Effect-directed analysis in food by thin-layer chromatography assays.

Thin-layer chromatography (TLC) is widely used for food analysis and quality control. As an open chromatographic system, TLC is compatible with microbial-, biochemical-, and chemical-based derivatization methods. This compatibility makes it possible to run in situ bioassays directly on the plate to obtain activity-profile chromatograms, i.e., the effect-directed analysis of the sample. Many of the properties that can be currently measured using this assay format are related to either desired or undesired features for food related products. The TLC assays can detect compounds related to the stability of foods (antioxidant, antimicrobial, antibrowning, etc.), contaminants (antibiotics, pesticides, estrogenic compounds, etc.), and compounds that affect the absorption, metabolism or excretion of nutrients and metabolites or could improve the consumers health (enzyme inhibitors). In this article, different food related TLC-assays are reviewed. The different detection systems used, the way in which they are applied as well as selected examples are discussed.

Effect-Directed Synthesis of PhoP/PhoQ Inhibitors to Regulate Salmonella Virulence.

Salmonella spp. are among the leading bacterial causes of foodborne infections. The PhoP/PhoQ two-component regulatory system serves as a master virulence regulator in Salmonella. Although PhoP/PhoQ represents an ideal target for disarming Salmonella virulence, it has very few inhibitors reported so far. We describe a novel platform by which an inhibitor was selected out of around 185 compounds directly from reaction media containing thiosemicarbazones and mono-, di-, and trihydrazones. To achieve this, tandem library preparation, thin-layer chromatography (TLC) bioautography, and effect-directed deconvolution were applied. We illustrate the potential of this effect-directed synthesis for the identification of new useful bioactive compounds for the food field.

Sulfur in Dynamic Covalent Chemistry.

Sulfur has been important in dynamic covalent chemistry (DCC) since the beginning of the field. Mainly as part of disulfides and thioesters, dynamic sulfur-based bonds (DSBs) have a leading role in several remarkable reactions. Part of this success is due to the almost ideal properties of DSBs for the preparation of dynamic covalent systems, including high reactivity and good reversibility under mild aqueous conditions, the possibility of exploiting supramolecular interactions, access to isolable structures, and easy experimental control to turn the reaction on/off. DCC is currently witnessing an increase in the importance of DSBs. The chemical flexibility offered by DSBs opens the door to multiple applications. This Review presents an overview of all the DSBs used in DCC, their applications, and remarks on the interesting properties that they confer on dynamic chemical systems, especially those containing several DSBs.

Artichoke extracts with potential application in chemoprevention and inflammatory processes

Abstract The aim of this work is to study three cultivars of artichoke (Cynara cardunculus var. scolymus): Gauchito, Guri and Oro Verde in terms of their in vitro chemoprevention and anti-inflammatory properties. These cultivars show good productive performance. The phenolic composition of their fresh leaves and edible bracts was analyzed by high performance liquid chromatography and high resolution mass spectrometry (HPLC-HRMS), showing mainly caffeoylquinic acids and flavonoids. Caffeoylquinic acids were quantified and the highest content was found in Gauchito cultivar. In this cultivar, the content of dicaffeoylquinic acids in fresh bracts was six times higher than that in fresh leaves (10064.5 ± 378.3 mg/kg versus 1451.0 ± 209.3 mg/kg respectively). Luteolin flavonoids were detected in leaves. The extracts from fresh bracts and leaves were assessed in their in vitro bioactivity against human neuroblastoma cells (SH-SY5Y). Inhibition of SH-SY5Y cells proliferation by Gauchito and Guri leaf extracts (8 µg/mL) was higher than 50 %. The leaf extracts of the same cultivars showed an inhibitory effect on human interferon IFN-I, decreasing its activity 50% at 40 µg/mL. Interestingly, the bract extracts did not show in vitro bioactivity at these concentrations, nor did the pure compounds chlorogenic acid, cynarin, apigenin and luteolin (at 2 µg/mL). These results suggest that Gauchito and Guri leaf extracts have potential for human neuroblastoma chemoprevention and treatment of inflammatory processes.

Effect directed synthesis of a new tyrosinase inhibitor with anti-browning activity.

