一种被毛孢(Hirsutella sp.)培养液中自由基清除剂的分析、分离和制备

[目的]在一次对虫生真菌代谢物进行大规模的清除自由基活性物质筛选中,发现一种被毛孢(Hirsutella sp.)菌株RCEF0881发酵液中存在有较强的清除自由基活性物质.本研究目的是初步搞清这些活性成分的具体组成,并制备出一定量的纯品用于进一步的结构鉴定.[方法]用有机溶剂法提取活性成分;用二苯基苦基苯肼自由基(DPPH)酶标仪法和薄层色谱法进行活性测定;用高分辨液质联用方法进行活性成分初步分析和鉴定;用反相制备色谱法制备活性组分.[结果]提取实验结果表明具清除自由基活性的物质能较好地被乙酸乙酯提取出来;液相色谱-质谱-活性测定分析表明提取物中活性组分的可能分子式分别为C7H6O4、C8H8O3和C12H14N2O.结合色谱特性、紫外光谱特征、质谱碎片和数据库查询可初步推断它们分别为二羟基苯甲酸、羟基甲基苯甲酸和生物碱类物质,但具体结构还有待于进一步确认.从高效液相色谱和质谱离子流的峰面积可知上述3种活性物质中C12H14N2O的含量最高.本研究成功地用反相制备色谱制备出该天然活性组分的纯品.该3种清除自由基活性物质都是首次发现存在于虫生真菌的代谢物中.

Analysis and isolation of radical scavengers from metabolites of Hirsutella sp.

[Objective] During a screening for free radical scavengers from metabolites of entomogenous fungi, we fond a fermentation broth of the strain RCEF 0881 of Hirsutella sp. exhibited strong radical scavenging activity. To make clear of the constituents of the active compounds, and prepare some pure active compound for further structure identification we launched this study. [Methods] We used organic solvent for active compounds extraction. DPPH-TLC and DPPH- Mi-croplate assay were used for activity analysis. Components analysis was carried out on a HPLC-DAD-HRMS, and bioac-tive compound preparation on a preparative RP-HPLC. [Results] Our extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD-HRMS-DPPH assay revealed that the molecular for-mular of the radical scavengers existed in the extract were possibly C7H6O4, C8H8O3 and C12H14N2O. From the chroma-tographic and Uv properties, and the MS fragments, and database consulting, the compounds could be deduced as dihy-droxybenzoic acid, methyl-hydroxyl benzoic acid, and an alkaloid, however, the structures are still needed to be confirmed. The pick area of HPLC and MS showed that the compound C12H14N2O was the main component of the extract. It was iso-lated via activity directed fractionation. The activity of the prepared compound was confirmed with DPPH-TLC assay and its purity was confirmed with HPLC-DAD-HR-ESIMS. The occurance of the three active compounds in entomogenous fungi was revealed for the first time.