Effects Of Lemon Peel Extracts On The Glucosyltransferase Gene Transcription Of Streptococcus Mutans

Objectives We use Real-time fluorescence quantitative PCR to detect gene expression level of gtfB,gtfC,gtfD that encoding glucosyltransferase(GTF) of Streptococcus mutans after adding lemom peel extracts.Through the studying the gene expression level of S.mutans treated by LPE that has three three concentrations,we can explore the molecular anti-caries mechanism for providing the basis of the future development of natural,non-toxic,new and ecological anticarious agents.Methods1. The experimental strains is Streptococcus mutans UA159, and we use the liquid dilution method to detect minimum inhibitory concentration (MIC)of LPE on Streptococcus mutans.2. Reference to MIC test results,diluting LPE with TPY liquid medium to get three different concentrations, that is lowerd than 4.5mg/ml which is 2.250mg/ml, 1.125mg/ml,0.562mg/ml.Taking bacterial suspension and TPY liquid medium with the ratio of 1:10 (v/v) into LPE diluted solution and TPY liquid medium Without LPE,and inoculated at anaerobic incubator 37℃.Taking TPY liquid medium containing 0.05% chlorhexidine as a positive control,and TPY liquid medium without LPE is a negative control, with three parallel tubes,extracting bacterial suspension witch is cultured respectively 0,0.5 and 1,2,4,6,1h,24h,then use CFU counting method to calculate the number of viable cells. At last, we can draw the grown curve of five experimental group with the number of colony forming units of log (log CFU/ml) for the longitudinal coordinates and time as the abscissa. P<0.05 was considered statistically significant.3. Effects of LPE on GTF transcription expression of Streptococcus mutansExperimental groups and control group design are same with the second experiment.Collecting 3 ml bacterial suspension of Streptococcus mutans UA159 when it has grown 16 hours in TPY liquid medium.Then using Omega bacterial total bacterial RNA extraction kit to extracted total RNA and reverse transcription into cDNA,.At last, real-time quantitative PCR experimental groups was determined gtfB, gtfC, gtfD gene expression to make further study about inhibiting GTF activity at genetic level of lemon peel extract.16SrRNA is the internal reference. Ct values Using2-ΔΔCT calculating method to make statistical analysis,using SPSS 13.0 software for data input and analysis and use single-factor analysis of variance (ANOVA) to compare the overall mean of each group; Student-Newman-Keuls test was used to compare bewteen every two groups; P＜0.05 was considered statistically significant.Results1. MIC of LPE on S.mutans is 4.500mg/ml.2.2.250mg/ml,1.125mg/ml,0.562mg/ml LPE groups,which are lower than MIC has obvious ability of inhibiting the growth of S.mutans,and the ability sustain in 24 hours; with the concentration of LPE solution increases, the ability inhibiting bacterial growth is more obvious,which is dependent on the concentration of LPE; the ability inhibiting bacterial growth of 0.05% chlorhexidine group is stronger than LPE groups,but 30 minutes later, S.mutans was completely killed.3. From 0.562mg/ml to 2.250mgl/ml LPE group, with the concentration of LPE gradually increased, the mRNA expression level of gtfB, gtfC, gtfD of LPE group decreased.Compared with the control group, the differences were statistically significant (P＜0.01).4. The gtfC's expression level of 0.562mg/ml LPE group is the highest,and gtfB's expression level is the lowest,There are highly significant differences between every two genes of the same LPE group (P＜0.01); expression level of gtfB, gtfD of 1.125mg/ml LPE group is less than gtfC, There are no significant difference between gtfB and gtfD(P＞0.05); There are no significant difference between the gene expression level of every two genes of 2.250mg/ml LPE group (P＜0.01).Conclusion1. Three concentration of LPE solution can effectively inhibit the growth of Streptococcus mutans, significant inhibitory effect after 30 minutes.2. Three concentrations of LPE solution can inhibit gtfB, gtfC, gtfD transcription expression of Streptococcus mutans.3. From 0.562mg/ml to 2.250mgl/ml LPE group, with the concentration of LPE group increase, the inhibitory effect of LPE on gtfB, gtfC, gtfD expression level strengthens gradually.4. The extent of inhibitory effect of 0.562mg/ml LPE group of three kind of genes is gtfB＞gtfD＞gtfC, the inhibitory effect of 1.125mg/ml LPE group on gtfB, gtfD expression is stronger than gtfC,effects on gtfB,gtfC,gtfD expression of 2.250mg/mlLPE group is similar.