High resolution screening of plant natural product extracts for estrogen receptor alpha and beta binding activity using an online HPLC-MS biochemicaldetection system

A new screening technology that combines biochemical analysis with the reso lution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capabi lity of the HRS technology to analyze biologically active compounds in comp lex mixtures is demonstrated by screening a plant natural product extract l ibrary for estrogen receptor (ER) alpha and beta binding activity. The simu ltaneous structure elucidation of biologically active components in crude e xtracts was achieved by operating the HRS system in combination with mass s pectrometry (MS). In contrast to conventional microtiter-type bioassays, th e interactions of the extracts with the ER and the employed label, coumestr ol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a RPLC separation colu mn. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Pri mary screening of the extract library was performed in the fast-flow inject ion analysis mode (FlowScreening) wherein the chromatographic separation sy stem was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, ind icating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the func tional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in c ombination with MS. Information on the number of active compounds, their re tention times, the molecular masses, and the MS/MS-fingerprints as a functi on of their biological activity was obtained from 50% of the functional act ive extracts in real time. This dramatically enhances the speed of biologic ally active compound characterization in natural product extracts compared to traditional fractionation approaches.