371 research outputs found
Separation and identification of antibacterial chamomile components using OPLC, bioautography and GC-MS
Components of 50% ethanolic chamomile (Matricaria recutica L.) flower extract, previously found antibacterial in a TLC-bioautographic study, were separated and isolated by the use of on-line OPLC, which consisted of an OPLC 50 BS system, an on-line coupled flow-through UV detector, and a manual fraction collector. The collected peaks were investigated by GC-MS analysis and by TLC re-chromatography with subsequent visualization, performed after use of the vanillin-sulphuric acid reagent, or under UV illumination, or applying bioautographic detection. The main compounds of the collected 11 fractions were identified by GC-MS. The results showed that the antibacterial effect of 50% ethanolic extract of chamomile is ascribable to cis-, trans-spiroethers, and the coumarins like herniarin and umbelliferone
In-situ Clean-up and OPLC Fractionation of Chamomile Flower Extract Searching Active Components by Bioautography
Bioassay-guided isolation of antibacterial components of chamomile flower methanol extract was performed by OPLC with on-line detection, fractionation combined with sample clean-up in-situ in the adsorbent bed after sample application. The antibacterial effect of the fractions and the separated compounds remained on the adsorbent layer (do not overrun during OPLC separation) was tested with direct bioautography (DB) against the bioluminescent Pseudomonas savastanoi pv. maculicola and Vibrio fischeri. The fractions with great biologically activity were analysed by SPME-GC-MS and LC-MS/MS and the two active uneluted compounds were characterized by OPLC-MS using interface. Mainly essential oil components, coumarins, flavonoids, phenolic acids and fatty acids were identified in the fractions
Az ĂĄltalunk leĂrt ĂĄltalĂĄnos növĂ©nyi rezisztencia patolĂłgiai, biokĂ©miai Ă©s molekulĂĄris mechanizmusĂĄnak tovĂĄbbi feltĂĄrĂĄsa = Further investigation of the pathological, biochemical and molecular mechanisms of the general plant resistance described by our group
A növĂ©nyi ĂĄltalĂĄnos rezisztencia (BR) a leggyorsabb vĂ©dekezĂ©si vĂĄlaszrendszer mikrobiĂĄlis fertĆzĂ©sre. Mi folytattuk a BR kutatĂĄsi hagyomĂĄnyainkat, immĂĄr molekulĂĄris szinten is. A BR erĆsen fĂŒggött bizonyos környezeti tĂ©nyezĆktĆl, elsĆsorban a hĆmĂ©rsĂ©klettĆl. A proteomikai vizsgĂĄlatok szĂĄmos Ășj fehĂ©rjĂ©t mutattak ki a sejtfalbĂłl, mely a baktĂ©riumokkal szembeni harc szĂntere. Egy BR-hez kapcsolhatĂł kitinĂĄzt sikerĂŒlt klĂłnozni, rĂ©szlegesen tisztĂtani Ă©s jellemezni, fĆleg ami baktĂ©rium-eredetƱ szubsztrĂĄt (pl. baktĂ©rium setfal összetevĆk) bontĂĄsi kĂ©pessĂ©geit illeti. A kitinĂĄz a BR egyik valĂłszĂnƱ effektora. A baktĂ©rium vizsgĂĄlatokban fĂ©ny derĂŒlt pl. arra, hogy a BR gĂĄtolja a baktĂ©riumok patogenitĂĄsi gĂ©njeinek kifejezĆdĂ©sĂ©t. SzubtraktĂĄlt cDNS könyvtĂĄr Ă©s a klĂłnok segĂtsĂ©gĂ©vel kĂ©szĂŒlt chippel sikerĂŒlt a dohĂĄny levĂ©lben nagyszĂĄmĂș BR alatt aktivĂĄlĂłdĂł gĂ©nt azonosĂtani (176 db), tovĂĄbbĂĄ nagy szĂĄmĂș gĂ©nt tartalmazĂł chip segĂtsĂ©gĂ©vel az eredmĂ©nyeket megerĆsĂteni, kiszĂ©lesĂteni. A megvĂĄltozott aktivitĂĄst mutatĂł azonosĂtott gĂ©nek funkciĂłi növĂ©nyi folyamatok szĂ©les körĂ©t Ă©rintik. A BR Ă©s mĂĄs biotikus Ă©s abiotikus stresszek közötti ĂĄtfedĂ©seket figyeltĂŒnk meg a gĂ©nkifejezĆdĂ©s szintjĂ©n. A jelĂĄtviteli folyamatok Ă©s a fenilpropanoid szintĂ©zis szerepĂ©t rĂ©szletesebben is tanulmĂĄnyoztuk. VizsgĂĄlataink szerint egyes szignĂĄl utak (pl. foszfolipĂĄz A Ă©s C Ă©s a foszforilĂĄciĂł) fontos szerepet töltenek be a BR szabĂĄlyozĂĄsĂĄban. A fenilpropanoid Ășt gĂĄtlĂĄsa a BR kifejlĆdĂ©sĂ©t gyengĂtette. | Basal resistance (BR) of plants is the quickest defence response to microbial infection. We continued our traditions of BR research, now on the molecular level as well. BR was heavily dependent on some environmental factors, especially temperature. Proteomics revealed several novel proteins from the cell wall, which is the playground for fighting bacteria. We succeeded in cloning, partially purifying and characterizing an EBR-related chitinase, with a focus on its ability to degrade substrates of bacterial origin (e.g. bacterial cell wall constituents). This chitinase probably represents an effector of BR. Studies on the bacterial side showed for example, that BR inhibits expression of bacterial pathogenicity genes. With a gene chip based on a subtraction cDNA library an clones we could identify a large number (176) of genes activated under BR and assert and widen these data using a high density gene array. The functions of genes with modified expression cover a wide range of physiological processes. On the transcriptional level we observed an overlap between BR, other biotic and abiotic stresses. Roles of certain signal transduction pathways and the phenylpropanoid synthesis pathway has been studied in more detail. Results indicated that signalling routes like phospholipase A, C and phosphorylation are important for controlling BR. Inhibition of the phenylpropanoid pathway attenuated development of BR
