Authors:Denise Spira, Georg Reifferscheid and Sebastian Buchinger
The objective of this study was to combine the Yeast Estrogen Screen (YES) for the detection of estrogenic compounds with thinlayer chromatography. Such an approach might serve as an effective tool in effect-directed analysis, i.e., the identification of bioactive compounds in mixtures. The proposed method (planar-YES) allows the detection of estrogenic effects on the surface of a TLC plate after an exposure time of 2-3 h instead of the usual 18 h which are used for the traditional microplate-YES. The estimated limits of quantification for the estrogenic model compound 17α-ethinylestradiol (EE2) were 0.8 pg without and 1.6 pg with a prior chromatographic development of the TLC plate. The hormones 17α- ethinylestradiol (EE2), 17β-estradiol (E2) and estrone (E1) could be separated by thin-layer chromatography and were detected by means of the specific bioassay subsequently. Finally, the applicability of the method on real samples was demonstrated with the detection of estrogenic effects in sediment extracts from sediments of the river Elbe.
Authors:Yvonne Anna Minar, Barbara Milz and Bernd Spangenberg
We present a planar chromatographic separation method for the phytoestrogenic active compound equol, separated on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic cattle manure extract contains this phytoestrogenic active compound to a larger amount. As solvents for the mobile phase, hexane, ethyl acetate, and acetone (45:15:10, v/v); acetone and water (15:10, v/v); and n-hexane, CH2Cl2, ethyl acetate, methanol, and formic acid (40:40:20:5:1, v/v) have been used. After separation, a modified yeast estrogen screen (YES) test was applied, using the yeast strain Saccharomyces cerevisiae BJ3505 containing an estrogen receptor. Its activation by equol induces the reporter gene lacZ which encodes the enzyme β-galactosidase. The enzyme activity is measured directly on the TLC plate by using the substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside) or the substrate X-β-Gal (5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside). β-Galactosidase cleaves MUG into a fluorescing compound. X-β- Gal is also hydrolyzed and then oxidized by oxygen forming the deep-blue dye 5,5′-dibromo-4,4′-dichloro-indigo. Both reactions in combination with a thin-layer chromatography (TLC) separation allow very specific detecting of equol in cattle manure, although that is a very challenging matrix. Preliminary results show that the average content of equol in liquid manure is roughly 60 μg g−1. The value for urine is 50 μg mL−1.
A method was developed for effect-directed analysis (EDA) of the root extract of Pimpinella saxifraga L. High-performance thin-layer chromatography (HPTLC) was hyphenated with microchemical, biochemical, and biological assays as well as electrospray ionization– mass spectrometry (ESI–MS). This HPTLC–UV/Vis/FLD– EDA–MS method directly pointed to multi-potent compounds in the P. saxifraga L. root extract. 2,2-Diphenyl-1-picrylhydrazyl radical scavengers, acetylcholinesterase inhibitors, estrogen-effective compounds, antimicrobials against Gram-positive Bacillus subtilis bacteria, and Gram-negative Aliivibrio fischeri bacteria were discovered in the root extract. A first targeted characterization of four unknown multi-potent compounds was performed by HPTLC–ESI–MS and microchemical derivatizations. This highly streamlined effect-directed profiling is recommended for a fast and cost-efficient natural product search.
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.
A new concept coupling sample preparation (using thin-layer chromatography) with effect-directed analysis (TLC-EDA) is proposed and demonstrated, using the planar-YES (a variation of the yeast estrogen screen, YES) as example. The concept allows performing on one single TLC layer all steps including sample preparation, sample separation and the detection of bio-effective substances and also offers a potential link to high-end chemical analytics. Estrogen standards were separated by TLC, distinguished and detected by a biological test system. Dose-response curves were produced for 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). In a field trial, bioactive compounds were extracted from a recirculating fish culture and from a wastewater treatment plant and tested. Low concentrations of a substance likely to be E2 were found in both samples. The level of detection for E2 and EE2 at the current stage of development was 0.5 pg per band (5 mm × 1 mm). The time demand for incubation, compared to the standard YES procedure of 1996, was reduced from >20 hours to 5 hours. If the level of detection can be lowered a little more, by less than one order of magnitude, a rapid screening of native aqueous samples on estrogenic activity is realistic. This will open a wide range of different fields of application.
