The Study Of Antibacterial Activity And Mechanism Of Silybin In Vitro

Objectives:1.To study the antibacterial effects and antibacterial spectrum of silybin on common bacterial infections in vitro.2.To study the combined effects of silybin and commonly used clinical antibiotics.3.To study the antibacterial mechanisms of silybin and provide scientific basis for the development of new antibacterials.Methods:1.The antibacterial activity test of silybin(1)Determined the minimum inhibitory concentration(MIC)of silybin and its isomers(Isosilybin,Silydianin and Silicristin)on the standard strains(Staphylococcus aureus,Staphylococcus epidermidis,Enterococcus faecalis,Enterococcus faecium,Escherichia coli and Pseudomonas aeruginosa)by the broth microdilution method.Repeated the test 3 times and take the average.(2)Determined the effect of different concentrations of silybin on the growth curve of standard strains of common clinical bacteria by plate counting method.Repeated the test 3 times.(3)Determined MIC of silybin on the clinically isolated isolates by the broth microdilution method.Repeated the test 3 times and take the average.(4)Checkerboard trace broth dilution method was used for combined sensitivity test of silybin and commonly used clinical antibiotics to determine the MICs of silybin and antibiotics against bacteria in the case of combination therapy and calculate the combined inhibitory index(FIC).Repeated the test 3 times.2.The antibacterial mechanism test of silybin.(1)The effect of silybin on cell membrane integrity of Staphylococcus epidermidis: After different concentrations(1 MIC,2 MIC)of drugs acting on bacteria(The control group used sterile saline instead of silybin)at 37?,regularly(0 h?0.5 h?1 h?2 h?3 h?5 h?7 h and 9 h)sampled and determined the concentration of extracellular protein in the bacterial culture fluid at each time point(3 times in parallel)by Coomassie brilliant blue-spectrophotometric method.Finally,ploted the protein concentration-time curve and compared the results of the drug group and the controlgroup.(2)The effect of silybin on the protein metabolism of Staphylococcus epidermidis:After different concentrations(1 MIC,2 MIC)of drugs acting on bacteria(The control group used sterile saline instead of silybin)at 37?,regularly(12 h)sampled and took an equal volume samples which had the same OD values and got the bacterial protein for SDS-PAGE electrophoresis.Finally,photographed and analysis the gel map.Repeated the test 3 times.(3)The effect of silybin on nucleic acid metabolism of Staphylococcus epidermidis: After different concentrations(1 MIC,2 MIC)of drugs acting on bacteria(The control group used sterile saline instead of silybin)at 37?,regularly(6 h?12 h?24 h and48 h)sampled and took an equal volume samples which had the same OD values to react with DAPI dye.Then,measured separately the fluorescence intensity of the DNA and RNA of the bacterial cells by using fluorescence spectrophotometer(3times in parallel).Finally,used software(SPSS 21.0)for ANOVA and P <0.05 was considered statistically significant.(4)The effect of silybin on DNA topoisomerase activity of Staphylococcus epider-midis: pBR322 DNA exists primarily in supercoiled form(Form?),and topoisomera-ses can unwind the Form I into open loop or linear DNA(Form ?).After different concentrations(1 MIC,2 MIC)of drug acting on bacterial DNA topoisomerase(The control group used DMSO instead of silybin),determined the effect of silybin on DNA topoisomerase activity of Staphylococcus epidermidis through observing the form of pBR322 DNA.Repeated the test 3 times.(5)The effect of silybin on Staphylococcus epidermidis biofilm: Different stages(6 h,24 h,and 48 h)of biofilms was treated with 1 MBIC and 2 MBIC silybin,respectively,and incubated overnight at 37 ?.On one hand,Semi-quantitatively determined the biofilm scavenging effect of silybin by crystal violet staining,and used software(SPSS 21.0)for ANOVA and P <0.05 was considered statistically significant.On the other hand,used the laser confocal technique to observe the effect of this drug on the biofilm structure directly.Repeated the test 3 times.Results:1.The antibacterial activity test of silybin.