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目的:从用于抗感染民间药物的泥土中,发现能代谢产多种类似物或结构类型,且次级代谢产物具有抗MRSA(耐甲氧西林金黄色葡萄球菌)活性的放线菌菌株。方法:采用平板法,在改良高氏培养基上进行放线菌菌株的选择性分离,分得菌株用HPLC-UV对其进行化学筛选;然后分别采用琼脂扩散法和微量肉汤稀释法,对目标菌株发酵提取物的抗MRSA活性和最低抑菌浓度进行测定;采用MTT法测定目标菌株对人结肠癌细胞株HCT-116的细胞毒性。结果:从采集于湖南省株洲县的土壤样品中分得61株放线菌菌株;HPLC-UV图谱分析显示:目标菌株ZZ027、ZZ021和ZZ044发酵提取物含有很多类似物和不同结构类型的化合物;其中菌株ZZ027和ZZ021发酵提取物(500μg)对MRSA指示菌的抑菌圈分别为29～35mm和17～24mm,最低抑菌浓度(MICS)分别为0.625～1.25mg·mL1和2.50mg·mL1;同时菌株ZZ021发酵提取物抑制人结肠癌细胞株HCT-116的IC50为6.4μg·mL1,而菌株ZZ027和ZZ044发酵提取物的IC50>20μg·mL1。结论:从用于抗感染民间药物的泥土样品中,经选择性分离、化学筛选和抗MRSA活性测定,得到能代谢产多种似物和结构类型,且具有抗MRSA活性的放线菌菌株ZZ027和ZZ021;并且菌株ZZ021发酵提取物对人结肠癌细胞株HCT-116显示明显的细胞毒性。
OBJECTIVE: To identify actinomycete strains capable of metabolizing many analogues or structural types and having secondary antimicrobial activity against MRSA (methicillin-resistant Staphylococcus aureus) from soil used for anti-infective folk medicine. METHODS: Selective isolation of actinomycete strains was carried out on modified Kao medium by plate method. The strains were screened by HPLC-UV. Then the agar diffusion method and micro-broth dilution method The anti-MRSA activity and the minimum inhibitory concentration of the fermented extracts of the target strains were determined. The cytotoxicity of the target strains on human colon cancer cell line HCT-116 was determined by MTT assay. Results: 61 strains of actinomycetes were isolated from soil samples collected in Zhuzhou County, Hunan Province. HPLC-UV spectrum analysis showed that the fermented extracts of ZZ027, ZZ021 and ZZ044 contained many analogs and compounds with different structure types. The inhibition zones of strains ZZ027 and ZZ021 (500μg) against MRSA were 29-35mm and 17-24mm, respectively, and the MICs were 0.625-1.25mg · mL1 and 2.50mg · mL1, respectively. At the same time, the strain ZZ021 fermentation extract inhibited the IC50 of human colon cancer cell line HCT-116 to 6.4μg · mL1, while the strain ZZ027 and ZZ044 fermentation extracts had IC50> 20μg · mL1. CONCLUSION: The actinomycete strain ZZ027, which can metabolize many kinds of mimics and structure types and has anti-MRSA activity, was obtained from soil samples used for anti-infective folk medicine through selective isolation, chemical screening and anti-MRSA activity assay And ZZ021; and strain ZZ021 fermentation extract showed significant cytotoxicity on human colon cancer cell line HCT-116.