Thin-layer chromatographic quantification of magnolol and honokiol in dietary supplements and selected biological properties of these preparations
Two isomeric biphenyl neolignans, magnolol and honokiol, are considered as constituents responsible for the healing effect of magnolia bark, a traditional Oriental medicine. To survey the increasing number of dietary supplements that contain magnolia bark or its extract, an affordable quantitative thin-layer chromatography (TLC) âdensitometry method was developed. The methanol extracts were analyzed on the silica gel plates after manual sample application using n -hexane âethyl acetate âethanol (16:3:1, v/v/v) as a mobile phase. For quantitation, the chromatograms were scanned in the absorbance mode at the wavelength λ= 290 nm. The limits of detection and quantitation were 90 and 280 ng/zone for magnolol and 70 and 200 ng/zone for honokiol, respectively. None of the two targeted neolignans were detected in two of the six analyzed supplements. In the other four samples, the measured amounts were between 0.95-114.69 mg g â1 for magnolol and 4.88-84.86 mg g â1 for honokiol. Moreover, separations of these two neolignans on the TLC and high-performance TLC (HPTLC) layers were compared and HPTLC was combined with antioxidant (DPPH) and antibacterial ( Bacillus subtilis and Aliivibrio fischeri ) assays and mass spectrometry (MS), using the elution-based interface. Both magnolol and honokiol exhibited effects in all bioactivity assays. The HPTLC-MS tests confirmed purity of neolignan zones in the extracts of dietary supplements and supported tentative identification of the alkaloid piperine and the isoflavone daidzein as additional bioactive components of the investigated dietary supplements. Using the same mobile phase in the orthogonal directions 2D-HPTLC-MS experiments proved degradation, i.e., instability of magnolol and honokiol on the silica gel adsorbent
Nine-Dimensional Bioprofiles of Tunisian Sages (Salvia officinalis, S. aegyptiaca and S. verbenaca) by High-Performance Thin-Layer Chromatography â Effect-Directed Analyses
Ethyl acetate extracts of Tunisian Salvia aegyptiaca and S. verbenaca aerial parts and S. officinalis leaves were examined via bioanalytical profiling using high-performance thin-layer chromatography (HPTLC) combined with nine bioactivity assays, namely antibacterial (Aliivibrio fischeri, Bacillus subtilis, and Rhodococcus fascians), antifungal (Bipolaris sorokiniana, and Fusarium avenaceum), radical scavenging (DPPH center dot), and enzyme inhibitory (alpha-glucosidase, acetylcholinesterase, and lipase) ones. The screening, us-ing toluene -ethyl acetate - methanol 6:3:0.5 (V/V/V) as a mobile phase, revealed five bioactive zones (a-e) that were analyzed by HPTLC-electrospray ionization-mass spectrometry (ESI-MS). Zones b and c, observed exclusively in S. officinalis, were active in all assays except alpha-glucosidase, and only c inhib-ited F. avenaceum. Compounds in these zones were identified by HPLC-high resolution tandem MS (LC-HRMS/MS) as rosmanol/epirosmanol and methyl carnosate, respectively. In the bioactive zones a and e, corosolic/maslinic acid and ursolic/oleanolic acid isomer pairs were present, which could be identified in all three Salvia species after their HPTLC separation using pre-chromatographic derivatization with iodine and MS detection. The triterpenes inhibited B. subtilis and R. fascians bacteria and alpha-glucosidase enzyme. Linoleic and linolenic acids were detected in zone d, which showed strong lipase inhibition in all three sage species
Bioassay-guided isolation and identification of antimicrobial compounds from thyme essential oil by means of overpressured layer chromatography, bioautography and GC-MS
A simple method is described for efficient isolation of compounds having an antibacterial effect.
