| Background and Objectives：In recent years, traditional Chinese medicine has played a more and moreimportant role in the medical field. Many scholars have found that it is feasible forChinese medicine to treat oral diseases. Chinese herbal medicine derives from naturalplants with little side effect and non-resistance. Grape seed procyanidins extracts is anatural powerful antioxidant, which has a strong inhibitory effect. Studies have shownthat porphyromonas gingivalis plays an important role in the dental pulp disease,periodontal disease and the production of halitosis. Porphyromonas gingivalis is akind of G-anaerobic Brevibacterium which secrets the endotoxin and other virulencefactors and biofilms can evade the host’s immune defense system and disrupt the hostperiodontal tissues. There are rare reports about the grape seed procyanidinsinhibiting porphyromonas gingivalis. The experiment would study the effect of Grapeseed proanthocyanidins extracts on the lipopolysacharides and bacterial biofilm ofporphyromonas gingivalis, and get the detail datas and experimental results, explorethe feasibility of the grape seed procyanidins in treatment of oral diseases. It willcarry out a theory basis for the developing of the high-efficient and low toxicity drugand correlative food in treating pulpitis periodontitis and bad breath of oral disease.Materials and Methods:Experiment1: The minimal inhibitory concentration about GSPE on porphyrom-onas gingivalisFreeze-dried porphyromonas gingivalis resurrected, enriched and subcultured inmedium containing tryptone soy broth (TSB), the prepared porphyromonas gingivalisdiluted into standard with PBS buffer. Grape seed proanthocyanidins extract wasdiluted into128mg/ml with sterile deionized water, then twice echelon dilution wasused to different concentrations. The medium mixed with1.50ml drug and13.50ml50°C TSB agar then poured into plates and maked the final concentration of 12.8mg/ml,6.4mg/ml,3.2mg/ml,1.6mg/ml,0.8mg/ml,0.4mg/ml,0.2mg/ml,0.1mg/ml,0.05mg/ml.2ul porphyromonas gingivalis were seeded in the differentconcentrations of plate. The porphyromonas gingivalis were incubated for48hours at37°C. The minimal inhibitory concentration (MIC) is the lowest concentration ofGSPE without bacteria.The experiment was repeated for three times.Experiment2: Inhibition of GSPE on LPS from bacteria with the Limulus testThe sterile inactivated tubes were poured with2ml medium with TSB alone ascontrol group or combined with0.8mg/ml，0.4mg/ml，0.2mg/ml，0.1mg/ml，0.05mg/ml，0.025mg/ml (final concentration)GSPE,0.1ml(1×106cfu/ml)porphyromonas gingivalis were seeded in each group, then incubated for72hours at37°C.Detected after LAL mixed with0.2ml sample of each group. The experimentwas repeated for three times.Experiment III: The impact of GSPE on biofilm of porphyromonas gingivalisThe prepared porphyromonas gingivalis (1×106cfu/ml) were seeded in96-wellplates, each hole was0.1ml. The biofilm formed in the inner wall of the96-wellplates after incubated for5days at37°C in anaerobic environment. The broth of the96-well plates was poured out.96-well plates was divided into four groups, theexperimental group was from1to3, which added the different concentration ofGSPE (ranged from3.2mg/ml to0.1mg/ml) in A to F.The positive control group wasfrom4to6, which added the different concentration of sodium hypochlorite rangedfrom0.14%to0.0045%, the negative control group was from7to9, the backgroundcontrol group was from10to12, which was added only liquid.96-well plates weregently was washed with PBS solution for three times after incubate24hours, andwashed again after stained with crystal violet for30minutes, the OD values weremeasured with microplate reader after decolorized by the sodium deoxycholate for30minutes.The experiment was repeated for six timesResult:There were a small amount of bacteria in the TSB agar medium with the GSPEsolution concentration of0.05mg/ml,0.1mg/ml,0.2mg/ml and0.4mg/ml. But there were no bacteria in the TSB agar medium with the GSPE solution concentration at0.8mg/ml,1.6mg/ml,3.2mg/ml,6.4mg/ml and12.8mg/ml. It demonstrated that theminimum inhibitory concentration of the GSPE on porphyromonas gingivalis was0.8mg/ml.Limulus test statistics showed that the reaction results were positive for theGSPE concentration from0.4mg/ml to0.025mg/ml. In contrast, they were negativefor the GSPE concentration of0.8mg/ml. The statistical results showed that theinhibition of GSPE on LPS enhanced as the increasing of the concentration in thescope of0.05mg/ml to0.4mg/ml。The absorbance value of biofilm in96-well plates reduced significantly aftertreating with the GSPE, and the biofilm inhibition rate increased with the increasingGSPE concentration. Compared with NaClO, the effect of GSPE on biofilm was notbetter than NaClO.Conclusion1. The inhibitory effect of grape seed proanthocyanidin extracts on porphyrom-onas gingivalis was obvious, and the minimal inhibitory concentration was0.8mg/ml.2.The content of LPS was litter as the increasing of the GSPE’s concentration.and the production and releasing of toxins of porphyromonas gingivalis can be inhibitedby the grape seed proanthocyanidins extracts when the concentration was ranged from0.05mg/ml to0.4mg/ml。3. The experiment of the inhibitor of biofilm can find that the inhibitory anddestructive effect of grape seed proanthocyanidin extracts on porphyromonasgingivalis was more pronounced, and the effect was stronger as the increasing of theconcentration which was ranged from0.1mg/ml to3.2mg/ml. |
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