Screening estrogenic effects of several dietary supplements with a fluoerescent ligand-based receptor binding assay


TOKER Y.

International Postgraduate students Meetings on Pharmaceutical Sciences, (IPSMPS 2010), Turkey, 01 July 2010, pp.60

  • Publication Type: Conference Paper / Full Text
  • Country: Turkey
  • Page Numbers: pp.60

Abstract

SCREENING ESTROGENIC ACTIVITY OF SEVERAL DIETARY SUPPLEMENTS WITH A FLUOERSCENT LIGAND-BASED RECEPTOR BINDING ASSAY Toker Y., Gurer-Orhan H. Ege University, Faculty of Pharmacy, Department of Toxicology, 35100 Izmir Conventionally in vitro binding affinity of chemicals to the estrogen receptor (ER) determined by a competitive binding assay in which radioactive ligand and test compound compete for binding. Radioactive ligand binding assay is a sensitive technique, although has limitations such as the need to use radiochemicals, facilities to handle such compounds, and experienced personnel. Our group developed and published a “receptor binding assay” where coumestrol is used as a tracer ligand. The compound itself is fluorescent, and its fluorescence intensity at 408 nm increases upon binding to the ER. The assay based on the competition between fluorescent tracer ligand coumestrol and estrogenic compounds for binding to the ligand binding domain (LBD) of the ER-alpha in 96-well plate format. Displacement of coumestrol from LBD is measured as a decrease in fluorescence intensity in microplate reader. It is an easy and inexpensive method, and allows quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening for ER ligands. In the present study we analyzed estradiol and a well known estrogenic phytoestrogen, genistein, as positive controls. Furthermore the method is applied for screening the estrogenic activity of several herbal dietary supplements such as St. John’s Wort, Saw Palmetto, Green Tea Leaf Extract Black Cohosh and Gotu Kola. Both acid hydrolysis and direct methanolic extracts of the products were screened and several of them found to have potential estrogen receptor binding affinity. This study is supported by the Scientific and Technical Research Council of Turkey (TUBITAK) by the project number 108S202. Keywords: Estrogen receptor binding assay, estrogenic activity, Phytoestrogens, dietary supplements