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香青兰提取物对临床来源病原菌的抑制活性及生物信息学研究 Δ

刘 云 ，刘 敏 ，于 慧 ，王占黎 （1.包头医学院研究生学院，内蒙古 包头 014060；2.包头医学院第二附属
1＊
2 #
1
2
医院检验科，内蒙古 包头 014030）
中图分类号 R285；R978.1 文献标志码 A 文章编号 1001-0408（2020）06-0666-05
DOI 10.6039/j.issn.1001-0408.2020.06.06

摘 要 目的：研究香青兰不同萃取部位对临床来源病原菌的体外抑制活性，并挖掘其可能抗菌机制。方法：香青兰药材经65％
乙醇提取后，依次以石油醚、二氯甲烷、乙酸乙酯、正丁醇萃取，得不同极性萃取部位。以肺炎克雷伯菌、金黄色葡萄球菌等临床常
见多重耐药病原菌为对象，采用纸片扩散法测定不同萃取部位的抑菌圈直径，筛选抑菌活性部位；采用琼脂倍比稀释法检测抑菌
活性部位对上述病原菌的最小抑菌浓度（MIC），并采用比浊法绘制该部位作用下多重耐药金黄色葡萄球菌的生长曲线；利用
PEAKS Q 8.5 软件筛选抑菌活性部位组与对照组的差异表达蛋白，借助 Blast 2 GO、KOBAS 3.0 在线软件对其进行基因本体
®
（GO）分析和KEGG信号通路富集分析。结果：香青兰石油醚、二氯甲烷、乙酸乙酯、正丁醇部位对革兰氏阴性杆菌均无明显的抑
制作用，乙酸乙酯部位对金黄色葡萄球菌、表皮葡萄球菌等多种革兰氏阳性球菌均具有不同程度的抑制活性（抑菌圈直径为10～
16 mm），为抑菌活性部位。该部位对金黄色葡萄球菌、表皮葡萄球菌、人葡萄球菌的MIC均为0.781 3 mg/mL，对腐生葡萄球菌的
MIC为0.390 7 mg/mL，对溶血葡萄球菌和金黄色葡萄球菌标准菌株的MIC均为1.562 5 mg/mL。1、2倍MIC的乙酸乙酯部位可抑
制多重耐药金黄色葡萄球菌的生长，且抑制活性有随剂量增加而增强的趋势。共筛选出差异表达蛋白300个（P＜0.01），其中表达
上调239个、表达下调61个。差异表达蛋白主要集中于细胞、细胞部位等细胞组成以及代谢过程、细胞过程和催化活性、蛋白结合
等生物过程和分子功能，且主要富集于不同环境中的微生物代谢和果糖与甘露糖代谢等两条信号通路上（P＜0.05）。结论：香青
兰乙酸乙酯部位为抑菌活性部位，其抑菌活性可能与影响微生物代谢和细菌糖代谢有关。
关键词 香青兰；乙酸乙酯部位；抑菌圈；最小抑菌浓度；差异表达蛋白；生物信息学分析

Study on Inhibitory Activity of Dracocephalum moldavica Extracts to Clinical Pathogenic Bacteria and Its
Bioinformatics
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LIU Yun ，LIU Min ，YU Hui ，WANG Zhanli（1. Graduate School，Baotou Medical College，Inner Mongolian
Baotou 014060，China；2. Dept. of Laboratory，the Second Affiliated Hospital，Baotou Medical College，Inner
Mongolian Baotou 014030，China）
ABSTRACT OBJECTIVE：To study inhibitory activity of different extracts of Dracocephalum moldavica to clinical pathogenic
bacteria，and to excavate its possible antibacterial mechanism. METHODS：After extraction by 65％ ethanol and extraction by
petroleum ether，dichloromethane，ethyl acetate，n-butanol，the different polar fractions of D. moldavica were obtained. Taking
Klebsiella pneumoniae，Staphylococcus aureus and other clinical multiple resistant pathogens as objects，the diameter of inhibition
zone of different extraction fractions was measured by paper diffusion method，and the antibacterial active fraction was screened.
The minimal inhibitory concentration（MIC）of antibacterial active fraction to common clinical pathogens was determined by agar
dilution method；the growth curve of MRSA was drawn by turbidimetric method. The differentially expressed protein between
antibacterial active fraction group and control group was screened by PEAK Q 8.5 software，and the gene ontology（GO）analysis
®
and KEGG signaling pathway enrichment analysis were carried out by Blast 2 GO and KOBAS 3.0 online software. RESULTS：
Petroleum ether，dichloromethane，ethyl acetate and n-butanol fraction of D. moldavica had no significant inhibitory effect on
Gram-negative bacteria. The ethyl acetate fraction of D. moldavica had antibacterial activity in varying degrees against several kinds
of Gram-positive bacteria as Staphylococcus aureus，S. epidermidis；the diameter of inhibition zone was 10-16 mm，which was
the active fraction. MICs of ethyl acetate fraction to S. aureus，S. epidermidis and S. hominis were all 0.781 3 mg/mL；MIC
to S. saprophyticus was 0.390 7 mg/mL；MICs to S. saprophyticus and standard strain of S. aureus were both 1.562 5 mg/mL. The 1，
2 times MIC of ethyl acetate could inhibit the growth of MRSA，and the inhibitory activity increased with the increase of dose.
A total of 300 differentially expressed proteins were screened （P＜0.01）， of which 239 were up-regulated and 61 were
down-regulated. The differentially expressed proteins were
Δ 基金项目：国家自然科学基金资助项目（No.81260478，No.816-
mainly concentrated in cell sites such as cells，cellular com-
60048）
ponent，etc.，and in metabolic process such as cell process，
＊硕士研究生。研究方向：天然药物、病原微生物感染与免疫。
biological process and molecular functions such as catalytic
E-mail：997487421@qq.com
activity，protein binding，etc. They were mainly concentrated
# 通信作者：教授，硕士生导师，博士。研究方向：天然药物、病原
微生物感染与免疫。E-mail：wang.zhanli@hotmail.com in microbial metabolism in different environments，fructose

·666 · China Pharmacy 2020 Vol. 31 No. 6 中国药房 2020年第31卷第6期