| The free radical scavenging activity and active components of fermentation mycelium from Ophiocordyceps formosana was systematically studied. Strain RCEF0868 and RCEF5491 of O. formosana were cultured by liquid fermentation and mycelia were freeze dried. Better strain and solvent were selected by comparing their scavenging activity of DPPH free radical, hydroxyl radical and superoxide radical. Better extracting agents were used to extract the strain RCEF0868 as tested; the freeze-dried powder were got to quantitatively analyze its scavenging activity of DPPH and hydroxyl radical. The molecular formula and chemical structure of the active components were preliminary analyzed by HPLC and LC-MS. The extraction method of active components in strain RCEF0868 were optimized based on DPPH free radical scavenging activity by software SAS.As the selecting result, strain RCEF0868 showed better free radical scavenging activity and there were large differences among the different solvent extracts. Water and methanol extracts showed higher free radical clearance rate. Thus, strain RCEF0868 were selected as the research object, and the DPPH free radical and hydroxyl radical scavenging activities of its water and methanol eatracts were further analyzed.Quantitative analysis of strain RCEF0868 showed that two kinds of free radical scavenging activities of water extract and metnanol extract were increased with the increasing of sample concentration. When sample concentration was 5.0mg/ml after 20 min, DPPH clearance rate of water extract and methanol extract reached 87.83% and 91.70% respectively, and their half maximal inhibitory concentrations were 0.85mg/ml and 1.23mg/ml respectively. While the hydroxyl radical clearance rate of water extract and methanol extract reached 90.04% and 61.60% respectively, and their half maximal inhibitory concentrations were 1.37mg/ml and 2.91mg/ml respectively. In conclusion, both water extract and metnanol extract of strain RCEF0868 showed obvious DPPH scavenging activity. In addition, the hydroxyl radical scavenging activity of water extract was also good, while the methanol extract showed relatively weaker activity.The results of HPLC separation and LC-MS analysis of active components showed that three active ingredients were detected in water extract, their molecular formula were C6H14O6(1), C14H17N5O8(2) and C20H33NsO9(3), respectively; While besides the compound 1, there were another three active components in methanol extract, i.e.,C30H22O11(4) io(5) and C30H18O10(6), respectively. The six active ingradients all showed DPPH free radical scavenging activity, and compounds 1,4 and 5 also showed hydroxyl radical scavenging activity. By querying the database of natural products and reference to the relevant reports, component 3 may be a new compound, the others were D-Mannitol, Succinoadenosine,4a-Oxyrugulosin, Rugulosin and Skyrin, respectively. Among them Succinoadenosine and 4a-Oxyrugulosin were first time found in entomogenous fungi.Single factor experiment of the extract optimization showed that material liquid ratio(mg/ml), temperature(’C) and methanol volume ratio(%) were relatively the main factors which influence DPPH free radical clearance rate. Further response surface optimization results showed that material liquid ratio(mg/ml) and methanol volume ratio(%) significantly effected DPPH free radical clearance rate(P<0.05), and interaction that significantly affect the clearance rate did not exist between the two factors. Although DPPH clearance rate increases with temperature increased, the influence of temperature on the clearance rate was not significant. Simulated by Software SAS 9.0, the optimal extracting condition was material liquid ratio 12mg/ml and methanol volume ratio 80%, under which the clearance rate could reach 81.08%. According to the optimal extracting condition, validation test result was 82.70±0.87(%), which reached the level of simulated optimization and further showed the accuracy of optimization. |
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