Authors:Yvonne Anna Witos, Barbara Milz and Bernd Spangenberg
We present a two-dimensional (2D) planar chromatographic separation of estrogenic active compounds on RP-18 W (Merck, 1.14296) phase. A mixture of 8 substances was separated using a solvent mix consisting of hexane, ethyl acetate, acetone (55:15:10, v/v) in the first direction and of acetone and water (15:10, v/v) in the second direction. Separation was performed on an RP-18 W plate over a distance of 70 mm. This 2D-separation method can be used to quantify 17α-ethinylestradiol (EE2) in an effect-directed analysis, 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).
Authors:Lena Stütz, Patricia Leitner, Wolfgang Schulz and Rudi Winzenbacher
The widespread use of the pharmaceutical metformin for diabetes therapy has led to finding its way into many surface waters up to the μg L−1 range and subsequently into different water treatment processes. In this study, metformin was treated with hypochlorite on the laboratory scale, and the resulting transformation products were investigated with the umu assay in a microtiter plate, where a genotoxic effect was detected. For the characterization of this genotoxic effect, the sample was separated using high-performance thin-layer chromatography (HPTLC), and 29 zones over the whole retardation area were extracted from the HPTLC plate with the thin-layer chromatography-mass spectrometry (TLC-MS) Interface. Then, the umu assay was performed again with each extracted zone, such that the genotoxic effect in the sample could be assigned to a certain zone. By the measurement of this effective zone with high-performance liquid chromatography with high-resolution mass spectrometry and by performing a non-target screening, the effective substance could be identified as a cyclic dehydro-1,2,4-triazole derivate with an intense yellow color. This substance formerly was found by Armbruster et al. (Water Research, 2015), which is a major transformation product of chlorine-treated metformin.
Authors:Ágnes M. Móricz, Dániel Krüzselyi and Péter G. Ott
In this study, the antibacterial profiling of the ethanolic leaf extract of greater burdock (Arctium lappa L.) is demonstrated, applying thin-layer chromatography (TLC) coupled bioassays against the Gram-positive soil bacterium Bacillus subtilis and the Gram-negative pepper pathogen Pseudomonas syringae pv. maculicola. The main active component was isolated by eluting from the adsorbent bed and subjected to a targeted characterization by high-performance liquid chromatography–diode array detection–electrospray ionisation–mass spectrometry. The identification of the germacranolide sesquiterpene lactone onopordopicrin was based on its retardation factor, bioactivity in TLC-based methods, and retention tim as well as ultraviolet (UV) and mass spectra, compared to those of the reference substance isolated earlier in our laboratory from Onopordum acanthium leaf.
Authors:Anne Klöppel, Wolfgang Grasse, Franz Brümmer and Gertrud Morlock
Rapid analysis by coupling HPTLC with bioluminescence and mass spectrometry enables very fast response to bioactive substances in unknown samples. In this study marine sponges were screened for new bioactive compounds. After chromatographic separation of twelve methanolic marine sponge extracts the HPTLC plates were coated with bioluminescent bacteria (
) by a simple dipping procedure. If separated compounds were bioactive they inhibited or enhanced the bacterial luminescence and could be identified as dark zones on the luminescent background. This micro-biological detection revealed new compounds compared with physical (absorbance or fluorescence measurement) or chemical (microchemical derivatization) detection techniques. Effect-directed analysis turned out to be superior to target analysis in the search for natural products with a distinct effect. For identification of unknown bioactive zones the HPTLC system was coupled to a high-resolution mass spectrometer to obtain the exact masses of the unknowns. Thus, a
-bioactive zone was identified as avarone, a bioactive metabolite so far only known to be synthesized by the sponge
. This methodology proved very effective not only for detection but also for identification of unknown bioactive metabolites in sponges.