Study On The Inhibitory Effect And Mechanism Of Catechin On Tyrosinase

Tyrosinase is the key rate-limiting enzyme in the regulation of melanin synthesis pathway,which participates in various physiological metabolic processes and diseases,such as aging,malignant cancer and neurodegenerative diseases.Tyrosinase mainly acts on the conversion of monophenol and o-diphenol to o-quinone in the melanin formation pathway,when its activity is too high or in excess,it may lead to the abnormal expression of melanin and its accumulation,leading to a series of skin disorders,such as chloasma,stains,etc.At present,tyrosinase inhibitors were used for skin-brightening and cosmetic effect enhancement,at the same time,tyrosinase inhibitors were also an important means to control browning of fruits and vegetables.However,the lack of understanding of the interaction between tyrosinase and inhibitors is not conducive to the practical application of natural tyrosinase inhibitors in cosmetic and pharmaceutical industries,and it will further affect the development prospects of new tyrosinase inhibitors.In this paper,the three most representative ester catechins in catechins,namely epigallocatechin gallate（EGCG）,gallocatechin gallate（GCG）and epigallocatechin gallate（ECG）,were selected as the main research object,the vitro inhibitory effect kinetics of enzymatic inhibition,binding properties,and conformational effects of these three catechins on tyrosinase were systematically investigated by combining modern analytical methods such as multispectroscopy and computer simulations.Meanwhile,the inhibition of tyrosinase activity by the combination of three catechins and kojic acid was investigated.Moreover,the changes and mechanism of the inhibition of tyrosinase activity by the combination of heating and ultrasound treatments on GCG were studied.The main research contents and results were as follows:1.EGCG,GCG and ECG had good inhibitory effect on tyrosinase,their half inhibition concentration(IC₅₀)values were（3.94±0.54）×10^-5 mol L^-1,（3.68±0.21）×10^-5 mol L^-1and（1.13±0.82）×10^-5 mol L^-1,respectively.The strength ordering of their inhibition ability was:ECG>GCG>EGCG.EGCG,GCG and ECG exhibited mixed-type inhibition of tyrosinase,and the inhibition constants of the three were2.15?×?10^-5 mol L^-1,1.01?×?10^-5 mol L^-1and 0.67?×?10^-5 mol L^-1,respectively.When EGCG,GCG and ECG inhibited tyrosinase activity combining with kojic acid,EGCG and kojic acid showed antagonism effect,while GCG and ECG and kojic acid showed additive effect and synergistic effect.Furthermore,the three catechins showed synergistic effect with their combinations.2.EGCG,GCG and ECG can chelate with copper ions and their binding constants were 2.07×?10⁴,1.05×?10⁴and 1.95×?10⁴,the order of binding ability was:EGCG>ECG>GCG.These catechins may induced the inactivation of tyrosinase through the copper ion in the chelatase activity center.EGCG GCG and ECG mainly formed ground state complex with tyrosinase under the dominance of hydrophobic forces and hydrogen bonding,causing the enzyme’s endogenous fluorescence to be quenched,and there was only one binding site of these three catechins on tyrosinase.At room temperature,the binding constant values of EGCG,GCG and ECG with tyrosinase were 1.59×?10⁴ L mol^-1,2.18×?10⁴ L mol^-1and 4.03×?10⁴ L mol^-1,respectively.ECG had the better affinity for tyrosinase,followed by GCG and EGCG.The results of molecular docking and molecular dynamics simulation showed that EGCG,GCG and ECG inserted into the active center of tyrosinase,bound to the copper ions and key amino acid residues,which prevented the binding of L-dopa at the active site.The formation of the complex induced the fluctuation of amino acid residues in the local areas,interfering with the stability of the structure of tyrosinase and inducing changes in the structure of the enzyme,thus reducing the catalytic ability of the enzyme.MM-PBSA analysis suggested that the hydrophobic force played a major role in this process,and the hydrogen bond mainly played a role in stabilizing the structure of the complex.3.The effects of the combination of EGCG,GCG and ECG with tyrosinase on its conformation were analyzed by synchronous,three-dimensional fluorescence spectroscopy,Fourier transform infrared（FT–IR）and circular dichroism（CD）.The three catechins had no significant difference in the effect of hydrophobicity and polarity around the tyrosine residues and tryptophan residues in tyrosinase.Synchronous fluorescence quenching ratio analysis proved that tyrosine residues mainly contributed to the binding of EGCG and ECG to tyrosinase,while tryptophan residues contributed more to the binding of GCG and tyrosinase.The results of 3D fluorescence spectroscopy confirmed that the combination of EGCG,GCG and ECG with tyrosinase led to the stretching of the polypeptide chains in the molecular structure of tyrosinase,thereby affecting the conformation of the enzyme.The results of CD and FT–IR spectra showed that the presence of EGCG,GCG and ECG induced a decrease inα-helix content and an increase inβ-sheet content in tyrosinase,which resulted in changes in the conformation of tyrosinase and made its molecular structure looser.The catalytic function of tyrosinase was therefore weakened.These results indicated that the intervention of EGCG,GCG and ECG induced changes in the secondary structure of tyrosinase,affecting the conformational stability,which was not conducive foe the substrate entry into the active site of the enzyme,limiting the performance of tyrosinase’s catalytic ability,thereby reducing the catalytic activity of tyrosinase.4.The effect of heating and ultrasonic pretreatment on the ability of GCG to inhibit TY activity was explored.Heat treatment（100℃,20 min）and ultrasonic treatment（630 W,60 min）can improve the ability of GCG to inhibit tyrosinase.And the inhibitory effect of heating and ultrasonic treatment together（100℃,20 min+630 W,60 min）was better than the single treatment group.The treated GCG had a stronger fluorescence quenching effect.The quenching constant before and after comparison was:K_sv（after treatment）>K_sv（before treatment）,but heating and ultrasonic treatment had no effect on the quenching mechanism of GCG,and the combination of GCG and tyrosinase was still static quenching.After heating and ultrasonic treatment,the affinity between GCG and tyrosinase increased,but the number of binding sites was still 1.Thermodynamic analysis showed that the binding force mode remained unchanged after treatment,and the hydrophobic force was still the main driving force.The qualitative analysis of high performance liquid chromatography further explained the internal reasons why heating and ultrasonic treatment enhanced the GCG inhibition ability.After heating and ultrasonic treatment,part of GCG was decomposed into EGCG and GA,the two decomposition products can cooperate with GCG to promote the inhibition of tyrosinase,as a result,GCG showed stronger tyrosinase inhibition ability.