds from the stems and leaves of Panax quinquefolius. By employing a highly innovative separation technique that combined multistage countercurrent chromatography (MRCC), high-speed countercurrent chromatography (HSCCC), and an advanced online-storage inner-recycling countercurrent chromatography (OS-IRCCC) mode for the first time, 12 compounds were successfully isolated, including 10 ginsenosides and 2 flavonoids. First, the crude extract was fractionated into five parts using D101 MRCC, with HPLC analysis revealing that 40% and 60% ethanol eluate contained the highest compound diversity. Overall, 40% ethanol eluate was separated using the solvent system of EtOAc/n-BuOH/H₂O (2:1:3, v/v), whereas 60% ethanol eluate underwent traditional countercurrent chromatography coupled with OS-IRCCC separation using the solvent system of methyl tert-butyl ether (MTBE)/n-BuOH/ACN/H₂O (4:2:3:8, v/v). Ultimately, various compounds were obtained, including kaempferol 3-O-β-d-glucopyranosyl-(1 → 2)-β-d-galactopyranosyl-7-O-α-l-rhamnopyranoside (13.2 mg), ginsenoside Rc (7.4 mg), 20(R)-ginsenoside Rh₁ (7.2 mg), ginsenoside Re (12.3 mg), kaempferol 3-O-β-d-glucopyranosyl-(1 → 2)-β-d-galactopyranoside (14.1 mg), ginsenoside Rb₁ (8.2 mg), ginsenoside Rb₂ (17.5 mg), ginsenoside Rb₃ (27.3 mg), ginsenoside Rg₁ (13.3 mg), ginsenoside Rg₂ (9.7 mg), ginsenoside Rd (11.4 mg), and pseudo-ginsenoside F₁₁ (16.7 mg). This research highlights the efficacy of the novel separation technique in isolating and purifying valuable compounds from P. quinquefolius stems and leaves.