The isolation and structure elucidation of secondary metabolites from local Streptomyces species and investigation on their biological activities

In recent years, bacterial and fungal infections threat human health worldwide, due to the development of new diseases and evidence of microorganisms resistant against to the presently used antibiotics. This situation requires discovery of new biologically active metabolites. Microbial secondary metabolites, having special structures and biological activities represent one of the productive sources of natural compounds. Streptomyces species, which is included in Actinomycetales order, are talented microorganisms having property of producing secondary metabolites most of which are biologically active. The main secondary metabolites isolated from these microorganisms which have commercial value are antibacterial, antifungal and antitumor agents. Screening of new compounds from well known and talented microorganisms such as Streptomyces, is one of the basic strategies for discovery of novel biologically active metabolites.For this purpose, effects of crude and different organic extracts which were obtained previously from 67 Streptomyces and one Nocardiopsis species that isolated from soil around root of endemic plants and identified by using 16S rRNA genes were tested in vitro on growth of Gram-positive and Gram-negative bacteria and that of Candida albicans yeast. It was determined that 25 of the 68 species tested during pre-screening processes based on biological activity have a preventive effect on the growth of test microorganisms. The interesting species were selected from these biologically active species according to the results obtained from chemical screening in which crude extracts of species were estimated by using TLC stained with different staining reagents. In order to obtain sufficient amount of pure secondary metabolites under optimum culture conditions, the interesting species encoded Streptomyces sp. BA2, Streptomyces sp. AA50, Streptomyces sp. BS40 and Streptomyces sp. AS42 that determined by biological and chemical screening, were cultured on a large-scale. Different chromatographic methods such as column chromatography, TLC, PTLC, molecular exclusion chromatography and HPLC were applied to purify these extracts. The structures of pure secondary metabolites were elucidated by 1H NMR and 13C NMR spectroscopic method and 2D NMR (COSY, HSQC, HMBC) and UV, IR and MS as well as several database such as AntiBase, Dictionary of Natural Products and Chemical Abstract.From the terrestrial Streptomyces sp. BA2, mixture of known compounds monensin B (171) and monensin A (172) and actinomycin D (170) and bis(2-ethylhexyl)phthalate (173) were isolated and their structure were elucidated. Antimicrobial activity of actinomycin D (170), which is used clinically for the treatment of some cancer types, and monensin B (171) and monensin A (172) mixture, which is a well-known representative of naturally polyether ionophore antibiotics, were investigated for their antimicrobial activity on proliferation of test microorganisms. It was shown that actinomycin D (170) and the mixture of monensin B (171) and monensin A (172) exhibited high antimicrobial activity.Chrysophanol (175) and 2-acetylchrysophanol (178) which were previously isolated from some Streptomyces species, and chrysophanol-10,10'-bianthron (174) which was formerly isolated from several plants but firstly isolated from microorganisms in this study are purified. Moreover, cyclic pentapeptide A (179) and cyclic pentapeptide B (180), which are known as endothelin-binding inhibitors, were isolated as a mixture. DNA cleavage protection activity of the chrysophanol-10,10'-bianthron (174) and chrysophanol (175) and DNA cleavage activity against OH radicals were studied by using Agarose Gel Electrophoresis. The antioxidant properties such as DPPH radical scavenging activity and reducing power of these compounds were also studied.Carbazomycin G (182) and mixture of teleocidin B2 (183) and isomer of teleocidin B2 (184) and mixture of novel teleocidin derivatives 14-O-acetylteleocidin B2 (195) and 14-O-acetylteleocidin B2 isomer (196) were also isolated from Streptomyces sp. BS40. Mixture of 14-O-acetylteleocidin B2 (195) and 14-O-acetylteleocidin B2 isomer (196) showed 100 % cytotoxic activity in the concentration range of 0.1 to 10 µg/mL on brine shrimp (Artemia salina L.). It was observed that this mixture cause strong skin irritation and vesication.Cyclic pentapeptide A (siklo(-D-Glu-L-Ala-D-Val-L-Leu-D-Trp-) (179) and cyclic pentapeptide B (siklo(-D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp-) (180), which are endothelin receptor antagonists, were isolated as a mixture from the terrestrial Streptomyces sp. AS42 and characterized by 1D NMR and 2D NMR and HPLC-ESI-MS2/MS3 techniques. This mixture of cyclic pentapeptide A and B were also isolated from Streptomyces sp. AA50 in the beginning.