| Bacterial infection is one of the important causes of human death.Antibiotics can resist bacterial infection,but bacteria are prone to resistance mutations under long-term survival pressure and have drug resistance,which reduces the therapeutic effect of antibiotics.In addition,the rate of bacterial resistance is much higher than the rate of development of new antibiotics,so it is urgent to find alternative methods to solve the problem of bacterial resistance.The production of pathogenic factors of pathogenic bacteria is related to the bacterial quorum sensing system.By interfering with the bacterial quorum sensing system,the pathogenic behavior of bacteria can be inhibited without killing the bacteria,which provides a new idea for solving the problem of bacterial resistance.In the early stage,our research group screened a marine fungi strain with quorum sensing inhibitory activity,Penicillium chrysogenum DXY-1,obtained from the sediment in the waters of Fujian Province in the Taiwan Strait.After the separation and purification of its metabolites,one of the active substances was identified as chrysin.On the basis of this research,this paper first comprehensively evaluated the anti-QS activity of chrysin,and preliminarily analyzed its QS inhibitory mechanism.In addition,the poor water solubility of chrysin restricts its application in biomedicine.Therefore,we loaded chrysin with microgel as a carrier,and conducted preliminary explorations on the drug release behavior and anti-QS activity of the resulting composite material.The results indicated that chrysin can be used as a potential quorum sensing inhibitor to solve the growing problem of bacterial resistance.The main contents of this dissertation are as follows:（1）Using Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PA01as the QS model bacteria,the ability of chrysin to quench the quorum sensing of bacteria was comprehensively evaluated.The experimental results showed that chrysin has a concentration-dependent inhibitory effect on the production of QS-regulated virulence factors（violacein）in CV026 and the production of QS-regulated virulence factors（pyocyanin,elastase and proteolytic enzyme）and the formation of biofilms in PA01.A 20μg/m L dose of chrysin inhibited violacein production in CV026 by 31.6%.A 40μg/m L dose of chrysin suppressed pyocyanin production,elastase activity,proteolytic activity,and biofilm formation regulated in PA01 by 41.4%,13.8%,8.3%,and 42.4%,respectively.Chrysin could also inhibit the swarming motility activity of bacteria.Further,the anti-quorum sensing mechanism of chrysin at the molecular level was explored by circular dichroism spectroscopy and molecular docking.The result of circular dichroism spectroscopy showed that chrysin changed the secondary structure of the receptor protein Cvi R,and the change in structure was different from that of the natural signal molecule C6HSL.The result of molecular docking showed that chrysin and C6HSL can competitively bind to the same pocket of the receptor protein Cvi R.（2）Natural polysaccharide welan gum,2-（2-methoxyethoxy）ethyl methacrylate and polyethylene glycol dimethacrylate were used as raw materials to prepare temperature-responsive and p H-responsive microgel by free radical precipitation polymerization.The WEO-1 microgel was used as the drug carrier to load chrysin,the loading rate was 27.8%,and the drug loading was 5.5%.The cumulative release curve of chrysin of the obtained composite microgel conformed to the First-order kinetic model,and the release rate in the release medium with p H=5.6 is lower than p H=7.4.The MIC values of the composite microgels for both CV026 and PA01 were 650μg/m L.A 20μg/m L dose of chrysin inhibited violacein production regulated by 40.0%using CV026 as model bacteria.Comparing the effect of the same concentration of chrysin before loading on the yield of CV026 violacein,it is found that the composite microgel had a more significant QS inhibitory effect.We thought this may be due to the increased local concentration of chrysin after the formation of the composite microgel,thereby enhancing the anti-quorum sensing activity. |
