Study On The Inhibition Mechanism Of Flavonoids Towards Tyrosinase And Structure-activity Relationship

Tyrosinase is the important enzyme in the melanogenesis pathway.It plays an important role in catalyzing oxidation of both monophenols and diphenols to?-quinone,and finally transforms to melanin.Excessive accumulation of melanin resulting from the overexpression of tyrosinase in the human body is responsible for a variety of pigmentation disorders.At present,kojic acid and hydroquinone are the mainly used as tyrosinase inhibitor,but some studies showed they have some side effects.Thus,finding out the effective,low toxicity and natural tyrosinase inhibitors is of great importance.Flavonoids widely consist in natural plant,possess a wide range of biological and therapeutic activities and can inhibit tyrosinase activity.The studies of inhibitory effect of flavonoids against tyrosinase have attracted growing attention.The article using the UV-vis absorption,fluorescence spectroscopy,circular dichroism?CD?,Fourier transform infrared?FT–IR?and molecular docking to study the inhibitory kinetics,binding characteristics and conformation change of myricetin,dihydromyricetin and quercetin on tyrosinase.According the different structure of flavonoids,the inhibitory effect and structure-activity relationship against tyrosinase were studied.The main research results in the article are summarized as follows:1.The experimental result of the inhibitory kinetics suggested that myricetin,dihydromyricetin and quercetin possess similar inhibitory capacities on tyrosinase activity.The IC₅₀0 values were?8.45±0.25?×10^-5??3.66±0.14?×10^-55 and?3.08±0.74?×10^-5mol L^-1,respectively.The IC₅₀0 values of positive control kojic acid is?4.32±0.37?×10^-55 mol L^-1.The inhibition effect of tyrsoianse is quercetin>dihydromyricetin>kojic acid>myricetin.Myricetin and dihydromyricetin induced mixed–type inhibition on tyrosinase,and quercetin is a competitive type of tyrosinase inhibitor.The values of inhibition contants K_i of myricetin,dihydromyricetin and quercetin were?7.26±0.45?×10^-5,?8.97±0.45?×10^-55 and?6.41±0.61?×10^-55 mol L^-1,respectively.2.Fluorescence spectroscopy and molecular docking were used to research interaction mecanisms of myricetin,dihydromyricetin and quercetin on tyrosinase.The results of fluorescence spectroscopy showed these three flavonoids could quench fluorescenceoftyrosinase,thebindingsite?n?1?betweenmyricetin,dihydromyricetin or quercetin and tyrosinase was 1.The binding distance r between myricetin,dihydromyricetin or quercetin and tyrosinase were 4.93,3.43 and 4.08 nm,r?27?8 nm and 0.5R₀?27?r?27?1.5R₀,revealing that there existed the radiation energy transfer between tyrosinase and these three flavonoids.At 298 K,binding constant?K_a?of myricetin,dihydromyricetin and quercetin were?2.18±0.32?×10⁴,?1.69±0.12?×10⁵and?4.59±0.14?×10⁴L mol^-1,respectively.The results shown that myricetin or quercetin can interact with tyrosinase to form ground complexes driven by hydrophobic interaction,and dihydromyricetin interacted with tyrosinase driven by hydrophobic interaction and hydrogen bonding.Molecular docking results suggested that these three flavonoids could insert into the active center of tyrosinase,through hydrogen bond and hydrophobic forces combined with tyrosinase.The binding induced the conformational change of tyrosinase which occupying the channel into the active center of tyrosinase,prevented the entrance of substrate,weakening the catalytic activity of tyrosinase.3.Synchronous fluorescence,CD and FT-IR were applied to analyze the conformational change of tyrosinase induced by myricetin,dihydromyricetin and quercetin.The results suggested that the binding of myricetin,dihydromyricetin or quercetin to tyrosinase resulted in the change of hydrophobicity and polarity around the tyrosine residue and the tryptophan residue.The addition of myricetin or quercetin induced the secondary structure of tyrosinase changed,resulting in decreasing of?-helix and random coil,increasing cotent of?-turn and?-sheet.The addition of myricetin induced the hydrophobicity around tryptophan residues in tyrosinase decreased,the polarity around tyrosine residues did not conspicuously change.The polarity and hydrophobicity around tryptophan and tyrosine residues in tyrosinase did not conspicuously change after addition of quercetin.The addition of dihydromyricetin resulted in decreasing of?-helix and?-turn,increasing cotent of random coil and?-sheet.Dihydromyricetin induced the hydrophobicity increased around tyrosine residues in tyrosinase,the polarity around tryptophan residues in tyrosinase did not conspicuously change.4.By collating and analyzing experimental data and referring literatures,structural aspects of inhibitory activity and binding affinity of flavonoids as tyrosinase inhibitors were studied.Flavonoids through hydrogen bond combined with tyrosinase,and the class of flavonols showed the positive correlation between affinities and inhibitory activities.Glycosylation of 3C-OH,the hydroxylation and methoxylation on ring B make against inhibitory effect against tyrosinase.Hydrogenation of the C2=C3 double bond of flavones might decrease both the affinities and inhibitory activities on tyrosinase,but the flavonols were just the opposite.