Experimental Study Of Rubrus Suarissimus S.Lee Saponin On Streptococcus Mutans In Vitro

OBJECTIVETo assess the effects of Rubrus Suarissimus S.Lee saponin(RSL) on growth,metabolism,adhesion, glucosyltransferase of S.mutans in vitro.In order to explore its anticariogenic activities,and lay a foundation for the exploitation of new agents on prevention of dental caries in the future.METHODS1. RSL and baeterial preparationsThe RSL was diluted into TTY liquid medium by two-fold dilution, of which concentration ranges from 40g/100ml to 0.125g/100ml. S.mutans were cultivated in TSB for 3h (37℃,80%N2,10%CO2, 10%H2)→cultivated in MSB for 48h(37℃,80%N2,10%CO2,10%H2)→Colonies picked in normal sodium and centrifuged 15min→washed twice with sterilized normal sodium→adapted the optical density (OD) at 540nm to 1.2. Effect of RSL on growth of S.mutansMinimal inhibitory concentration(MIC) was determined by tube diffusion method using TTY liquid medium.S.mutans were cultivated in TTY with RSL for 24h(37℃,80% N2,10% CO2,10% H2)→centrifuged 15min→washed twice with sterilized normal sodium→adapted the optieal density at 540nm. 3. Effect of RSL on acidogenicity of S.mutansSelected five different concentration of RSL which were sub-MIC→l% suerose TTY→adapted every of the minitial PH to 7.04 with PH meter→added identical bacteria→anaerobic cultivated 48h(37℃)→measured PH of supernatants and calculated△PH. Xylitol were designed to compare with Rubrus suarissimus S.Lee.4. Effect of RSL on adherence of S.mutansSelected five different concentration of RSL which were sub-MIC→1% sucrose TTY→added identical bacteria→anaerobic cultivated with an angle of 30°for 48h→washed and resurspended in 0.01M NaOH→measured OD at 540nm→calculated the rate of bacterial adherence inhibition. Xylitol were designed to compare with Rubrus suarissimus S.Lee.5. Glucosyltransferase(GTF) extraction of S.mutans and the insoluble exopolysaccharides (IS) detectionDifferent concentrations RSL was divided into 5 treatment groups,each group added 3ml Rubrus Suarissimus S.Lee saponin and 0.3ml Streptococcus Mutans.After anaerobic cultured 48h, centrifugation to separate supernatant and bacteria. The supernatant was purified by ammonium sulfate precipitation and left at 4℃for one night,followed by centrifugation again. The precipitation was solute into sodium phosphate bufer,and dialyzed for 48 hours for obtaining crude GTF. Bradford method was used to measure the content of total protein of GTF and Neson-Somogyi method was used to measure glucan polysaccharide. One unit of GTF was defined as the amount of enzyme required to incorporate lμmol of glucose into glucan polysaccharide per minute understandard assay conditions,and specific activity was calculated.The part of bacteria was rinsed with 0.5M NaOH for three times.The supernatant was collected.Then the ethanol which is 3 times volume of the supernatant was added. Reserved at 4℃,followed by centrifugation,precipitation was added 5ml 0.1M NaOH.Anthron method was used to measure the insoluble exopolysaccharides. Xylitol were designed to compare with Rubrus suarissimus S.Lee.RESULTS1. S.mutans didn't appear in TTY which concentration of RSL is over 20g/100ml. S.mutans grew differently in TTY which concentration is sub 10g/100ml.They were concentration dependent. The lower concentration,the more S.mutans.MIC of RSL on S.mutans was 20g/100ml according to determining by tube dilution test. The inhibitory zone was appeared in concentration ranges from 0.165g/100ml to 40g/100ml .2.△PH gradually decreased with the increasing concentration of RSL. There were highly significant difference about△pH of each groups.△PH of RSL groups were lower than those of control groups and Xylitol groups.3. The inhibition rate of adherence increased dramatically with the increasing concentration of RSL . OD number of S.mutans in RSL groups were lower than those of control groups and xylitol groups. Except for the group of 5g/100ml,2.5 g/100ml,1.25g/100ml,there was highly significant difference among group of RSL.4. The results of total protein,GTF activity,insoluble exopolysaccharides in group of RSL4.1 The content of total protein increased with the increasing of concentration of RSL. The total protein of GTF in RSL groups were higher than those of control groups and xylitol groups. Except for the group of 0.625g/100ml,0.313 g/100ml,there was highly significant difference among group of RSL.4.2 With the concentration of RSL increasing, GTF activity descended at the same time. The glucan polysaccharide,GTF activity,specific activity in RSL groups were lower than those of control groups and xylitol groups. Except for the group of 5g/100ml,2.5 g/100ml,1.25 g/100ml ,there was highly significant difference among group of RSL.4.3 The IS of S.mutans decreased gradually with the increased concentration of RSL. The content of IS in control groups were higher than those of RSL groups and xylitol groups.There was approximately straight correlationship between the GTF activity and insoluble exopolysaccharides. Except for the group of 5g/100ml,2.5 g/100ml,1.25 g/100ml ,there was highly significant difference among group of RSL.CONCLUSION1. The inhibitory effect of RSL on growth of S.mutans is very outstanding.2. RSL could inhibit the acidogenieity and adherence of S.mutans significantly,it's much lower than xylitol groups.3. RSL can inhibit the GTF activity of S.mutans,and lower the synthesis of IS, it's much lower than xylitol groups.4. RSL as the extract from natural plant,has some promising future in the field of dental caries prevention.