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本文采用组织分离法从片栗叶部分离获得一株内生真菌,通过28s r DNA序列测定和系统发育树分析对此菌株进行了鉴定。用PD培养基和YM培养基分别发酵培养此菌株,其次级代谢产物通过高效液相色谱(HPLC)和质谱(MS)进行了分析。在YM培养基条件下对此菌株大量培养,利用硅胶层析等手段对其次级代谢产物进行分离纯化,采用MTT法测定了次级代谢产物对癌细胞的毒性。结果表明,从片栗叶部分离得到的内生真菌鉴定为Eupenicillium sp.NF2_PD013。经HPLC和MS分析表明,此内生真菌的次级代谢产物在YM培养基条件下比在PD培养基条件下丰富且产量大。从YM培养基条件下的次级代谢产物中分离得到4个化合物,分别为geodin,dihydrogeodin,3’-chlorotrypacidin和monochlorosulochrin,4个化合物均含有氯元素。化合物geodin和3’-chlorotrypacidin对乳腺癌细胞MCF-7和肝癌细胞Hep G2都有明显的增殖抑制作用。
In this paper, an endophytic fungus was isolated from the leaves of chestnut by tissue isolation method. The strain was identified by 28s r DNA sequence analysis and phylogenetic tree analysis. The strain was fermented separately with PD medium and YM medium, and its secondary metabolites were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry (MS). Under YM medium, the strain was cultured in large quantities, and its secondary metabolites were isolated and purified by silica gel chromatography. The toxicity of secondary metabolites to cancer cells was determined by MTT assay. The results showed that the endophytic fungi isolated from the chestnut leaves were identified as Eupenicillium sp. NF2_PD013. Analysis by HPLC and MS showed that the secondary metabolites of this endophytic fungus were abundant and yieldable in the YM medium condition than in the PD medium condition. Four compounds, geodin, dihydrogeodin, 3’-chlorotrypacidin and monochlorosulochrin, were isolated from the secondary metabolites of YM medium. All four compounds contained chlorine. The compounds geodin and 3’-chlorotrypacidin have significant inhibitory effects on breast cancer cell line MCF-7 and Hep G2 cell line.