Studies On Antioxidative Secondary Metabolites Of Aspergillus Sp. Fungi Isolated From Lilium Davidii Var.unicolor

Most of the natural medicines are extracted and isolated directly from plants.However,with the destruction of the environment and the increasing demand of people,it is urgent to develop new natural medicinal resources.In recent years,the study of endophytic fungi are mostly focused on species distribution and diversity,the impact on host plants and the resistance to stress,etc.More researches is focused on the isolation of secondary metabolites of endophytic fungi,and a growing number of bioactive compounds found,make it become another major source of natural products.However,there are few researches on endophytic fungi of Lilium.Therefore,in this study,secondary metabolites of endophytic fungus GBH2J4 isolated from bulbs of Lilium davidii var.unicolor Cotton were studied,and the results were as follows:1.Endophytic fungus GBH2J4 was identified as Aspergillus sp.by morphological characteristics combined with molecular biological methods.2.The fermentation broth and mycelium were obtained by filtering potato dextrose broth which was cultivated under controlled conditions of temperature and rotational speed.The fermentation broth was concentrated and extracted with ethyl acetate and n-butanol successively to obtain the corresponding ethyl acetate soluble parts samples and n-butanol soluble parts samples.The mycelium was dried and extracted with ethanol to obtain ethanol extract.Sixteen compounds were isolated and purified by silica gel chromatography,gel chromatography and thin layer chromatography.By ¹H-NMR,¹³C-NMR and mass spectrometry,combined with TLC analysis and physicochemical properties of compounds,twelve compounds were identified as succinic acid（1）,fumaric acid（2）,methyl hydrogen succinate（3）,kojic acid（4）,β-sitosterol（5）,methyl pyroglutamate（6）,fructose（7）,dibutyl succinate（8）,ethyl oleate（9）,glycerol 1,3-dioleate-2-stearate（10）,stearic acid（11）and oleic acid（12）.Among them,1-4,6 and 8-12 are carboxylic acids and ester compounds,5 is a steroidal compound,7 is a carbohydrate compound.Compound 3,8 and 10 were isolated from Aspergillus sp.for the first time.3.The antioxidant activity of compounds 1-12 was evaluated by TLC-DPPH-bioautogra-phy with 2 mg/m L sample solution and 0.75 mg/m L vitamin C（Vc）ethanol solution as positive control.The results showed that compounds 1,2 and 4 had antioxidant activity.In order to further determine the activity of the three compounds,DPPH method,salicylic acid method and Prussian blue method were used to determine the antioxidant activity.The sample solution was 1 mg/m L,Vc solution of the same concentration was used as positive control,and Vc solution of 0.035 mg/m L was used as positive control for Prussian blue method.The DPPH·scavenging activity（%）of compound 4 was 38.80%,while that of the positive control was 96.74%,indicating that compound 4 had moderate DPPH·scavenging activity.The scavenging ability of hydroxyl radical was determined by salicylic acid method,and compounds 1,2 and 4 were inactive.The results of Prussian blue assay showed that the absorbance values of compound 1 and 2 were 0.026 and 0.034,respectively,and the total reducing power were weak.The absorbance value of compound 4 was 0.975,while that of the positive control was 0.968,indicating that compound 4 had strong activity of total reducing power.In this study,the endophytic fungus GBH2J4 with antioxidant activity was identified as Aspergillus sp.Sixteen compounds were isolated and purified from the fermentation liquor and the mycelium,and twelve compounds were identified.The antioxidant activity tests showed that compound 4 had moderate DPPH·scavenging activity and strong total reducing power activity.