Study On The Screening Of Tyrosinase Inhibitors And Their Interaction Mechanism

Tyrosinase is the key rate-limiting enzyme in melanin synthesis which determines the color of skin and hair in the human body.Research had shown that the production of melanin in the body could be significantly reduced by inhibiting the activity of tyrosinase in the body,thereby achieving the effect of spot-fading and whitening.Therefore,looking for natural,high efficiency,and low toxicity tyrosinase inhibitors has broad application prospects.In this study,nine compounds were selected as the research object,trying to establish a simple and effective platform for the screening of tyrosinase inhibitors,and to explore the mechanism of compounds that could inhibit tyrosinase activity.The main findings were as follows:1.Screening of the tyrosinase inhibitors.The effect of the compounds on the intracellular tyrosinase activity and melanin content were measured using the L-DOPA oxidation method and sodium hydroxide lysis within a concentration range where the compound did not substantially affect cell growth.The results of the study showed that resveratrol,oxyresveratrol,acetyl-trans-resveratrol,trimethoxystilbene,cinnamic acid,cinnamaldehyde,4-Hydroxycinnamic acid these seven compounds all inhibited the activity of tyrosinase in the cells,thus reducing the production of melanin.2.Study on the effect of compounds on tyrosinase activity and type of inhibition.Dopamine oxidation method had been applied to the determination of seven compounds effects on tyrosinase activity.The results showed that seven compounds have significant inhibitory activity of tyrosinase.Kojic acid were used as the positive control.Inhibitory activities were as follows: oxyresveratrol > resveratrol >acetyl-trans-resveratrol > kojic acid > trimethoxystilbene,4-hydroxycinnamic acid >cinnamaldehyde>kojic acid>cinnamic acid.Among them,resveratrol,oxyresveratrol,trimethoxystilbene and 4-hydroxycinnamic acid,this four compounds on tyrosinase were competitive inhibitiors.Competitively combined the active center of enzyme with substrate,reducing the catalytic activity of enzyme.Acetyl-trans-resveratrol,cinnamaldehyde and cinnamic acid were non-competitive inhibitiors.It didn't compete with substrate for the active center of enzyme,but combined other sites other than the active site of enzyme to change the structure of enzyme and inhibited the activity of enzyme.3.Study on the interaction mechanism between compounds and tyrosinase.The results of spectroscopic method and molecular docking showed that all seven compounds entered the active center of tyrosinase.These seven compounds mainly interacted with the amino acid residues in the active center of tyrosinase through hydrophobic force,electrostatic force and hydrogen bound force.Altering the microenvironment of amino acid residues in the active center of enzyme,resulting in the endogenous fluorescence quenching of tyrosinase.And altering the secondary structure of tyrosinase resulted in a decrease in the catalytic activity of enzyme.