Study On Antimicrobial Secondary Metabolites Of Xylaria Striata

Higher fungi, which can produce polysaccharide, alkaloid, flavonoid,steroid etc with complex chemical structure and a variety of biological activity under natural conditions, plays an important role in biological invasion and is a potential resource for developing new drugs or pesticides in the future. This dissertation is consisted of four parts:(1)Screening of antimicrobial and antioxidant activities from 12 higher fungi named Antrodia subxantha, Xylaria striata, Leucocoprinus cepistipes, Daldinia eschscholtzii, Lycoperdon excipuliforme, Scorias spongiosa, Xylaria sp. 808, Xylaria pedunculata, Xylaria sp.507,Xylaria longipes, Xylaria schweinitzii and Xylaria sp. X338;(2)Optimization of submerged culture conditions for X. striata;(3)Antimicrobial and antioxidant activities of crude extracts from X.striata mycelium;(4) Activity-guided fractionation and evaluation of antimicrobial active substances from X. striata extracts.The results are as follows:(1) The antibacterial and antioxidant activities of 12 higher fungi were determined by TLC-bioautography analysis. The results of antibacterial tests showed the extracts from A. subxantha, X. striat, D.eschscholtzii, L. excipuliforme, Xylaria sp. 808, X.longipes had different inhibitary effects against the tested bacteria. The mycelial growth rate of Fusarium graminearum was evaluated for antifungal activity of the extracts from higher fungi mycelium and fermentation broth. The methanol extracts from A. subxantha and X. striata mycelium showed strong inhibitory effects against F. graminearum, and the inhibition ratio were 85.41 % and 83.58 %, respectively. The ethyl acetate extracts from Xylaria sp. 808 fermentation broth had a complete growth inhibition(inhibition ratio 100 %) against F. graminearum at the concentration of 0.5 mg/ml. The results of antioxidant analysis indicated that all of the extracts from 12 higher fungi mycelium andfermentation broth had the ability of scavenging DPPH free radical.(2) The optimum submerged culture condition of X. striata was established through One-factor-at-a-time method and orthogonal experiment design and the dry weight of mycelium was determined for evaluation the biomass of the growth of X. striata mycelium. The results from one-factor-at-a-time experiment showed that maltose,glucose or corn powder was the best suitable carbon source and the soybean powder fine was optimum nitrogen source. The optimum ratio of carbon to nitrogen source was 5:1. Under the optimum carbon,nitrogen source and the ratio between them, the highest dry weight of mycelium was obtained at p H 6 of medium when inoculated with 2mycelial discs and fermented for 7 days. Moreover, the growth of mycelium was observed and it was promoted significantly by addition of K, Mg, P and VB1. The consequence of orthogonal experiment showed that the optimum carbon source and nitrogen source were 4 %maltose and 0.8 % soybean powder fine, respectively, and the highest mycelium biomass could be obtained when shaking cultivated at p H 7,25°C for 13 days.(3) Antibacterial activity of the extracts from X. striata against 10 bacteria was determined with oxford cup method and microplate assay,and antifungal activity of the extracts against 9 plant pathogenic fungi was tested by mycelial growth rate method. The results of antibacterial analysis showed that inhibition zone diameter of the mycelium extract was from 13.0 mm to 17.5 mm and from 10.5 mm to 12.7 mm for the fermentation broth extract. Enterobacter cloacae was the most sensitive to both extracts and the inhibition zone diameter of mycelium extract and fermentation broth extract were 17.1 mm and 12.7 mm respectively,while the minimum inhibitory concentration were 2.5 mg/ml and 5mg/ml. The highest inhibitory effect against pathogenic fungus was observed in F. graminearum and the inhibition ratios of mycelium extract and fermentation broth extract from X. striata were 85.88 % and69.53 %, respectively.The antioxidant activity of the extracts from mycelium and fermentation broth was measured by analyzing the ability of scavenging free radicals, DPPH·, ·OH and O2-·, the reducing power and the chelation of the iron ion. The results displayed that both extracts from X. striata mycelium extract and fermentation broth had greater clearing ability on DPPH·, · OH and ABTS free radical(clearance rate higher than 75 %) and strong reducing power and chelating ability were also found.(4) Two compounds with strong antimicrobial activity were isolated from the mycelium extracts of X. striata by silica gel column chromatography and Sephadex LH-20 column chromatography and identified as ergosterol and ergosterol peroxide by spectroscopic analysis with1 H NMR and13 C NMR. Erwinia carotovora, one of the tested phytopathogen bacteria, was the most sensitive strain to both two compounds and the minimum inhibitory concentration of ergosterol and ergosterol peroxide were 5 and 18 μg/ml, respectively. At the concentration of 20 μg/ml, ergosterol showed complete growth inhibition effect against Helminthosporium maydis, Valsa mali Miyabe et Yamade, Sclerotinia sclerotiorum and F. graminearum, while ergosterol peroxide had the 100% inhibitory effect against H. maydis alone.