The food supplement market is growing as many consumers wish to complement their nutrient intake. Despite all the regulations in place to ensure food supplements safety, there are still many cases of ...irregularities reported especially connected to internet sales. Twenty resveratrol food supplement products sold on the Slovenian market were evaluated on their compliance of declared vs. determined resveratrol content, as well as the compliance of labels with the European Union (EU) and Slovenian regulatory requirements. Both the ingredient contents and food information are important parts of food safety. Analyses of 20 food supplements performed using high-performance thin-layer chromatography (HPTLC) coupled with densitometry showed that 95% of products had contents different from what was declared and 55% of products contained higher contents than declared. In 25% of the products the determined content per unit exceeded the maximum level (150 mg/day) specified in EU novel food conditions for food supplement with
-resveratrol. Evaluation of the 20 food supplement labels included mandatory and voluntary food information, food supplement information, novel food information, health claims and nutrition claims. Most labels contained the necessary information, but multiple errors were observed ranging from typos to misleading practices. From a food safety perspective there is still a lot of improvement needed in the field of food supplements.
Invasive alien plant species Canadian goldenrod (Solidago canadensis L.) and giant goldenrod (Solidago gigantea Aiton) were investigated as a source of phytochemicals and yellow dyes. Flavonoids and ...phenolic acids were extracted from the inflorescence of Canadian goldenrod with thirteen extraction solvents ethanol, methanol, acetone, water, and mixtures of organic solvents (70%, 80%, and 90%) with water. High performance thin-layer chromatography (HPTLC) coupled to densitometry and high-performance liquid chromatography with photo-diode array detector (HPLC-PDA) were used for analyses of the obtained sample test solutions (STSs), which showed the best and comparable extraction efficiencies for 70% acetone(aq), 70% methanol(aq), and 70% ethanol(aq). HPTLC combined with image analyses in fluorescent mode resulted in different chromatographic fingerprints for Canadian goldenrod and giant goldenrod STSs (70% acetone(aq)) after development, after post-chromatographic derivatization with NP reagent and after use of PEG reagent. The developed HPLC methods enabled analyses of phenolic acids and flavonoids (aglycones and glycosylated) in STSs and hydrolyzed STSs form inflorescence of Canadian and giant goldenrod. Different contents of chlorogenic acid, rutin, hyperoside, isoquercetin, and quercetin were observed in STSs of both goldenrod species. The analyses of hydrolyzed STSs confirmed that glycosylated flavonoids in Canadian and giant goldenrod inflorescence are mainly glycosides of quercetin, kaempferol, and isorhamnetin. Additional analyses using HPTLC and HPLC coupled to tandem mass spectrometry (MS/MS; HPTLC-MS/MS and LC-MS/MS) enabled tentative identification of phenolic acids and flavonoids (10 with HPTLC-MS/MS and 15 with LC-MS/MS), from which several were identified in Canadian (4 with HPTLC-MS/MS and 8 with LC-MS/MS) and in giant (7 with HPTLC-MS/MS and 9 with LC-MS/MS) goldenrod for the first time.
Anthraquinones (yellow dyes) were extracted from Japanese knotweed rhizomes with twelve extraction solvents (water; ethanol
(20%, 40%, 60%, 70% and 80%), ethanol, 70% methanol
, methanol, 70% acetone
..., acetone and dichloromethane). The obtained sample test solutions (STSs) were analyzed using high-performance thin-layer chromatography (HPTLC) coupled to densitometry and mass spectrometry (HPTLC-MS/MS) on HPTLC silica gel plates. Identical qualitative densitometric profiles (with anthraquinone aglycones and glycosylated anthraquinones) were obtained for STSs in all the solvents except for the STS in dichloromethane, which enabled the most selective extractions of anthraquinone aglycones emodin and physcion. The highest extraction efficiency, evaluated by comparison of the total peak areas in the densitograms of all STSs scanned at 442 nm, was achieved for 70% acetone
. In STS prepared with 70% acetone
, the separation of non-glycosylated and glycosylated anthraquinones was achieved with developing solvents toluene-acetone-formic acid (6:6:1, 3:6:1 and 3:3:1
/
) and dichloromethane-acetone-formic acid (1:1:0.1,
/
). Non-glycosylated anthraquinones were separated only with toluene-acetone-formic acid, among which the best resolution between emodin and physcion gave the ratio 6:6:1 (
/
). This solvent and dichloromethane-acetone-formic acid (1:1:0.1,
/
) enabled the best separation of glycosylated anthraquinones. Four HPTLC-MS/MS methods enabled the identification of emodin and tentative identification of its three glycosylated analogs (emodin-8-
-hexoside, emodin-
-acetyl-hexoside and emodin-
-malonyl-hexoside), while only the HPTLC-MS/MS method with toluene-acetone-formic acid (6:6:1,
/
) enabled the identification of physcion. Changes of the shapes and the absorption maxima (bathochromic shifts) in the absorption spectra after post-chromatographic derivatization provided additional proof for the detection of physcion and rejection of the presence of chrysophanol in STS.
Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and ...signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.
