鞣花酸对变异链球菌的体外抑菌作用及机制研究
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篇名: 鞣花酸对变异链球菌的体外抑菌作用及机制研究
TITLE: Study on in vitro Anti-bacterial Activity and Mechanism of Ellagic Acid on Streptococcus mutans
摘要: 目的:研究鞣花酸对变异链球菌的体外抑菌作用及可能机制,为其防治龋病的应用提供实验依据。方法:以复方氯己定含漱液为阳性对照、5%二甲基亚砜(DMSO)溶液为阴性对照,采用打孔法抑菌试验测定抑菌圈直径考察50、25、12.5、6.25、3.125、1.5625mg/mL鞣花酸对变异链球菌的抑菌效果,并采用微量稀释法测定其对变异链球菌的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。以5%DMSO溶液为阴性对照,采用结晶紫染色法测定1/8MIC、1/4MIC、1/2MIC、MIC鞣花酸对变异链球菌生物膜形成的影响,并在荧光染色后通过激光扫描共聚焦显微镜观察1/2MIC鞣花酸作用后生物膜的结构变化;分别采用苯酚硫酸法和还原型辅酶Ⅰ氧化法考察1/8MIC、1/4MIC、1/2MIC、MIC鞣花酸对变异链球菌细胞外多糖(EPS)的抑制率和细胞外基质中乳酸脱氢酶(LDH)活力的影响。结果:12.5~50mg/mL鞣花酸对变异链球菌产生了直径均大于15mm的抑菌圈,50mg/mL鞣花酸作用下抑菌圈直径与复方氯己定含漱液相当。鞣花酸对变异链球菌的MIC、MBC分别为12.5、25mg/mL。1/8MIC~MIC鞣花酸作用后细菌生物膜的存活率较阴性对照显著降低(P<0.01),且呈剂量依赖趋势,MIC鞣花酸作用后细菌生物膜的存活率仅为(16.41±1.346)%;荧光染色后显微镜下可见,1/2MIC鞣花酸作用后细菌生物膜结构被破坏;1/8MIC~MIC鞣花酸作用后细菌水不溶性EPS和水溶性EPS的抑制率较阴性对照均显著升高(P<0.01),1/4MIC~MIC鞣花酸作用后细胞外基质中LDH活力较阴性对照均显著升高(P<0.01),且呈剂量依赖趋势。结论:鞣花酸对变异链球菌的生长具有一定的抑制作用;其机制可能与抑制EPS产生、降低细菌的黏附、破坏细菌细胞膜有关。
ABSTRACT: OBJECTIVE:To study the in vitro anti-bacterial activity and potential mechanism of ellagic acid on Streptococcus mutans,and to provide evidence for its prevention and treatment of dental caries. METHODS :Using Compound chlorhexidine gargle as positive control ,5%DMSO as negative control ,bacteriostasis experiment was conducted by the method of drilling hole , and bacteriostatic effects of 50,25,12.5,6.25,3.125,1.562 5 mg/mL ellagic acid on S. mutans was preliminarily determined by measuring the diameter of bacteriostatic ring. The minimal inhibitory concentration (MIC)and minimal bactericidal concentration (MBC)of ellagic acid on S. mutans were determined by microdilution method. Using 5% DMSO as negative control ,the effects of 1/8 MIC,1/4 MIC,1/2 MIC and MIC ellagic acid on the formation of S. mutans biomembrane was determined by crystal violet staining. The changes of the biomembrane structure under the action of 1/2 MIC ellagic acid were observed by microscopy after fluorescence staining. Phenol sulfuric acid method and reducing coenzyme Ⅰoxidation method were used to determine inhibitory effects of 1/8MIC,1/4MIC,1/2MIC,MIC ellagic acid on S. mutans on extracellular polysaccharide (EPS)as well as effect on the activity of lactate dehydrogenase (LDH)in extracellular matrix. RESULTS :Ellagic acid with concentration of 12.5~50 mg/mL produced an inhibitory ring on S. mutans with diameter greater than 15 mm. Under the action of 50 mg/mL ellagic acid ,the diameter of bacteriostatic ring was the same as that of Compound chlorhexidine gargle . MIC and MBC of ellagic acid to S. mutans were 12.5 mg/mL and 25 mg/mL. The survival rate of bacterial biomembrane after 1/8MIC-MIC ellagic acid treatment was significantly lower than that of the negative control (P<0.01),and had a certain dose-response trend. After MIC ellagic acid treatment,the survival rate of bacterial biomembrane was (16.41±1.346)%. After fluorescence staining ,the structure of bacterial biomembrane was destroyed by 1/2 MIC ellagic acid. After treated with 1/8MIC-MIC ellagic acid ,its inhibitory rates on water-soluble EPS and water-insoluble EPS were increased significantly ,compared with negative control (P<0.01). After treated with 1/4MIC-MIC ellagic acid ,the activity of LDH in the extracellular matrix of bacteria increased significantly ,compared with negative control (P<0.01),in dose-effect dependent trend. CONCLUSIONS :Ellagic acid can inhibit the growth of S. mutans ,the mechanism of which may be associated with inhibiting EPS production ,reducing bacterial adhesion ,destroying bacterial cell membrane.
期刊: 2020年第31卷第05期
作者: 闫莉,周晓英
AUTHORS: YAN Li,ZHOU Xiaoying
关键字: 鞣花酸;变异链球菌;细菌生物膜;体外;抑菌作用;机制
KEYWORDS: Ellagic acid ;Streptococcus mutans ;Biomem-
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