Design, Synthesis And Biological Actives Of Novel Hydroxypyridinone Derivatives And Their Preservative Effect On Shrimp

Shrimp is a kind of very popular sea food due to its delicious and nutritious properties. However, the sales and the storage of shrimp are seriously affected because of spoilage and browning. Therefore, the development of novel shrimp preservatives with high safety and efficiency has become one of the hotspots in food preservation field. Kojic acid, a natural product, namely5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, has been used as a food additive for food preservative in the food industry, due to the relatively strong tyrosinase inhibitory activity, some antibacterial and antioxidant properties. Studies indicated that kojic acid is not stable enough and its activity is unsatisfactory. Thus it is is an important way to search for the compounds with better biological activity by the modification of kojic acid. In this thesis, two groups of hydroxypyridinone derivatives were designed by using a principle of bioisosterism and synthesized starting from kojic acid. Based on the results of the tyrosinase inhibitory effects of these hydroxypyridinone derivatives, in vitro antioxidant activity, antimicrobial activity and cytotoxicity of several compounds were investigated. Finally, one hydroxypyridinone derivative with multi-functions was investigated for the preservative effect on shrimp.The main results of this thesis are summarized as follows:1. Two groups of novel hydroxypyridinone derivatives,5(a-e) and8(f-h), were designed and synthesized using kojic acid as a starting material. Structures of the derivatives have been confirmed by1H NMR,13C NMR, and ESI-HRMS.2. The mushroom tyrosinase inhibitory effect (on both monophenolase and diphenolase activity) and inhibitory kinetics of these compounds were investigated. L-tyrosine was used as a substrate for the monophenolase activity assay, and the results indicated that compounds5e and8f possess a stronger inhibitory activity against monophenolase of tyrosinase than kojic acid. The IC50values (half inhibitory concentration) of5e and8f on the monophenolase activity were determined as1.95μM and2.79μM, respectively. Thus,5e is stronger than kojic acid (IC50=12.50μM) by six-fold. Then compounds5e and8f were selected to investigate for the inhibitory effect on diphenolase activity using L-DOPA as a substrate. The results showed that the inhibitory mechanism of these two compounds was reversible and the inhibitory type was competitive-uncompetitive mixed-type I. The IC50values of5e and8f on the diphenolase activity of tyrosinase were determined to be8.97μM and 26.20μM, respectively. The inhibitory constants (KI and KIS) of5e were determained as17.17μM and22.09μM, respectively. While the Ki and Kis of8f were34.41μM and79.02μM, respectively.3. Inhibitors5e and8f were found to be able to scavenge DPPH, hydroxyl and superoxide anion free radicals. The total antioxidant capacity assay also indicated that both5e and8f possess a certain antioxidant activity.4. The antimicrobial evaluation of inhibitors5e and8f was performed by employing agar disc diffusion method to determine the inhibition zone diameter, and by using the method of serial2-fold dilutions to measure the minimum inhibitory concentrations (MIC). Bacillus subtilis, Staphylococus aureus, Escherichia coli, Pseudomonas (?) aeruginosa and Salmonella spp. were the tested strains. The results of the inhibition zone indicated that both compound5e and8f exhibited stronger antimicrobial activity than kojic acid. Among the tested bacteria, Salmonella spp. was the most sensitive strain, followed by Pseudomonas aeruginosa. The MIC assay demonstrated that compound5e showed the best antibacterial activity in comparision with compound8f and kojic acid. The MICs of compound5e against B.subtilis, S.aureus, E.coli, Salmonella spp. and P.aeruginosa were32μg/mL,32μg/mL,32μg/mL,8μg/mL,16μg/mL, respectively. The cytotoxicity of inhibitors (including5e,8f and kojic acid) on three kind of cell lines (Hela, A549and MCF-7) was measured by MTT assay. It was found that all the three conpounds had a very weak inhibitory effect on the three cell lines, compound5e lacks obvious toxicity up to10μM.5. The preservative effect of inhibitors5e on Penaeus vannmei was evaluated by the sensory evaluation and measurement of pH value, volatile basic nitrogen (TVB-N), aerobic plate count and color change. The results indicate that inhibitor5e possesses better preservative effect than kojic acid and and4-hexylresorcinol (active component in commercial preservative "fresh shrimp Po"). It has also been demonstrated that treatment with inhibitor5e could slow down the increase of pH value and TVB-N, retard the growth of microbes and inhibit the formation of melanosis, thereby extending the shelf life of Penaeus vannmei to11days, whereas the shelf life of shrimp treated with4-hexylresorcinol, kojic acid and the control group was10,8and5days, respectively.