Comprehensive two-dimensional HepG2/cell membrane chromatography/monolithic column/time-of-flight mass spectrometry system for screening anti-tumor components from herbal medicines

Cell membrane chromatography (CMC) is a biological affinity chromatographic method using specific cell membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and membrane receptors. In this study, a novel comprehensive two-dimensional (2D) chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p < 0.05). The result demonstrated that the proposed comprehensive 2D HepG2/CMC/monolithic column/TOFMS system has the advantages of strong recognition and rapid analysis abilities for the total screening procedure, which will be selectable and practical in drug discovery from complex HM samples and can also be applied to other biochromatography models.

► A new HepG2 cell membrane model was developed.
► Novel comprehensive two-dimensional chromatography approach was established and optimized for fast screening procedure.
► Four anti-tumor active components from herbal medicines were screened and reliably identified by TOFMS.
► EGFR was preliminary selected as the target membrane receptor via competitive displacement assays.
► BBR and THP revealed inhibiting effects on cell proliferation in vitro.