Preparative Separation and Isolation of Three Flavonoids and Three Phloroglucinol Derivatives from Hypericum japonicum Thumb. using High‐Speed Countercurrent Chromatography by Stepwise Increasing the Flow Rate of the Mobile Phase

Abstract

A preparative high‐speed counter‐current chromatography (HSCCC) was used to isolate and separate chemical compounds from the medicinal plant H. japonicum. First, the ethanol extract of H. japonicum was directly isolated by HSCCC without any preparation and three flavonoid glycosides including isoquercitrin, quercitrin, and quercetin‐7‐O‐rhamnoside were successfully purified using a two‐phase solvent system composed of ethyl acetate–ethanol–water at the volume ratio of 5:1:5 (v/v) by increasing the flow rate of the mobile phase from 1.0 mL/min to 2.0 mL/min after 120 min. After 400 min, the remaining compounds, mainly containing three phloroglucinol derivatives including sarothialen A, sarothralen B, and sarothalin G in the HSCCC column were forced out by pressurized nitrogen gas, which were further successfully separated by HSCCC with a two‐phase solvent system composed of n‐hexane–ethyl acetate–ethanol–water at the volume ratio of 1:1.2:1.2:1 (v/v) by increasing the flow rate of the mobile phase from 1.2 mL/min to 2.2 mL/min after 120 min. The fractions obtained from HSCCC were analyzed by high performance liquid chromatography, and the separation produced a total of 124 mg isoquercitrin, 85 mg quercitrin, 68 mg quercetin‐7‐O‐rhamnoside, 24 mg sarothialen A, 18 mg sarothralen B, and 58 mg sarothalin G from 750 mg ethanol extract, with the purities of 98.6%, 95.8%, 95.6%, 96.5%, 95.4%, and 98.6%, respectively. The chemical structural identification was carried out by MS, 1H NMR, and 13C NMR.