Design,Synthesis And Biological Actives Of Multifunctional Hydroxypyridinone Derivatives And Their Application In Shrimp Preservation

In the aquatic products,shrimp is a kind of nutritious healthy food.Due to its tender,delicious properties and unique flavor,shrimp has been very popular,becoming a kind of indispensable high nutrition healthy food in the dietary structure.However,after the fresh shrimp were harvested,due to the microbial contamination and the catalysis of enzymes,shrimp body is extremely easy to decay and become dark during storage,which greatly affects the appearance of shrimp products,resulting in the loss of commercial value.Thus,it has currently become one of the hot research topics to develop efficient and safe shrimp preservatives for experts and scholars in the field of aquatic products.The browning of shrimp is mainly caused by the melanosis initiated by the oxidation of tyrosine or its derivatives,which was catalyzed by tyrosinase.Kojic acid,a kind of natural products of aspergillus fermentation,not only has strong tyrosinase inhibitory activity,but also has some antibacterial and antioxidant activities.Thus,kojic acid has been used as a food additive in food processing and production.However,the thermal stability and biological activities of kojic acid are not ideal,thus its application has been limited.The structural modification of kojic acid is a promising protacol to obtain shrimp preservatives with high efficiency,safety,and better biological activity.In this thesis,a series of novel hydroxypyridinone derivatives with double inhibition action on tyrosinase,were designed and synthesized staring from kojic acid.These compounds were investigated for the tyrosinase inhibitory effect,inhibitory mechanism,antioxidant and antibacterial activities.Finally,the preservative effect of such hydroxypyridinone derivative with multi-functions on Penaeus vannamei was investigated.Main results are as follows:1.A series of novel hydroxypyridinone derivatives,5(a-g),were designed and synthesized starting from kojic acid,and their structures have been confirmed by 1H NMR and ESI-HRMS.2.The mushroom tyrosinase monophenolase activity assay demonstrated that compound 5f has a stronger inhibitory activity against monophenolase of tyrosinase than kojic acid.The IC5O value of 5f on the monophenolase activity was determined as 1.33 ?M,indicating that 5f is stronger than kojic acid(IC50=15.89 ?M)by twelve-fold.Then 5f was selected to investigate for the inhibitory effect on diphenolase activity.The IC50 value of 5f on diphenolase activity of tyrosinase was determined as 7.83 pM.3.Investigation of inhibition mechanism:(1)Inhibition kinetics assay of 5f on diphenolase activity of mushroom tyrosinase indicated that the inhibitory mechanism of 5f was reversible and the inhibitory type was competitive-uncompetitive mixed-type I.The inhibitory constants K1 and KIS of 5f were determained to be 11.37 ?M and 20.01 ?M,respectively.(2)Copper ions reducting ability and chelating ability of compound 5f were dertermined to further explore the mechanism of tyrosinase inhibition.4.The radical(DPPH free radicals,hydroxyl free radicals and superoxide anion free radicals)scavenging abilities of 5f were measured to evaluate the antioxidant activity.Results indicated that 5f possesses a certain antioxidant activity,but it is weaker than vitamin C.5.The antimicrobial evaluation of 5f was performed by the determination of inhibition zone diameter,minimum inhibitory concentrations(MIC)and minimum bactericidal concentration(MBC).Escherichia coli,Staphylococus aureus,Salmonella spp.,Bacillus subtilis and Pseudomonas aeruginosa were chose as test strains.Inhibition zone assay demonstrated that 5f has more stronger antimicrobial activity than kojic acid.Among the tested bacteria,P.aeruginosa and B.subtilis were the most sensitive strains,followed by E.coli and S.aureus.The MIC assay and MBC assay also indicated that 5f has better antibacterial activity than kojic acid.The MICs of 5f against E.coli,S.aureus,P.aeruginosa,B.subtilis and Salmonella spp.were 31.25,31.25,31.25,31.25 and 62.5 ?g/mL,respectively.The MBCs of 5f were 62.5,62.5,62.5,62.5 and 125 ?g/mL,respectively.6.The preservative effect of 5f on Penaeus vannmei was evaluated by the odor,tissue and sensory evaluation,and the measurement of volatile basic nitrogen(TVB-N),aerobic plate count,pH value and color change during the storage.Results indicate that 5f possesses better preservative effect than kojic acid and 4-hexylresorcinol(4-HR).It has also been demonstrated that 5f not only could slow down TVB-N,increase of pH value and inhibit the growth of microbes,but also inhibit the formation of melanosis.The shelf life of Penaeus vannmei treated with 5f could be extended to 11 days,whereas those of shrimp treated with sterilized water,kojic acid and 4-HR were 5,7 and 9 days,respectively.