Authors:L. Vlase, D.-S. Popa, A. Tero-Vescan, and N. Olah
A new liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of seven isoflavones (daidzin, genistin, ononin, daidzein, glycitein, genistein, and formononetin) and coumestrol in vegetable extracts was developed. The separation was performed on a Zorbax SB-C18 column with a mixture of methanol (solvent A) and 0.1% (υ/υ) acetic acid in water (solvent B) under gradient conditions at 50°C with a flow rate of 1 mL min−1. The detection of analytes was performed by electrospray ionization, negative ionisation, in non-reactive MS2 mode for aglycons or in reactive MS2 mode for glycosides. The method shows a good linearity (r2 > 0.9948) over the concentration range of 40–4000 ng mL−1 for all analytes, a good precision (CV < 11%) and accuracy (<10%). The method was successfully applied to quantify the isoflavones and coumestrol in vegetable extracts obtained from red clover (Trifolium pratense L., Fabaceae) and dyer's greenweed (Genista tinctoria L., Fabaceae) and can be used in the chemical characterization of vegetables with phytoestrogen content.
Authors:Marek Opalka, Justyna Kugla-Owczarska, Barbara Kaminska, Helena Puchajda-Skowronska, Wioletta Hryniewicka, and Luiza Dusza
., Ciereszko, R. E., Skarzynski, D. J., Nogowski, L., Opalka, M., Kaminska, B., Nynca, A., Kraszewska, O., Slomczynska, M., Woclawek-Potocka, I., Korzekwa, A., Pruszynska-Oszmialek, E. and Szkudelska, K. (2006): Mechanism of phytoestrogen action in reproductive
also natural EDs contained by plants that are consumed by human beings. The most important forms of these latter are the phytoestrogens.
The well-known disruptor effects of the aforementioned molecules are manifested in the sexual area, the
., Szewczykowski, M. A., Sato, T., Woods, J. A., Chang, J. S., Allred, C. D., Helferich, W. G. and Cooke, P. S. (2002): The phytoestrogen genistein induces thymic and immune changes: a human health concern? Proc. Natl. Acad. Sci. USA 99 , 7616
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-methylumbelliferyl-β-d-galactopyranoside). The enzyme can also hydrolyse X-β-Gal (5-bromo-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-dichloro-indigo which allows to detect phytoestrogenic activity more specific in the presence of native fluorescing compounds.