从蛹虫草小麦培养基残渣分离虫草素及提取物对肝癌细胞的抑制效果

Extraction of cordycepin from wheat medium waste of Cordyceps militaris cultivation and inhibitory effects of the extracted product on Hep G2 cells

从蛹虫草Cordyceps militaris小麦培养基残渣中分离和纯化虫草素,并进行了提取物体外人肝癌细胞Hep G2(human hepatoma cell line,Hep G2)的抑制作用研究。以蛹虫草小麦培养基残渣为原料,热超声水提取虫草素,探究p H值、温度、浸提时间和超声功率对虫草素的提取效果,用苯乙烯型弱极性大孔吸附树脂(AB-8)柱层析分离、重结晶纯化虫草素,并用高效液相色谱法(high performance liquid chromatography,HPLC)、红外光谱法(infrared spectroscopy,IR)鉴定。虫草素处理肝癌细胞Hep G2后,倒置相差显微镜下观察肝癌细胞形态变化,并用四甲基偶氮唑蓝(methylthiazolyltetrazolium,MTT)法测定细胞生长情况。结果表明:固液比1:50、pH值6.0、温度60℃、浸提时间3 h和超声功率300 W时提取效果好。经鉴定,分离提取物的虫草素纯度达到99%以上。不同浓度的虫草素对肝癌细胞Hep G2有明显的抑制作用,且与虫草素浓度和作用时间呈正相关,被抑制的细胞凝集、变圆、碎裂增加。本提取工艺得到的虫草素纯度高,工艺简单易扩大,且提取物对肝癌细胞增殖作用具有明显的抑制作用,为其在功能性食品添加剂方向的应用提供了数据基础。

Cordycepin was extracted and purified from wheat medium waste of Cordyceps militaris cultivation and its inhibitory effect on cells of human hepatoma cell line(Hep G2)was examined in vitro.The extraction of cordycepin was optimized by using a thermosonic water bath at varying pH values,temperatures,extraction times and ultrasonic power.The results showed that the extraction was optimal under solid to liquid ratio of 1:50,pH value of 6.0,extraction temperature of 60°C,extraction duration of 3 hours,and ultrasonic power of 300 W.The purity of cordycepin was over 99%under such conditions.The extracted cordycepin was purified by employing macroporous adsorptive resin(AB-8),and was identified by high performance liquid chromatography(HPLC)and infrared spectroscopy(IR).The growth of Hep G2 cells treated with the purified cordycepin was measured using the methylthiazolyltetrazolium(MTT)assay,and the morphological changes of Hep G2 cells were observed by means of inverted differential microscopy.Different concentrations of cordycepin showed significant inhibitory effect on Hep G2 cells,manifesting a positive correlation between cordycepin concentration and inhibitory time.The inhibited cells became round,agglutinated and fragmented.This extraction process is simple and expandable,and the cordycepin with high purity obtained also has obvious inhibitory effect on the proliferation of liver cancer cells,which is worth popularizing in the production of functional food additives.