Mohseni-Motlagh, A.; Alereza, T.; Mozaffari, L.; Rozbeh, M.

Flavonoids have been widely investigated for their antiviral and anti-inflammatory properties, but their mechanisms of action often remain insufficiently defined. In this study, high-purity flavonoids were evaluated using an integrated workflow combining molecular docking, LigPlot+ interaction mapping, surface plasmon resonance (SPR), fluorescence-based TMPRSS2 inhibition assays, and cell-based viability studies. Hesperidin emerged as the strongest candidate, demonstrating active-site engagement with TMPRSS2 catalytic residues and stable binding within the SARS-CoV-2 main protease (Mpro). Experimental validation confirmed protease inhibition and cellular protection against Spike-induced toxicity. These findings support a multi-target antiviral mechanism.