| 林思敏,徐广进,高海,等.十字孢碱产生菌筛选及其肿瘤细胞毒活性产物研究[J].中国海洋药物,2023,42(3):36-42. |
| 十字孢碱产生菌筛选及其肿瘤细胞毒活性产物研究 |
| Screening for Staurosporine-producing Strains and the Identification of Staurosporine analogues |
| 投稿时间：2022-04-19 修订日期：2022-04-28 |
| DOI：10.13400/j.cnki.cjmd.2023.03.003 |
| 中文关键词: 海洋放线菌 十字孢碱产生菌 吲哚咔唑 细胞毒活性 |
| English Keywords:Marine actinobacteria staurosporine-producing strains indolocarbazoles cytotoxic activity |
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| 中文摘要: |
| 目的 寻找海洋放线菌来源的十字孢碱产生菌,筛选获得目标菌株,分离鉴定其具有吲哚咔唑母核的产物。方法 以十字孢碱在λmax290 nm附近尖锐的特征紫外吸收为标准,利用HPLC-UV对不同来源的海洋放线菌的发酵提取物进行定向筛选,筛选出十字孢碱产生菌,利用16S rRNA序列对筛选得到的菌株进行种属鉴定。通过比较各产生菌的十字孢碱类产物的丰度,选择能够产生更多该类化合物的菌株进行规模化发酵,并采用硅胶柱层析、Toyopearl HW-40F凝胶柱层析、高效液相色谱(HPLC)等方法对其发酵提取物进行分离、纯化,运用质谱(MS)、核磁共振(NMR)等技术鉴定化合物的结构。采用CCK-8法测定化合物的细胞毒活性。 结果 从42株放线菌中筛选出12株十字孢碱的产生菌,其中Streptomyces sp. OUCMDZ-5380的产物最为丰富,从该菌株发酵提取物中分离鉴定了3个吲哚咔唑类化合物,分别为K252c (1)、十字孢碱 (2)和4"-O-去甲基十字孢碱 (3)。细胞毒活性测试表明,化合物3对12株肿瘤细胞株的IC50值达到纳摩尔、介于0.0003–0.623 μM之间。 结论 筛选得到12株主产十字孢碱的海洋放线菌,从OUCMDZ-5380的发酵产物中鉴定了3个吲哚咔唑类化合物,化合物3具有广谱的肿瘤细胞毒活性(半数抑制浓度IC50为0.0003–0.623 μM.)和对内部串联复制突变的人急性髓细胞性白血病细胞MV4-11的选择性(相对于人正常细胞株L-02和其它人肿瘤细胞株的选择指数分别为1120和50–2080)。 |
| English Summary: |
| Objective To find staurosporine-producing strains derived from marine actinobacteria and obtain staurosporine analogues. Methods Based on the characteristic UV sharp absorption of staurosporine around λmax 290 nm, the staurosporine-producing strains were screened by analyzing their extracts of fermentation broth with HPLC-UV and identified by 16S rRNA gene sequences. By comparing the abundance of indolocarbazoles in the extracts, a strain identified as Streptomyces sp. OUCMDZ-5380 was selected for large-scale fermentation. The indolocarbazoles metabolites were separated and identified by column chromatography on silica gel, Toyopearl HW-40F gel and HPLC, MS and NMR. The cytotoxic activity was assayed by CCK-8 method. Results Twelve staurosporine-producing strains were screened out from 42 marine-derived actinobacterial strains. Streptomyces sp. OUCMDZ-5380 was selected for chemical study because of the more abundance of indolocarbazoles in the fermentation broth, from which three indolocarbazoles were isolated and identified as K252c (1), staurosporine (2) and 4"-O-demethylstaurosporine (3). Compound 3 showed potent cytotoxic activity against 12 tumor cell lines with IC50 values ranging from 0.0003 to 0.623 μM. Conclusion Twelve staurosporine-producing strains were obtained and three indolocarbazoles were identified from Streptomyces strain OUCMDZ-5380. Compound 3 displayed broad-spectrum anti-proliferation of the tumor cells and a good selectivity for MV4-11, a human acute myeloid leukemia cell line with internal tandem duplications mutation. The selective index is 1120 and 50–2080 compared with the human normal cell L-02 and the other human tumor cells, respectively. |
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