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目的用五倍子和蜂房的有效成分作为实验药物研究中药对口腔细菌生物膜结构、活性的影响及对生物膜中葡萄糖转移酶(GTF)活性的影响,探讨中药防龋的可能性。方法用荧光显微镜结合特异荧光染料标记口腔细菌生物膜中死菌和活菌的方法,研究实验药物对口腔细菌生物膜结构和活性的影响。硫酸铵沉淀法提取粗酶,Neson-Somogyi 法测定还原糖,G250微量蛋白定量 GTF还原糖计算 GTF 活性单位,评价实验药物对口腔细菌生物膜中 GTF 活性的影响。结果早期细菌定植黏附到形成成熟生物膜结构的过程中都存在一定数量的死菌。24h 常态生物膜结构中活菌面积占主导地位,有少量死菌存在,细菌互相紧密黏附在一起,生物膜结构清晰。0.05%氯己定对24h 细菌生物膜作用后,细菌总量明显下降,残存滞留的细菌基本为死菌。0.023%的 NaF 作用于细菌生物膜后,生物膜结构发生明显变化,细菌总量明显减少,存在的细菌以活菌为主。经4g/L 五倍子多酚性化合物(GCE)、五倍子化学组分 B(GCE-B)及32g/L 蜂房化学组分1(NVE1)作用后,生物膜结构模糊不清,细菌散开,生物膜结构密度下降,其中死菌和活菌量基本相同。生物膜细菌总量减少,与24h 常态生物膜相比,差异有统计学意义(P<0.05)。各实验组活菌百分比与阴性对照组活菌百分比相比差异有统计学意义(P<0.05)。24h 口腔细菌生物膜经4 g/L GCE 以及32 g/L NVE1作用后,GTF 的活性受到明显抑制,与阴性对照组相比差异均有统计学意义(P<0.05)。GCE-B 在实验浓度下对 GTF 的活性未产生明显的抑制作用。结论五倍子和蜂房有效成分组成的实验药物对口腔细菌生物膜的抑制作用不单纯是对口腔生物膜细菌有杀灭作用,还可能通过对口腔细菌生物膜结构的改变调整其内部的细菌组成及抑制细菌生物膜中 GTF 活性而发挥作用。
Objective To study the effect of traditional Chinese medicine on the structure and activity of oral bacterial biofilms and the effect of Glucose Transferase (GTF) activity in biofilms using the active ingredients of Galla chinensis and Hive as experimental drugs, and to explore the possibility of Chinese herbal medicines against phlegm. Methods The method of labeling dead bacteria and viable bacteria in oral bacterial biofilms with fluorescence microscopy combined with specific fluorescent dyes was used to study the effect of experimental drugs on the structure and activity of oral bacterial biofilms. Ammonium sulfate precipitation method was used to extract crude enzyme, Neson-Somogyi method was used to measure reducing sugar, and G250 microprotein was used to calculate GTF reducing sugar. GTF activity unit was used to evaluate the effect of experimental drugs on GTF activity in oral bacterial biofilms. Results A certain number of dead bacteria existed in the process of early bacterial colonization and the formation of mature biofilm structure. At 24h, the area of live bacteria in the normal biofilm structure dominated, and a small amount of dead bacteria existed. The bacteria adhered closely to each other and the biofilm structure was clear. After the effect of 0.05% chlorhexidine on the biofilm of bacteria for 24 hours, the total amount of bacteria decreased significantly, and the residual bacteria were basically dead bacteria. After 0.023% of NaF acted on the bacterial biofilm, the biofilm structure changed significantly, the total amount of bacteria was significantly reduced, and the presence of bacteria was mainly viable. After 4g/L gall polyphenolic compounds (GCE), gallnut chemical components B (GCE-B) and 32g/L honeycomb chemical component 1 (NVE1), the structure of biofilm was unclear and the bacteria spread out. The density of membrane structure decreases, and the amount of dead bacteria and viable cells is basically the same. The total amount of biofilm bacteria decreased, compared with 24h normal biofilm, the difference was statistically significant (P<0.05). The percentage of viable cells in each experimental group was significantly different from the percentage of viable cells in the negative control group (P<0.05). After 24h oral bacterial biofilm was treated with 4 g/L GCE and 32 g/L NVE1, the activity of GTF was significantly inhibited, and the difference was statistically significant compared with the negative control group (P<0.05). GCE-B did not produce a significant inhibitory effect on GTF activity at experimental concentrations. Conclusion The inhibitory effect of experimental drugs consisting of Chinese medicinal ingredients and Chinese medicinal ingredients on the oral bacterial biofilm is not only a killing effect on the oral biofilm bacteria, but also may adjust its internal bacterial composition and inhibition by changing the structure of the oral bacterial biofilm. GTF activity plays a role in bacterial biofilms.