药用植物雷公藤内生真菌分离及其抑菌活性筛选研究

Study on Isolation of Endophytes from the Medicinal Plant Tripterygium Wilfordii and Screening of Its Antibacterial Activity

关键词:  雷公藤  内生真菌  抑菌活性  最低抑菌浓度

Key words:  Tripterygium Wilfordii  endophytic fungi  antibacterial activity  MICs

摘要:

目的 研究药用植物雷公藤内生真菌的分离及其抑菌活性，并预测次级代谢化合物种类，为进一步筛选雷公藤内生真菌产天然抑菌物质提供依据。方法 采用组织块法和反复划线法对雷公藤原植物进行微生物的分离纯化；采用滤纸片法检测雷公藤内生真菌液体发酵液对金黄色葡萄球菌、白色念珠菌和铜绿假单胞菌的抑菌活性，并测定其最小抑菌浓度(minimum inhibitory concentration，MIC)；采用UPLC/Q-TOF-MS法对具有抑菌功能的菌株发酵液进行分析，得到各样品总离子流图，结合相关数据库及软件对其成分进行质谱网络化分析，预测微生物次级代谢化合物种类。结果 共获得20株内生真菌；5株内生真菌发酵液对金黄色葡萄球菌具有显著抑菌活性，其中LGT-15对金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌均有高度抑菌活性，抑菌圈直径分别为38.0，34.0，13.1 mm，MIC分别为0.49，0.61，1.46 μg·mL^-1。根据质谱数据分析结果，LGT-3和LGT-15代谢产物数量和类型较多，均含有氮杂螺环酮类化合物，而LGT-15另含有结构新颖的喹唑啉类生物碱。结论 采用菌落形态学和分子生物学方法鉴定了5株活性菌株，其中LGT-15被鉴定为烟曲霉菌。质谱数据提示LGT-15的次级代谢产物值得进一步挖掘。

Abstract:

OBJECTIVE To study the isolation and antibacterial activity of endophytic fungi from medicinal plant Tripterygium wilfordii and predict the types of secondary metabolites, provide the basis for further screening of natural antibacterial substances produced by endophytic fungi of Tripterygium wilfordii. METHODS The methods of tissue block and repeated line were used to isolate and purify the endophyte from T. wilfordii. The activity of the liquid fermentation broth of endophyte against the tested strains of S. aureus, C. albicans and P. aeruginosa was detected through the filter paper method, and the minimum inhibitory concentration(MIC) of active strains was detected by microdilution method. The fermentation broth of strains with antibacterial function was analyzed by UPLC/Q-TOF-MS method, and the total ion chromatogram of each sample was obtained. Combined with relevant databases and software, mass spectrometry network analysis was carried out on its components to predict the types of microbial secondary metabolites. RESULTS There were 20 strains of endophytic fungi obtained from T. wilfordii. The fermentation broth of 5 endophytic fungi had significant antibacterial activity against S. aureus. Among them, LGT-15 had high antibacterial activity against S. aureus, P. aeruginosa and C. albicans with the inhibition zones diameters 38.0, 34.0, 13.1 mm, and the MICs 0.49, 0.61, 1.46 μg·mL^-1, respectively. According to the results of mass spectrometry data analysis, LGT-3 and LGT-15 metabolites were more numerous in number and types, and both contained azaspiroketones, while LGT-15 also contained novel quinazoline alkaloids. CONCLUSION Five active strains are identified by colony morphology and molecular biology. LGT-15 is identified aspergillus fumigatus. The secondary metabolites of LGT-15 should further be explored according the mass spectrometry data.