| Objective:This study employs the tea polyphenol and chlorogenic acid as main ingredients to develop a mouthwash,through the observation of its inhibitory effect on common pathogenic bacteria of oral,the SD rat experimental dental caries and gingivitis prevention effect,for clinical provide theoretical basis to prevent tooth decay and gum disease.Methods:1.Preparation of mouthwash:the minimum bactericidal concentration（MBC）values of tea polyphenol and chlorogenic acid against Streptococcus mutans（S.mutans）and Porphyromonas gingivalis（P.gingivalis）were 2 mg/mL and 12.5 mg/mL respectively by liquid dilution method;therefore,tea polyphenol was prepared into 2 mg/mL,1 mg/mLand 0.5 mg/mL.The concentrations of chlorogenic acid were 12.5 mg/mL,6.25 mg/mL and 3.125 mg/mL.after the three concentrations of the above two drugs were cross proportioned,1mL of each drug was sucked into a sterile EP tube to obtain 9 concentrations of mouthwash,appropriate amount of glycyrrhizic acid（0.2 mg/mL）and peppermint oil（0.2 mg/mL）were added into the mixture to make mouthwash.2.Bacteriostatic experiment:the bacteriostatic effects of 9 concentrations of mouthwash on S.mutans and P.gingivalis were detected.2.1 Antibacterial test of mouthwash on S.mutans:experimental group:new mouthwash+bacterial solution,positive control group:Xipayi gum firming solution+bacterial solution,negative control group:LB liquid medium+bacterial solution,blank control group:bacterial solution.9 kinds of new mouthwash,Xipayi gum firming solution and LB liquid medium with 10μL each were added into 96 well plate.S.mutans bacterial solution(OD600=1)with 100μL was added into each well.After constant temperature cultivation（37℃,16h）,50μL mixed solution was absorbed from each well and coated on LB solid medium plate.After constant temperature cultivation（37℃,16h）,MBC value was calculated.2.2 Antibacterial experiment of mouthwash on P.gingivalis:experimental group:new mouthwash+bacterial solution,positive control group:Xipayi gingival consolidating solution+bacterial solution,negative control group:BHI liquid medium+bacterial solution,blank control group:bacterial solution.Nine kinds of concentration of new mouthwash,Xipayi gum firming solution and BHI liquid medium were added into 96 well plate,and 100μL of P.gingivalis bacterial solution(OD600=1)was added into each well.After anaerobic culture（37℃,4d）,50μL mixed solution was absorbed from each well and coated on BHI solid medium plate.After anaerobic culture（37℃,4d）,MBC value was calculated.3.Safety experiment:15 male SD rats were randomly divided into 3 groups with 5 rats in each group.The experimental group was coated with mouthwash,the positive control group was coated with Xipayi gum firming solution,and the negative control group was coated with normal saline.The medication method was 40μL three times a day for 1month.During the experiment,the body weight,mental state,color and shape of oral mucosa of SD rats were observed.One month later,SD rats were killed.The pathological conditions of heart,liver,spleen,lung and kidney were observed by histopathological section.The organ coefficient ratio was recorded by measuring the quality of each organ.4.Preventive effect of mouthwash on experimental dental caries in SD rats:Twelve 18 day old male SD rats were randomly divided into two groups,with 6 rats in each group.The experimental group:oral mucosa coated with mouthwash,the negative control group:oral mucosa coated with normal saline;medication:40μL,3 times a day,for 3 months.Three months later,the rats were killed and the maxilla was taken out.Under the cooling condition,the occlusal surface of the maxillary molars of the rats was sectioned along the mesial and distal direction with emery tablets.The rats were observed under the stereomicroscope and photographed.The caries situation was judged according to the Keyes classic scoring method.5.Preventive effect of mouthwash on experimental gingivitis in SD rats:18 male SD rats were randomly divided into 3 groups,6 in each group.The animal model of gingivitis was established by ligating the second molar with silk thread.The experimental group:oral mucosa coated with mouthwash,the positive control group:oral mucosa coated with Xipayi gingival firming solution,the negative control group:oral mucosa coated with normal saline;medication:40μL,3 times a day,for 7 days.0.5 mL of saliva was collected2,4 and 6 days after the successful establishment of gingivitis model,and aspartate aminotransferase（AST）was measured by automatic biochemical analyzer.After 7 days of continuous administration,the gingival tissue of the second molar was taken to detect the inhibition rate of superoxide dismutase（SOD）and the activity of catalase（CAT）.Results:1.Preparation and bacteriostatic test of a new mouthwashThe liquid dilution method was used to determine MBC.The results showed that there was no growth of S.mutans and P.gingivalis in the mixed solution of tea polyphenols 1mg/mL and chlorogenic acid 6.25 mg/mL.Therefore,the MBC value of mouthwash to S.mutans and P.gingivalis was tea polyphenols 1 mg/mL and chlorogenic acid 6.25 mg/mL.The MBC values of S.mutans and P.gingivalis were determined by liquid dilution method.The optimal ratio of each component in the new mouthwash was found to be 1 mg/mL tea polyphenols,6.25 mg/mL chlorogenic acid,0.2 mg/mL glycyrrhizic acid and 0.2 mg/mL peppermint oil.2.Safety test results of mouthwashDuring the experiment,the rats in each group had no anorexia and were in good mental state.From the first week to the fourth week,the body weight of SD rats in each group showed an increasing trend,and the difference was not statistically significant（P>0.05）.After the rats were killed at the fourth week,the organ coefficient ratio of heart,liver,spleen,lung and kidney showed no significant difference among the groups（P>0.05）.The results of histopathological examination showed that there were no obvious pathological changes in the organs of rats in each group.3.Preventive effect of mouthwash on experimental dental caries in SD ratsThe maxilla of rats was observed by stereomicroscope.The results showed that the caries area was stained pink,and the non caries area was hardly stained or light stained.The Keyes score results showed that:compared with the control group,the Keyes score of the experimental group was significantly lower than that of the control group,and the difference was statistically significant（P<0.05）;the number of E and DS in the experimental group was more than that in the control group,but the number of DM and DX in the experimental group was less than that in the control group,and the difference was statistically significant（P<0.05）.The results showed that mouthwash had preventive and therapeutic effect on experimental caries in SD rats.4.Preventive effect of mouthwash on experimental gingivitis in SD ratsOne week after silk thread ligation,gingival swelling and bleeding were observed in the second molar.The salivary ast test results showed that the salivary ast test results of the three groups were increased before treatment,indicating that gingivitis modeling was successful,but the difference was not statistically significant（P>0.05）;The results of saliva AST showed that there was significant difference between the experimental group and the positive control group（P<0.05）,the negative control group and the pre-drug group（PP<0.05）.The AST value of saliva in the experimental group and the positive control group was lower than that in the negative control group（P<0.05）.The results showed that the new mouthwash and Xipayi gum firming solution could reduce the ast value of gingivitis.The results showed that:compared with the negative control group,the sod inhibition rate and CAT activity of the experimental group and the positive control group were increased,and the difference was statistically significant（P<0.05）;but there was no significant difference between the experimental group and the positive control group（P>0.05）.These results indicate that mouthwash has therapeutic effect on experimental gingivitis in SD rats.Conclusion:The mouthwash developed by our team the best ratio of each component is:TP 1 mg/mL,chlorogenic acid 6.25 mg/mL,glycyrrhizic acid 0.2 mg/mL,peppermint oil0.2 mg/mL,it can effectively restrain S.mutans and P.gingivalis,experimental of SD rats dental caries and gingivitis have a certain preventive effect,and it is preliminarily proved to be safe and non-toxic. |
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