Two-Dimensional Thin-Layer Chromatography of Phytoestrogens on RP-18 W Plate, Detected by Effect-Directed Analysis Using the Yeast Estrogen Screen Test

Summary

We present a two-dimensional (2D) planar chromatographic separation method for phytoestrogenic active compounds on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic extract of liquorice (Glycyrrhiza glabra) roots contains four phytoestrogenic active compounds. As solvent, in the first direction, the mix of hexane, ethyl acetate, and acetone (45:15:10, v/v) was used, and, in the second direction, that of acetone and water (15:10, v/v) was used. After separation, a modified yeast estrogen screen (YES) test was applied, using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducing the reporter gene lacZ which encodes the enzyme β-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylum belliferyl-β-D-galactopyranoside). The enzyme can also hydrolyse X-β-Gal (5-bro-mo-4-chloro-3-indoxyl-β-D-galactopyranosid) into β-galactose and 5-bromo-4-chloro-3-indoxyl. The indoxyl compound is oxidized by oxygen forming the deep-blue dye 5,5β-dibromo-4,4β-di-chloro-indigo which allows to detect phytoestrogenic activity more specific in the presence of native fluorescing compounds.