牡蛎共生菌Chaetomium globosum ML-4发酵液的化学成分及其体外抗肿瘤活性研究

Chemical constituents of liquid culture of symbiotic Chaetomium globosum ML-4 of oyster and their in vitro antitumor activity

采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、制备薄层色谱和高效液相色谱等方法对牡蛎共生菌Chaetomium globosum ML-4发酵液的化学成分进行分离纯化,获得5个化合物。利用高分辨质谱、核磁共振谱及文献比对的方法,将上述化合物分别鉴定为chaetoglobosin V(1),chaetoglobosin Vb(2),tyrosol(3),5-methyluracil(4),uracil(5)。采用MTT法测定化合物对人肝癌细胞株SMMC-7721的体外细胞毒活性,结果发现,化合物1对SMMC-7721细胞具有明显的增殖抑制活性,其IC50为60.5 mg·L-1,阳性对照药顺铂的IC50为19.96 mg·L-1。进一步研究发现,化合物1可引起SMMC-7721细胞G2期细胞周期阻滞,并能诱导SMMC-7721细胞凋亡;SMMC-7721细胞中Bcl-2/Bax的比值下降,Caspases-3,-8,-9的表达增加,Akt蛋白的表达和磷酸化水平下降。上述结果表明,化合物1对SMMC-7721细胞的体外抗肿瘤活性与诱导细胞周期G2期阻滞以及细胞凋亡相关;其诱导凋亡作用同时涉及线粒体途径和死亡受体途径,且与PI3K/Akt信号通路活性下降相关。

Isolation and purification of chemical constituents of liquid culture of symbiotic Chaetomium globosum ML-4 of oyster was performed through silica gel column chromatography, gel filtration over Sephadex LH-20, preparative TLC and HPLC. Five compounds were obtained and their structures were determined as chaetoglobosin V （ 1 ）, chaetoglobosin Vb （ 2 ）, tyrosol （ 3 ）, 5-methyluracil （ 4 ） and uracil（S）, respectively, based on HR-MS and NMR data and comparison with literatures. In vitro cytotoxicity of compounds against human hepatocellular carcinoma cell line SMMC-7721 were measured byMTT method, and results showed that compound 1 could obviously inhibit the proliferation of SMMC-7721 cells with an IC^0 value of 60. 5 mg~ L-l, while the ICs0 value of positive con- trol cisplatin was 19. 96 mg ~ L 1. Further studies discovered that compound 1 could lead to G2 phase arrest in SMMC-7721 cells and induce SMMC-7721 ceils apoptosis. The ratio of Bcl-2/Bax in SMMC-7721 ceils was decreased. The expression of protein Caspases-3, -8 ,-9 was improved and the expression and phosphorylation level of Akt were reduced. Aforementioned results revealed that in vitro antitumor activity of compound 1 against SMMC-7721 cells were related to G2 phase cell cycle arrest and induced-apoptosis. The induced- apoptosis was involved in both the mitochondrial pathway and the death receptor pathway and connected with activity decline of PI3K/Akt signaling pathway.