Study On The Allkaloid Of Indolecarbazole In Marine Actinomycetes

As particularity about their survival environment,marine actinomycetes form the unique way of metabolic pathways,survival and adaptation mechanisms,and thereby produce a lot of secondary metabolites with unique structures,which have become one of the important sources of marine active natural products.Indolecarbazoles are a kind of natural alkaloid with important bioactivity,and their good pharmacological activity has attracted more and more attention from researchers.In this paper,350 strains of marine actinomycetes were isolated from a variety of marine samples collected from Wenchang in Hainan by SC,M2 and 1/10ATCC172 culture mediums.According to UV absorption characteristics and HPLC fingerprint analysis,13 strains were found to produce indolecarbazole alkaloids and selected for comparative analysis of 16S rRNA gene sequence,MLS A multi-site gene sequence and richness of fermentation product and so on.The strain HNM0089 was selected to further isolate and identify their metabolites,also to evaluate activity of compounds and optimize the fermentation condition.15 compounds were isolated from strains HNM0089 by normal-phase silica gel,reverse-phase silica gel,LH-20 gel chromatography,high performance liquid chromatography(HPLC),which included 8 indolecarbazole alkaloids and 7 non-indolecarbazoles compounds.The main compound,TL-1,was identified as staurosporine(ST)by the high-resolution mass spectrometry,one-dimensional and two-dimensional nuclear magnetic spectrum technique.Compound TL-2,TL-3,TL-5,TL-6 and TL-7 belonged to known indolecarbazoles,and compound TL-4 and TL-8 were suspected to be new indolecarbazoles.Bioactivity assay shows that compound TL-1 exhibited antitumor activity against human cancer cells(A549 cells),human leukemia cells(HL 60 cells)and human breast cancer cells(MCF-7 cells)with IC50 values of 2.061 nM,22.33 nM and 398.6 nM respectively,which means it has excellent antitumor activity.At the same time,compound TL-1 also showed strong toxicity to human normal liver cells(L-02 cells)with IC50 value of 4.051 nM.In addition,compound TL-1 has significant antifungal activities against Colletotrichum asianum T0408,Colletotrichum gloeosporioides Cg9,Magnaporthe grisea,Colletotrichum gloeosporioides RC178 and Fusarium oxysporum f.sp.cubense race 4 with MIC values of 0.125 μg/mL.8 μg/mL.8μg/mL、8 μg/mL and 8 μg/mL respectively.The result shows that compound TL-1 has the potential of developing as candidate agents of antifungal agrochemicals.The strain HNM0089 was identified as Streptomyces fradiae by 16S rRNA gene sequence of phylogenetic analysis,morphological,physiological and biochemical characteristics.It is found that the strain grows under the conditions such as 14-45 ℃,pH 5-12 and 0-7.5%salt,and its carbon and nitrogen source utility are very broad.By response surface experiment,five variables which included soluble starch,soybean powder,yeast extract,CaCO3 and sea salt were selected to optimize the fermentation conditions of the strain HNM0089,and the optimal medium is composed of soluble starch 3.4%,soybean flour 0.5%,yeast powder 0.3%,CaCO3 0.1%,sea salt 3.6%and pH 8.0.The yield of staurosporine increased to 182.57 μg/mL by 4.5 times,which provides a material basis for the development of agrochemicals in the agricultural field in the future.