György Kátay

Micro-preparative OPLC — rapid isolation by transfusion and infusion-transfusion processes

A new OPLC procedure, infusion—transfusion OPLC, has been developed and compared with conventional transfusion OPLC. Spot and/or band deformation caused by the total wetness front (which results from pore filling) was reduced, as was the bubble effect in on-line detection. Both techniques were used for rapid micro-preparative OPLC isolation on analytical adsorbent layers. In-situ clean-up and separation were used to isolate trigonelline from Leuzea extract. Modeling of loading capacity for isolation of ascorbigen was accomplished by fully off-line OPLC. Under optimized conditions ascorbigen of high purity was isolated from cabbage extract by transfusion and infusion—transfusion OPLC.

BioArena: An unlimited possibility of biochemical interactions in the adsorbent layer after chromatographic separation

The BioArena system, which integrates the modern technique and biological results of bioautography with layer liquid chromatography is especially suitable for investigating biochemical interactions. Formaldehyde (HCHO) and its reaction products play a crucial role in the antibiotic activity of trans-resveratrol and other molecules–when HCHO-capturing molecules are used in culture media the antimicrobial activity of antibiotic-like compounds decreases substantially. HCHO and hydrogen peroxide are present as normal endogenous compounds in cells, so there is a possibility of interaction in which singlet oxygen (1O2) and excited HCHO can be formed. The 1O2 can oxidize water molecules and so H2O3 can be formed, from which, by disproportion, among other reactions, ozone (O3) also can be formed in the chromatographic spots. Elimination of HCHO and/or O3 from the spots results in a decrease in the antiproliferative effect.

The potential of BioArena in the study of the formaldehydome

New results with BioArena as a complex separation and detection (evaluation) system support earlier observations that formaldehyde (HCHO) and its reaction products play a special and crucial role in the effects of antibiotic in general. It has been established that antibiotic-like compounds (e.g. trans-resveratrol, Cu(II) ions) have a duplicate inhibiting effect on pathogen cells as a result of the action of HCHO. HCHO as a key molecule of the formaldehydome participates in series of interactions which can be screened by means of different spectroscopic and chromatographic techniques; accumulation of HCHO and its reaction products in TLC spots is limited, however, so indirect detection is advantageous. In BioArena the planar stationary phase bed after TLC/HPTLC and, mainly, OPLC separation can be used for manifestation of deprivation of HCHO from antibiotic spots, for observation of the duplicate effect of substances with a direct effect, and for demonstration of cell proliferation promotion and retardation.

Antibiosis, Antibiotics, and the Formaldehyde Cycle: The Unique Importance of Planar Chromatographic Techniques to Progress in these Fields

The BioArena system, which integrates the up-to-date methodology and biological results of bioautography with OPLC as an efficient planar separation technique (compact spots, etc.), is especially suitable for investigating biochemical interactions in an adsorbent bed after chromatographic separation. The first results from BioArena show that formaldehyde (HCHO), which can originate from pathogen cells in some situations, can play a special role in the antibiotic activity of trans-resveratrol. When L-arginine and glutathione were used as endogenous HCHO-capturing molecules in the culture medium the antimicrobial activity of trans-resveratrol on the adsorbent layer decreased substantially. It has been observed that trans-resveratrol generates a time-dependent, and therefore concentration-dependent, duplicate inhibiting effect on the pathogen, and that the BioArena system was suitable for illustration of this new phenomenon. It is probable that this effect occurs as a result of HCHO, with special emphasis on the possibility of interaction between the HCHO and H2O2 of endogenous origin on the adsorbent layer incubated with pathogen cells. It seems that the BioArena system will be an important, indispensable complement to the basic separation technique OPLC.

Comparison of Components from Red and White Wines for Antimicrobial Activity by Biodetection after OPLC Separation

Abstract Using OPLC separation and subsequent biodetection with Pseudomonas savastanoi pv. phaseolicola antibacterial compounds have been detected in both red and white wine extracts. However, the microbicidal power of red wine extracts was, in all cases, higher than that of white wines. The red wines contain different types of antimicrobial compounds but trans-resveratrol (TR) and its reduced form (bibenzyl-3,5,4′-triol, BB) in some cases are determining components. When the formaldehyde (HCHO) capturing molecule was used in culture medium (BioArena system) the antimicrobial activity of all antibiotic-like compounds was decreased characteristically, that is, HCHO plays a role in the antibacterial activity of these known and unknown compounds.

