Hydroxyl substitution patterns affect flavonoid-HSA binding: Mechanistic insights from multispectral spectroscopy and...

Flavonoids, diverse plant-derived compounds, show distinct binding behaviors with human serum albumin (HSA), influencing their pharmacokinetics. We examined three flavonoids-chrysin, baicalin, glycitein-using multispectral analysis and molecular simulations. Fluorescence (time-resolved, synchronous, steady-state), circular dichroism, dynamic light scattering and 3D fluorescence confirmed static quenching, ground-state complex formation, increased α-helix content, reduced protein size, and enhanced hydrophobic exposure. Thermodynamic results (ΔG < 0) showed spontaneous, Hydrogen bonds and van der Waals forces driven binding exhibits strong, moderate, and weak affinity, respectively(Kb ≈ 10³-10⁵ M^-1); chrysin had the strongest affinity due to optimal 5,7-dihydroxyl substitution, while glycitein's isoflavone structure decreased its affinity. Docking pinpointed Sudlow site III as the binding site. Molecular dynamics revealed increased helicity and decreased HSA flexibility, implying conformational tightening. These findings link flavonoid structural features-hydroxyl pattern and ring connectivity-to binding strength, site specificity, and HSA stability, offering mechanistic insight for designing flavonoid-based therapeutics and improving pharmacokinetic predictions.