This is the first report on identification of all B-type proanthocyanidins from monomers to decamers (monomers-flavan-3-ols, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, ...nonamers, and decamers) and some of their gallates in leaves of Japanese knotweed (
Houtt.), giant knotweed (
F. Schmidt) and Bohemian knotweed (
×
(Chrtek & Chrtkova) J.P. Bailey). Flavan-3-ols and proanthocyanidins were investigated using high performance thin-layer chromatography (HPTLC) coupled to densitometry, image analysis, and mass spectrometry (HPTLC-MS/MS). All species contained (-)-epicatechin and procyanidin B2, while (+)-catechin was only detected in Bohemian and giant knotweed. (-)-Epicatechin gallate, procyanidin B1 and procyanidin C1 was only confirmed in giant knotweed. Leaves of all three knotweeds have the same chemical profiles of proanthocyanidins with respect to the degree of polymerization but differ with respect to gallates. Therefore, chromatographic fingerprint profiles of proanthocyanidins enabled differentiation among leaves of studied knotweeds, and between Japanese knotweed leaves and rhizomes. Leaves of all three species proved to be a rich source of proanthocyanidins (based on the total peak areas), with the highest content in giant and the lowest in Japanese knotweed. The contents of monomers in Japanese, Bohemian and giant knotweed were 0.84 kg/t of dry weight (DW), 1.39 kg/t DW, 2.36 kg/t, respectively, while the contents of dimers were 0.99 kg/t DW, 1.40 kg/t, 2.06 kg/t, respectively. Giant knotweed leaves showed the highest variety of gallates (dimer gallates, dimer digallates, trimer gallates, tetramer gallates, pentamer gallates, and hexamer gallates), while only monomer gallates and dimer gallates were confirmed in Japanese knotweed and monomer gallates, dimer gallates, and dimer digallates were detected in leaves of Bohemian knotweed. The profile of the Bohemian knotweed clearly showed the traits inherited from Japanese and giant knotweed from which it originated.
Flavan-3-ols and proanthocyanidins of invasive alien plants Japanese knotweed (
Houtt.), giant knotweed (
F. Schmidt) and Bohemian knotweed (
×
(Chrtek & Chrtkova) J.P. Bailey) were investigated ...using high performance thin-layer chromatography (HPTLC) coupled to densitometry, image analysis and mass spectrometry (HPTLC-MS/MS). (+)-Catechin, (-)-epicatechin, (-)-epicatechin gallate and procyanidin B2 were found in rhizomes of these three species, and for the first time in Bohemian knotweed. (-)-Epicatechin gallate, procyanidin B1, procyanidin B2 and procyanidin C1 were found in giant knotweed rhizomes for the first time. Rhizomes of Bohemian and giant knotweed have the same chemical profiles of proanthocyanidins with respect to the degree of polymerization and with respect to gallates. Japanese and Bohemian knotweed have equal chromatographic fingerprint profiles with the additional peak not present in giant knotweed. Within the individual species giant knotweed rhizomes and leaves have the most similar fingerprints, while the fingerprints of Japanese and Bohemian knotweed rhizomes have additional peaks not found in leaves. Rhizomes of all three species proved to be a rich source of proanthocyanidins, with the highest content in Japanese and the lowest in Bohemian knotweed (based on the total peak areas). The contents of monomers in Japanese, Bohemian and giant knotweed rhizomes were 2.99 kg/t of dry mass (DM), 1.52 kg/t DM, 2.36 kg/t DM, respectively, while the contents of dimers were 2.81 kg/t DM, 1.09 kg/t DM, 2.17 kg/t DM, respectively. All B-type proanthocyanidins from monomers to decamers (monomers-flavan-3-ols, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers and decamers) and some of their gallates (monomer gallates, dimer gallates, dimer digallates, trimer gallates, tetramer gallates, pentamer gallates and hexamer gallates) were identified in rhizomes of Bohemian knotweed and giant knotweed. Pentamer gallates, hexamers, hexamer gallates, nonamers and decamers were identified for the first time in this study in Bohemian and giant knotweed rhizomes.
Bee pollen, known as a 'life-giving dust', is a product of honeybees using flower pollen grains and combining them with their saliva secretions. Thus, flower pollen could be an indicator of the bee ...pollen botanical source. Identification of bee pollen sources is a highly crucial process for the evaluation of its health benefits, as chemical composition is directly related to its pharmacological activity. In this study, the chemical profiles, contents of phenolic marker compounds and pharmacological activities of
L. (ivy) bee pollen samples from Türkiye and Slovenia, as well as ivy flower pollen grains, were compared. High-performance thin-layer chromatography (HPTLC) analyses revealed that pollen samples, regardless of where they were collected, have similar chemical profiles due to the fact that they have the same botanical origins. Marker compounds afzelin, platanoside and quercetin-3-
-β-glucopyranosyl-(1→2)-β-galactopyranoside, common to both bee pollen and flower pollen, were isolated from bee pollen, and their structures were elucidated by nuclear magnetic resonance (NMR) and mass spectrometry (MS). These three compounds, as well as chlorogenic acid and 3,5-dicaffeoylquinic acid (found in flower pollen), were quantified using high-performance liquid chromatography (HPLC) analyses.
tests and effect-directed analyses were used to evaluate the xanthine oxidase inhibition and antioxidant activity of the marker compounds and extracts from flower pollen and bee pollen. This is the first report comparing chemical profiles and related bioactivities of the flower pollen and bee pollen of the same botanical origin, as well as the first report of the chemical profile and related bioactivities of ivy flower pollen.
