Magnetic ligand fishing combination with high-performance liquid chromatography-diode array detector-mass spectrometry to screen and characterize cyclooxygenase-2 inhibitors from green tea.

Cyclooxygenase-2 (COX-2) inhibitors may be used to efficiently treat inflammation or cancer diseases. In the present study, we established a new screening assay based on magnetic Fe3O4@SiO2-COX-2 ligand fishing combination with high-performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MSn) to screen and identify COX-2 inhibitors from green tea. Optimized conditions (pH at 7.4, temperature at 30°C, and incubation time for 30min) for fishing out COX-2 inhibitors were achieved by testing positive control, celecoxib, with active and inactive COX-2. Notably, immobilized COX-2 showed high stability (remained 94.7% after ten consecutive cycles), reproducibility (RSD<10% for batch-to-batch evaluation). Finally, eight catechins with COX-2 binding activity were screened in green tea, and their structures were characterized by ultraviolet (UV), accurate molecular weight, diagnostic fragment ions and nuclear magnetic resonance (NMR). Particularly, the COX-2 inhibitory activities of two rare catechins, [(-)-epigallocatechin-3-(3″-O-methyl)-gallate (3″-O-methyl-EGCG, IC50=0.17±0.03μM 0.16±0.01), (-)-epicatechin-3-(3″-O-methyl)-gallate (3″-O-methyl-ECG, IC50=0.16±0.02μM)], were reported for the first time. The results indicated that the proposed method was a simple, robust and reproducible approach for the discovery of COX-2 inhibitors from complex matrix.