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

Abstract:

[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.