北虫草培养残基中虫草素的提取纯化及抗肿瘤活性

Extraction,Purification,and Antitumor Activity of Cordycepin from Cordyceps Militaris Residue Medium

以北虫草固体培养残基为实验材料,比较闪式提取、超声波提取和超高压提取培养残基中虫草素的效果,用陶瓷膜过滤提取液,获得虫草素粗提液,利用高速逆流色谱分离制备虫草素,通过LC-MS/MS对产物结构进行鉴定,并验证了虫草素对小鼠S180肉瘤的抑制作用。结果显示,超高压提取培养基中虫草素较好,高速逆流色谱分离纯化条件是以V(乙酸乙酯)∶V(正丁醇)∶V(水)=2∶3∶5的两相溶剂系统,上相为固定相,下相为流动相,流速3.0 m L/min,主机转速900 r/min,分离温度28℃,以此分离条件经一步洗脱,高速逆流色谱收集馏分,其中峰Ⅲ产物经HPLC检测纯度达97.6%,结构经LC-MS/MS鉴定为虫草素。从100 g北虫草培养残基中可制备得虫草素181.90mg,并对其活性验证发现剂量为100 mg/kg时小鼠S180肉瘤瘤重抑制率为64.48%。

Flash extraction,ultrasonic extraction,and ultra-high pressure extraction technology were used to extract cordycepin from Cordyceps militaris residue medium. The extracting solution was filtered with ceramic membrane and cordycepin was separated and prepared by using high-speed countercurrent chromatography（ HSCCC）. The product structure was identified by LC-MS/MS. Antitumor activity of cordycepin was tested in mice S180 tumor. The results showed that ultra-high pressure extraction technology was better for cordycepin extraction. Combining a two-phase solvent system composed of ethyl acetate-n-butanol-water（ 2∶ 3 ∶ 5,V/V/V） with high-speed counter-current chromatography（ HSCCC）,the cordycepin was purified at 28 ℃ with flow rate of 3. 0 m L/min and revolution speed was 900 r/min,while the upper phase was stationary phase and the lower phase was mobile phase. Cordycepin identified was identified by LC-MS/MS and the purity of cordycepin was 97. 6%. About 181. 90 mg of cordycepin was obtained from 100 g of Cordyceps militaris residue medium. The inhibition rate of cordycepin was 64. 48%at doses of 100 mg in vitro test.