The inhibition of enzymatic browning is an attractive target to elevate the quality of foods. The objective of this work is to describe a novel platform for the discovery of tyrosinase inhibitors, based on (a) one-pot preparation of a library of thiosemicarbazide compounds, (b) biological evaluation using tyrosinase TLC bioautography, (c) inhibitor identification via mass spectrometry coupled to bioautography. During these proof-of-concept experiments, the approach led to the straightforward identification of a new thiosemicarbazone with improved tyrosinase inhibition properties and fresh-cut apple slices antibrowning effect when compared to kojic acid. In conclusion, the platform represents an interesting strategy for the discovery of this type of inhibitors.

Enzymatic Bioautographic Methods.

Enzymatic bioautography enables the detection of enzyme inhibitors absorbed on a thin-layer chromatography plate. Therefore, it is an assay format that is particularly useful for the detection of inhibitors present in complex mixtures. The inhibition properties of compounds separated by thin-layer chromatography can be directly analyzed to produce an inhibition profile. Here, we describe the conditions to detect inhibitor of the enzymes xanthine oxidase and ß-glucosidase immobilized on agar gel.

Engineering multilayer chemical networks.

Dynamic multilevel systems emerged in the last few years as new platforms to study thermodynamic systems. In this work, unprecedented fully communicated three-level systems are studied. First, different conditions were screened to selectively activate thiol/dithioacetal, thiol/thioester, and thiol/disulfide exchanges, individually or in pairs. Some of those conditions were applied, sequentially, to build multilayer dynamic systems wherein information, in the form of relative amounts of building blocks, can be directionally transmitted between different exchange pools. As far as we know, this is the first report of one synthetic dynamic chemical system where relationships between layers can be changed through network operations.

A Tyrosinase Inhibitor from a Nitrogen-Enriched Chemically Engineered Extract.

The enzyme tyrosinase is involved in the biosynthesis of melanin and the enzymatic browning of fruits and vegetables, and therefore, its inhibitors have potential to treat hyperpigmentary disorders or to function as food antibrowning agents. The use of hydrazine monohydrate as a reagent to prepare chemically engineered extracts can lead to semisynthetic compounds that contain the portion N-N, a fragment rarely found in natural products and present in some tyrosinase inhibitors. Here, we report the tyrosinase inhibition screening of a series of chemically engineered extracts that are diversified by reaction with hydrazine. LC-MS was used to evaluate the change in composition produced by the reaction. Bioguided fractionation of the most active chemically engineered extract, prepared from Matricaria recutita L., led to the discovery of a pyrazole that inhibits tyrosinase with an IC50 value of 28.20 ± 1.13 µM. This compound was produced by a one-pot double chemical transformation of its natural precursor, which includes an unexpected selective removal of one -OH group.

Molecular Networks in Dynamic Multilevel Systems.

Dynamic multilevel systems can be assembled from molecular building blocks through two or more reversible reactions that form covalent bonds. Molecular networks of dynamic multilevel systems can exhibit different connectivities between nodes. The design and creation of molecular networks in multilevel systems require control of the crossed reactivity of the functional groups (how to connect nodes) and the conditions of the reactions (when to connect nodes). In recent years, the combination of orthogonal and communicating reactions, which can be simultaneous or individually activated, has produced a variety of systems that have given rise to macrocycles and cages, as well as molecular motors and multicomponent architectures on surfaces. A given set of reactions can lead to systems with unique responsiveness, compositions, and functions as a result of the relative reactivities. In this Concept article, different molecular networks from synthetic systems that can be produced by combinations of different reaction types are discussed. Moreover, applications of this chemistry are highlighted, and future perspectives are envisioned.

Discovery of a Biologically Active Bromodomain Inhibitor by Target-Directed Dynamic Combinatorial Chemistry.

Target-directed dynamic combinatorial chemistry (DCC) has emerged as a strategy for the identification of inhibitors of relevant therapeutic targets. In this contribution, we use this strategy for the identification of a high-affinity binder of a parasite target, the Trypanosoma cruzi bromodomain-containing protein TcBDF3. This protein is essential for viability of T. cruzi, the protozoan parasite that causes Chagas disease. A small dynamic library of acylhydrazones was prepared from aldehydes and acylhydrazides at neutral pH in the presence of aniline. The most amplified library member shows (a) high affinity for the template, (b) interesting antiparasitic activity against different parasite forms, and (c) low toxicity against Vero cells. In addition, parasites are rescued from the compound toxicity by TcBDF3 overexpression, suggesting that the toxicity of this compound is due to the TcBDF3 inhibition, i.e., the binding event that initially drives the molecular amplification is reproduced in the parasite, leading to selective toxicity.

