一株韦司梅拟盘多毛孢的鉴定及其发酵液抗乳腺癌活性

Identification of a Pestalotiopsis vismiae strain and anti-breast cancer activity of its fermentation broth

【目的】对1株分离自桃树枝的内生真菌N1进行分类鉴定与系统发育分析,探讨其深层发酵液的抗乳腺癌活性,为该菌资源的进一步开发利用提供依据。【方法】通过形态学特征和ITS rDNA序列对野生菌株N1进行分类鉴定;基于ITS1-5.8S-ITS2序列的变异程度对菌株N1遗传多样性进行分析;采用MTT法检测菌株发酵液石油醚、乙酸乙酯、正丁醇和甲醇提取物的抗乳腺癌活性;观察乙酸乙酯提取物不同作用时间下乳腺癌细胞的形态变化;采用LC-MS/MS对乙酸乙酯活性提取物的成分进行分析。【结果】根据菌株形态学和ITS rDNA序列特征,将该野生菌株N1鉴定为韦司梅拟盘多毛孢Pestalotiopsis vismiae;其ITS1-5.8S-ITS2序列总变异率为10.3%,表明韦司梅拟盘多毛孢在自然界中具有丰富的遗传多样性。菌株N1深层发酵液乙酸乙酯提取物对人乳腺癌MDA-MB-231细胞生长的抑制作用最强(P<0.05),作用24h的半数抑制浓度(IC50)为131.39μg/mL,作用48h时,细胞呈现凋亡状态。LC-MS/MS数据表征了其中的13个化学成分,确定了6种物质,对其他未能鉴定的7个物质的分子式进行了初步表征。【结论】获得了韦司梅拟盘多毛孢遗传多样性的基础信息,其深层发酵液乙酸乙酯提取物是发挥抗乳腺癌细胞的主要活性物质。

【Objective】A strain of endophytic fungi N1 isolated from peach twig was identified and phylogenetically analyzed and the anti-breast cancer activity of its deep fermentation broth was studied to provide basis for the further development and utilization of this fungus resource.【Method】The wild strain N1 was identified by morphological characteristics and ITS rDNA sequences.The genetic diversity of strain N1 was analyzed based on the variation degree of ITS1-5.8S-ITS2 sequences.The anti-breast cancer activities of petroleum ether,acetidin,n-butanol and methanol extracts of the fermentation broth were determined by MTT assay.The changes of morphology of breast cancer cells were observed under different treatments of ethyl acetate extract.LC-MS/MS was used to analyze the functional constituents.【Result】The wild strain N1 was identified as Pestalotiopsis vismiae according to its morphology and ITS rDNA sequence.The total variation rate of ITS1-5.8S-ITS2 was 10.3%in Pestalotiopsis vismiae,indicating that Pestalotiopsis vismiae had abundant genetic diversity in the natural world.Ethyl acetate extract fractions of strain N1 had the strongest inhibitory effect on the growth of human breast cancer MDA-MB-231 cells(P<0.05)with the IC 50 value for 24 h of 131.39μg/mL.When the action time was 48 h,the cells showed apoptotic state.LC-MS/MS preliminarily characterized 13 chemical components,of which 6 components were identified and the molecular formulas of the other 7 unidentified substances were preliminary characterized.【Conclusion】This study obtained basic information about the genetic diversity of Pestalotiopsis vismiae,and showed that its ethyl acetate extract was as the main anti-breast cancer active substance.