Mechanism Of Inhibition Of Melanogenesis By Pyrones As Tyrosinase Inhibitor

Tyrosinase(TYR)belongs to the copper-containing oxidase,which is widely found in natural organisms such as fungi,plants and animals.It is an important target for the treatment of abnormal melanin deposition.This paper studied the inhibitory effect of ethyl maltol and KAD4 on tyrosinase activity.Ethyl maltol is a kind of food additives,and KAD4 is a kind of lab-synthesized compounds based on the structure of kojic acid.The results of enzymatic experiments showed that the IC50 of ethyl maltol and KAD4 on monophenase activity were 4.294 mmol/L and 11.819 μmol/L,respectively.The IC50 of ethyl maltol and KAD4 on diphenolase activity were 0.853 mmol/L and 9.733 μmol/L,respectively.The inhibition mechanism of them were reversible inhibition.Then,the results of fluorescence quenching,copper ion chelation experiment and molecular docking showed that compounds interacted with copper ions and amino acid residues of tyrosinase through hydrogen bond and other interactions to affect tyrosinase activity.At the cellular level,we set B16F10 as the research object to study the effects of compounds on melanogenesis.When the concentration of ethyl maltol was 200 μmol/L,the activity of tyrosinase of B16F10 was decreased to 77.64%,the melanin content was decreased to 61.19%,When KAD4 concentration was 100 μmol/L,the activity of tyrosinase of B16F10 was decreased to 49.37%and the melanin content decreased to 58.76%.Besides,except KAD4 had no effect on DCT expression,two compounds could promote the phosphorylation of AKT,inhibit the phosphorylation of CREB,and reduce the protein expression of MITF and TYR families in cells.In combination with the results of the transcription level changes of MITF and TYR gene families detected by Q-PCR,we speculated that the compounds mainly reduced the expression of MITF and TYR gene families by affecting the translation,thus inhibiting melanogenesis in cells.Then we set zebrafish as the research object to study at the individual level.When the concentration of ethyl maltol was 2 mmol/L,the activity of tyrosinase in zebrafish was decreased to 67.57%,the melanin content was decreased to 38.26%.When KAD4 concentration was 400 μmol/L,the melanin content of zebrafish was decreased to 60.53%,but the activity of TYR had no obvious changes.Both of them can reduce the TYR expression,and the inhibition degree of the compounds on TYR expression was stronger than that on the TYR catalytic activity.Under the action of the highest concentration,the inhibition effect of KAD4 was worse than that of ethyl maltol.Finally,the results of ABTS+and DPPH showed two compounds both had antioxidant capacity.The results of this paper indicated that both ethyl maltol and KAD4 had the potential to be developed as TYR inhibitors and antioxidants,and provided a theoretical reference for the development of other compounds.