Rapid autographic method for detection of enzymatic browning inhibitors based on enzyme immobilization.

This work describes a TLC-coupled bioautographic assay suitable for the separation and detection of apple polyphenol oxidase (PPO) inhibitors from natural extracts. PPO was immobilised in agar containing l-DOPA as substrate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) to enhance colour development. The inhibition was detected as white spots on reddish background. Minimum amount of PPO inhibitors detected was 0.0125 µg of 4-hexylresorcinol, 0.025 µg of ascorbic acid, 0.5 µg of cysteine and 1 µg of kojic acid. The assay was compatible with normal and reverse phase TLC systems and allows detecting compounds that directly had action on the enzyme as well as agents that could convert quinones back to their reduced form. The chromatographic run evidenced the different nature of enzymatic browning inhibitory compounds from garlic and onion extracts. Using natural enzymes will provide a fast and cheap alternative for target specific exploration of natural enzymatic inhibitors.

A Bioactive Trypanosoma cruzi Bromodomain Inhibitor from Chemically Engineered Extracts.

A set of chemically engineered extracts enriched in compounds including N-N and N-O fragments in their structures was prepared. Bromodomain binding screening and bioguided fractionation led to the identification of one oxime hit that interacts with TcBDF3 with affinity in the submicromolar range and that shows interesting antiparasitic properties against the different life cycle stages of T. cruzi.

Rewiring Chemical Networks Based on Dynamic Dithioacetal and Disulfide Bonds.

The control of the connectivity between nodes of synthetic networks is still largely unexplored. To address this point we take advantage of a simple dynamic chemical system with two exchange levels that are mutually connected and can be activated simultaneously or sequentially. Dithioacetals and disulfides can be exchanged simultaneously under UV light in the presence of a sensitizer. Crossover reactions between both exchange processes produce a fully connected chemical network. On the other hand, the use of acid, base or UV light connects different nodes allowing network rewiring.

Host Amplification in a Dithioacetal-Based Dynamic Covalent Library.

Molecular amplification in a dithioacetal-based dynamic library is described for the first time. The homatropine induced selection, amplification, and isolation of one cyclophane host demonstrates the utility of dithioacetal exchange for preparing responsive dynamic libraries. Nuclear magnetic resonance and isothermal titration calorimetry analysis suggest that the amplified macrocycle forms a 1:1 complex with the template. This is the first report about a host/guest system involving a dithioacetal cyclophane.

Reverse Phase Compatible TLC-Bioautography for Detection of Tyrosinase Inhibitors.

INTRODUCTION: Reverse phase chromatography and bioautographic assays are key tools for natural product bioguided isolation; however, their direct coupling has not been fully achieved. OBJECTIVES: To develop a bioautographic assay to detect tyrosinase inhibitors present in complex matrices sorbed on reverse phase (RP) TLC-plates that can be used for bioguided isolation of bioactive compounds. METHODS: Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The gel turns into a brown "skin like" colour due to tyrosinase catalysed oxidation of l-tyrosine. The inhibitors are visualised as clear spots against a brown coloured background. RESULTS: The assay was able to localise cinnamaldehyde in Cinnamomum cassia essential oil, as its main constituent with known tyrosinase inhibition properties. The assay allowed the detection of 0.03% (w/w) of kojic acid co-spotted with a methanolic extract of Sphaeralcea bonariensis and chromatographed on RP-TLC. CONCLUSION: The developed assay is able to detect, with high sensitivity, tyrosinase inhibitors present in complex matrices that were chromatographed in RP-TLC. Results can be easily read by colour change, inhibitors appear as clear spots in a darker background. Copyright © 2016 John Wiley & Sons, Ltd.

A New Fluorinated Tyrosinase Inhibitor from a Chemically Engineered Essential Oil.

The generation of fluorinated essential oils as a source of bioactive compounds is described. Most of the components of the natural mixtures were altered, leading to the discovery of a new fluorinated tyrosinase inhibitor.

Dithioacetal Exchange: A New Reversible Reaction for Dynamic Combinatorial Chemistry.

