The Construction And Mechanism Study Of Novel Multifunctional Tyrosinase Inhibitors And Their Application In Food Preservation

Tyrosinase is a copper-containing oxidase,which widely exists in microorganisms,animals,and plants.It is a key rate-limiting enzyme in the melanin synthesis pathway.In the food industry,the enzymatic oxidation of phenolic compounds catalyzed by tyrosinase is the main reason for the browning of fresh fruits,vegetables,and beverages.Inhibition of tyrosinase activity with inhibitor is an effective method to maintain the nutritional value and prolong the shelf life of fresh food.However,due to low efficiency,safety and cost,only a few tyrosinase inhibitors have been used in the food industry.Therefore,the development of new,stable,safe,and efficient tyrosinase inhibitors has a wide application prospect in the field of the food industry.Focusing on the three major factors leading to food deterioration（enzymatic browning reaction,microbial putrefaction,and oxidative deterioration）,this paper designs and synthesizes a new multifunctional tyrosinase inhibitors with antibacterial,antioxidant,and anti-tyrosinase activities,clarify its mechanism of anti-tyrosinase,antioxidant and antibacterial,and explores its efficacy and application prospect in the field of food preservation.The main research contents and results are as follows:（1）Design,synthesis and biological activity study of novel single functional tyrosinase inhibitors.Based on the molecular hybridization,a novel class of kojic acid-aromatic aldehyde derivatives（series Ⅰ）was designed and synthesized by combining kojic acid and aromatic aldehydes,and their tyrosinase inhibitory activities were evaluated.Most of the target compounds showed strong tyrosinase inhibitory activity,among which compound Ⅰ-j showed the strongest inhibitory activity with IC₅₀ of 5.32±0.23μM,9times that of kojic acid.The inhibition mechanism of compound Ⅰ-j on tyrosinase was studied by enzyme inhibition kinetic,fluorescence quenching spectroscopy,ANS binding fluorescence spectroscopy,and molecular docking method.It was confirmed that compound Ⅰ-j can target the active site of tyrosinase and is a new non-competitive inhibitor tyrosinase inhibitor.Moreover,the hydroxyl group in compound Ⅰ-j is the key group interacting with the active center of enzyme,and the linear structure is more active,which points out the direction for the follow-up research.In the study of the anti-browning effect on fresh-cut apples,compound Ⅰ-j showed a stronger anti-browning effect than positive control kojic acid.Moreover,the cytotoxicity test confirmed that compound Ⅰ-j had low toxicity on the normal human cell lines and has the potential for further research and development.（2）Design,synthesis and biological activity study of novel bifunctional tyrosinase inhibitors with antibacterial function.Based on the series Ⅰ,a new class of kojic acid-thiocarbamides（series Ⅱ）was designed and synthesized by introducing the active functional groups of thiosemicarbazone with antibacterial activity,and their structures were identified by ¹H NMR,¹³C NMR,and HRMS.All the target compounds showed potent tyrosinase inhibitory activity with IC₅₀ values ranging from 1.99±0.19μM to 45.14±6.25μM,as compared to the positive control kojic acid.Among them,compound Ⅱ-j(IC₅₀=1.99±0.19μM)displayed the highest activity,which was 23 times that of kojic acid.The inhibition mechanism of compound Ⅱ-j on tyrosinase was studied by enzyme inhibition kinetic,copper ion chelation experiment,fluorescence quenching experiment,UV spectrum experiment,AFM experiment,and molecular docking experiment.The results of the antibacterial experiment showed that compound Ⅱ-j had good antibacterial activity against V.Parahaemolyticus with MIC of 0.13 m M.Time-kill kinetics analysis,integrity and permeability of bacterial cell membrane,membrane protein polyacrylamide gel electrophoresis,and membrane protein fluorescence spectra confirmed that it could break the bacterial cell membrane and inhibit the growth of bacteria.The fresh-keeping experiment of Penaeus vannamei showed that compound Ⅱ-j had the dual effects of inhibiting bacterial growth and preventing shrimp browning.In addition,compound Ⅱ-j has shown low toxicity on human normal cell lines.Therefore,a new type of tyrosinase inhibitors with antibacterial activity were prepared in this study,which can be used as the lead compounds for the development of new bifunctional tyrosinase inhibitors,which laid a foundation for future research and development.（3）Design,synthesis and biological activity study of novel bifunctional tyrosinase inhibitors with antioxidant function.Based on the linear structural characteristics of series Ⅰ compounds,a new class of1,2,4-triazole-hydrazones（series Ⅲ）was designed and synthesized by introducing antioxidant active functional group 3-mercapto-4-amino-1,2,4-triazole.The study of tyrosinase inhibitory activity showed that most compounds showed potent tyrosinase inhibitory activity.Thereinto,compound Ⅲ-i showed the highest inhibitory activity(IC₅₀=0.9±0.1μM),and its inhibitory activity was about 70 times that of kojic acid.On the other hand,the antioxidant activity experiments showed that most of the target compounds had strong antioxidant capacity,which proves that the introduction of antioxidant groups can improve the antioxidant effect of the compounds.The anti-browning experiment results showed that compound Ⅲ-i had a significant anti-browning effect on fresh fruits and vegetables,and could be used as an anti-browning agent for fresh-cut fruits and vegetables.In addition,compound Ⅲ-I exhibited low cytotoxic activity against human normal cell lines.Therefore,this study found a new tyrosinase inhibitor with antioxidant function,which is expected to be used in the food industry.（4）Design,synthesis and biological activity study of novel three functional tyrosinase inhibitorsBased on the results of these three series of compounds,a class of gallic acid-phenylhydrazone（series Ⅳ）with linear structural characteristics was designed and synthesized by introducing the structural characteristics of para-hydroxyl,hydroxyl and phenylhydrazone with anti-tyrosinase,antioxidant and antibacterial activities,respectively.Among them,compounds Ⅳ-d and Ⅳ-f exhibited potent tyrosinase inhibitory activity with IC₅₀ of 15.28μM and 3.29μM,respectively,which was better than kojic acid.DPPH free radical scavenging assay,ABTS free radical scavenging assay,and Fe³⁺reducing power assay confirmed that compounds Ⅳ-d and Ⅳ-f had strong antioxidant activities,and their antioxidant activities were superior to positive control Vc.Compared with the positive control kojic acid,Ⅳ-d and Ⅳ-f exhibited stronger antibacterial activities against V.parahaemolyticu and S.aureus.Moreover,compounds Ⅳ-d and Ⅳ-f displayed significant anti-browning and fresh-keeping effects on fresh fruits and vegetables,Penaeus vannamei,and fresh shrimps.Meanwhile,compounds Ⅳ-d and Ⅳ-f showed low toxicity to human normal cell lines（HEK293）,and compound Ⅳ-f also showed good safety in acute toxicity tests in mice.Therefore,these compounds have tyrosinase inhibitory,antioxidant,and antibacterial activities at the same time,and can be used as the lead compounds for the research and development of new multifunctional tyrosinase inhibitors.