The In Vitro Adhesion Inhibition Effects Of B-type Procyanidine Oligomers On S.sobrinus 6715 And Its Possible Mechanism

Dental caries, which does great harm to people’s oral health, is a disease caused by bacterial infection. Cariogenic bacteria adhere to tooth surface and participate in the formation of dental plaque, then use sucrose to produce acid cause tooth decay. If we can inhibit the adherence and accumulation of cariogenic bacteria to tooth surface, we can prevent dental caries completely. Procyanidine is a kind of natural active polyphenols separated from the plants and attracts more and more attention in anti-caries research. However, the inhibition effect and mechanism of procyanidine to S.sobrinus is still unclear. Thus, our paper selected three kinds of B-type procyanidine oligomers from sorghum episperm(SEP) to investigate the inhibition effect of SEP on the growth and preliminary adherence of S.sobrinus ATCC 6715 in order to elect the highly anti-caries active B-type procyanidine oligomer. Through methods of gene clone and recombinant expression to gain S.sobrinus ATCC 6715 GTF-I’s CAT protein. The inhibition mechanism of SEP Ⅳ to GTF-I/CAT’s enzyme activity was investigated by spectrographic method. In this paper, the research contents and results are as follows:1. Methods for extraction, isolation and identification of B-type procyanidine oligomers.Use 70% ethanol water solution to extract procyanidine,then purify the product by AB-8 macroporous resin, extracted by acetic ether and separated by Toyopearl HW-40 s gel chromatography to obtain three target fractions, the result of RP-HPLC-ESI-MS/MS showed that the three fractions were B-type procyanidines dimer, trimer and tetramer, respectively. They were named as SEPⅡ, SEPⅢ and SEPⅣ.2. Effect of SEPⅡ, SEPⅢ and SEPⅣ on the growth and preliminary adherence of S.sobrinus ATCC 6715.By using the double dilution method, the MIC of SEPⅡ, SEPⅢ and SEPⅣ to S.sobrinus ATCC 6715 was 16.00 mg/m L, 16.00 mg/m L, 8.00 mg/m L, respectively. The result showed that all of SEP Ⅱ, SEPⅢ and SEPⅣ could inhibit the growth of S.sobrinus ATCC 6715. An in-vitro model of saliva coated hydroxyapatites(SHA)was used to detect the inhibition effect of SEPⅡ, SEPⅢ and SEPⅣ on S.sobrinus ATCC 6715 preliminary adherence. The result showed that the adherence inhibition rates of SEPⅡ, SEPⅢ and SEPⅣ pretreated salivary acquried pellicle were 7%-35%, 10%-47% and 20%-54%.The adherence inhibition rates of SEPⅡ, SEPⅢ and SEPⅣ pretreated saliva were 10%-42%, 30%-42% and 20%-45%.The adherence inhibition rate of SEPⅡ, SEPⅢ and SEPⅣ pretreated bacteria itself were 33%-63%, 42%-66% and 50%-75%. It suggested that SEPⅡ, SEPⅢ and SEPⅣ could decrease the preliminary adherence of S.sobrinus ATCC 6715 to tooth surface. SEPⅣ had the best inhibition among the three B-type procyanidine oligomers. The major pathway of the three B-type procyanidine oligomers inhibited S.sobrinus ATCC 6715 preliminary adherence was treated bacteria.3. Clone, expression and identification of CAT region of S.sobrinus ATCC 6715 GTF-I.In order to investigate the inhibition effect and mechanism of SEPⅣ to GTF-I, we used gene clone and recombinant expression methods to gain S.sobrinus ATCC 6715 GTF-I’s CAT protein. CAT gene was amplified by PCR and cloned into vector p GEX-4T-1, after being identified, recombinant plasmids was transformed into T7 Express Competent E.coli(High Efficiency) and introduced by 1 mmol/L IPTG for 4 h at 37℃, then purified and concentrated to get the target protein GTF-I/CAT, the purity was 85%, expression amount was 1.22 mg/m L. The result of Nano LC-MS/MS shows that the expressed protein GTF-I/CAT was S.sobrinus ATCC 6715’s GTF-I,appraisal score was 103.75. The enzyme activity and WIG formation ability of GTF-I/CAT was 8.446 m IU and 1.603 mg/(mg·h).4. Interaction between SEPⅣ and GTF-I/CAT.The inhibition effect of SEPⅣ on GTF-I/CAT enzyme activity was quantified by the amount of WIG that GTF-I/CAT formed, by using UV absorption spectra, fluorescence quenching spectra and synchronous fluorescence spectroscopy methods to investigate the inhibition mechanism. The result shows that SEPⅣ could remarkably inhibit GTF-I/CAT forming WIG(P<0.01). SEP-Ⅳ could cause fluorescence quenching to GTF-I/CAT, the quenching mechanism was static quenching. The binding-sitenumbers were 0.7682(289K), 0.7924(299K), 0.8617(309K), approximately equal to 1. The thermodynamic parameter were △G<0, △S>0, △H>0, indicating the binding force was mainly hydrophobic force. Synchronous fluorescence spectroscopy results showed that the bind site was close to Trp. The UV spectra results suggested that SEPⅣ could change GTF-I/CAT absorption spectra, with the increase of SEPⅣ concentration, the absorption intensity of GTF-I/CAT was increased and the peak positions were red shift, which suggested that SEPⅣ could change the conformation of GTF-I/CAT, and further confirmed the fact that the quenching mechanism of SEPⅣ and GTF-I/CAT was static quenching. All the result showed that the interaction between SEPⅣ and GTF-I/CAT could change the protein conformation; this change can affect the function and activity of enzyme and reduce the WIG production. Therefore, SEP Ⅳ decreased cariogenic bacterial adhering and gathering on tooth surface and prevented dental caries.