Reversibility of dithioacetal bond formation is reported under acidic mild conditions. Its utility for dynamic combinatorial chemistry was explored by combining it with orthogonal disulfide exchange. In such a setup, thiols are positioned at the intersection of both chemistries, constituting a connecting node between temporally separated networks.

A reversed-phase compatible thin-layer chromatography autography for the detection of acetylcholinesterase inhibitors.

A dual readout autographic assay to detect acetylcholinesterase inhibitors present in complex matrices adsorbed on reversed-phase or normal-phase thin-layer chromatography plates is described. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The effects of substrate and enzyme concentrations, pH, incubation time, and incubation temperature on the sensitivity and the detection limit of the assay were evaluated. Experimental design and response surface methodology were used to optimize conditions with a minimum number of experiments. The assay allowed the detection of 0.01% w/w of physostigmine in both a spiked Sonchus oleraceus L. extract chromatographed on normal phase and a spiked Pimenta racemosa (Mill.) J.W. Moore leaf essential oil chromatographed on reversed phase. Finally, the reversed-phase thin-layer chromatography assay was applied to reveal the presence of an inhibitor in the Cymbopogon citratus (DC.) Stapf essential oil. The developed assay is able to detect acetylcholinesterase inhibitors present in complex matrixes that were chromatographed in normal phase or reversed-phase thin-layer chromatography. The detection limit for physostigmine on both normal and reversed phase was of 1×10(-4) µg. The results can be read by a change in color and/or a change in fluorescence.

Thin Layer Chromatography-Autography-High Resolution Mass Spectrometry Analysis: Accelerating the Identification of Acetylcholinesterase Inhibitors.

INTRODUCTION: The prevailing treatment for Alzheimer's disease is the use of acetylcholinesterase (AChE) inhibitors. Natural extracts are the principal source of AChE's inhibitors. However, their chemical complexity demands for simple, selective and rapid assays. OBJECTIVE: To develop a strategy for identification of AChE inhibitors present in mixtures employing high resolution mass spectrometry (HRMS) and thin layer chromatography (TLC)-biological staining. METHODOLOGY: The strategy uses an autographic assay based on the α-naphthyl acetate - fast blue B system for the detection of AChE activity. The immobilisation of AChE in agar allowed the extraction of the compounds for analysis by HRMS. Three TLC experiments employing different solvent systems were used in parallel and the mass spectra of the compounds extracted from the inhibition halos, were compared. The analysis was performed under MatLab environment. RESULTS: The strategy was used to detect the presence of physostigmine in an extract of Brassica rapa L. spiked with the inhibitor. Similarly, caffeine was straightforwardly spotted as responsible for the inhibitory properties of an extract of Ilex paraguariensis Saint-Hilaire. Comparison of the HRMS profiles lead to the facile identification of the [M+H](+) and [M+Na](+) of the compounds responsible for the inhibition. CONCLUSION: The proposed methodology, coupling TLC-AChE autography-HRMS, illustrates the feasibility of assigning molecular formulas of active compounds present in complex mixtures directly from autography. The new AChE agar-immobilised assay presented a more homogenous colour and a better definition than direct spraying methods, reducing the cost of the assay and improving its sensitivity.

Multiresponse Optimisation Applied to the Development of a TLC Autography for the Detection of Tyrosinase Inhibitors.

INTRODUCTION: Autographic methods are useful tools to detect bioactive compounds in complex matrixes. Experimental design and optimisation techniques were implemented for the development of an autographic assay suitable for the detection of tyrosinase inhibitors. OBJECTIVES: To develop an autographic assay to detect tyrosinase inhibitors using gel entrapped enzyme, experimental design and response surface methodology (RSM) to optimise conditions with a minimum number of experiments. METHODS: Gel entrapment was used for the assay and the effects of four factors on the sensitivity and the detection limit for known inhibitors of the enzyme were evaluated. The factors were: tyrosinase amount (TA), L-tyrosine amount (LTA), incubation time and incubation temperature. RESULTS: The assay allowed the detection of kojic acid in an extract of Calamagrostis viridiflavescens (Poir.) Steud spiked with 0.1% w/w. CONCLUSION: The developed assay is able to detect tyrosinase inhibitors present in complex matrixes in a reproducible way.