Study On The Bioactivity Metabolite Of Actinomycetes NEAU-Ht9and Streptomyces Microflavus Neau3Y-3

Soil microbial secondary metabolites have always been animportant source of bioactive leads. and for the sake ofsearching more microorganism-derived metabolites from soil microorganisms, the active Actinomycetes NEAU-Ht9and Streptomyces microflavus neau3was isolated by our laboratory. For the sake of making the strain produce more metabolites, many efforts have been taken to improve the productivity of nemadectin by S. microflavus neau3. Subsequently, a mutant strain Y-3, which showed significantly different phenotypes including the morphology of aerial mycelia and the metabolite HPLC profiles to the wild-type strain, was generated through the treatment of the spores with UV and N-methyl-N’-nitro-N-nitrosoguanidine. In the course of investigating the metabolites of this mutant strain,the result shows that there are novle compounds produced by the mutant strain. in the article the secondary metabolites of the two strains were studied carefully as following:Streptomyces microflavus neau3was fermented with the volume of30liters, fermentation medium was made of glucose1%, cotton seed powder2.5%, lactose2.5%, CaCO30.03%, pH7.0. After configured with distilled water, the fermentation medium was sterilized for20minutes at121℃. With the inoculum size of8%, the seed liquid was shifted to50liter fermentor and cultured following conditions, including28℃, a ventilation rate of120m3/h and a stirring rate of100r/min and for7-8days, the fermentation broth was filtered. The resulting cake was washed with water. Then the resulting cake was washed with water, and both filtrate and wash were discarded. The washed cake was extracted for about24h with MeOH. The MeOH extract was evaporated under reduced pressure to suitable volume at45℃. The resulting concentrate was extracted three times using an equal volume of EtOAc. The combined EtOAc phase was concentrated under reduced pressure to yield oily substances. The residual oily substance was chromatographed by silica gel column, sephadex LH-20column, semi-preparative HPLC and other chromatographic means and yielded a series of compounds. Then, the structure of the compounds were elucidated on the basis of spectroscopic analysis, including1D and2D NMR, ESI-MS, UV and IR, and compared with the data from the literature. The results shows that there are two novle compounds,5-Methoxy-8-demethyl Nemadectin α2(1),23-hydroxy-25-(1,3-dimethy1-1-butenyl)-27-Ester carbonyl-Milbemycin ai (2) and six are known compounds, Nemadectin02(3), Nemadectin a (4), LL-F28249η (5), Nemadectin β (6), LL-F28249ξ (7), milbemycin B deriv (8). Both of compounds1and2exhibited potent acaricidal activity and nemtocidal activity. Especially, compound2demonstrated impressive acaricidal activity against adult mites with an IC50of 2.3±0.9μg/mL and mite eggs with an IC50of17.5±2.1μg/mL and nemtocidal activity against Caenorhabditis elegans with an IC50of0.7±0.2μg/mL, which are higher than those of nemadectin and the known commercial acaricide and nematocide milbemycin A3/A.4.Thirty fermentation broth was given by large-scale fermentation of the Actinomycetes NEAU-Ht. By using Diaion HP20column, silica gel column, and semi-preparative HPLC with bioassay-guided fractionation, a known compound Resistomycin was obtained. Then, the structure of the compound was elucidated on the basis of spectroscopic analysis, including1D and2D NMR, ESI-MS, UV and IR, and compared with the data from the literature.