Bioassays | Effects-Detection in Chromatography

doi:10.1016/B978-0-12-409547-2.13959-9

Reference Module in Chemistry, Molecular Sciences and Chemical Engineering

Encyclopedia of Analytical Science (Third Edition)

2019, Pages 261-270

https://doi.org/10.1016/B978-0-12-409547-2.13959-9 Get rights and content

Abstract

The importance of a fast effect-directed profiling of samples is triggered by the global threat to ecosystems, global product chains and unknown active principles of food, cosmetics, and traditional medicines. Chromatography combined with effect-directed detections points to active compounds in complex samples. Effect-directed assays were performed in situ, postcolumn in flow and offline as standard in vitro assay after postcolumn fractionation. Among the possible combinations of chromatography with effect-directed assays, high-performance thin-layer chromatography turned out to be the most flexible chromatographic technique in combination with biochemical and especially biological assays. Powerful multidetection is used to characterize unknown active compounds discovered.

References (0)

Cited by (10)

Nine-dimensional bioprofiles of Tunisian sages (Salvia officinalis, S. aegyptiaca and S. verbenaca) by high-performance thin-layer chromatography – effect-directed analyses
2023, Journal of Chromatography A
Citation Excerpt :
The association of high-performance thin-layer chromatography (HPTLC) with biological, biochemical, and microchemical assays (effect-directed analysis, EDA) is an efficient tool for the non-target screening of natural bioactive compounds in complex samples [1,2]. HPTLC-EDA ensures several benefits such as uncomplicated sample preparation, the ability to quickly analyze many samples at the same time, and the targeted identification of biologically active substances [3]. Due to the presence of active substances in medicinal plants, their bioactivity and therapeutic effects (treatment of diseases as well as improvement of health and well-being) have been exploited around the world [4].
Ethyl acetate extracts of Tunisian Salvia aegyptiaca and S. verbenaca aerial parts and S. officinalis leaves were examined via bioanalytical profiling using high-performance thin-layer chromatography (HPTLC) combined with nine bioactivity assays, namely antibacterial (Aliivibrio fischeri, Bacillus subtilis, and Rhodococcus fascians), antifungal (Bipolaris sorokiniana, and Fusarium avenaceum), radical scavenging (DPPH•), and enzyme inhibitory (α-glucosidase, acetylcholinesterase, and lipase) ones. The screening, using toluene - ethyl acetate – methanol 6:3:0.5 (V/V/V) as a mobile phase, revealed five bioactive zones (a–e) that were analyzed by HPTLC-electrospray ionization-mass spectrometry (ESI-MS). Zones b and c, observed exclusively in S. officinalis, were active in all assays except α-glucosidase, and only c inhibited F. avenaceum. Compounds in these zones were identified by HPLC-high resolution tandem MS (LC-HRMS/MS) as rosmanol/epi-rosmanol and methyl carnosate, respectively. In the bioactive zones a and e, corosolic/maslinic acid and ursolic/oleanolic acid isomer pairs were present, which could be identified in all three Salvia species after their HPTLC separation using pre-chromatographic derivatization with iodine and MS detection. The triterpenes inhibited B. subtilis and R. fascians bacteria and α-glucosidase enzyme. Linoleic and linolenic acids were detected in zone d, which showed strong lipase inhibition in all three sage species.
Profile comparison and valorization of Tunisian Salvia aegyptiaca and S. verbenaca aerial part extracts via hyphenated high-performance thin-layer chromatography
2022, Journal of Chromatography A
Citation Excerpt :
It resolves the complexity of multi-component mixtures into meaningful patterns, all image-based and side-by-side on the same planar surface [2]. Compared to the status quo (chromatographic fractionation combined with in vitro assay testing), advantages of this hyphenated bioanalytical method are, for example, method greenness, no loss in compound transfer, no assay problem with water-insoluble substances, eased sample preparation, shorter analysis time, stepwise automation, and analysis of many samples in parallel [1,2]. A reliable quantification based on the biological [3] and biochemical [4] on-surface responses is possible.
Aerial parts of the rare species Salvia aegyptiaca L. and S. verbenaca L. were collected from arid habitats in southern Tunisia. Their polar (ethanol−water) and mid-polar (ethyl acetate) extracts were analyzed non-targeted via a developed high-performance thin-layer chromatography profiling hyphenated with 12 effect-directed assays and 8 different physico-chemical detections. Bioactive compound zones were observed with inhibiting activities on α-glucosidase, ß-glucosidase, ß-glucuronidase, acetylcholinesterase, butyrylcholinesterase and tyrosinase, with radical scavenging (antioxidative) effects, and with activities against Gram-negative and Gram-positive bacteria. The effect-directed profile patterns showed common bioactive zones for different collection sites of the same species and distinct differences between species. Such characteristic profiles can be used to prove authenticity. Genotoxic, estrogen-like and androgen-like compounds were not detected even at higher amounts applied (for extracts from 1.6 mg sample). In the physico-chemical profiling, further organic substances were selectively detected, which highlighted the complexity of the multi-component mixture. The Tunisian sage profiles were further compared to the frequently used S. folium L. and S. officinalis L. leaves, and to reference mixtures containing phenolic acids and tanshinones. Selected bioactive zones in the S. verbenaca extracts were characterized by high-resolution mass spectrometry, and some mass signals were attributed to a caffeic acid derivative and to oleanolic and ursolic acids. Such effect-directed non-target profiling allows straightforward comparison not only of sage but of plant extracts in general.
