UPLC Q-TOF/MS法研究艾叶抑制EGFR激酶的活性部位

Study on the active part for anti-EGFR chemical constituents of Artemisiae argyi folium by UPLC Q-TOF/MS

目的应用高通量药物筛选寻找具有EGFR激酶抑制活性的中药提取物,同时采用超高效液相色谱与串联四级杆飞行时间质谱仪联用技术(UPLCQ-TOF/MS)对其活性部位进行分析和鉴别。方法艾叶药材经石油醚渗滤、乙醇提取、乙酸乙酯萃取和水煎煮后,得到4个相应提取部位;采用均相时间分辨荧光法检测抑制表皮生长因子受体(EGFR)的激酶活性,于620、665 nm处测定荧光信号的变化及信号值,计算提取物对EGFR激酶的抑制率;采用Acquity UPLC BEH C18色谱柱,以0.1%甲酸水溶液-乙腈为流动相梯度洗脱,在210~400 nm扫描,使用ESI离子源,在正离子模式下采集数据。结果艾叶的乙酸乙酯部位显示较强的EGFR激酶抑制活性,IC50=8.848μg·mL-1;Q-TOF/MS测定的一级、二级质谱信息及结合文献鉴定艾叶乙酸乙酯提取物中7个化合物,其中6个分别是芹菜素、棕矢车菊素、木犀草素、异泽兰黄素、β-谷甾醇、槲皮素,1个未知成分。其中棕矢车菊素、异泽兰黄素、β-谷甾醇为主要成分。结论首次发现艾叶具有抑制EGFR的激酶活性,推测其乙酸乙酯提取物中的黄酮类和甾醇类为抑制EGFR激酶活性的主要活性成分。

OBJECTIVE To extract the anti -EGFR active constitutent and determine the constituents in Artemisiae argyifolium by UPLC Q - TOF/MS, METHODS Herbs were percolated with petroleum ether, extracted with ethanol and ethyl acetate, and then boiled with water, and then the corresponding parts of four extracts were obtain. The avtivity of EGFR was detected based on HTRF. The variation of fluorescence signal and signal value at 620 nm and 665 nm were determined, and the inhibition rate of extracts were calcu- lated. The chromatographic separation was performed on a C^s column with a gradient elution of 0.1% formic acid aqueous solution- acetonitrile . The mass spectrometer equipped with eleetrospray ionization source was used as detector and the data was collected under the positive ion modes. RESULTS The strong inhibitory activity of EGFR was observed in the ethyl acetate part of Artemisiae argyi folium. And the ICs0 values was 8. 848 Ixg＂ mL- 1. The information from references and the relative molecular mass distinguished by Q - TOF/MS, was used to identify the corresponding parts of ethyl acetate of Artemisiae argyifolium. They were apigenin, jaceosidin, luteo- lin, eupatilin, β -sitosterol, quercetin and one unknown substance. Jaceosidin, eupatilin, β -sitosterol, were the main component. CONCLUSION The results indicated that Artemisiae argyi folium contained flavonoids and sterols speculation to inhibit EGFR.