Study Of Effect Of Propolis Combined With Ornidazole For Porphyromonas Gingival And Human Gingival Fibroblast

Background:Periodontitis is an infection of the tissues around the teeth. Dental plaque is the key pathogenic factor of periodontitis.Fibroblast is the main cell in the periodontal tissues, showing the most important function. Any destroy to fibroblast will affect health of periodontal tissue. So an ideal medicine for periodontitis not only can kill or inhibit the growth of bacteria, but also is safe for periodontal tissue, lower toxicity.At present , nitromidazole medicine, like ornidazole is main antibacterial which shows ideal anti-anaerobic effect. The emergence of antibiotic resistance in pathogenic bacteria will happen with long-term use of antibiotic.And periodontitis is caused by multi-bacterium, so one anti-anaerobic medicine alone can not show the ideal therapeutic efficacy. So the combinaion of antibacterials for periodontitis is advocated highly.Recently, special attention has been paid to natural propolis, which . has versatile pharmacological activities. And some studies have found that propolis shows antimicrobial synergistic action with some antibiotics, such as macrolides, polypeptide antibiotics, aminoglycosides, ambramycin and amphemycin. These studies are mainly regarded aerobic bacteria.Now there is no study about propolis combined with periodontal antibacterial for anaerobic. Combining propolis and ornidazole possess value of being studied in treatment of periodontitis, which may a new type of compatibility program.Objective:Based on some domestic and abroad studies, we combine advantage of propolis and ornizaloe and explore potentiality of mixture combined them for treating periodontal disease. The synergism of propolis and ornidazole on porphyromonas gingival (Pg) and toxic action on human gingival fibroblast(HGF)will be evaluated, so that we can get experimental information of composition of propolis and ornidazole treating periodontitis.Material & Methods:1.Experiment of propolis to inhibit growth of Pg in vitro. With brain-heart infusion (BHI) medium added hemin, propolis was diluted to follow 8 concentrations by fluit delution method and ornidazole was diluted to 14 concentrations. 0.5 ml of bacteria suspension (106～107CFU/mL) was added into every test tube added above test agents respectively. After thoroughly mixtured, Pg was incubated anaerobicly for 48 hours, then streakly inoculated on BHI blood agar plates. After 48 hours, the minimal bactericidal concentration (MBC) of propolis and ornidazole were determined. The experiment was repeated three times and positive control group and negative control group were set up every time.2.Experiment of composition of propolis and ornidazole to inhibit growth of Pg in vitro. By the disc diffusion methods, paper discs respectively containing ornidazole (0.5, 0.4, 0.3, 0.2 or 0.1g/L) , propolis (100, 50 or 25g/L) ,or composition (containing propolis and ornidazole ) were pasted on BHI blood agar plates inoculating Pg. The plates were incubated anaerobicly at 35℃for 48 h. The diameters of the inhibitory zones were measured. Every medicine paper discs were repeated 4 times.3.Cytotoxicity of composition of propolis and ornidazole on HGF. The fifth generation cells were grown in 96-well for 24 h in condition of 37°C, 95% humidity, and 5% CO2, then culture medium in every well was removed, culture medium respectively containing propolis or ornidazole or composition was added into wells, and 96-well was cultured again for 24 h. Absorbance (A) value of each well was determined by MTT colorimetric method and relative growth rate (RGR) of HGF was calculated. We chose the concentration groups of every agents , RGR of which was all about 50%, then carried out experiment of determining the growth curve of Pg.. The sixth generation cells were grown in 96-well for 24 h (all 8 groups with each group 15 wells, each agent 3 wells). Then cells were cultured in culture medium containing required concentration of test agent for 24 h, A value of each well of the first group was determined by MTT colorimetric method. The wells of other 7 groups were added with fresh culture medium without test agents after the medium with test agent was removed from all wells. One group was selected out every day in the 7 days and A value was determined. Then the growth curves of Pg of every test agent group and negative control group were drew.Results:1.Propolis showed the good inhibiting effect on growth of Pg, with MBC value is 1.56 g/L. And MBC value of ornidazole to Pg was ranging from 0.125mg/L to 0.25mg/L.2.The mean of inhibiting zone of propolis group at concentration of 100g/L was just 10.25 mm. While after ornidazole combined with propolis, the inhibiting zones of composition groups with 100g/L propolis were all greater than those of ornidazole groups when comparing between the same concentration of ornidazole groups. This results also were showen in composition groups combined 50 g/L or 25 g/L propolis with low concentration of ornidazole. The inhibiting zones became big with increasing of concentration in each group. The statistic analytic result showed synergistic effect existed between propolis and ornidazole for inhibiting growth of Pg.3.The relative growth rate (RGR) of HGF was up to 100% and the class of toxicity was low to zero with decreasing of agent concentration. When concentration of ornidazole was 0.4g/L or 0.8g/L, the average A values of composite agent groups all exceeded those of oenidazole groups alone, and there were all statistical differences ( P < 0.05 ) .When concentration of ornidazole was 0.2g/L or 1.2g/L, there were no statistical differences between composite agent groups and ornidazole groups(P>0.05).4.RGRs of HGF all were about 50%,after 24-hour culture in the culture medium containing 1.2g/L ornidazole ,composite agent 1 (containing 1.2% ornidazole and 1g/L propolis ), composite agent 2 (containing 1.2% ornidazole and 0.5g/L propolis ) or 4g/L propolis. When cells continued to be cultured in the culture medium without agents, the A values of above four groups and negative control group would increased as time gone. There were statistical differences between time points of examination for the same group(P<0.05).At each point in time, there were statistical differences between each agent group and negative control group(P<0.05),but the A values of above four agent groups all closed to that of negative control group with passage of time.Conclusion:1.Propolis can inhibit the growth of Pg.2.The composition of propolis and ornidazole shows better growth inhibiting effect for Pg than ornidazole alone or propolis alone. Propolis and ornidazole can synergistically enhance growth inhibiting effect on Pg.3. 1g/L and 0.5g/L propolis can not enhance cytotoxic effect of ornidazole for HGF and can weak cytotoxic effect of ornidazole at some concentrations of ornidazole.4.About half of HGF could normally grow after cultured in the culture medium containing 1.2g/L ornidazole ,composite agent 1 (containing 1.2% ornidazole and 1g/L propolis ), composite agent 2 (containing 1.2% ornidazole and 0.5g/L propolis ) or 4g/L propolis for 24 hours. And the cell number in every group kept on increasing in 7 days, which indicated the cytotoxic effect of above agents for HGF is reversible.5.The composition of propolis and ornidazole is worth of be further studied in the respect of therapy of periodontitis.