| Natural products are important sources of bioactive substances and lead compounds for drugs,and play a vital role in human health life.The ever-rising incidence of infection,tumor and hypercholesterolemia make it urgently need to develop new drugs.Soil environments harbor abundant actinomycetes,which are important resources for drug development because of its unique evolution,the diversity of species and the diversity of secondary metabolites.Soil actinomycetes have been evolved in the process of ecological interactions between animal,plant and microorganism in environment to produce new classes of secondary metabolites with novel structures,many of which have a wide range of biological activities,such as,anti-infection,antitumor,antiviral activitiesand antihypercholesterolemic.Actinomycetes are thought to produce a treasure trove of bioactive secondary metabolites useful for treatment of various diseases.Unfortunately,the separation of new bioactive secondary metabolites from actinomycetes source have gradually cut down,and the probability of new discoveries is greatly reduced(plummeted)as the result of intolerably repetitive and cumbersome process,so the isolation of novel lead compounds for drugs from actinomycetes still exist many problems.Hence,selective isolation approachs under bioactivity-assays,and dereplication technologies through UPLC-Q-TOF-MS may improve the efficiency of the discovery of new lead compounds for drugs from actinomycetes.In order to obtain new biologically active compounds from actinomycetes,antimicrobial activities of fermentation broths were first accessed by 96-well plate method.1280 strains were obtained,which isolated from soil in farming,sand,colliery,saline-alkali and wetlands in Anhui Province.The antimicrobial activity of the fermentation broths from 1280 strains was determined by 96-well plate with the tested strains of Gram-positive bacteria,Gram-negative bacteria and pathogenic fungi.In this study,Methicillin resistant Staphylococcus aureus(MRSA),Vancomycin resistant enterococcus(VRE),Escherichia coli and Candida albicans and other pathogenic bacteria model were used to determine the bioactivity and antimicrobial activity in the bioactive screening for the antimicrobial activities of the secondary metabolites.The results showed that61 strains showed antimicrobial activity,15 of which showed stronger inhibition against the growth of E.coli 13 of which showed stronger inhibition against the growth of C.albicans,9 of which showed stronger inhibition against the growth of VRE,and 9 of which presented stronger inhibition against the growth of MRS A.According to their antimicrobial type and activities,9 strains having better antimicrobial activity were selected for further study of chemical composition and structures of their fermentation products.Among them,3 actinomycete strains(HCCB11494,HCCB11495,HCCB11402)presented the stronger inhibition against the growth of MRSA were selected because multidrug-resistant(MDR)bacteriais the greatest health threats;5 actinomycete strains(HCCB11835,HCCB11876,HCCB12080,HCCB12384,HCCB10836)presented the stronger inhibition against the growth of E.coli were selected because MDR Gram-negative bacteria have emerged to be one of the world's greatest health threats;1 actinomycete strains(HCCB11343)presented the stronger inhibition against the growth of C.albicans were selected because the number of antifungal agentis limited.Guided by bioassays,the dereplication technology of UPLC-Q-TOF-MS was used to screen chemical constituents and determine the known-constituents of fermentation broths from the 9 actinomycete strains rapidly,to avoid the time-consuming isolation of common natural products.At the same time,16SrRNA genes of the 9 actinomycete strains were amplified and sequenced,and the phylogenetic tree was derived from the distance matrices using the neighbor-joining method.The 9 strains were identified to be genus Streptomyces.UPLC-Q-TOF-MS with the capabilities of high throughput,high selectivity,high sensitivity and fast screening is a powerful analytical method for rapid detecting known compounds from the fermentation broth to avoid repeated isolation.Guided by bioassays,dereplication technologies of UPLC-Q-TOF-MS were applied to explore the chemical compositions for the diversity,novelty and types of the secondary metabolites.In this process,UPLC-Q-TOF-MS was used to rapidly screen out chemical constituents of fermentation broths through precursor ion scan,and summarize their cracking law in the ESI ion source mode,and then identify compounds structures through MS2 mass spectrum scan.Fermentation broths from four bioactive actinomycetes were identified through UPLC-Q-TOF-MS combining with literature research,SciFinder datebase and Dictionary of Natural Products research,and 19 known compounds were identified rapidly.The results showed that:HCCB11494 mainly produced Macrotetrolides compounds including nonactin,monactin,dinactin,trinactin,tetranactin,Macrotetrolide C and Macrotetrolide B;HCCB 11495mainly produced coumarins compounds including novobiocin and TPU 0031B;HCCB11835 mainly produced Tunicamycins compounds including Tunicamycin B1,Tunicamycin C1,Tunicamycin B2,Tunicamycin B3 and Tunicamycin D;HCCB11876 mainly produced Qunomycins compounds including 7-S-Oxide-Quinomycin A,Qunomycin A,Qunomycin B,Qunomycin C and Qunomycin E.Directed by bioassays and chemical screening,five strains with abundant bioactive substances have been selected for further study.Selective isolation,purification and identification of bioactive substances were performed after the comprehensive analysis of the biological activity and mass results.Sixty liters of strain broth were prepared respectively after fermentation of these five soil actinomycetes strains,and the whole broths were extracted with methanol to give ferment extracts.In order to separate and identify the bioactive ingredients,the crude extract was fractionated by silica gel column chromatography,Sephadex-LH20 chromatography and HPLC.Twenty compounds were separated and purified from the extraction on the basis of chromatography experiments from five strains.By means of modern spectral analysis(MS,OR,UV,IR,1H NMR,13C NMR,DEPT,COSY,HMQC,HMBC,NOESY,CD),X-ray,the structures of twenty compounds were determined,among them six compounds were new.Types of new compounds include polyene macrolides(15-glycidyl-filipin?(2),16a,17a-epoxy-filipinV(3),16?,17?-epoxy-filipinV(4),25?,26?-epoxy-filipinV(5)),unnatural amino acid 6-(2-carbonyl-hydroxymethyl-1H-pyrrol-1-yl)-L-norleucine(7),cyclic peptidecyclo-(Leu-Pro-Phe-Pro)(10).The bioactivity and antimicrobial activity of these compounds were further evaluated.New compounds 1-5 showed the ability to inhibit growth of C.albicans with MIC values of 6.25,6.25,100,100 and 100 ?g/mL,respectively,and showed the ability to inhibit growth of A.thaliana with IC50 values of 32.78,16.36,16.36,52.37,67.63 and 54.48 ?g/mL,respectively;cyclo-(Leu-Pro-Phe-Pro)(10)showed stronger inhibition against S.aureus with MIC values of 2.5 ?g/mL.Compounds 6-(2-carbonyl-hydroxymethyl-1H-pyrrol-1-yl)-L-norleucine(6),Chartreusin(19)and 3''-dimethyl-chartreusin(20)showed significant activity against A.thaliana seedling with IC50 values of 32.13,37.92 and 36.21 ?g/mL.This study indicates thatthe combination ofbioassay-guided selective fractionation and rapid dereplication technologieshas proven to be a powerful tool to aid in the identification of new bioactive componets from soil actinomycetes effectively. |
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