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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Ernő Tyihák, Ágnes Móricz, Péter Ott, György Kátay and Zsuzsa Király-Véghely

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.

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Bioautography can be extended to a complex system called BioArena by linking different steps to it, for example dissolving a variety of compounds in the cell suspension to affect biological activity, measuring putative mediators of antibiosis, for example formaldehyde (HCHO) and hydrogen peroxide (H 2 O 2 ) in the inoculated layer, and performing densitometric and ex and in situ spectroscopic examination to follow the changes in the inhibition zones and active compounds (e.g. antibiotics and toxins). Possibilities of the basic elements of BioArena system are illustrated in this paper by results with aflatoxin B1 (AFB1). Target bacterial cells in the logarithmic growth phase were found to be the most sensitive for direct bioautography. Densitometric signals of bioautograms (negative densitometry) of 0.125–1 μg AFB1 spots showed logarithmic correlation with the amount of AFB1. The HCHO capturer L-arginine decreased whereas the HCHO generator-mobilizer Cu(II) ions increased the antibacterial-toxic effect of AFB1. The latter effect was also confirmed by negative densitometry. Besides higher levels of HCHO, a decrease of H 2 O 2 in the toxin spot was found. HCHO could also originate, among other sources, from demethylation of AFB1, which is apparent from the Fourier transform Raman spectra obtained in situ from the AFB1-containing spots. These results support the suggested role of HCHO and its reaction products with H 2 O 2 (e.g. singlet oxygen ( 1 O 2 ), ozone (O 3 )) in the antibacterial-toxic effect of AFB1.

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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 ( 1 O 2 ) and excited HCHO can be formed. The 1 O 2 can oxidize water molecules and so H 2 O 3 can be formed, from which, by disproportion, among other reactions, ozone (O 3 ) also can be formed in the chromatographic spots. Elimination of HCHO and/or O 3 from the spots results in a decrease in the antiproliferative effect.

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The most current in vitro and in vivo results in the BioArena system and under greenhouse conditions provide a good opportunity for a fundamental renewal of biological detections and interactions in layer liquid chromatography. The adsorbent bed in a column liquid arrangement is not suitable for biological detection because the living cells do not grow there. Contrarily, the planar adsorbent layer enables the in situ biodetection of antimicrobials and the interactions among separated compounds, cells, and further various cofactors (molecules), making the study of mechanisms of action possible. The basic elements of the time- and dose-dependent quadruple immune response of plants to pathogens in relation to the function and reactions of formaldehyde and its reaction products (mainly endogenous ozone) were demonstrated. This finding opens a new horizon in the field of disease resistance in plants and perhaps in general in the biological world. These results give a good basis and possibility for studying and understanding the unique high-dilution phenomena as well, and at that time, they promise the elimination of century contradictions in this field.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Ágnes Móricz, Györgyi Horváth, Péter Molnár, Béla Kocsis, Andrea Böszörményi, Éva Lemberkovics and Péter Ott

The composition of the essential oil of Thymus vulgaris L. has been determined by GC-FID and GC-MS. Because separation of thymol, carvacrol, and linalool, components of the essential oil, was more efficient by overpressured layer chromatography (OPLC) than by conventional thin-layer chromatography (TLC), the forced flow technique was used before biological detection. All three test compounds had antibacterial effect against the phytopathogenic bacterium Pseudomonas syringae pv. maculicola, in bioautography, although in essential oil thymol was present in sufficient quantity to produce an inhibiting zone in the adsorbent layer. In BioArena investigations, when reduced glutathione as a formaldehyde (HCHO) capturer was dissolved in the cell suspension before bioautographic exposure to the essential oil, the characteristic inhibiting activity of thymol and carvacrol against Bacillus subtilis soil bacteria was reduced, whereas the presence of the HCHO precursors NGmonomethyl-l-arginine or N ɛ-monomethyl-l-lysine enhanced their antibacterial effect. These results suggest that HCHO and its reaction products may be involved in the antibacterial activity of thymol and carvacrol.