(1)The MICs of silybin against Gram-positive bacteria standard strains ranged from 50 to 400 ?g/mL,and showed the strongest antibacterial activity on Staphyloco-ccus epidermidis with MIC of 50 ?g/mL.The MICs of silybin against Gram-negative bacteria standard strains were> 400 ?g/mL.Among the isomers,the MICs of isosilybi-n against Staphylococcus epidermidis,Enterococcus faecium and Enterococcus faeca-lis were 400 ?g/mL,while the MICs of other drugs on common clinical strains were> 400 ?g/mL.(2)Silybin had obvious inhibitory effect on the growth curve of Staphylococcus epidermidis,Staphylococcus aureus,Enterococcus faecalis and Enterococcus faecium-s,and the effect was more obvious with the increase of drug concentration.However,the growth curves of Escherichia coli and Pseudomonas aeruginosa were not affected by it at all.(3)For clinical isolates,the MICs of silybin against Staphylococcus epidermidis were ?100 ?g/mL,the MICs of silybin against Staphylococcus aureus,Enterococcus faecalis and Enterococcus faeciums were ?400 ?g/mL,and the MICs of Gram-negative strains were all> 400 ug /mL.(4)In the combined susceptibility test,the combination indices of silybin and penicillin / erythromycin were 0.5 <FIC?1 and 1 <FIC?2,with no FIC?0.5 or FIC> 2for Gram-positive bacteria.For Gram-negative bacteria,the combined indices of silybin and ciprofloxacin were mainly FIC> 2,with less 1 <FIC?2,no FIC?0.5 or 0.5<FIC?1;The combined indices of silybin and gentamicin were mainly 1 <FIC?2,with less FIC> 2,without FIC?0.5 or 0.5 <FIC?1.2.The antibacterial mechanism test of silybin.(1)There was no significant difference in the concentration of soluble protein in the bacterial suspension between the control group and the drug group at each time point(p> 0.05).(2)Comparedwith the control group,the protein bands of drug group were finer,shallower or even absent.For example,the protein bands between 63 kD-75 kD and17 kD of the bacteria disappeared under the action of 1 MIC and 2 MIC silybin.(3)In the determination of DNA content,the fluorescence intensity of the 1 MIC and 2 MIC silybin dosages groups decreased by 16% and 21%(p <0.05)respectively at different time points compared with the control group;in the determination of RNA content,the fluorescence intensities of the 1 MIC and 2 MIC silybin dosages groups decreased by 15% and 21%(p <0.05)respectively.(4)Compared with the 1 U topoisomerase group,the number of deficient or linear DNA(Form II)decreased and the number of supercoiled DNA increased indrug group.(5)Under the effect of 1 MBIC and 2 MBIC silybin,the amount of 6h biofilm was lower by 16.0% and 25.0% compared with the control groups(P<0.05),respecti-vely;the amount of 24 h biofilm was lower by 7.8% and 9.0% compared with the control groups(P<0.05),respectively;the amount of 48 h biofilm was lower by 17.6%and 25.9% compared with the control groups(P<0.05),respectively.(6)Compared with the control groups,the structures of the Staphylococcus epidermidis biofilm showed different degrees of sparseness,and the dead bacteria had a wider distribution area and a more accumulated amount than the live bacteria on it after using1 MBIC and 2 MBIC silybin at different time points.And its thickness also showed a decline with the increase in the amount of medication.Conclusions:1.The antibacterial activity of silybin.(1)The antibacterial effect of silybin against Gram-positive bacteria(standard strains and clinical strains)was better than Gram-negative bacteria,and showed the strongest antibacterial activity on Staphylococcus epidermidis with MIC of 50 ?g/mL.In addition,the antibacterial activity of silybin is significantly higher than its isomers.(2)In the combined susceptibility test,for the Gram-positive bacteria,the combined effects of silybin and penicillin/erythromycin were mainly additive or unrelated,with no synergistic or antagonistic effects.For gram-negative bacteria,the combined effect of silybin and ciprofloxacin is mainly antagonistic,with no synergistic or additive effects;the combined effects of silybin and gentamicin is mainly irrelevant,and a few can produce antagonism,with no synergistic or additive effects.2.The antibacterial mechanism test of silybin.Silybin can achieve antibacterial effect through inhibiting protein and nucleic acid metabolism,topoisomerase activity,biofilm formation of Staphylococcus epidermidis,but it has no effect on bacterial cell membrane.