This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (27) Citing Articles via Google Scholar Google Scholar Articles by Schobel, U. Articles by Irth, H. Search for Related Content PubMed PubMed Citation Articles by Schobel, U. Articles by Irth, H. Social Bookmarking                   What's this?

High Resolution Screening of Plant Natural Product Extracts for Estrogen Receptor a and f3 Binding Activity Using an Online HPLC-MS Biochemical Detection System

ScreenTec B.V., Leiden, The Netherlands

Michel Frenay

ScreenTec B.V., Leiden, The Netherlands

Danny A. Van Elswijk

ScreenTec B.V., Leiden, The Netherlands

Joanne M. McAndrews

Pharmacia Corporation, Monsanto Company, Nutrition and Consumer Products, Saint Louis, MO

Kelly R. Long

Pharmacia Corporation, Monsanto Company, Nutrition and Consumer Products, Saint Louis, MO

Lisa M. Olson

Pharmacia Corporation, Monsanto Company, Nutrition and Consumer Products, Saint Louis, MO

Steven C. Bobzin

Pharmacia Corporation, Monsanto Company, Nutrition and Consumer Products, Saint Louis, MO

Hubertus Irth

ScreenTec B.V., Leiden, The Netherlands

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) a and fi binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system 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, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				E. V. Doukhanina, N. R. Apuya, H.-D. Yoo, C.-Y. Wu, P. Davidow, S. Krueger, R. B. Flavell, R. Hamilton, and S. C. Bobzin Expression of Human Nuclear Receptors in Plants for the Discovery of Plant-Derived Ligands J Biomol Screen, April 1, 2007; 12(3): 385 - 395. [Abstract] [PDF]

				J. Kool, S. M. van Liempd, H. van Rossum, D. A. van Elswijk, H. Irth, J. N. M. Commandeur, and N. P. E. Vermeulen Development of Three Parallel Cytochrome P450 Enzyme Affinity Detection Systems Coupled On-line to Gradient High-Performance Liquid Chromatography Drug Metab. Dispos., April 1, 2007; 35(4): 640 - 648. [Abstract] [Full Text] [PDF]

				J. Kool, M. Eggink, H. van Rossum, S. M. van Liempd, D. A. van Elswijk, H. Irth, J. N.M. Commandeur, J. H.N. Meerman, and N. P.E. Vermeulen Online Biochemical Detection of Glutathione-S-Transferase P1-Specific Inhibitors in Complex Mixtures J Biomol Screen, April 1, 2007; 12(3): 396 - 405. [Abstract] [PDF]

				S. M. van Liempd, J. Kool, W. M. A. Niessen, D. E. van Elswijk, H. Irth, and N. P. E. Vermeulen On-line Formation, Separation, and Estrogen Receptor Affinity Screening of Cytochrome P450-Derived Metabolites of Selective Estrogen Receptor Modulators Drug Metab. Dispos., September 1, 2006; 34(9): 1640 - 1649. [Abstract] [Full Text] [PDF]

				J. Kool, S. M. van Liempd, R. Ramautar, T. Schenk, J. H. N. Meerman, H. Irth, Jan. N. M. Commandeur, and N. P. E. Vermeulen Development of a Novel Cytochrome P450 Bioaffinity Detection System Coupled Online to Gradient Reversed-Phase High-Performance Liquid Chromatography J Biomol Screen, August 1, 2005; 10(5): 427 - 436. [Abstract] [PDF]

				T. Schenk, G. J. Breel, P. Koevoets, S. van Den Berg, A. C. Hogenboom, H. Irth, U. R. Tjaden, and J. van Der Greef Screening of Natural Products Extracts for the Presence of Phosphodiesterase Inhibitors Using Liquid Chromatography Coupled Online to Parallel Biochemical Detection and Chemical Characterization J Biomol Screen, August 1, 2003; 8(4): 421 - 429. [Abstract] [PDF]

				R. L. Rich, L. R. Hoth, K. F. Geoghegan, T. A. Brown, P. K. LeMotte, S. P. Simons, P. Hensley, and D. G. Myszka Kinetic analysis of estrogen receptor/ligand interactions PNAS, June 25, 2002; 99(13): 8562 - 8567. [Abstract] [Full Text] [PDF]