Two thyme (Thymus vulgaris) essential oils, obtained from the market, were chosen as
prospective materials likely to feature several bioactive components when examined by thin layer
chromatography coupled with direct bioautography as a screening method. The newly developed
infusion overpressured layer chromatographic separation method coupled with direct
bioautography assured that only the active components were isolated by means of overrun
overpressured layer chromatography with online detection and fractionation. Each of the 5
collected fractions represented one of the five antimicrobial essential oil components designated
at the screening. The purity and the activity of the fractions were confirmed with chromatography
coupled various detection methods (UV, vanillin-sulphuric acid reagent, direct bioautography).
The antibacterial components were identified with GC-MS as thymol, carvacrol, linalool, diethylphthalate,
and alpha-terpineol. The oil component diethyl-phthalate is an artificial compound,
used as plasticizer or detergent bases in the industry. Our results support that exploiting its
flexibility and the possible hyphenations, overpressured layer chromatography is especially
attractive for isolation of antimicrobial components from various matrixes
High-performance thin-layer chromatography - antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.)
The present work introduces a high-performance thin-layer chromatography (HPTLC)âdirect bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLCâEDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLCâmass spectrometry (MS), and bioassay-guided column chromatographic
(preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform â ethyl acetate â
methanol 15:3:2, V/V/V) was changed (to n-hexane â isopropyl acetate â methanol â acetic acid 29:20:1:1,
V/V/V/V) to achieve the separation of the closely related isomer pairs (1â2 and 3â4). Compounds 1 and
3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R.
fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC50 ) values
in the range of 32.3â64.4 ÎŒg/mL. The mass spectrometric fragmentation of the isolated compounds was
interpreted and their previously published NMR assignments lacking certain resonances were completed
Study of trace elements in BioArena system and in in vivo conditions
The adsorbent layer system is especially suitable for the biological evaluation of different compounds and trace elements as well. Present experiments showed that formaldehyde (HCHO) molecules participate in the antibiotic activity of Cu (II) ion, an âold antibioticâ. The elimination of HCHO from the chromatographic spots (e.g. by reduction or capturing) resulted in a characteristic decrease of the antibiotic effect of trace elements. The trace elements are HCHO carriers and generate a double effect (first step: deprivation of HCHO as also biological effect; second step: release of HCHO with big killing activity). These features offer good opportunities for influencing fundamental biochemical pathways. It has been established that the trace elements (mainly transition metal ions as e.g. Ni(II) ion) always generate quadruple, bioequivalent, specific immune-stimulating activity in plants with a non-linear dose-response. HCHO and its reaction products (mainly O3) are responsible also for this latter activity
Layer chromatography-bioassays directed screening and identification of antibacterial compounds from Scotch thistle
The antibacterial profiling of Onopordum acanthium L. leaf extract and subsequent targeted identification of active compounds is demonstrated. Thin-layer chromatography (TLC) and off-line overpressured layer chromatography (OPLC) coupled with direct bioautography were utilized for investigation of the extract against eight bacterial strains including two plant and three human pathogens and a soil, a marine and a probiotic human gut bacteria. Antibacterial fractions obtaining infusion-transfusion OPLC were transferred to HPLC-MS/MS analysis that resulted in the characterization of three active compounds and two of them were identified as, linoleic and linolenic acid. OPLC method was adopted to preparative-scale flash chromatography for the isolation of the third active compound, which was identified after a further semi-preparative HPLC purification as the germacranolide sesquiterpene lactone onopordopicrin. Pure onopordopicrin exhibited antibacterial activity that was specified as minimal inhibitory concentration in the liquid phase as well
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