Study On Chemical Constituents Of Secondary Metabolites From Four Streptomyces And Their Anti-Inflammatory Activities

As an important source of natural products,Streptomyces have attracted much attention due to their extensive distribution and abundant secondary metabolites.A variety of novel compounds produced by Streptomyces play a vital role in the development of new drugs,which showed remarkable biologic activities.In the course of our screening for new bioactive compounds from streptomycetes source,four Streptomyces have been selected in this study,including Streptomyces violaceoruber derivatived from animal feces,Streptomyces gramineus and Streptomyces pratensis associated with lichens,Streptomyces levis isolated from soil.The chemical composition of the secondary metabolites produced by them were systematically investigated.Additionally,according to the results of evaluating anti-inflammatory bioactivities of new compounds,the molecular mechanisms of which with remarkable anti-inflammatory activity were then explored.The biotransform pathway of isoflavone rhamnoside in S.pratensis were also explored.Biological activity screening phase and chemical diversity screening were used to investigate thess four streptomycetes.The optimal medium were determined according to the results.The ethyl acetate crude extract was separated by chromatographic techniques,including silica gel column chromatography,reversed-phase silica gel column chromatography,Sephadex LH-20,prepared silica gel thin-layer chromatography and semiproparative HPLC method.Fifty-six compounds were obtained from the secondary metabolites of four Streptomyces.All the structures of these compounds were determined on the basis of spectral analysis(MS,IR,UV,NMR,CD,X-ray crystal diffraction)as well as physicochemical properties and derivatization,including fifteen new compounds.Various compounds were isolated from fermentation of these four Streptomyces,including polyketides(1,2,4-5,8,22-30),alkaloids(3,7,11-14,17-18,32-34,54),furans(15-16,37-43),flavonoids(19,33,44-47),actinomycins(48-55),anthraquinones(6,31),terpenes(9,10),aromatics(20,55),nucleosides(35,36),sterides(21,56).We also investigated the biotransformation pathway of rhamnosides(44-47).The anti-inflammatory activities of new compounds(1-4,22-27)and their analogues(5,28-30)were evaluated by Griess assay.Compounds 1,2,25-26 and 29-30 displayed good inhibitory effect on the release of NO in LPS-induced RAW 264.7 cells.The effcts of compound 1 on mRNA expression of inflammatory cytokines,for instance inducible nitric oxide synthase(iNOS),interleukin-1?(IL-1?),interleukin-6(IL-6),and tumor necrosis factor alpha(TNF-?)were evaluated by RT-PCR.Meanwhile,iNOS protein expression as well as activation of NF-?B signaling pathway were examined by Western blot.The results showed that compound 1(3.3,10,30 ?M)inhibited the production of nitric oxide(NO)by suppressing iNOS mRNA and protein expression at both transcription and translation level in LPS-induced RAW 264.7 cells.Stimutainously,compound 1 inhibited the mRNA expression of IL-1?,IL-6 and TNF-? in LPS-induced RAW 264.7 cells.Molecular mechanism research showed that compound 1(3.3,10,30?M)significantly inhibited the expression of phosphorylated I?B-? and the degration of I?B-? to attenuate p65 expression in nucleus in LPS-induced RAW 264.7 cells.Meanwhile,compound 1 downregulated the expression of phosphorylated p65.All the evidences demonstrated that compound 1 inhibited the expression of pro-inflammatory cytokines via suppressing the activacation of NF-?B signaling pathway in LPS-induced RAW 264.7 cells,which may serve as a potential therapeutic candidate for the treatment of inflammation-related diseases.Additionally,Western blot results showed that compounds 25,26,29,30(15,30,60 ?M)inhibited the production of NO by downregulating the protein expression of iNOS in LPS-induced RAW 264.7 cells.ELISA results demonstrated that compounds 25,26,29,30(15,30,60 ?M)suppressed the LPS-induced production of tumor necrosis factor alpha(TNF-?)and interleukin-6(IL-6)in RAW 264.7 cells.Duing the investigation of the glycosylation in Streptomyces pratensis,we found that aglycone was from soybean and carbonyl group was metabolited by itself,which were catalyted by glycosyl transferase to form isoflavone rhamnoside.