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L. Botz, S. Nagy, B. Kocsis , and Gy. Horváth , Planar Chromatographic Aspects of Direct Bioautography, in Sz. Nyiredy (Ed.) Proc. Int. Symp. Planar Separations — Planar Chromatography 2000, Hungary, June 2000, pp. 77

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The isolation and characterization of antibacterial chamomile components were performed by the use of direct bioautography and solid phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS). Four ingredients, active against Vibrio fischeri, were identified as the polyacetylene geometric isomers cisand trans-spiroethers, the coumarin related herniarin, and the sesquiterpene alcohol (−)-alpha-bisabolol.

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Fluoroquinolones are a relatively new group of synthetic antibiotics (chemotherapeutics) widely used both in human and animal treatment. Because veterinary enrofloxacin and its main metabolite ciprofloxacin, a widely used human antibiotic, can be present as residues in milk, there is a need for methods for their separation and determination. Matrix solid-phase dispersion (MSPD) on a siliceous sorbent was used for isolation and concentration of enrofloxacin and ciprofloxacin residues from cows’ milk. This preseparation method was combined with high-performance liquid chromatography and with thin-layer chromatography-direct bioautography. Mean recoveries were calculated for different levels of the antibiotics in milk. The results revealed that thin-layer chromatography-direct bioautography can be used for the screening of enrofloxacin and ciprofloxacin at the maximum residue level stipulated for milk by the European Union.

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The main volatile compounds from three medicinal plants belonging to Lamiaceae family were screened for their biological properties. The plants were Salvia officinalis, Thymus vulgaris, and Mentha × piperita containing as the main volatile constituents thujone, thymol, and menthol, respectively. The applied chromatographic system was silica gel developed with toluene-ethyl acetate (93:7). Thin-layer chromatography — direct bioautography (TLC-DB) against Escherichia coli and Bacillus subtilis was used for detection of antibacterial activity of the plant extracts and essential oils. The bioautographic fingerprints were compared with the fingerprints obtained after derivatization with anisaldehyde.

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Certain classes of bioactive compounds can be separated using planar chromatography. Some biological effects (e.g. antibacterial) of these compounds can be investigated directly by examining the growth of a test organism on a specially treated sorbent of thin layer chromatography (TLC). A special method of detection, direct bioautography, is suitable for studying the antimicrobial activity of plant extracts of natural origin by using TLC. Zones of inhibition are visualised by use of a dehydrogenase-activity-detecting, tetrazol-type reagent. Zones of inhibition appeared as pale spots separating well from the dark background. The antibacterial effect of the main essential oil components of some Thymus taxa, as well as that of two antibiotics (streptomycin sulphate and gentamycin) known and applied in practice was investigated against plant pathogenic bacteria. Results showed that thyme essential oil and its components inhibited the growth of test bacteria, but not so considerably as the antibiotics applied. Compositions of the essential oils were analysed by gas chromatography (GC). It could be verified that among the essential oil components, thymol and carvacrol had the strongest inhibitory effect.

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Summary

Schizonepeta annua (Pall.) Schischk. is an endemic annual plant from the Lamiaceae family and it has been employed to cure tracheitis in traditional herbal medicine. Its essential oil exhibited a strong antimicrobial and antioxidative effect. Next, high-performance thin-layer chromatography-bioautography was applied for investigation of the bioactive compounds of S. annua, and gas chromatography-quadrupole time-of-flight mass spectrometry was used to perform subsequent targeted identification of compounds. Three active components were characterized, and two of them were tentatively identified as thymol and carvacrol. S. annua has the potential to be a good alternative for synthetic disinfectants and antioxidants.

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In this study, thin-layer chromatography—direct bioautography (TLC—DB) was used for guiding the isolation and identification of antibacterial constituents of Thymus vulgaris L. ethanol extract. This TLC—bioassay method enables the separation and detection of active components directly on the surface of chromatographic plates. They can be identified by comparison with reference substances or using physicochemical methods, preferably spectroscopic ones (liquid chromatography—tandem mass spectrometry [LC—MS/MS], in the presented paper). The described method belongs to the effect-directed analyses (EDA). Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, including methicillin-resistant Staphylococcus aureus as well as luminescent bacteria like Aliivibrio fischeri. Five fractions with the widest antimicrobial spectra were detected using TLC—DB, isolated by semi-preparative TLC and subjected to LC—MS/MS analyses. Finally, two bioactive components were tentatively identified, basing on their fragmentation pattern, as eriodictyol and 4,4′-dihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-bifenylodion.

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Thin-layer chromatography—direct bioautography (TLC—DB) followed by liquid chromatography—tandem mass spectrometry (LC—MS/MS) was used for screening and tentative identification of the antibacterial constituents of Salvia officinalis L. ethanol extract. Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, that is, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, luminescence gene-tagged Pseudomonas syringae pv. maculicola, and naturally luminescent marine bacterium Aliivibrio fischeri. Eight fractions with the widest antimicrobial spectrum were detected using TLC—DB, isolated by semi-preparative TLC, and subjected to LC—MS/MS analyses. Finally, five bioactive components were tentatively identified, based on their fragmentation pattern, such as salvigenin, cirsimaritin, rosmanol, carnosic acid, and 12-O-methyl carnosic acid.

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Thin-layer chromatography with microbiological detection (direct bioautography) of amphotericin B has never been reported. The combination of these methods can be used advantageously, especially when not only chemical identification of samples is required, but also when antifungal activity is of interest. In this paper a fast and easy-to-perform method is introduced in which major ( R F 0.46) and minor ( R F 0.31) components can be separated from amphotericin B, which itself is not a homogenous substance but mixture of polyenes. Thin-layer chromatography is performed on silica gel layers with chloroform-methanol-borate buffer 4:5:1 ( v/v ) as optimized mobile phase, and the microbiological activity of amphotericin B can be measured sensitively by direct bioautography. Candida albicans (ATCC 90028) and Saccharomyces cerevisiae (ATCC 9763) fungus strains were tested. Among the detection methods investigated, direct bioautography with Candida albicans proved to be the most sensitive, with a detection limit of 0.8 ng per spot. For densitometric evaluation of plates with (385 nm) ten times more substance is required, and with a UV lamp (366 nm) as much as 50 ng AmB per spot is needed to visualize the main component.

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Commercially available hop pellets of different origins were extracted by use of ethanol and water, chromatographed on silica layers by use of nonaqueous eluents, chemically derivatized and observed in ultraviolet (UV) light for the localization of component bands. The plates were developed in optimized systems, and direct bioautographic method by use of Bacillus subtilis and Escherichia coli strains was applied for the examination of the antimicrobial activities of hop components. The method enables for the identification of bactericidal/bacteriostatic components in the extracts of different polarities and shows differences in the composition of extracts from various varieties from an antimicrobial point of view.

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