古尼拟青霉中抗氧化活性组分的分离纯化和液质联用分析

Purification and HPLC-MS analysis of antioxidant components in Paecilomyces gunnii

在虫生真菌代谢物抗氧化活性物质筛选中,发现一种古尼拟青霉菌株RCEF4117固体培养物中存在有较强的抗氧化活性物质。该研究目的是分离纯化这些活性组分,并制备纯品进行结构鉴定。采用二苯代苦味肼基自由基—薄层色谱法(1,1-dipheny1-2-picrlyhydrazyl-thin layer chromatography,DPPH-TLC)和二苯代苦味肼基自由基—酶标仪法(DPPH-Microplate)对古尼拟青霉固体培养菌丝的甲醇提取物进行抗氧化活性测定。液质联用DPPH法(high performance liquid chromatography-mass spectrometry,HPLC-MS-DPPH)对活性物质进行初步鉴定;用反相高效液相色谱(reversed-phase-HPLC,RP-HPLC)制备分离活性成分。研究发现供试样品的甲醇提取物具有较强的抗氧化活性,进一步分离纯化从中得到2个高纯度的强抗氧化活性成分PG-1(纯度为97.31%,活性为89.62%)和PG-2(纯度为97.12%,活性为75.45%)。HPLC-MS-DPPH分析得PG-1、PG-2的分子式分别为C17H15NO6和C16H14O6。该研究首次从古尼拟青霉中成功分离纯化出2个抗氧化活性组分的纯品。

In the screening of antioxidant active substances for metabolites of entomogenous fungi,a strong antioxidant active substance was found in the solid culture of Paecilomyces gunnii strain RCEF4117. This study aimed to isolate and purify these active components and prepare pure products for structural identification. The antioxidant activity of methanol extracts from solid cultured of Paecilomyces gunnii mycelia was determined by 1,1-dipheny1-2-picryhydrazyl-thin layer chromatography (DPPH-TLC) and 1,1-dipheny1-2-picryhydrazyl hydrate-microplate (DPPH-Microplate). The active substances were preliminarily identified by high performance liquid chromatography-mass spectrometry DPPH (HPLC-MS-DPPH) and separated by reversed-phase high-performance liquid chromatography (RP-HPLC). We found that the methanol extract of the sample had strong antioxidant activity. Furthermore,two highly purified components,PG-1 (purity 97. 31%,activity 89. 62%) and PG-2 (purity 97. 12%,activity 75. 45%) were obtained from further separation and purification. The molecular formulas of PG-1 and PG-2 were C17H15NO6 and C16H14O6,respectively using HPLC-MS-DPPH analysis methods. In this study,two pure antioxidant components were successfully isolated and purified from Paecilomyces gunnii for the first time.