Rapid screening and detection of XOD inhibitors from S. tamariscina by ultrafiltration LC-PDA-ESI-MS combined with HPCCC.

Xanthine oxidase (XOD) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction of which could cause hyperuricemia, a risk factor for gout. Inhibition of XOD is a major treatment for gout, and biflavonoids have been found to act as XOD-inhibitory compounds. In this study, ultrafiltration liquid chromatography with photodiode-array detection coupled to electrospray-ionization tandem mass spectrometry (UF-LC-PDA-ESI-MS) was used to screen and identify XOD inhibitors from S. tamariscina. High-performance counter-current chromatography (HPCCC) was used to separate and isolate the active constituents of these XOD inhibitors. Furthermore, ultrahigh-performance liquid chromatography (UPLC) and triple-quadrupole mass spectrometry (TQ-MS) was used to determine the XOD-inhibitory activity of the obtained XOD inhibitors, and enzyme kinetics was performed with Lineweaver-Burk (LB) plots using xanthine as the substrate. As a result, two compounds in S. tamariscina were screened as XOD inhibitors: 65.31 mg amentoflavone and 0.76 mg robustaflavone were isolated from approximately 2.5 g S. tamariscina by use of HPCCC. The purities of the two compounds obtained were over 98 % and 95 %, respectively, as determined by high-performance liquid chromatography (HPLC). Lineweaver-Burk plot analysis indicated that amentoflavone and robustaflavone were non-competitive inhibitors of XOD, and the IC 50 values of amentoflavone and robustaflavone for XOD inhibition were 16.26 μg mL(-1) (30.22 μmol L(-1)) and 11.98 μg mL(-1) (22.27 μmol L(-1)), respectively. The IC 50 value of allopurinol, used as the standard, was 7.49 μg mL(-1) (46.23 μmol L(-1)). The results reveal that the method for systematic screening, identification, and isolation of bioactive components in S. tamariscina and for detecting their inhibitory activity using ultrafiltration LC-ESI-MS, HPCCC, and UPLC-TQ-MS is feasible and efficient, and could be expected to extend to screening and separation of other enzyme inhibitors.