Phytochemical Profiling and Computational Unveiling of Thymus algeriensis Boiss. Et Reut. Bioactive Metabolites as Dual-Mechanism Inhibitors of NADPH-Cytochrome P450 Reductase.

In this study, ultrasound-assisted and maceration extractions, phytochemical profiling, and computational modeling were used to investigate the inhibitory potential of its metabolites against NADPH-cytochrome P450 reductase (CPR) and experimentally validate the antioxidant capacity of leaves, flowers, stems and roots of Thymus algeriensis. The phytochemical profile was characterized using high-performance liquid chromatography coupled with a diode-array detector (HPLC-DAD), identifying 10 major bioactive constituents in all extracts. The leaf extract exhibited significant antioxidant activity (DPPH: IC₅₀ = 165.96 µg/mL; ferric reducing antioxidant power [FRAP] = 1.045 µM BHT/g DW). To decipher the molecular interactions with the CPR enzyme, a rigorous in silico workflow was applied, utilizing deep learning-based molecular docking (GNINA 1.3) followed by duplicate 50 ns molecular dynamics (MD) simulations. A refined MM/PBSA binding free energy analysis, focused on the thermodynamic equilibrium plateau (30-50 ns), revealed distinct binding strategies. Indeed, the glycosylated flavonoid rutin acted as a potent structural anchor (ΔH ≈ -19.6 kcal/mol), leveraging an extensive eight-residue contact footprint to rigidify the protein backbone, whereas the aglycone (+)-catechin exhibited a "lock-and-key" binding mode characterized by thermodynamic efficiency (ΔG ≈ -3.6 kcal/mol) and a minimal entropic penalty. These findings provide structural evidence that T. algeriensis metabolites can target CPR via complementary enthalpic and entropic mechanisms, highlighting the dual bioactivity of the species and suggesting these flavonoids as promising bioactive scaffolds.

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