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目的研究山麻黄Ephedra equisetina内生真菌Myrothecium roridum LLY固体发酵产物的化学成分,并进行体外细胞毒活性研究。方法采用硅胶柱、Sephadex LH-20凝胶柱色谱和高效液相色谱法进行分离纯化,利用高分辨质谱、核磁共振谱等方法鉴定化合物的结构。MTT法进行化合物体外细胞毒活性筛选,并采用流式细胞术和Western blotting法进行化合物细胞毒活性机制初步研究。结果从M.roridum LLY固体发酵产物中分离得到9个化合物,分别鉴定为吩嗪-1-羧酸(1)、1-甲氧基吩嗪(2)、N-methyl-1H-indole-2-carboxamaide(3)、甲基硫赭曲菌素(4)、ditryptophenaline(5)、7,8-二甲基异咯嗪(6)、deoxyleporin B(7)、尿嘧啶(8)和胸腺嘧啶(9)。MTT测定发现,化合物3对人肝癌细胞株SMMC-7721增殖具有一定的抑制活性,其IC50值为38.0μg/m L,阳性对照顺铂的IC_(50)值为11.5μg/m L。细胞周期分析发现,化合物3可导致SMMC-7721细胞S期阻滞;Western blotting分析发现,化合物3可使SMMC-7721细胞中细胞周期负调控蛋白p27的表达上调,并且Cleaved-PARP蛋白被明显活化。结论化合物7为1个新的天然产物。化合物3能诱导SMMC-7721细胞凋亡;其对SMMC-7721细胞的增殖抑制作用与S期细胞周期阻滞相关,并受到p27蛋白的调控。
Objective To study the chemical constituents of the solid fermentation product of endophytic Myrothecium roridum LLY from Ephedra equisetina and to study its cytotoxicity in vitro. Methods The compounds were separated and purified by silica gel column, Sephadex LH-20 column chromatography and high performance liquid chromatography (HPLC). The structures of the compounds were identified by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. MTT assay of compounds in vitro cytotoxic activity, and the use of flow cytometry and Western blotting compounds cytotoxic activity of the preliminary study. Results Nine compounds were isolated from the solid fermentation products of M.roridum LLY and identified as phenazine-1-carboxylic acid (1), 1-methoxyphenazine (2), N-methyl-1H- indole-2 (4), ditryptophenaline (5), 7,8-dimethylisoxazine (6), deoxyleporin B (7), uracil (8) and thymine (9). The results of MTT showed that compound 3 could inhibit the proliferation of human hepatocellular carcinoma cell line SMMC-7721 with the IC50 value of 38.0 μg / mL and the IC 50 of positive control cisplatin of 11.5 μg / mL. Cell cycle analysis showed that compound 3 could induce S phase arrest in SMMC-7721 cells. Western blotting analysis showed that compound 3 could up-regulate the expression of cell cycle negative regulator p27 in SMMC-7721 cells and cleaved-PARP protein was significantly activated . Conclusion Compound 7 is a new natural product. Compound 3 can induce apoptosis of SMMC-7721 cells; its inhibitory effect on proliferation of SMMC-7721 cells is related to S phase cell cycle arrest and is regulated by p27 protein.