| OBJECTIVECaries is the most common disease among human beings. WHO has already taken it as one of the major diseases that should be paid more attention to prevent and cure. Streptococcus mutans (S.mutans) is the primary bacterium causing dental caries in humans. Chelerythrine (CHE) is a major extract that comes from a Chinese traditional medicine Chelidonium majus L. The effects of CHE on growth, metabolism, hydrophobicity and adhesion of S.mutans were studied in vitro experiment in this paper, in order to explore its cariostatic effect, and lay a foundation for the exploitation of new agents on prevention of dental caries in the future.METHODS1. CHE and bacterial preparationsThe CHE was diluted into brain heart infusion (BHI) broth culture medium by two-fold dilution, of which concentration ranges from 100mg/ml to 24.4μg/ml, and BHI broth was used as control group. Cells were grown in BHI broth medium for 24 hours at 37℃(80％N2,20％CO2). At the end of growth the culture was vigorously vibrated using a vortex mixer for a few minutes, then followed by centrifugation, washing twice using sterilized normal sodium, adapt the optical density(OD)at 540nm to 1.0.2. The inhibitory effect of CHE on growth of S. mutansSlip diffusion method was used to measure the inhibition zone of each concentration of CHE, and 1.6mg/ml chlorhexidine was used as positive control group. Minimal inhibitory concentration (MIC) was determined by tube diffusion method using liquid BHI broth. 3. The effect of acidogenicity of CHE on S.mutansEach initial pH was adapted to 7.4 using pH meter, S. mutans were inoculated into each CHE solution of which concentration was sub-MIC, and cultured for 3, 6, 12, 24 and 48 hours at 37℃(80％N2,20％CO2). After centrifugation, the pH of supernatants was measured, and the△pH (difference between the initial pH and the cultured pH) was calculated.4. The effect of CHE on cell surface hydrophobicity of S.mutansThe CHE was doubly diluted to different concentrations from 781.3μg/ml to 24.4μg/ml. Microbial Adhesion to Hydrophobicity (MATH) method was used to measure the cell surface hydrophobicity (CSH) of S.mutans, and inhibitory rate of hydrophobicity was calculated.5. Effect of CHE on adherence of growing S. mutans cell to a glass surfaceThe sub-MIC concentration 24.4μg/ml, 48.8μg/ml, 97.7μg/ml 195.3μg/ml and 390.6μg/ml CHE solution were used in this study. 0.1ml S.mutans of which had adapted the OD to 1.0 was added to 0.05ml CHE and 2.1ml BHI broth plus 0.25ml 50％glucose-sodium phosphate buffer(G-PBS).Organisms were grown at 37℃(80％N2,20％CO2) with an angle of 30°for 18 hours in test tubes as detailed in Hottorri. After inoculation, the adhering cells were washed and resurspended in water. The amount of adherent cells was measured spectrophotometrically at 540nm. The rate of bacterial adherence inhibition was calculated.6. Glucosyltransferase (GTF) extraction and the extra-cellular Water-insoluble glucan (WIG) detectionCHE was used as the tested group of which concentrations range from 24.4μg/ml to 390.6μg/ml prepared with BHI broth medium contained 2％glucose. Each group was added 0.3ml S.mutans which had been adjusted OD to 1.0, cultured at 37℃(80％N2,20％CO2) for 24 hours in test tubes. The supernatant were divided into two batches after centrifugation. One batch of solution was purified by ammonium sulfate precipitation before adding the ethanol and left at 4℃for one night, followed by centrifugation again. The precipitation was solute into sodium phosphate buffer, and dialyzed for 48 hours for obtaining crude GTF. Bradford method was used to measure the content of total protein of GTF, and anthrone method was used to measure glucan polysaccharide. One unit of GTF was defined as the amount of enzyme required to incorporate 1μmol of glucose into glucan polysaccharide per minute under standard assay conditions, and specific activity was calculated. Another batch of solutions was rinsed with 0.5M NaOH for three times. The supematant was collected, and the ethanol 3 times volume of the supematant was added. Reserved at 4℃, followed by centrifugation, precipitation was added 5ml 0.1M NaOH. Anthrone method was used to measure the content of WIG.7. Statistical analysisStatistical analysis was use by SPSS13.0 software. Comparison of total sample of all groups was used by Analysis of variance (ANOVA), and Student-Newman-Keuls method was used to compare the difference between each two groups. Correlationship analysis was used by Spearman correlation test. The significant level was set at 0.05.RESULTS1. The inhibitory zone was appeared in concentration ranges from 6.25μg/ml to 100mg/ml by clip diffusion method. The diameter of inhibitory zone of 100mg/ml CHE was the largest (25.8mm) among all the test groups, and larger than that of 1.6mg/ml chlorhexidine (19.4mm). There was approximately straight correlationship between the inhibitory zone and the concentration of CHE(r=0.99, P＜0.01). MIC of CHE on S. mutans was 0.78mg/ml as determined by tube dilution method.2.△pH gradually decreased with the increasing of concentration of CHE. Except for the group of 24.4μg/ml,△pH of other groups were higher than that of control groups (P＜0.05). Except for time interval of 3h there were significant differences among groups of CHE compared with the control group,△pH gradually increased with the lasting of culture time interval. Compared with control group, the time interval for attainding to the maximum△pH was various, and it need longer time with the increasing of CHE concentration. The△pH variance was the largest for 6h interval in the 48.8μg/ml group, for 12h interval in the 97.7μg/ml group, for 24h interval in the 195.3μg/ml and 390.6μg/ml respectively.3. There was decreasing trend of CSH for S. mutans with the concentration ascent of CHE. There was highly significant difference between each test group and control group (P＜0.01). There were not statistical differences among test groups of 24.4μg/ml, 48.8μg/ml, 97.7μg/ml and 195.3μg/ml (P＞0.05). 4. The inhibition rate of adherence increased dramatically with the increasing of the concentration of CHE. There was highly significant difference between group of 195.3μg/ml or 390.6μg/ml and control group, also there were significant differences between the two tested of 195.31μ/ml, 390.6μg/ml and remained groups of 24.4μg/ml, 48.8μg/ml as well (P＜0.01). There was not statistical difference between group of 48.8μg/ml or 97.7μg/ml and control group (P＞0.05).5. The GTF of S.mutans decreased gradually with the increased concentration of CHE. There was highly significant difference between each test group and control group (P＜0.01). There was not significant difference between group of 24.4μg/ml and 48.8μg/ml.It is also the same among the groups of 97.7μg/ml, 195.3μg/ml and 390.6μg/ml(P＞0.05).With the concentration of CHE increasing, the WIG of S.mutans also deceased gradually. Except for group of 24.4μg/ml, there were all highly significant difference between the other test group and control group (P＜0.01). There was not significant difference between the group of 195.3μg/ml and 390.6μg/ml (P＞0.05).CONCLUSIONS1. The inhibitory effect of CHE on growth of S.mutans is very outstanding.2. CHE could lower the acidogenicity of S. mutans significantly.3. CHE could lower the CSH of S.mutans, and thus inhibit the adherence of S.mutans cell on glass surface.4. CHE can inhibit GTF of S. mutans, and lower the synthesis of WIG.5. CHE as the extract of Chinese traditional medicine Chelidonium Majus L., has some promising future in the field of dental caries prevention.
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