Screening Of Marine Actinomycetes With Antitumor Activity And Isolation And Identification Of The Cytotoxic Compounds Produced By Strain N350

The marine environment is being exploited for new antitumor drugs with unique structure and excellent bioactivity. Recently, marine microorganism was more attractive to researchers, since the resource of this kind for new drug discovery is more reachable with higher hit rate and can be easily reproduced in large scale in industry. Additionally, more and more documents demonstrated that many marine natural products previously ascribed to other organisms are secondary metabolites of bacteria associated to the organisms.In this study, the biodiversity of the actinomycetes associated to the marine plants and animals living in intertidal area in Xiamen Island was investigated. From this particular marine microbial flora, many strains with excellent antitumor activity were screened out using MIT assay and biochemical induction assay. Strain N350, noted for its high cytotoxic and DNA-target activity, was investigated in detail. Several compounds with significant antitumor activity were purified from the fermentation broth of strain N350 and their structures were elucidated as well.Microbiological population density of lO2~4O5 was revealed in the actinomycetes flora associated to surface, epidermis and intestines of the investigated marine plants and animals. Density of marine actinomycetes varied in different marine plants and animals, upon the species and the collection site of sample. Totally, 439 actinomycetes were isolated. The majority was streptomyces, accounting for 74.7% of all isolates, while micromonospora occupied 15.5% and the other actinomycetes not being to these two genera comprised the left 9.8%, in which actinoplanes was involved. Compared to the previously investigated actinomycetes, which collected from nearshore sea sediment, the actinomycetes associated to the marine plants and animals, here we studied, was denser in population and more diverse taxonomically.Among all 439 actinomycetes isolated, 10 (2.3%) strains exhibited excellent cytotoxic activity, their fermentation broth inhibited 50% growth of P388 and/or KBcell lines at the dilution of 1:10240. 3 out of these 10 strains were also observed to have significant DNA-target activity. This result demonstrated that actinomycetes associated with intertidal marine plants and animals this project explored is indeed a prolific resource of new antitumor drugs.Strain N350 was investigated in detail for its outstanding cytotoxic and DNA-target activity. ID50 of the fermentation broth of strain N350 against 7 tumor cell lines was overwhelmingly less than 1:20480, if evaluated using 9 cell lines consisting of 7 tumors. DNA-target activity of strain N350 was approximately equal to 1mg/mi cisplatin. Competitive antibacterial activity of strain N350 was also observed, however, no antifungal activity was detected. Strain N350 demonstrated obvious marine nature, both seawater and artificial seawater increased its biomass and biological activity significantly. Seawater was the optimum for its growth, whereas artifical seawater more favored the enhancement of the DNA-target activity. According to the cultural, morphological and chemotaxonomic characteristics of strain N350, a name of Streptomyces tauricus var. marinus was proposed, referring to its sea origin.From the fermentation broth of strain N350, four lipophilic compounds were isolated and identified as 7, 4'-dihydroxyisoflavone, 6-methoxy-7,4'-dihydroxyisoflavone, cis-13-docosenamide and ~3-sitosteroI-j3-D-glucopyranoside, respectively. All these compounds were, for the first time, isolated from the products of marine actinomycetes. 7, 4'-dihydroxyisoflavone, 6-methoxy-7, 4' -dihydroxyisoflavone, cis- 1 3-docosenamide demonstrated significant cytotoxic activity in vitro. Against human leukemia cell line, HL-60, their 1C50 were 8.1, 5.2, l5j.tg/ml, respectively, and against human epidermal carcinoma of oral cavity, KB cell line, 1C50 were 2.8, 1.9, 7j~tg/ml, respectively. Similar data on the antituinor activity of 6-methoxy-7, 4'-dihydroxyisoflavone and cis...