| Long-range researches prove that caries is an infectious diseases caused by the plaque gathering on the tooth surface. Streptococci mutans (S.mutans), which is the primary pathogenic factor of caries in humans and animals, show high selectivity and affinity to tooth surface and mainly distributed on tooth surface in the mouth. Over the years, people have tried to use drugs to control and prevent caries, but the use of antibiotics, such as penicillin, erythromycin and fluoride, showed some certain limitations. Therefore looking for a safe anti-caries drugs, which can effectively opsonize the balance of oral bacteria, become the main emphasis. Many scholars have started to pay attention to the research of natural products. Modern scientific researches showed that tea polyphenols can effectively inhibit the growth of anaerobic bacteria in dental plaque. Procyanidins which have the similar structure with tea polyphenols are a part of natural polyphenol compounds. Its excellent antibacterial effects and inhibition of acid production to S.mutans contribute an abroader development space in clinical prevention, caries-treatment and exploitation of relevant functional food. However, the relationships and mechanism between proanthocyanidins-intervened pathway of dental caries, the molecular weight (degree of polymerization) and the effects are still unclarified. This article used several oligomeric proanthocyanidins in sorghum episperm as materials, compared the relationship of different proanthocyanidins molecular weight (degree of polymerization) and inhibition of S. mutans Ingbritt(c) adherent effects, to filter the high active oligomeric proanthocyanidins to inhibite S. mutans Ingbritt(c)’s adhesion. Based on this, explore the main intervened approach in adhesion, and look forward its mechanism of action. The main research and experimental results are as follows:1.Procyanidins in sorghum episperm were extracted by70%ethanol as solvent and purified by ADS-17macroporous resin. Combined with UV, ESI-MS, and RP-HPLC-ESI-MS, the procyanidins and its fractions after Toyopearl HW-40gel chromatography could be separated and identified. The purity of procyanidins was98.5%(Porter’s assay). The results of ESI-MS showed that the composition of SPC purified by macroporous resin were mixture containing catechin/epicatechin monomers, dimmers, trimers and tetrames of SPC. RP-HPLC-ESI-MS-MS analysis results of SPC fractions proved that besides SPC-U was flavonoids, SPC-Ⅰ, SPC-Ⅱ and SPC-Ⅲ were high purity dimers, trimers and tetramers in turn. 2.OD value test results showed that when SPC-Ⅰ, SPC-Ⅱ and SPC-Ⅲ concentration were1.0mg/mL, they all exhibited strong inhibitory effect of the adhesion of S. mutans Ingbritt(c) on the inclined glass surface. Compared with the control group, dosing group showed a significant difference (P＜0.01), in which SPC-Ⅲ demonstrated the most conspicuous inhibitory effect. When SPC-Ⅰ was in the range of0.01to0.05mg/mL, SPC-Ⅱ and SPC-Ⅲ were both in the range of0.002to0.02mg/mL, these three kinds of drugs were all showing outstanding inhibitory ability in the adhesion of S.mutans Ingbritt(c) to the inclined glass surface, and the experimental group showed a significant difference (P＜0.01) to the control group. The data demonstrated that SPC-Ⅰ, SPC-Ⅱ and SPC-Ⅲ showed a notabal concentration-effect relationship in the inhibition of S.mutans Ingbritt(c) adhere to the glass surface in vitro, and we measured IC50values were0.033,0.016,0.012mg/mL respectively. The above data suggested that SPC-Ⅲ had the strongest ability to inhibit S.mutans Ingbritt(c) adhere to the sloping glass surface.In order to explore the major pathway of SPC intervened S.mutans Ingbritt(c) adhere to SHA in vitro, setting NaF as a negative control, while combined of SPC, SPC-Ⅰ, SPC-Ⅱ and SPC-Ⅲ, this study used fluorescence labeling method to detect the fluorescence intensity effected by different active compositions-treated S. mutans Ingbritt(c) and drug-treated salivary acquired pellicle of adhesion with the active compositions concentration at the range of7.8125～500.0μg/mL. The results showed that:with the increasing active compositions concentration, the adherent inhibition rate of active compositions-treated S. mutans Ingbritt(c) adhere to SHA was rising, especially SPC-Ⅲ showed the strongest inhibiting capacity, and its highest adherent inhibition rate was67.76%. Compared all drug test groups in each concentration with the control group, the fluorescence intensities of test groups were highly significant differences (P＜0.01). When the active compositions concentration were greater than or equal to62.5μg/mL, and still represented clearly concentration-effect relationship. While with the active compositions concentration increasing, though displayed a certain concentration-effect relationship, the highest adherence inhibition rate of active compositions-treated salivary acquired pellicle were less than35%, SPC-Ⅲ still showed the strongest inhibiting capacity. The results indicated that active compositions-treated salivary acquired pellicle significantly less effective than drug-treated S. mutans Ingbritt(c). It also suggested that the major pathway of SPC intervened S.mutans Ingbritt(c) adhere to SHA in vitro was it interacted with adhensins existed on the S.mutans Ingbritt(c) cell surface, and SPC-Ⅲ (tetramers) had the better inhibition than other4active compositions. 3.Glucosyltransferase was extracted and purified from the culture supernatant o f S.mutans Ingbritt(c) TSB by adjusting the supernatant to60%saturation with sol id (NH4)2SO4and isoelectric precipitation method. Properties of the protein were d etected by the following methods:Bradford for the estimation of its protein conten t, Somogyi method for the determination of its enzyme activity, Phenol-sulphoaci d method for mensurating its polysaccharide-producted capacity. After further purifi ed through Sepharose6B gel respectively, we obtained In-Ⅰ and In-Ⅱfrom protei ns named Initial,3.10-Ⅰ and3.10-Ⅱ from proteins named3.10,3.40-Ⅰ and3.40-Ⅱ named3.40finally, in which speculated by the measurement data,3.10were more likely had GTFs activity in all of the six protein components. Theses six pr otein components above-mentioned, detected by SDS-PAGE, were hybrid proteins, which containing different molecular-weight proteins in the range of35to170KD. MALDI-TOF-MS analysis of170KD protein in3.10showed that there were10credible proteins, in which the molecular weight of4matching proteins were arou nd150KD. These four matching proteins were derived from a strain, which belo nging to Proteobacteria,(3-proteobacteria.4. UV spectrogram showed that with the concentration of SPC-III increasing, the characteristic absorption peak of GTFs-like proteins (containing3.10,3.10-Ⅰ and3.10-Ⅱ) treated by SPC-Ⅲ in different concentrations exhibited red shift amplitude successively lager. The obvious interaction between SPC-Ⅲ and target protein could be inferred by hyperchromicity. SDS-PAGE and MALDI-TOF-MS method were adopted to study the interaction of SPC-Ⅲ with3.10. Located170KD of the protein bands were cut and retrieved on-line, and the two protein molecular weight of153462Da and153246Da were the same before and after treated by SPC-Ⅲ. For the molecular weight of two proteins forementioned were close to GTFs’, speculation was rife as they might be a member of GTFs family or had the same function with GTFs. The combination of2-DE and MALDI-TOF-MS two kinds of method to analyze the proteins of large gray value, which were maximum points of difference before and after reaction between GTFs-like proteins (3.10-Ⅰ and3.10-Ⅱ) and SPC-III respectively, showed that all different points were different proteases and derived from S. mutans. After the whole bacterisl database retrieval, A24was a outer membrane protein, which suggested that the protein is a homologous protein or a new protein never detected in S. mutans previously. |
PDF Full Text Request