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  • Author or Editor: Ernő Tyihák x
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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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In the twenty-two years since a similar review in the first issue of this journal there has been much progress in forced-flow planar liquid chromatography (FFPLC). Innovative developments have resulted in extremely diverse new technical solutions. This review briefly summarizes techniques which are already in use as a result of this progress (overpressured-layer chromatography, OPLC, and rotation planar chromatography, RPC) and the future potential of other FFPLC techniques, for example electrochromatographic techniques and shear-driven chromatography (SDC). It seems that efforts in FFPLC provide real possibilities of efficient analytical and preparative separations of different complex mixtures exploiting, with increasing success, the objective advantages of planar layer chromatography. This review summarizes, first, the unique opportunities resulting from modern biological detection on the adsorbent layer (BioArena) and some recent results obtained. These results include — among others — the indirect detection of small key molecules (e.g. formaldehyde, HCHO, and ozone, O3) in chromatographic spots. Practical applications are mainly in-vivo investigations — already without chromatographic separations — with special emphasis on disease resistance and cell proliferation. It seems, however, that in the future BioArena-guided in-vivo investigation will enable characterization of biologically active substances.

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Fumonisins were produced by six strains of Fusarium moniliforme and Fusarium proliferatum on inoculated rice culture. For study of fumonisins of the B series (B1, B2, B3, and B4) a quick and efficient two-step reversed-phase OPLC method was developed in which the first step serves for cleaning of the samples. Measurements were performed with the automatic OPLC instrument. Results showed that one isolate of F. moniliforme and one of F. proliferatum produced all the fumonisins investigated – the other isolates produced no fumonisins. This work is the first report of the separation of all the fumonisins of the B series by use of a planar layer liquid chromatographic technique.

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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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Direct bioautography performed with luminescence gene-tagged bacteria enables almost real-time detection of antimicrobial compounds in plant extracts. This method for the detection of chamomile ( Matricaria recutita ) components with antibacterial effect against Bacillus subtilis soil bacteria was more sensitive than commonly used bioautographic visualization by staining with a tetrazolium salt. Some compounds had a strong inhibiting effect only via the bioluminescence measurement. Extraction of antibacterial components of chamomile flowers was most effective with 50% ethanol; slightly lower efficiency was achieved with acetone and methanol, and hexane was least effective. The results were confirmed by using luminescent Pseudomonas syringae pv. maculicola plant pathogen bacteria.

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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.

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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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The greater celandine ( Chelidonium majus L.), a well-known source of isoquinoline alkaloids, has a long history of use as a medicinal plant. Although the antimicrobial activity of Chelidonium alkaloids against pathogenic bacteria has been reported, the mechanism of this action is almost unknown. The BioArena system, which integrates the modern method and biological results of bioautography with TLC and/or OPLC, is especially suitable for investigating biochemical interactions in the adsorbent layer after chromatographic separation. The antimicrobial effect of alkaloids obtained from Chelidonium root has been demonstrated by use of this system. It was assumed that the antibiotic activity of chelidonium alkaloids was a result of formation of formaldehyde. It was also assumed that addition of endogenous HCHO-capture molecules, for example l -arginine and glutathione, to the culture medium reduces the antibacterial activity of Chelidonium alkaloids whereas Cu(II) ions enhance the effect. The results obtained support these assumptions and our earlier observations that HCHO and its reaction products are very important in the antibiotic action of these compounds. These small molecules (l -arginine and glutathione) can capture HCHO molecules mobilized by alkaloids and possibly by pathogen cells, and may be responsible for reduced antibacterial effect. The HCHO-mobilizing power of Cu(II) ions dramatically enhanced the antibiotic effect. The BioArena system is highly suitable for studying special interactions in the adsorbent layer.

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