An online coupled cell membrane chromatography with LC/MS method for screening compounds from Aconitum carmichaeli Debx. acting on VEGFR-2

Abstract

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.