Screening for active substances with TLC bioautography : challenges with artefacts

TLC, and its modern form HPTLC, is a standard method for identification and quality control of herbals [1]. Its combination with bioassays offers simultaneous separation and screening of plant extracts and other multi-compound mixtures for active substances. The intriguing simplicity and rapidness of the method might lead to underestimation of potential artefacts, first reported by Taibon et al. for HPTLC-Tyrosinase Inhibition [2]. In assays based on cells or enzymes in hydrophilic media, high lipophilic substances prevent contact with the potentially active substance and might lead to false-positive or false-negative results. Additionally, diffusion problems from the layer to the biological assay agents can also lead to wrong interpretation of results, due to difference in the physico-chemical properties of the molecules in the sample mixture. The planar Yeast Estrogen Screen Assay (pYES) is used for detection of human estrogen receptor alpha agonists in mixtures [3]. The hER-alpha is expressing beta-galactosidase in presence of estrogenic substances. Substances with a strong blue autofluorescence at 366 nm detection and low estrogenic activity are difficult to differentiate from real estrogenic activity, when using the blue fluorescent dye 4-methylumbelliferyl-beta-D-galactopyranosid (MUG) as substarte for beta galactosidase. Results of blue fluorescing phenolic acid rich plant extracts with known estrogen activity are demonstrated and a control assay using beta-D-galactopyranoside (RGP) as substrate is proposed [4]. The poster summarizes potential artefacts from pYES and other antimicrobial assays and reviews the theoretical considerations and suggests further investigation to overcome the challenges and improve bioautographic methods.