质谱学报2017，Vol.38Issue(4)：425-432,8.DOI:10.7538/zpxb.2016.0203
细胞膜色谱在线HPLC-IT-TOF MS联用系统筛选分析射干中次野鸢尾黄素的抗EGFR活性作用
EGFR Antagonism of the Irisflorentin from Rhizoma Belamcandae Using CMC-Online-HPLC-IT-TOF MS System
摘要
Abstract
Rhizoma Belamcandae (Shegan), obtained from the dried rhizome of Belamcanda chinensis (L.) DC., is one of the most important traditional Chinese medicines (TCMs).It has many pharmacological effects, such as eliminating toxins, heat, phlegmand dyspnea, antiviral effect, anti-inflammatory, etc.In recent years, studies on Shegan were increasing, and reports showed that tectorigenin and tectoridin separated from Shegan had the activity of inhibiting angiogenesis, however, the targets which they act with were uncertain.Hence, screening receptor-specific components with anti-tumor effect from Shegan is necessary.Epidermal growth factor receptor (EGFR) is a part of the tyrosine kinase family of receptors responsible for intracellular signalling and cell growth, and overexpression of EGFR is associated with angiogenesis, invasion and metastasis of cancer cells.Cell membrane chromatography (CMC) has its history of more than 20 years, it was established in 1996 by professor He and his colleagues.As a kind of biological affinity chromatography, CMC has been developed and confirmed to be an effective method for screening bioactive components from complex systems.In this study, a two-dimensional online system was built to screen active compounds acting on epidermal growth factor receptor from Rhizoma Belamcandae.The first dimension was EGFR/CMC system, and the second was HPLC-IT-TOF MS system.Gefitinib was selected as the positive control drug to investigate the suitability of the EGFR/CMC-online-HPLC-IT-TOF MS system.For the EGFR/CMC system, the EGFR HEK293 engineered cell line was cultured and used to prepare the EGFR/CMC column according to the wet packing method, after analysing total extracts of Shegan through the EGFR/CMC system, the retained fractions were switched to the second dimension for identification.Molecular docking assay was performed using the SYBYL-X1.1 software to determine the strength of binding between the screened components and EGFR, and methyl thiazolyl tetrazolium (MTT) assay were used to evaluate the in vitro inhibition activity of the target components.The screening results showed that irisflorentin from Rhizoma Belamcandae was the targeted component which could specifically act on EGFR.Molecular docking assay showed irisflorentin could act on EGFR in similar manner with gefitinib as a positive control drug.The results of MTT assay showed gefitinib and irisflorentin inhibited high expressed EGFR cell growth in a dose-dependent manner from 0.4 to 50.0 μmol/L.These results indicated that EGFR cell membrane chromatography online high performance liquid chromatography-ion trap-time of flight-mass spectrometry (EGFR/CMC-online-HPLC-IT-TOF MS) system would be a useful method in drug discovery with natural medicinal herbs for searching potential anti-tumor candidates.关键词
细胞膜色谱/表皮生长因子受体(EGFR)/高效液相色谱-离子阱-飞行时间质谱(HPLC-IT-TOFMS)/次野鸢尾黄素/射干/抗肿瘤作用Key words
cell membrane chromatography/epidermal growth factor receptor (EGFR)/high performance liquid chromatography-ion trap-time of flight-mass spectrometry (HPLC-IT-TOF MS)/irisflorentin/Rhizoma Belamcandae/anti-tumor分类
化学化工引用本文复制引用
吕艳妮,付佳,孔利云,韩省力,张涛,贺浪冲..细胞膜色谱在线HPLC-IT-TOF MS联用系统筛选分析射干中次野鸢尾黄素的抗EGFR活性作用[J].质谱学报,2017,38(4):425-432,8.基金项目
国家自然科学基金(81503032、 81230079、81227802) （81503032、 81230079、81227802）
国家博士后基金项目(2015 M570844、2016 T90930) （2015 M570844、2016 T90930）
陕西省博士后科学基金 （）
中央高校基本科研业务费资助 （）
