中国弯颈霉提取物抗氧化和抗肿瘤体外活性研究

中国弯颈霉(Tolypocladium sinense)分离自冬虫夏草，其生长最适温度25～28℃，最适pH6～7。在液体培养基(葡萄糖 4%、蛋白胨 0.5%、酵母膏1%、KH2PO4 0.1%、MgSO4 0.05%)中，经过28℃下摇床(150r/min)培养4～10d 后，菌丝体生物量达8.49～9.73g/L。其菌丝体石油醚、乙酸乙酯、无水乙醇和蒸馏水提取率分别为3.51%、1.62%、4.84%、23.33%，菌丝体多糖提取率为5.31%。各提取物对DPPH 自由基均具有清除作用，2mg/ml 多糖具有较强的清除作用，清除率为49.13%；菌丝体多糖(0.08mg/ml)能显著地清除羟自由基，清除率为82.5%。MTT 法检测发现乙酸乙酯提取物具有较强的抑制白血病细胞增殖作用；作用于K562 和SHI-1 细胞2d 后，乙酸乙酯提取物IC50 分别为26.55、17.75μg/ml；该提取物对正常细胞(人PBMC 和小鼠骨髓细胞)的抑制作用较弱；AO/EB 双染和TUNEL 法检测提示菌丝体的乙酸乙酯提取物可以诱导K562 细胞凋亡。上述实验结果表明，中国弯颈霉具有抗氧化和抗肿瘤方面的应用开发前景。

Study on Anti-oxidant and Anti-tumor Activities in vitro of Tolypocladium sinense Extracts

Tolypocladium sinense was isolated from the fruiting body of a wild Cordyceps sinensis. After growing for 4 to 10 days at 25 to 28 ℃ by shake culture (150 r/min) in a liquid medium (pH 6 to 7) containing glucose 4%, yeast extract 1%, peptone 0.5%, KH2PO4 0.1% and MgSO4 0.05%, the mycelial biomass of Tolypocladium sinense reaches 8.49 to 9.73 g/L. The mycelia extraction rates with petroleum, ethyl acetate, ethanol and hot water are 3.51%, 1.62%, 4.84% and 23.33%, respectively, and the extraction rate of mycelium polysaccharide is 5.31 %. All these extracts of Tolypocladium sinense have scavenging ability, especially mycelium polysaccharide presents the strongest scavenging rates of 49.13% to DPPH·at 2 mg/ml and 82.5% to·OH at 0.08 mg/ml. Moreover, the cytotoxicity of the ethyl acetate extract against leukemia cells such as K562 and SHI-1 line was observed in a MTT assay. The IC50 values on K562 and SHI-1 cell line are 26.55 and 17.75μg/ml, respectively. However, the ethyl acetate extract has low inhibitory effects against PBMC and mouse bone marrow cells. AO/EB staining and TUNEL test demonstrated that the ethyl acetate extract can induce apoptosis of K562 cells. In conclusion, the extracts of Tolypocladium sinense mycelium are potential anti-oxidant and anti-tumor sources.