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

中药射干具有多种药理作用并得到广泛研究,但对其抗肿瘤活性组分的研究较少。本研究建立了一种用于筛选射干中抗肿瘤活性组分的方法。利用高表达表皮生长因子受体(EGFR)细胞制备细胞膜色谱柱,并通过十通切换阀与高效液相色谱-离子阱-飞行时间质谱(HPLC-IT-TOF MS)构建成二维在线联用系统。利用吉非替尼验证该系统的适用性,分子模拟对接实验研究活性组分与受体的相互作用方式,并利用MTT实验研究所筛选组分对EGFR细胞的体外抑制作用。结果表明,利用该系统可从射干总提取物中筛选出能够作用于EGFR的活性成分,并鉴定为次野鸢尾黄素;次野鸢尾黄素与EGFR的相互作用方式与阳性药吉非替尼相似;在浓度为0.4~50μmol/L范围内,吉非替尼、次野鸢尾黄素对高表达EGFR细胞的体外抑制作用具有剂量依赖性。EGFR细胞膜色谱在线HPLC-IT-TOF MS法有望成为从中草药中发现潜在抗肿瘤候选药物的有效方法。

EGFR Antagonism of the Irisflorentin from Rhizoma Belamcandae Using CMC-Online-HPLC-IT-TOF MS System

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.