药用真菌粗毛纤孔菌的分子甄别及其发酵液抗乳腺癌活性研究

从分子水平上探究粗毛纤孔菌与桑黄孔菌属菌株的差异,同时以人乳腺癌MDA-MB-231细胞筛选粗毛纤孔菌深层发酵液抗乳腺癌活性部位,并对其成分进行初步分析。基于rDNA ITS序列分子系统发育树和拆分网络（splits network）分析研究粗毛纤孔菌与桑黄孔菌属菌株的差异;采用MTT法检测发酵液不同提取物抗乳腺癌活性;通过细胞形态观察进一步了解活性提取物对细胞生长的影响;采用LC-MS/MS对活性提取物的成分进行分析。系统发育树表明粗毛纤孔菌与桑黄孔菌属菌株存在一定的遗传差异,粗毛纤孔菌GC含量较桑黄孔菌属菌株低,同时拆分网络分析法能更好地区分粗毛纤孔菌与桑黄;粗毛纤孔菌深层发酵液正丁醇提取物对MDA-MB-231细胞生长的抑制作用最强且呈量效和时效关系（ P<0.05）,作用24h的IC₅₀值为245.44μg/mL;细胞形态观察显示正丁醇提取物浓度在250μg/mL时,对MDA-MB-231细胞的凋亡作用随着时间的延长而加强;LC-MS/MS检测到正丁醇萃取部分的物质有2个,这2个物质均首次在粗毛纤孔菌中鉴定得到。结果表明,拆分网络分析法可较好地用于粗毛纤孔菌与桑黄孔菌属菌株的分子甄别,正丁醇提取物是粗毛纤孔菌深层发酵液抗乳腺癌MDA-MB-231细胞的主要活性部位,为进一步开发利用粗毛纤孔菌药用资源提供科学依据。

Molecular screening of medicinal fungus Inonotus hispidus and anti-breast cancer activities of its submerged fermentation broth

The difference of Inonotus hispidus and Sanghuangporus spp . was revealed at molecular level. The active constituents of Inonotus hispidus against breast cancer cell line MDA-MB-231 were screened and their active ingredients were analysed. The phylogenetic tree and split network based on rDNA ITS sequence was used to analyse the difference of Inonotus hispidus and Sanghuangporus spp. MTT method was used to detect the anti-breast cancer activity of different extracts of Inonotus hispidus fermentation broth. The effect of extraction fractions on cancer cell growth was detected by morphological observation. LC-MS/MS was used to analyze the active constituents of the extracts. The phylogenetic tree showed that Inonotus hispidus and Sanghuangporus spp. had certain genetic differences. The GC content of Inonotus hispidus were lower than that of Sanghuangporus spp. and the split network analysis could distinguish Inonotus hispidus from Sanghuangporus spp. N-butanol extract of Inonotus hispidus fermentation broth had the strongest inhibitory effect on the growth of MDA-MB-231 cells and the cell proliferation was inhibited in a dose and time dependent manner ( P<0.05) and the IC₅₀ value for 24 hours was 245.44μg/mL. Cell morphology observation showed that the effect of n-butanol extract at 250μg/mL concentration on the apoptosis of MDA-MB-231 cells increased with the prolong of treatment time. LC-MS/MS data had identified two chemical components, and they were recognized from Inonotus hispidus for the first time. The n-butanol extract of Inonotus hispidus fermentation broth could be considered having anti-breast cancer activity.