Studies On The Anti-Tyrosinase Mechanism Of Five Coumarins And Their Anti-Browning Mechanism In Fresh-Cut Apple

Tyrosinase is a key enzyme in melanin production,and the browning of fruits and vegetables.Inhibiting its catalytic activity and reducing the production of melanin has become the main way to treat skin pigmentation diseases and control the enzymatic browning of food.In this paper,the inhibitory effect and mechanism of five coumarins on tyrosinase were investigated.On this basis,the anti-browning effect and mechanism of esculetin on fresh-cut apple were studied.The main research results are as follows:1.In this paper,the five coumarins exhibited highly potent tyrosinase inhibitory activity.Their IC₅₀values were 43±0.5μM（esculetin）,275±1.4μM（7-（diethylamino）coumarin-3-carboxylicacid）,286±1.2μM（7-（diethylamino）coumarin-3-carboxylic acid ethyl ester）,364±1.3μM（7-hydroxycoumarin-3-carboxylic acid）,and 908±2.1μM（7-hydroxycoumarin-3-carboxylic acid ethyl ester）,which were stronger than arbutin(IC₅₀=1830μM).Furthermore,the inhibitory potency of esculetin,7-（diethylamino）coumarin-3-carboxylicacid,and7-（diethylamino）coumarin-3-carboxylic acid ethyl ester were stronger than kojic acid(IC₅₀=320μM).2.In this paper,enzyme kinetics,multiple fluorescence spectroscopy methods（including fluorescence quenching spectroscopy,synchronous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy）and molecular simulation techniques（molecular docking and molecular dynamics simulation）are used to further explore the mechanism mechanism between coumarins and tyrosinase.The results declared that esculetin,7-（diethylamino）coumarin-3-carboxylic acid,7-（diethylamino）coumarin-3-carboxylicacidethylester,7-hydroxycoumarin-3-carboxylic acid,7-hydroxycoumarin-3-carboxylic acid ethyl ester inhibited the activity of tyrosinase in a reversible competitive manner.They formed stable complexes with tyrosinase driven by hydrophobic interactions,hydrogen bonding,electrostatic interaction and van der Waals forces.The binding of these five coumarin compounds with tyrosinase affected the microenvironment polarity around tyrosine（Tyr）and tryptophan（Trp）residues,changed the polypeptide skeleton of the enzyme,and caused the structural stretching of the enzyme,ultimately leading to the conformational change of the enzyme and a diminution in catalytic activity.3.In this paper,the anti-browning effect of esculetin on fresh-cut apple was studied,and its anti-browning mechanism was elucidated from the perspective of phenolic metabolism and antioxidant system.The results manifested that esculetin could effectively reduce the mass loss and browning degree of fresh-cut apples.It reduced the oxidation of total phenols and inhibited the activities of the browning-related enzymes polyphenol oxidase（PPO）,peroxidase（POD）,and phenylalanine ammonia lyase（PAL）in fruit and vegetable cells.Furthermore,it enhances the antioxidant capacity of apple and reduces cell membrane damage by suppressing the activity of lipoxygenase（LOX）and increasing the activities of catalase（CAT）,ascorbic acid peroxidase（APX）and superoxide dismutase（SOD）,thereby reducing ROS-induced membrane lipid peroxidation.In summary,esculetin,7-（diethylamino）coumarin-3-carboxylic acid,7-（diethylamino）coumarin-3-carboxylicacidethylester,7-hydroxycoumarin-3-carboxylic acid,and 7-hydroxycoumarin-3-carboxylic acid ethyl ester were all effective tyrosinase inhibitors.What’s more,esculetin has a good anti-browning effect.It suppressed the browning reaction of fresh-cut apple by inhibiting phenolic metabolism,enhancing antioxidant systems and alleviating membrane lipid peroxidation.This study not only provides guidance for the search of novel and efficient tyrosinase inhibitors,but also provides a theoretical basis for developing new fruit and vegetable preservatives.