Overpressured layer chromatography: from the pressurized ultramicro chamber to BioArena system

The pressurized ultramicro (UM) chamber as a closed adsorbent layer chamber enables the use of a special chromatoplate and a pump to increase and optimize the mobile phase flow velocity through an optional development distance in an adsorbent layer. This chamber is the basic instrument of overpressured-layer chromatography (OPLC), which is a separation technique that combines the advantages of conventional TLC/HPTLC with those of HPLC. The versions of OPLC instrument, the character and achievement of off-line and on-line OPLC systems in analytical and preparative use are described. The development of BioArena as a complex bioautographic system means an exploitation of the unique advantages of planar-layer system for detection, isolation and identification of new antimicrobials, antineoplastics, biopesticides and other biologically active substances as well as for studying fundamental biochemical reactions and mechanisms

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

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

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

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) –densitometry method was developed. The methanol extracts were analyzed on the silica gel plates after manual sample application using n -hexane –ethyl acetate –ethanol (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 −1 for magnolol and 4.88-84.86 mg g −1 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

Detection of antibacterial activity of essential oil components by TLC-bioautography using luminescent bacteria

The aim of the present study was the chemical characterization of some medically relevant essential oils (tea tree, clove, cinnamon bark, thyme and eucalyptus) and the investigation of antibacterial effect of the components of these oils by use of a direct bioautographic method. Thin layer chromatography (TLC) was combined with biological detection in this process. The chemical composition of the oils was determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Eucalyptol (84.2%) was the main component of the essential oil of eucalyptus, eugenol (83.7%) of clove oil, and trans-cinnamic aldehyde (73.2%), thymol (49.9%) and terpinen-4-ol (45.8%) of cinnamon bark, thyme and tea tree oils, respectively. Antibacterial activity of the separated components of these oils, as well as their pure main components (eucalyptol, eugenol, trans-cinnamic aldehyde and thymol) was observed against the Gram-negative luminescence tagged plant pathogenic bacterium Pseudomonas syringae pv. maculicola (Psmlux) and the Gram-negative, naturally luminescent marine bacterium Vibrio fischeri. On the whole, the antibacterial activity of the essential oils could be related to their main components, but the minor constituents may be involved in this process. Trans-cinnamic aldehyde and eugenol were the most active compounds in TLC-bioautography. The sensitivity of TLC-bioautographic method can be improved with using luminescent test bacteria. This method is more cost-effective and provides more reliable results in comparison with conventional microbiological methods, e.g. disc-diffusion technique

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

Application of direct bioautography and SPME-GC-MS for the study of antibacterial chamomile ingredients

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

Does The Glucocorticoid Stress Response Make Toads More Toxic? An Experimental Study on The Regulation of Bufadienolide Toxin Synthesis

Chemical defense is a crucial component of fitness in many organisms, yet the physiological regulation of defensive toxin synthesis is poorly understood, especially in vertebrates. Bufadienolides, the main defensive compounds of toads, are toxic to many predators and other natural enemies, and their synthesis can be upregulated by stressors, including predation risk, high conspecific density, and pollutants. Thus, higher toxin content may be the consequence of a general endocrine stress response in toads. Therefore, we hypothesized that bufadienolide synthesis may be stimulated by elevated levels of corticosterone (CORT), the main glucocorticoid hormone of amphibians, or by upstream regulators that stimulate CORT production. To test these alternatives, we treated common toad tadpoles with exogenous CORT (exoCORT) or metyrapone (MTP, a CORT-synthesis inhibitor that stimulates upstream regulators of CORT by negative feedback) in the presence or absence of predation cues for 2 or 6 days, and subsequently measured their CORT release rates and bufadienolide content. We found that CORT release rates were elevated by exoCORT, and to a lesser extent also by MTP, regardless of treatment length. Bufadienolide content was significantly decreased by treatment with exoCORT for 6 days but was unaffected by exposure to exoCORT for 2 days or to MTP for either 6 or 2 days. The presence or absence of predation cues affected neither CORT release rate nor bufadienolide content. Our results suggest that changes in bufadienolide synthesis in response to environmental challenges are not driven by CORT but may rather be regulated by upstream hormones of the stress response

Chemical defence effective against multiple enemies: Does the response to conspecifics alleviate the response to predators?

1. Animals living in groups with high conspecific densities typically decrease their level of plastic anti-predatory defence because its benefits diminish with reduced per capita predation risk (a benefit of aggregation), whereas its costs increase due to intensifying competition and increased infection risk. Furthermore, phenotypic responses that provide protection from predators are also often disadvantageous against competitors and infections. 2. Such a trade-off may be absent when the same phenotype provides an effective defence against both predators and competitors, as is the case with some chemical defences. For such multifunctional defensive traits, both predation risk and high conspecific density are expected to increase defence expression while exposure to both predators and conspecifics may result in non-additive effects whereby the defence level induced by two enemies is lower than the sum of responses induced by either of them alone. 3. We tested this theoretical prediction by studying the effects of multiple enemies on chemical defence in a vertebrate animal. We investigated patterns of change in toxin production of common toad Bufo bufo tadpoles following exposure to different conspecific densities and the simultaneous presence or absence of chemical cues on predation risk. 4. We found that tadpoles significantly increased their production of bufadienolide toxins in response to high tadpole density, as well as to predation risk when tadpole density was low. Although the response in bufadienolide production to predation risk was not significant at high tadpole density, the magnitude of anti-predatory response did not differ significantly between low and high tadpole densities. 5. These results show that toad tadpoles adjust their chemical defence to conspecific density and to predation risk simultaneously, and these two effects are more likely additive than non-additive, at least within the range of densities and predation-risk levels studied here. Nevertheless, the trend we found suggests that toxin levels induced by very high conspecific density might weaken the chemical response to predators, which is relevant for the evolutionary ecology of chemical defences, as well as for the conservation of fauna impacted by toxic invaders. A free Plain Language Summary can be found within the Supporting Information of this article