Screening For Cytotoxic Marine Actinomycetes And Study On Its Secondary Metabolites Of Two Selected Strains

Because of the special living environment, marine-derived actinomycetes possess distinct and complex metabolic capabilities, resulting in wide diversity of their secondary metabolites in chemical structure and biological activity. Thus, actinomycete becomes one of the important resources of active lead compounds. This thesis describes the screening of cytotoxic marine actinomycetes and the secondary metabolites produced by two selected marine actinomycetes. Studies include isolation of actinomycetes from marine sediments samples and screening of cytotoxic strains, selecting aimed strains, fermentation studies, bioassay-guided fractionation, structural elucidation and preliminary evaluation in vitro for anti-tumor activities of pure compounds.262 actinomycete strains are isolated from 10 marine sediment samples which is collected from the seashore of Weihai, Fujian and the mangroove root sediment of Guangdong. Using brine shrimp Lethality assay, antibiotics test and K562 cell line as bioactive screen model, these 262 strains are detected for the cycle inhibitory, apoptosis and cytotoxic activity and 17 of them exhibited obvious cytotoxicities. These strains and one offered by the lab are chosen for stability test and TLC, HPLC evaluation. Following these results, 2 strains (WH1-2216-6, THW-7) are selected for further study of secondary metabolites.After choosing appropriate fermentation condition, large-scale fermentation and preparation of the active fractions were performed to obtain the active fractions of the bioactive strains. By means of chromatography over silica gel column, Sephadex LH20, preparative TLC and semi-preparative HPLC, 20 compounds (1-17,19,21,22) and 6 compounds (14-15,19-20,23-24) were isolated from actinomycete strains WH1-2216-6 and THW-7, respectively. The separation procedure is guided by a bioassay using K562 cell lines. By means of physico-chemical properties and spectral analysis (IR, UV, MS, NMR, etc.), their structures were elucidated as caerulomycine G (1), caerulomycine F (2), caerulomycine H (3), caerulomycins D (4), caerulomycine A (5), caerulomycinomitile (6), caerulomycinamide (7), caerulomycins C (8), 1,4-Benzenedicarboxylic acid dimethyl ester (9), Benzoic acid (10), Ethanone (11), 4-Hydroxybenzaldehyde (12), N-hydroxybenzisoxazolone (13), cyclo-( Pro- Leu) (14), cyclo-( Val- Pro) (15), cyclo(Val-Leu) (16), cyclo(Val-Ile) (17), cyclo-(Pro-Ile) (18), thymine (19), Thymidine (20). The identified obtained compounds include 8 pyridine alkaloids (1-8), 5 dicyclopeptides (14-17), five benzene derivates (9-13) and two nucleosides (18,22). Compounds 1-3 were new compounds.The anti-tumor activity in vitro against several cancer cell lines of these compounds was assayed by MTT, SRB methods and among them 5 compounds (1, 2, 4, 5, 8) with anti-tumor activity are found. Compound 2 shows high inhibition activity against P388 with IC50=0.05μM. Compound 4 and 5 show high inhibition activity against P388, A-549, HL60, BEL-7402. Compound 1 and 8 show moderate inhibition activity against P388 or K562. In addition, our study demonstrated for the first time that the pyridine alkaloids show inhibitory activity against K562, P388, A-549, HL60, BEL-7402.