Extraction, Purification And Structural Identification Of Secondary Metabolites From Streptomyces Microflavus Neau3

Nemadectin is a kind of 16-membered ring macrolide antibiotics, and it is mainly produced by Streptomyces cyaneogriseus subspecies noncyanogenus. It has many virtues in common with avermectin and milbemycin, such as a broad spectrum insecticide, easily degradable, low-residual, no drug resistance, harmless to humans and animals, little environmental pollution and so on. As a member of milbemycins, nemadectin is used as precursor for synthesis of more active moxidectin except using as veterinary drug. This research would not only enrich the structural diversity of nemadectin and milbemycin analog libraries, but also provide the necessary foundation for the further studying of the potential structure-activity relationship, optimizing their molecular structure, developing more-efficient, low-toxic and low-residual macrolide antibiotics.In the process of screening new anti-parasite antibiotics, we obtained a new nemadectin producing strain (Streptomyces microflavus neau3). Then the secondary metabolites of this strain were compared with others'by HPLC, the results showed more nemadectin analogue's characteristic absorption peaks. In this paper, we attempt to study the secondary metabolites of the strain in detail with the purpose of finding more and better anti-parasite antibiotics.First, the producing strain was fermented by shake-flask. 8.0 ml of the culture was transferred into a 1-L Erlenmeyer flask containing 100 ml of the producing medium consisting of glucose 1%, lactose 2.5%, cotton seed powder 2.5% and CaCO3 0.03%, pH 7.0, before sterilization. Fermentation was carried out at 28℃for 7-8 days on a rotary shaker at 250 r.p.m.Second, 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 about 24 h with MeOH. The MeOH extract was evaporated under reduced pressure to suitable volume at 45℃. 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-20 column, semi-preparative HPLC and other chromatographic means and yielded a series of compounds at last.At last, the structures of the compounds were determined on the basis of spectroscopic analysis, including 1D and 2D NMR, as well as HR-ESI-MS, UV and IR, and compared with the data reported in the literature. Two compounds of them as a member of milbemycins were first discovered, and named:8-demethyl-25-(1,3-dimethyl-1-butenyl)-23-hydroxy Milbemycinβ3 (1), Seco-nemadectin (2).The other compounds were:Nemadectin (3), Doramectin (4), Avermectin B1a (5), Avermectin B1b (6), LL-F28249ν(7), 23- Desoxy Factor A (8), VM44857 (9), LL- F28249γ(10), 25-(1,3-dimethyl-1-butenyl)-23-hydroxy Milbemycinβ3 (11) and LL- F28249ω(12).