Exploring the effect of a series of flavonoids on tyrosinase using integrated enzyme kinetics, multispectroscopic, and molecular modelling analyses.

The control of food browning can be achieved by inhibiting tyrosinase (TY) activity, but current studies on the interaction of flavonoids as potent inhibitors with TY are inadequate. Herein, the effect of a library of flavonoids on TY was investigated using enzyme kinetics, multispectroscopic methods, and molecular modelling. Some flavonoids including 4, 8, 10, 17, 18, 28, 30, 33, and 34 exhibited potent TY inhibitory activity, with compound 10 demonstrating reversible inhibition in a mixed-competitive manner. Ultraviolet-visible spectral changes confirmed the formation of flavonoid-TY complexes. Fluorescence quenching analysis suggested effective intrinsic fluorescence quenching by flavonoids through static quenching with the ground-state complex formation. Synchronous fluorescence spectra showed the microenvironment change around the fluorophores induced by flavonoids. ANS-binding fluorescence assay indicated TY's surface hydrophobicity change by flavonoids and highlighted the change in secondary structure conformation, which was further confirmed by Fourier-transform infrared spectra. Molecular modelling results helped visualize the preferred binding conformation at the active site of TY, and demonstrated the important role of hydrophobic interaction and hydrogen bonding in stabilizing the flavonoid-TY complexes. These findings prove that diverse flavonoid structures distinctly impact their binding behavior on TY and contribute to understanding flavonoids' potential as TY inhibitors in controlling food browning.