School of Medicine, Xi'an Jiaotong University, 76 Yanta Westroad, Xi'an, China.
J Pharm Biomed Anal. 2010 Dec 1;53(4):1063-9. doi: 10.1016/j.jpba.2010.07.011. Epub 2010 Jul 16.
An online analytical method coupling high expression vascular endothelial growth factor receptor (VEGFR) cell membrane chromatography (VEGFR-CMC) with high performance liquid chromatography mass spectrometry (LC/MS) for screening and identification of active component from traditional Chinese herb Aconitum carmichaeli Debx. acting on VEGFR-2 was established. Through a 10-port column switcher, factions separated by VEGFR-CMC column (first dimension) were transferred and were adsorbed on an enrichment column. Then, these fractions were sent into LC/MS system (second dimension) immediately and directly for separation and preliminary identification, respectively. Sunitinib malate (SN) was used as positive control, while nifedipine (NF), dexamethasone acetate (DX), methoxyamine hydrochloride (MT) and atenolol (AT) as negative controls. The specification of this VEGFR-CMC-online-LC/MS method was validated by competitive displacement test. As a result, mesaconitine (MSC), aconitine (AC), and hypaconitine (HPC) were identified as the active constituents acting on VEGFR-2. The in vitro inhibition activity of starting extract of Aconitum carmichaeli Debx., MSC, AC, and HPC on HEK293/VEGFR cell viability by MTT test, separately. The in vitro inhibition activity of MSC, AC, and HPC on vascular endothelial growth factor (VEGF) secretion of HEK293/VEGFR cell was tested by VEGF-ELISA assay. The screening results given by the system offered additional exemplification supporting this online coupling method and gave new evidence to the development of anti-tumor drug from natural products.
建立了一种在线分析方法，将高表达血管内皮生长因子受体 (VEGFR) 细胞膜色谱 (VEGFR-CMC) 与高效液相色谱-质谱联用 (LC/MS) 相结合，用于筛选和鉴定来自传统中药乌头的作用于 VEGFR-2 的活性成分。通过 10 通阀柱切换器，VEGFR-CMC 柱（第一维）分离的馏分被转移并吸附在富集柱上。然后，这些馏分立即直接进入 LC/MS 系统（第二维）进行分离和初步鉴定，分别。马来酸舒尼替尼 (SN) 用作阳性对照，硝苯地平 (NF)、醋酸地塞米松 (DX)、盐酸甲氧胺 (MT) 和阿替洛尔 (AT) 用作阴性对照。通过竞争置换试验对该 VEGFR-CMC 在线-LC/MS 方法的规格进行了验证。结果表明，中乌头碱 (MSC)、乌头碱 (AC) 和次乌头碱 (HPC) 是作用于 VEGFR-2 的活性成分。通过 MTT 试验分别检测乌头的起始提取物、MSC、AC 和 HPC 对 HEK293/VEGFR 细胞活力的体外抑制活性，通过 VEGF-ELISA 测定 MSC、AC 和 HPC 对 HEK293/VEGFR 细胞血管内皮生长因子 (VEGF) 分泌的体外抑制活性。该系统提供的筛选结果为这种在线偶联方法提供了额外的例证，并为天然产物抗肿瘤药物的开发提供了新的证据。
