Identification of Potential COX-2 Inhibitors from Eleusine indica Through In Vitro Cyclooxygenase Assay, LC-MS/MS-based Profiling, Molecular Docking, and Molecular Dynamics Simulation

Introduction: Cyclooxygenase-2 (COX-2) is a key enzyme involved in the production of pro-inflammatory mediators and is an important target for anti-inflammatory drug development. This study investigated the anti-inflammatory potential of Eleusine indica through in vitro COX inhibition, LC–MS-based phytochemical profiling, and computational analyses to identify potential COX-2 inhibitory metabolites. Methods: The methanolic leaf extract of E. indica was evaluated for COX-1 and COX-2 inhibitory activities. Metabolites were identified using liquid chromatography–mass spectrometry (LC–MS) and screened for drug-likeness, ADMET, and toxicity properties using SwissADME and ProTox-3.0. Selected metabolites were subjected to molecular docking against COX-2 (PDB ID: 4COX), and the top-ranked compound was further evaluated through a 100 ns molecular dynamics simulation. Results: The extract exhibited significantly greater inhibition of COX-2 (IC_₅₀ = 4.0 μg/mL) than COX-1 (IC_₅₀ = 12.5 μg/mL; p = 0.0005), indicating preferential COX-2 inhibition. LC–MS analysis tentatively identified ten metabolites, among which diosmetin, sinapine, and senkyunolide I showed favorable drug-likeness, pharmacokinetic, and toxicity profiles. Molecular docking revealed that diosmetin exhibited the strongest binding affinity toward COX-2 (−7.8 kcal/mol), followed by senkyunolide I (−6.6 kcal/mol) and sinapine (−6.3 kcal/mol). Molecular dynamics simulations confirmed the stability of the diosmetin–COX-2 complex through reduced structural fluctuations, maintained protein compactness, and persistent hydrogen-bond interactions. Conclusions: Diosmetin emerged as the most promising metabolite from E. indica, exhibiting favorable pharmacokinetic, safety, docking, and molecular dynamics profiles. These findings suggest that diosmetin is a promising natural COX-2 inhibitor that warrants further experimental validation.