五倍子抑制表皮生长因子受体活性及活性部位的UPLC/Q-TOF-MS分析

Activity of anti-epidermal growth factor receptor in Calla Chinensis and UPLC/Q-TOF-MS analysis on its effective part

目的 应用均相时间分辨荧光（HTRF）技术，筛选发现具有抑制表皮生长因子受体（EGFR）活性的五倍子提取物，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（UPLC/Q-TOF-MS）对其活性部位进行分析和鉴别。方法 药材经石油醚渗滤、乙醇提取、醋酸乙酯萃取和水煎煮后，得到4个提取部位。采用HTRF法检测五倍子醋酸乙酯提取物对EGFR的抑制作用，并计算抑制率。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，200～400 nm扫描，使用ESI离子源，在正离子模式下采集数据。结果 五倍子的醋酸乙酯部位显示较强的EGFR抑制活性，并从其活性部位中分析鉴定出14个化合物，主要成分为鞣质类化合物，其中11个分别为石榴皮鞣素、香草醛、六羟基二苯酰胺葡萄糖、鞣花鞣质、杨梅苷、原儿茶素、五倍子鞣质倍酰葡萄糖、五倍子鞣质、鞣花酸、3, 5-二咖啡酰奎宁酸、monogalloyl-glucose，另外3个为未知成分。结论 本研究首次发现五倍子醋酸乙酯提取部位具有较强的EGFR抑制活性，IC50为5.528 μg/mL，Q-TOF-MS测定的相对分子质量及正离子信息鉴定表明五倍子醋酸乙酯提取物中主要为鞣质类成分，其中五倍子鞣质、鞣花酸、鞣花鞣质为最主要成分。五倍子中含有的鞣质类成分为抑制EGFR活性的主要成分，为其在抗癌方面的应用提供理论依据，同时为进一步跟踪分离活性成分奠定基础。

Objective Using homogeneous time-resolved fluorescence （HTRF） technology to screen the Calla Chinensis extracts with anti-epidermal growth factor receptor activity and to analyze the active ingredients in Calla Chinensis by ultra-performance liquid chromatography/quadrupole-time of flight mass-spectrometry （UPLC/Q-TOF-MS）. Methods After percolation with petroleum ether, ethanol extract, ethyl acetate extraction, and boiling with water, four fractions were obtained. HTRF method was applied to detecting the inhibition of ethyl acetate fraction on EGFR, and the inhibitory rate was calculated. The chromatographic separation was performed on Acquity UPLC BEH C18 column with a gradient elufion of 0.1% formic acid water-acetonitrile. The mass spectrometer equipped with electrospay ionization source was used as defector, data were collected under the positive ion modes, and screened at 200—400 nm. Results The ethyl acetate fraction of Calla Chinensis showed the strong inhibitory activity on EGFR. Fourteen compounds were analyzed and identified, among which tannins were the main active components, the 11 tannin compounds were punicalin, vanillin, di-HHDP-glucose, ellagitannin, myricetin 3-O-rhamnoside, protocatechuic acid, gallotannin-glucose, ellagic acid-hexose, 3, 5-dicaffeoylquinic acid, monogalloyl-glucose, and gallotannin, and the three others were unknown. Conclusion The ethyl acetate fraction of Calla Chinensis has the significant EGFR inhibitory activity, and the IC50 value is 5.528 μg/mL. Tannins, including Chinese tannin, gallogen, and ellagitannin as main constituents, are identified through the information of positive ion and relative molecular mass determined by Q-TOF-MS. The results indicate that Calla Chinensis contains tannin ingredients to inhibit EGFR, in the hope to provide a theoretical basis of the application in anticancer and to lay the foundation for the further tracking separation of the active ingredients.