Separation of Stilbene Isomers from Red Wine by Overpressured-Layer Chromatography

A simple method is described for separation and determination of the isomers of trans-resveratrol and related compounds from different types of red wine by overpressured layer chromatography (OPLC). Comparison of OPLC with TLC clearly showed the advantages of the forced-flow technique (higher theoretical plate number, good resolution, etc.) over conventional planar layer liquid chromatography. It was established that the glycosides of resveratrol isomers were always present in higher concentrations than free stilbene isomers in red wine samples. This was especially true for the Pinot Noir wine. Exploitation of the advantages of OPLC provides further possibilities of analysis and isolation of stilbene isomers from grapes and wine.

Overpressured-layer chromatographic determination of ascorbigen (bound vitamin C) in Brassica vegetables

A simple and efficient method is described for separation and determination of ascorbigen in Brassica vegetables by overpressured-layer chromatography. Natural ascorbigen was identified from chromatographic retention, UV spectral, and mass spectrometric data using the authentic substance for comparison. Quantification of ascorbigen revealed significant differences (4.52-26.81 mg kg-1) among the species studied. The highest level of endogenous ascorbigen was observed in the sample prepared from broccoli (26.81 mg kg1).

Spectroscopic and OPLC identification and measurement of formaldehyde and potential formaldehyde generators in macroscopic fungi

Optimum-performance laminar chromatography (previously known as overpressured-layer chromatography; OPLC) exploits the advantages of the optimum laminar flow of mobile phase obtained by use of pump to introduce the mobile phase to the adsorbent layer in an automated, microprocessor-controlled separation system. The optimized flow profile in OPLC is the basis of the efficiency of this new technique. The attractiveness of OPLC is particularly apparent from the width of the separation surface (large number of samples). OPLC has enabled improvement of the velocity profile with a decrease in eddy diffusion. OPLC can be used for efficient separation of formaldehyde (HCHO) and some betaines, potential HCHO generators, in macroscopic fungi. Endogenous HCHO is determined, after conversion to formaldemethone, and characterized from proton magnetic resonance spectroscopic (1H NMR) and electron-impact mass spectro-metric (EIMS) data. The results show that macroscopic fungi contain moderate levels of HCHO compared with, for example, the leaves of certain higher plant species. Among the betaines, l-carni-tine and glycinebetaine were identified by matrix-assisted laser-desorption ionization mass spectrometry (MALDI MS), 1H NMR spectroscopy, and OPLC. Data prove that automated OPLC, a new separation technique, is suitable for efficient separation of natural substances from a large number of samples in one separation; it is, therefore, a prospective complementary methodological direction among separation techniques.

Biological Characterization of Ingredients in OPLC-BioArena- Greenhouse-System: Unique Reactions of Endogenous HCHO and O3 in In Vitro and In Vivo Conditions

In the case of two “old medicines”, the extension of the results from the in vitro BioArena studies was successfully solved for in vivo (greenhouse) conditions. These preliminary results of in vitro and in vivo studies confirmed the unique role and function of HCHO and O3 in the antibiotic effect of these two chemical substances, and in the basal and induced resistance. In the future, for the characterization of known and new active substances, in vitro and in vivo biological studies will be recommended in addition to chemical and physical characterization.

Identification of 1'-Methylascorbigen in Broccoli

The presence of 1’-methylascorbigen, the most biologically active indole derivative of L-ascorbic acid, in broccoli has been proved by use of planar liquid and column liquid chromatography and mass spectrometry. Endogenous 1’-methylascorbigen was identified on the basis of its retention behavior, by means of the modified PRIMA method, and from its characteristic MALDI MS data. This paper is the first report of identification of 1’-methylascorbigen in the plant kingdom.