HPLC micro-fractionation coupled to a cell-based assay for automated on-line primary screening of calcium antagonistic components in plant extracts.

High performance liquid chromatography (HPLC) micro-fractionation was successfully coupled to an automated 45Ca2+ uptake assay using GH4C1 cells for the separation of natural product extracts and for the primary detection of their calcium antagonistic components. The reliability of the procedure was first established with a reference solution consisting of pure compounds with a known effect on the Ca2+ uptake. No loss of activity was observed to occur after HPLC micro-fractionation. Extracts of Peucedanum palustre and Pinus sylvestris, showing high and no inhibition of Ca2+ uptake as total extracts, respectively, were analysed and the inhibitory activity of the P. palustre extract could be traced to two components, identified as columbianadin and isoimperatorin. As expected, no significant inhibition was observed with the micro-fractionated P. sylvestris samples. In summary, the procedure was found to be applicable for primary detection of calcium antagonistic components in complex matrices and to significantly reduce the time previously needed for bioactivity-guided isolation.