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The accumulation of hydrogen peroxide (H 2 O 2 ) is an early and crucial step in paclitaxel-induced cancer cell death before commitment of the cells to apoptosis. In these intracellular events formaldehyde (HCHO) as endogenous, indispensable component may be present mainly as hydroxymethyl groups and so there is a possibility of its endogenous interaction with H 2 O 2 in which singlet oxygen ( 1 O 2 ) and excited HCHO (H*CHO) can be formed. 1 O 2 can interact with H 2 O molecules and in this interaction dihydrogen trioxide (H 2 O 3 ) is formed. The disproportion of this molecule — among others — results in ozone (O 3 ). It is supposed that this reaction series is also valid for the conditions in layer chromatographic spots after inoculation. Results with paclitaxel support this idea. Using BioArena as a complex bioautographic system the HCHO molecules could be captured with well-known endogenous HCHO capture molecules (l -arginine, glutathione) in the spots of paclitaxel on the TLC/OPLC adsorbent layer after inoculation. Capture of HCHO resulted in a dose-dependent decrease of the antibacterial activity of paclitaxel. The antibacterial activity of paclitaxel in the chromatographic spots can be increased dramatically by using Cu(II) ions as HCHO-mobilizing and carrier ions in the culture medium. The HCHO molecule can N -hydroxymethylate the C3’ amide of paclitaxel. By applying an O 3 scavenger (e.g. indigo carmine) this oxidant, as a key reaction product of HCHO, could be detected indirectly in chromatographic spots of paclitaxel. It seems that these small molecules — from HCHO to endogenous O 3 — may be crucial factors of the mechanism of antiproliferative action of the paclitaxel including killing of bystander cancer cells also. It seems that the basic molecule (paclitaxel) does not have a direct effect on the bacterial cells; its induction of the formation of H 2 O 2 molecules (and indirectly HCHO molecules) may, however, be resulting in this complicated process.

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Singlet oxygen ( 1 O 2 )-catalyzed oxidation of water leads to a variety of oxidants (e.g. ozone, O 3 ) in host-parasite relationships as well. It can be detected indirectly in TLC or OPLC zones by use of the simple BioArena system and O 3 -eliminating molecules (e.g. d -limonene and indigo carmine) in the culture medium. It follows from these new findings that not only formaldehyde but also O 3 and related bioreactive compounds may play a crucial role in the mechanism of antibacterial activity of antibiotic-like compounds. The toxic potential of a molecule, however, originates from the ratio of the oxidants produced in the chromatographic spots.

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On the basis of our earlier observations, preliminary experiments were conducted with the fire blight pathogen Erwinia amylovora with the BioArena system using the potential antibacterial substances 1′-methylascorbigen (MeAG) as formaldehyde (HCHO) precursor and trans-resveratrol (RES) as HCHO mobilizer, capturer, and scavenger. Results showed characteristic, strong, dose-dependent antibacterial effects of both MeAG and RES present as chromatographic spots on the adsorbent layer. It is supposed that the weak antibacterial effect of AG may have originated from MeAG formed in situ on the layer by partial enzymatic methylation of AG. Addition of HCHO capture compounds (l-arginine, glutathione, glucosamine) to the culture medium before inoculation partially or totally reduced the antibacterial effect of both molecules, that is, this simplest aliphatic aldehyde, the endogenous HCHO molecule, participates in the antibacterial activity of these compounds. It follows from these results that there is a possibility of interaction between endogenous H2O2 and HCHO in the chromatographic spots and it is supposed that these reactive molecules and potential derivatives, e.g. singlet oxygen (1O2) and ozone (O3), may be the causes of the destructive effect of the fire blight pathogen on fruit trees.

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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 H 2 O 2 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.

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A group of thirteen newly synthesized potential herbicides, N -aryltrichloroacetamides or 2-(chlorophenoxy)acyl derivatives, have been initially investigated by reversed phase (RP) TLC. The lipophilicity of the substances was described by retention factors in water, log k w , calculated from experimental RP TLC data, and by log P values calculated by use of software. Biological activity was examined by use of the BioArena system of TLC separation then (micro)biological detection. The potential role of formaldehyde (HCHO) in the toxic antibacterial action of substances against Pseudomonas savastanoi pv. phaseolicola bacterial cells was investigated. The effect of HCHO capturers (L -arginine and reduced glutathione) and Cu 2+ ions on the bioactivity and mechanism of toxicity of the substances was examined. It was established that HCHO and its potential reaction products (e.g. 1 O 2 , O 3 ) are crucial in the mechanism of action of these molecules. Correlations between the lipophilicity and bioactivity of the test compounds were also analyzed. It seems that hydrophobicity is not the decisive factor affecting the bioactivity of these substances.

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