Studies On Free Radical Scavenging Activities Of Extracts From A Strain Of Paecilomyces Gunnii

Paecilomyces gunnii is a fungus with high medicative value and promising utilization perspectives. A free radical scavenging assay revealed that extracts from mycelia of Paecilomyces gunnii had a strong scavenging activity. To enhance the yield of free radical scavengers, further experiments were done, including the optimization of culture conditions, extraction and purification processes of the bioactive components, as well as the structural analysis and identifications of bioactive compounds.A DPPH-TLC and a DPPH-Microplate assay were made to determine the free radical scavenging activities of methanol extracts from the mycelia of Paecilomyces gunnii by solid culture. The results revealed that the extracts had strong free radical scavenging activities, at the concentration of 30 mg/ml the methanol extracts could decrease 94.91% percent of 0.2 mg/ml DPPH radicals after incubated at 37℃for 10 minutes. HPLC-MS analysis showed that the methanol extracts from the mycelia of Paecilomyces gunnii was relatively complicated. Its chromatogram had more then ten peaks. Bioactivity combined analysis showed that two of the peaks had radical scavenging activity. The retention time of the two peaks were 10.8 min (11.2 min on MS ion flow chromatogram) and 15.8 min (16.2 min on MS ion flow chromatogram). The two bioactive products were the main components of the methanol extracts.Based on scavenging activity through orthogonal experiments of culture conditions, an optimized medium composition was obtained as follows:sucrose 20g/L, corn flour 10 g/L, yeast extract 5g/L, NaNO33 g/L, MgSO4·7H2O 0.5g/L, NaC10.5 g/L, CaCl21.0 g/L and 0.2 g/L. The optimal culture condition was optimized as:seed age 4d, inoculation amount 10%, culture peried 8d and culture temperature 25℃。Based on scavenging activity through orthogonal test and response surface experiment of extraction conditions, an optimal extraction process was obtained as follows:the optimal solvent methanol, the optimal temperature 37℃, the ultrasonic power 80KHz, the extraction time 34min; the ratio of material to solvent 1:30. Under those conditions, the predicted value of DPPH free radical scavenging activity from the mycelia of Paecilomyces gunnii was 96.61%, which was in consistent well with the measured value.Two kinds of the radical scavenging components were isolated from RCEF4117 by semi-preparative high performance liquid chromatography, which were respectively named pg-1 and pg-2. The purity of the two obtained components was determined with HPLC as 97.3%(pg-1) and 97.1%(pg-2). HPLC-DAD-HRMS analysis revealed that the molecular formula of the two radical scavengers existed in the extract were possibly C17H15NO6 and C16H14O6. DPPH-Microplate assay showed that the two purified compounds had strong free radical scavenging activities with values of 89.62% and 75.45% respectively. It suggested that the separation and purification of the bioactive components from the extract was successful.