In this study, a new method based on ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) combined with
enzyme channel blocking (ECB) was developed to discover bioactive components from
herbal medicines.
Xanthine oxidase (XOD), a critical
enzyme for treating
gout, was employed as the target
protein for screening. By comparing chromatographic profiles of the compounds binding to XOD before and after the ECB experiment, the selective
ligands could be distinguished from the non-selective binders. In this experiment,
febuxostat bound to the channel entering into the active site of the
enzyme and prevented potential
ligands from binding. Finally, four compounds, namely,
luteolin-7-O-glucoside, apigenin-7-O-glucoside,
luteolin and
apigenin were screened and identified as the candidate XOD inhibitors based on the ultrafiltration chromatogram of Flos Chrysanthemum, a famous
traditional Chinese medicine used in many prescriptions for
gout treatment. To verify the compounds screened further, a microplate method was applied to evaluate their
enzyme inhibitory activities. The IC50 values of the above 4 compounds were 23.61, 38.80, 1.54 and 1.96μM, respectively. The structure-function relationship was also estimated according to the in vitro assay. The results were in favor of the hypothesis that the Flos Chrysanthemum extract might be used for
gout treatment by inhibiting XOD.