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鞣花酸对变异链球菌的体外抑菌作用及机制研究 Δ

闫 莉 ，周晓英 （1.新疆医科大学中心实验室，乌鲁木齐 830011；2.新疆医科大学药学院，乌鲁木齐
1＊
2
830011）

中图分类号 R965 文献标志码 A 文章编号 1001-0408（2020）05-0607-05
DOI 10.6039/j.issn.1001-0408.2020.05.20

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、
1.562 5 mg/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：12.5～50 mg/mL鞣花酸对变异链球菌产生了直径均大于15 mm的抑菌圈，50 mg/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、25 mg/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。
关键词 鞣花酸；变异链球菌；细菌生物膜；体外；抑菌作用；机制
Study on in vitro Anti-bacterial Activity and Mechanism of Ellagic Acid on Streptococcus mutans
2
1
YAN Li ，ZHOU Xiaoying（1.Central Laboratory，Xinjiang Medical University，Urumqi 830011，China；2.
School of Pharmacy，Xinjiang Medical University，Urumqi 830011，China）
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 coenzymeⅠoxidation 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.
Δ 基 金 项 目 ：新 疆 维 吾 尔 自 治 区 自 然 科 学 基 金 资 助 项 目
KEYWORDS Ellagic acid；Streptococcus mutans；Biomem-
（No.2017D01C238）
＊助理研究员，硕士。研究方向：中药与民族药。电话：0991- brane；in vitro；Anti-bacterial activity；Mechanism
4366434。E-mail：597205931@qq.com

中国药房 2020年第31卷第5期 China Pharmacy 2020 Vol. 31 No. 5 ·607 ·