Fluorescent labeled enzyme immobilized on chitosan-coated magnetic microspheres for potential cyclooxygenase-2 inhibitors screening accompanied with molecular modeling and in situ cell imaging

Fluorescein isothiocyanate (FITC)-labeled cyclooxygenase-2 (COX-2) immobilized chitosan (CS) coated Fe₃O₄ (FITC-COX-2-CS@Fe₃O₄, FCICFs) nanocomposites were accurately prepared by solvothermal and crosslinking methods, and employed as drug discovery platforms and fluorescent tracers to specifically screen and evaluate the activity of potential COX-2 inhibitors derived from Yaobitong capsules (YBTC). The FCICFs nanocomposites demonstrated a significant capacity for the selective recognition of positive compounds from artificial mixtures, indicating their substantial utility. Under optimal conditions, the prepared nanocomposites screened fourteen potential COX-2 inhibitors from YBTC, which were identified by UHPLC-MS/MS as tetrahydrocolumbamine, protopine, trahydocoptisine, yuanhunine, rotundine, tetrahydroberberine, thalictrimine, palmatine, dehydrocorydaline, mudanpioside D, astringin, nodakenin, paeoniflorin sulfite and ligustilide. The molecular docking analysis demonstrated a strong binding affinity between 14 small molecule ligands and the COX-2. Furthermore, the screened compounds exhibited significantly enhanced inhibitory activity against COX-2 compared to the YBTC crude extract. Fluorescence cell imaging results indicated that the screened compounds effectively inhibited synovial cell proliferation, suggesting their potential therapeutic efficacy in the treatment of rheumatoid arthritis (RA). The proposed methodology, integrating highly specific screening with in-situ visual imaging, established a robust platform for the efficient and selective screening of plant-derived biologically active molecules.