Panax japlcus var is a typical Chinese herb with a large number of
saponins existing in all parts of it. The common methods of screening and isolating
saponins are mostly labor-intensive and time-consuming. In this study, a new assay based on ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) was developed for the rapid screening and identifying of the
ligands for
xanthine oxidase from the extract of P. japlcus. Six
saponins were identified as
xanthine oxidase inhibitors from the extract. Subsequently, the specific binding
ligands, namely, 24 (R)-majoroside R1,
chikusetsusaponin IVa, oleanolic acid-28-O-β-D-glucopyranoside, notoginsenoside Fe,
ginsenoside Rb2 and
ginsenoside Rd (the purities of them were 95.74%, 96.12%, 93.19%, 94.83%, 95.07% and 94.62%, respectively) were separated by high-speed counter-current chromatography (HSCCC). The component ratio of the
solvent system of HSCCC was calculated with the help of a multiexponential function model was optimized. The partition coefficient (K) values of the target compounds and resolutions of peaks were employed as the research indicators, and exponential function and binomial formulas were used to optimize the
solvent system and flow rate of the mobile phases in a two-stage separation. An optimized two-phase
solvent system composed of
ethyl acetate,
isopropanol, 0.1% aqueous
formic acid (1.9:1.0:1.3, v/v/v, for the first-stage) and that composed of
methylene chloride,
acetonitrile,
isopropanol, 0.1% aqueous
formic acid (5.6:1.0:2.4:5.2, v/v/v/v, for the second-stage) were used to isolate the six compounds from P. japlcus. The targeted compounds isolated, collected and purified by HSCCC were analyzed by high performance liquid chromatography (UPLC), and the chemical structures of all the six compounds were identified by UV, MS and NMR. The results demonstrate that UF-LC-MS combined with HSCCC might provide not only a powerful tool for screening and isolating
xanthine oxidase inhibitors in complex samples but also a useful platform for discovering bioactive compounds for the prevention and treatment of
gout.