Journal Article

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

Chen, Lina, Ma, Bing, Liu, Chun-ming, Wang, Jing, Song, Fengrui, Liu, Zhiqiang, Liu, Shu

2014

Overview

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. Graphical Abstract ᅟ

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: Allopurinol / Analysis / Analytical Chemistry / Animals / bioactive properties / Biochemistry / Cattle / Characterization and Evaluation of Materials / Chemistry / Chemistry and Materials Science / Chromatography / Chromatography, High Pressure Liquid - methods / countercurrent chromatography / Countercurrent Distribution / Drug Discovery / electrospray ionization mass spectrometry / Enzyme inhibitors / Enzyme Inhibitors - pharmacology / Enzyme kinetics / Enzymes / Flavonoids - pharmacology / Food Science / Gout / High performance liquid chromatography / Hyperuricemia / Hypoxanthine / Inhibition / Inhibitors / Ionization / Ions / Laboratory Medicine / Liquid chromatography / Mass spectrometry / Mass spectroscopy / metabolism / Molybdenum compounds / Monitoring/Environmental Analysis / Oxidases / Photodiodes / Physiological aspects / Quadrupoles / rapid methods / Research Paper / Rheumatism / Risk analysis / Risk factors / Scientific imaging / Screening / Selaginellaceae - chemistry / Spectrometry, Mass, Electrospray Ionization - instrumentation / Spectrometry, Mass, Electrospray Ionization - methods / Spectroscopy / Substrates / ultra-performance liquid chromatography / Ultrafiltration / Ultrafiltration - methods / Uric acid / xanthine / Xanthine oxidase / Xanthine Oxidase - antagonists & inhibitors / Xanthine Oxidase - metabolism / Xanthines