Modern direct bioautography for fast screening and characterization of active compounds in plant extracts used in cosmetics

High-performance thin-layer chromatography (HPTLC) can ideally be combined with biological, biochemical and chemical assays without much effort [1]. The direct link to effective compounds is achieved in the same chromatographic separation bed, so an assignment to bioactive compounds can easily be made. Sophisticated technical equipment for the bioassay coupling is not necessary. By a simple automatic immersion step, the assay suspension or solution can homogeneously be applied. Among the many advantages is the direct accessibility of bioactive compounds for structure elucidating techniques like HRMS and NMR [2]. The sample extract is applied as natural as possible and the assay can comprehensively detect the whole sample extract. Even such bioactive compounds left at the starting zone are detectable – a clear benefit in contrast to column techniques.

For direct bioautography, new concepts of using water-wettable reversed layers were introduced recently [3 – 5]. On such layers, the bioassay medium showed no elution power and thus the analytes remained detectable as sharp bands, even after hours of incubation. So the quality of the bioautograms improved substantially. Such an effect-directed screening can be used to obtain target-orientated results rapidly (bioprofiling) and allows the evaluation of the plant's fingerprint with regard to stability, degradation and adulteration. Such a hyphenated screening concept like HPTLC-UV/Vis/FLD-(bio) assay-MS is shown for extracts of Salvia officinalis L., Taxus baccata L. and Foeniculum vulgare (L.) Mill. is exemplarily demonstrated using the DPPH* reagent for detection of radical scavenging compounds, the tyrosinase assay for skin whitening agents, the Bacillus subtilis for antimicrobials, the pYES bioassay for estrogen-effective compounds and the Aliivibrio fischeri bioassay for bioactive compounds in general.

Keywords: High-performance thin-layer chromatography, HPTLC hyphenations, mass spectrometry, bioautography, effect-directed analysis, bioassays.

References:

[1] Morlock G. Chromatography combined with bioassays and other hyphenations – the direct link to the compound indicating the effect, ACS Syposium Series 2013; 1185: 101 – 121

[2] Morlock G, Schwack W. Hyphenations in planar chromatography, J Chromatogr A 2010; 1217: 6600 – 6609

[3] Morlock GE, Klingelhöfer I. Liquid chromatography-bioassay-mass spectrometry for profiling of physiologically active food, Anal Chem 2014; 86: 8289 – 8295

[4] Klingelhöfer I, Morlock GE. Sharp-bounded zones link to the effect in planar chromatography-bioassay-mass spectrometry, J Chromatogr A 2014; 1360: 288 – 295

[5] Klingelhöfer I, Morlock GE. Bioprofiling of surface/wastewater and bioquantitation of discovered endocrine-active compounds by streamlined direct bioautography, Anal Chem 2015; 87: 11098 – 11104