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The laboratory is separated from China into an aloe plant endophyte A11, the strain Nengchan resistant Staphylococcus aureus, Bacillus subtilis and other Gram-positive bacteria, secondary metabolites, for a variety of plant pathogenic fungi also has varying degrees of resistance to antimicrobial spectrum is wide. But not yet pure, and their physicochemical properties are also poorly understood, in order to determine the molecular structure of the active ingredient, this experiment Staphylococcus aureus indicator bacteria, purified through two different routes for the enrichment of active substances , get a certain purity of the active substance, a preliminary understanding of some of the molecular structure information. Research Methods I. endophyte A11 inoculated in PDA medium, the shaker was the initial 48 hours fermentation broth, fermentation broth through sonication, 105 ℃ sterilization, 10000r/min centrifuged for 5 minutes, and the supernatants , supernatant was dialyzed (molecular weight cutoff of 3500), to take dialysate through the dialysate temperature and concentrated in vacuo to a volume, the volume of 1:1 n-butanol extraction, the organic phase was concentrated to dryness in vacuo temperature, with a small amount of methanol dissolving the crude sample. Gradient elution through silica gel column purification of the active substance, GC-MS spectra collected for analysis of the active ingredient. The results were as follows: silica gel column chromatography, methanol: ethyl acetate gradient elution in 4:6,5:5 were eluted with two A, B two active ingredients. Spectral analysis by GC, A, B two active ingredients spectrum contains the same two components, molecular weights were: 475.2,499.5. Using silica gel separation did not make the active ingredients separately. Methods II endophyte A11 PDA culture medium inoculated by shaking 48 hours have initial fermentation broth, the fermentation broth by using a 6M / L hydrochloric acid to adjust PH to about 3, after standing for some time, 10000r / min centrifugation for 5 min to remove contaminating proteins, NaOH to adjust the PH 7 fermentation supernatant, and then with 3 volumes of absolute ethanol fermentation supernatant removed again contaminating proteins and polysaccharides, and filtration, the filtrate after the cryogenic vacuum pump After drying, a small amount of concentrated under reduced pressure to give the crude sample was dissolved in methanol, and purified by silica gel column chromatography, concentration crystallization, Sephadex LH-20 separated by molecular size. By liquid chromatography-mass spectrometry and nuclear magnetic resonance spectrum of the molecular weight of the active substance and molecular structure of the initial identification. The results were as follows: silica gel column chromatography, methanol: ethyl acetate gradient elution gradient elution in 2:8,3:7 were eluted with two No. A-1, A-2 active ingredients of two types. No. A-1 was again separated by silica gel column, high activity combined eluate was concentrated and allowed to stand for precipitation of crystals. A-2 号 by two successive silica gel column chromatography, Sephadex LH-20 separated by molecular size, high activity combined eluate was concentrated and allowed to stand, and finally also been a certain purity of the active substances. No. A-1, A-2, respectively, of two types active substances through the LC-MS spectrum analysis of their molecular weights were: 460.17,460.2. A-2 号 active material by NMR analysis, suggesting that it is does not contain a benzene ring and aldehyde, may contain an amide bond, an alkyl group, a carbon-carbon double bond of the compound of chemical groups.
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