•First four HPTLC methods for the separation of proanthocyanidins on diol plates.•First four on-line HPTLC-MS methods on diol for proanthocyanidins up to decamers.•A-type (procyanidins A1, A2) ...separated from B-type dimers (procyanidins B1, B2, B3).•Stability testing after development revealed a phenomenon of enhanced absorption.•Enhanced absorption is influenced by developing solvent, stationary phase and light.
We developed the first four HPTLC methods for the separation of proanthocyanidins according to degree of polymerization on HPTLC diol F254S plates. Acetonitrile, ethyl acetate, ethyl acetate–formic acid (9:0.1, v/v) and toluene–acetone–formic acid (3:6:1, v/v) were used as developing solvents and 4-dimethylaminocinnamaldehyde (DMACA) as the detection reagent. Each of these methods enables separation of standards of procyanidin dimers from procyanidin trimer (procyanidin C1) and separation of B-type dimers (procyanidins B1, B2, B3) from A-type dimers (procyanidins A1, A2). Based on these HPTLC methods we developed four new HPTLC-MS/MS methods for analyses of proanthocyanidins on HPTLC diol F254S plates and we identified B-type proanthocyanidins from monomers up to decamers in crude extracts of invasive Japanese knotweed (Fallopia japonica Houtt., Polygonum cuspidatum Sieb. & Zucc.) rhizomes. Monomers, monomer gallates, dimers, dimer gallates, dimer digallates, trimers, trimer gallates, tetramers, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes using developing solvents acetonitrile and toluene–acetone–formic acid (3:6:1, v/v). Ethyl acetate enabled separation from monomers up to hexamer gallates and ethyl acetate–formic acid (9:0.1, v/v) from monomers up to hexamers. During the five hours of stability testing of (–)-epicatechin and procyanidin B2 standards on HPTLC diol plates developed with all solvents we observed enhanced absorption at 280 nm. This was a totally unexpected phenomenon. This new discovery confirmed what we reported in our previous study on HPTLC silica gel. Enhanced absorption is influenced by the developing solvent (more than 30%), the stationary phase (up to 24%) and the light (up to 15%).
•First on-line HPTLC-MS methods on silica gel for proanthocyanidins up to decamers.•Ionization suppression was solved by two pre-developments of HPTLC plates.•Stability testing after development ...revealed a phenomenon of enhanced absorption.•Free and galloylated B-type proanthocyanidins were identified in Japanese knotweed.•HPTLC-MS methods enabled also identification of stilbenes and anthraquinones.
On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol–formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MSn methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6′-O-malonyl)-hexoside).
•New efficient sample preparation procedure for physalin extraction.•First HPTLC method for characterization of physalins in crude extracts.•First HPTLC–MS/(MS) method for characterization of ...physalins in crude extracts.•Innovative simultaneous hyphenation of two distinctive MS analyzers with HPTLC.•Characterization of physalins in different parts of P. alkekengi L.
We developed first HPTLC and HPTLC–MS/MS methods which enable characterization of structurally similar and complex biologically active compounds – physalins – from crude extracts of Chinese lantern (Physalis alkekengi L.). Separation on HPTLC silica gel plates developed with ethyl acetate–toluene–formic acid (7:3:0.2, v/v) enabled densitometric screening of physalins in absorption and, after post-chromatographic derivatization with sulfuric acid reagent, also in fluorescence mode. Compared to existing (U)HPLC methods, in this case TLC provides an alternative selectivity, better sensitivity and higher resolution, which was exemplified by the separation of physalin L standard and its impurity, identified as 2,3,25,27-tetrahydrophysalin A. Strong ion suppression caused by the developing solvent additive – formic acid – was efficiently solved by two successive plate pre-developments with methanol–formic acid (9:1, v/v) and methanol. This significantly improved the sensitivity of HPTLC–MS/MS method, but also required a slightly modified developing solvent ethyl acetate–toluene–formic acid (6:4:0.2, v/v). Simultaneous hyphenation of HPTLC with a triple quadrupole and an ion trap mass analyzer enabled a reliable and straightforward non-targeted characterization of physalins from the same chromatographic zone (band) and determination of physalin types. The performance of developed HPTLC-densitometric and HPTLC–MS/MS methods was demonstrated by the analysis of physalins from the aqueous extracts, prepared by an optimized fast and simple extraction method under reflux. Variations in physalin profiles and abundances in different parts of P. alkekengi L. harvested at different stages of maturity were observed. This indicates that not all parts of the plant, or plant as a whole, are appropriate for specific medicinal applications. Husks are proposed as the most suitable plant part for P. alkekengi L. quality control, because they exhibited the most obvious MS2 fingerprints of physalins with minimal interferences.
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