Studies On Screening Of Actinomycetes With Antitumor Activities From Marine Sponges And The Active Compounds Produded By Strain A01059

Medication, as the main treatment for tumors, is always the focus of research by the scientists of biology, medicine and pharmaceutics. The marine environment is being exploited for novel antitumor drugs with unique structure and excellent bioactivity. Marine microorganisms have been becoming an important resource because they are with various species, higher hit rate and can be easily reproduced in large scale in industry. Especially, marine sponges are more attractive to researchers and have been becoming a novel resource since abundant microorganisms exist in them.In this study, research was carried out as following: isolation of actinomycete from marine sponges, screening and identification of antitumor strains, optimizing fermentation condition and medium, separation and purification of active compounds and structure identification of the active compounds and so on.A total of 335 actinomycete isolates were separated from 20 marine sponge samples collected from Sanya, Qionghai and Zhanjiang. Based the models of Pyricularia oryzae and MTT, 49 strains of antitumor activity were screened.Three strains with high bioactivity and genetic stabilization were polyphasic studied. Genotypic and phenotypic data indicated that strain A01054, A01013 and A01059 were coincided with those of the genus Streptomycetes. By studying the morphology, cultural characteristics, physiological and biochemical properties, 16S rDNA and phylogenetic tree, A01054 was proposed as Streptomyces lomondensis A01054. Strain A01013 showed closest 16S rRNA gene sequence similarity to Streptomyces fradiae (99.4%), but constituted a unattached branch in the phylogenetic tree. It suggested that strain A01013 maybe a novel specie of genus Streptomyces and proposed Streptomyces sp. A01013 for the moment. A01059 shared a high homology with Streptomyces griseocarneus (98.4%) and constituted a branch in the phylogenetic tree. So strain A01059 was identified primarily to Streptomyces griseocarneus A01059. However, there were some differences between them in physiological and biochemical characterization. The 16S rDNA GenBank accession numbers of strain A01013 and A01059 are DQ304115 and DQ319186, respectively.The best optimal liquid fermentation condition and medium component were researched. The effects of carbon source, nitrogen source, inorganic salt, pH value and incubation volume on the production of antitumor bioactive compounds of A01059 were studied and the ratio of nutrition component was optimized by orthogonal experiment. The optimum culture medium includes: maize meal 1.0%, soluble starch 1.0%, soybean flour 0.5%, glucose 0.5%, sea salt 1%. The optimum inoculation condition is: initial pH7.0⁷.5, seed cultural time 48h, 6% inoculation amount, 28℃and 150r/min for 6d. Under the optimized conditions, the inhibitory rates against human liver cancer cell SMMC-7721 and rat sarcoma cell S180 were 95% and 94%, respectively. Furthermore this strain was fermented by 10L fermentor.It was showed that the antitumor products were hydrophilic and raw extract was obtained from the fermentation broth of A01059 by ethyl acetate. 2 bioactive compounds were purified based on HPLC and half-preparation chromatography and the content reached 97.32% and 97.85%, respectively.Based on the results of ¹H-NMR, ¹³C-NMR, H-H NMR, HMBC, HSQC, ESIMS and UV spectra, the structures of the two active compounds were identified. It suggested that they are both novel structure compounds based on the correlative data.