TLC bioprofiling—A tool for quality evaluation of medicinal plants
2022, Evidence-Based Validation of Herbal Medicine: Translational Research on Botanicals
Thin-layer chromatography (TLC) is a useful tool for qualitative analysis of natural compounds due to the fast separation of many samples in parallel and convenient visualization directly on a TLC plate. In herbal analysis, TLC profiling enables comparing samples of various species, as well as samples within the same species but of different origin, harvesting, storage conditions, or producer. So-called “fingerprints” allow to find differences among the samples and to reject those of other types, poor quality or adulterated. However, the most important are pharmacological properties of medicinal plants, in a particular correlation of bioactivity of their constituents with structural information.TLC can be hyphenated with bioassays performed directly on a TLC plate (TLC-direct bioautography) to detect bioactive constituents of plant extracts. TLC-DB followed by spectroscopic identification belongs to the effect directed analysis (EDA) and provides complete information of sample components showing a given effect on a TLC plate.
High-performance thin-layer chromatography combined with effect-directed assays and high-resolution mass spectrometry as an emerging hyphenated technology: A tutorial review
2021, Analytica Chimica Acta
Citation Excerpt :
After 75 years of bioautography, Goodall and Levi [102] would be delighted with the instrumental playground for planar hyphenations that we have today (Fig. 8 [63,66]). The terminology has already been addressed [63], not to use terms incorrectly [103–106]. Briefly, the umbrella term is effect-directed assays.
Among the analytical advances, hyphenated HPTLC offers great potential for solving pressing questions. It provides straightforward information about effects arising from individual compounds in complex or natural samples separated in parallel. The chromatographic separation is combined with effect-directed detection using enzymatic or biological assays. This helps to select from the thousands of compounds in a sample the important ones that need to be further characterized using high-resolution mass spectrometry. Unique benefits are discussed exemplarily arising from its super-hyphenation, minimum requirements for sample preparation, detection of multi-modulating compounds or agonistic versus antagonistic effects, and miniaturized on-surface metabolization. HPTLC stands for a versatile, creative and flexible open-format technique. As miniaturized open-source LabToGo system, it shows the potential to be applied as Citizen Science.
High-performance thin-layer chromatography hyphenated to high-performance liquid chromatography-diode array detection-mass spectrometry for characterization of coeluting isomers
2020, Talanta
The effect-directed analysis on a planar chromatogram allows for fast non-target screening, multi-imaging detection of effects (bioprofiling) and highly targeted characterization and isolation of bioactive compounds. For direct characterization by high-resolution mass spectrometry (HRMS), however, the orthogonal hyphenation of two different liquid chromatographic techniques (planar and column chromatography) is still underexplored. In particular, it can be helpful in case of coeluting compounds. Exemplarily, lemon balm (Melissa officinalis L.) leaf extract was analysed by high-performance thin-layer chromatography in combination with bioactivity assays for antibacterial (against the Gram-positive Bacillus subtilis and the Gram-negative Aliivibrio fischeri) and α-glucosidase-inhibitory compounds (HPTLC-UV/Vis/FLD-EDA). High-resolution mass spectra of two bioactive compound zones were directly recorded via an elution head-based interface. By HPTLC-HESI-HRMS, the compound in zone a inhibited A. fischeri and was identified as linolenic acid, whereas the two closely related constitutional isomers oleanolic acid and ursolic acid were present in zone b. This was proven by two-dimensional liquid chromatography. Heart-cutting HPTLC-UV/Vis/FLD-HPLC-DAD-MS allowed the separation of the two isomers and proved both to be present in the bioactive zone with ursolic acid at a much higher abundance.
Effect-directed analysis of bioactive compounds in Cannabis sativa L. by high-performance thin-layer chromatography
2020, Journal of Chromatography A
The scientific interest on the plant Cannabis sativa L., and in particular on its non-psychoactive or fibre-type variety (hemp), has been highly increasing in recent years, due to the pharmaceutical and nutraceutical potential of its bioactive compounds. This plant is indeed characterized by a very rich chemical composition, which encompasses different classes of constituents, such as cannabinoids and terpenes. In this context, the bioanalytical testing of hemp extracts can be difficult and time-consuming. Effect-directed analysis (EDA) by the combination of high-performance thin-layer chromatography (HPTLC) with biological and enzymatic assays represents one of the latest tools available for the rapid bioprofiling of complex matrices, such as plant extracts. In this ambit, the aim of this project was the non-targeted screening of inflorescence extracts from ten different hemp varieties for components exhibiting radical scavenging, antibacterial, enzyme inhibiting and estrogen-like effects. By HPTLC-EDA, the hemp samples exhibited strong antibacterial activities against both Gram-positive Bacillus subtilis and especially Gram-negative Aliivibrio fischeri bacteria, and also estrogen-like activity. They also inhibited α- and β-glucosidase, tyrosinase and acetylcholinesterase. The characterization of two prominently multipotent bioactive compound zones was finally achieved by HPTLC-HRMS and preliminary assigned as cannabidiolic acid and cannabidivarinic acid.

View all citing articles on Scopus

View full text