Dániel Krüzselyi

Effect-Directed Discovery of Bioactive Compounds Followed by Highly Targeted Characterization, Isolation and Identification, Exemplarily Shown for Solidago virgaurea

A nontargeted, effect-directed screening (bioprofiling) and a subsequent highly targeted characterization of antibacterial compounds from plant matrices is demonstrated on the example of Solidago virgaurea root extracts. The procedure comprises high-performance thin-layer chromatography (HPTLC) coupled with six bacterial bioassays including two plant pathogens, a radical scavenging assay, an acetylcholinesterase assay as well as in situ and ex situ mass spectrometric analyses. In situ mass spectra were directly recorded from the adsorbent using the Direct Analysis in Real Time interface (HPTLC-DART-MS), whereas ex situ mass spectra were recorded using an elution head-based interface (HPTLC-ESI-MS). For further bioassay-guided isolation of the main antimicrobial compounds, flash chromatographic fractionation and semipreparative high-performance liquid chromatographic purification were used and nuclear magnetic resonance data allowed the identification of the unknown antimicrobial compounds as 2Z,8Z- and 2E,8Z-matricaria esters. The discovered antibacterial activity was confirmed and specified by a luminometric assay and as minimal inhibitory concentration in the liquid phase.

Variation in Chemical Defense Among Natural Populations of Common Toad, Bufo bufo, Tadpoles: the Role of Environmental Factors

Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles’ toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.

Age- and environment-dependent changes in chemical defences of larval and post-metamorphic toads

BackgroundChemical defences are widespread in animals, but how their production is adjusted to ecological conditions is poorly known. Optimal defence theory predicts that inducible defences are favoured over constitutive defences when toxin production is costly and the need for it varies across environments. However, if some environmental changes occur predictably (e.g. coupled to transitions during ontogeny), whereas others are unpredictable (e.g. predation, food availability), changes in defences may have constitutive as well as plastic elements. To investigate this phenomenon, we raised common toad (Bufo bufo) tadpoles with ad libitum or limited food and in the presence or absence of chemical cues on predation risk, and measured their toxin content on 5 occasions during early ontogeny.ResultsThe number of compounds showed limited variation with age in tadpoles and was unaffected by food limitation and predator cues. The total amount of bufadienolides first increased and later decreased during development, and it was elevated in young and mid-aged tadpoles with limited food availability compared to their ad libitum fed conspecifics, whereas it did not change in response to cues on predation risk. We provide the first evidence for the active synthesis of defensive toxin compounds this early during ontogeny in amphibians. Furthermore, the observation of increased quantities of bufadienolides in food-restricted tadpoles is the first experimental demonstration of resource-dependent induction of elevated de novo toxin production, suggesting a role for bufadienolides in allelopathy.ConclusionsOur study shows that the evolution of phenotypic plasticity in chemical defences may depend on the ecological context (i.e. predation vs. competition). Our results furthermore suggest that the age-dependent changes in the diversity of toxin compounds in developing toads may be fixed (i.e., constitutive), timed for the developmental stages in which they are most reliant on their chemical arsenal, whereas inducible plasticity may prevail in the amount of synthesized compounds.

Chronic exposure to a glyphosate-based herbicide makes toad larvae more toxic

Chemical pollutants can exert various sublethal effects on wildlife, leading to complex fitness consequences. Many animals use defensive chemicals as protection from predators and diseases, yet the effects of chemical contaminants on this important fitness component are poorly known. Understanding such effects is especially relevant for amphibians, the globally most threatened group of vertebrates, because they are particularly vulnerable to chemical pollution. We conducted two experiments to investigate how exposure to glyphosate-based herbicides, the most widespread agrochemicals worldwide, affects the production of bufadienolides, the main compounds of chemical defence in common toads (Bufo bufo). In both experiments, herbicide exposure increased the amount of bufadienolides in toad tadpoles. In the laboratory, individuals exposed to 4 mg a.e./L glyphosate throughout their larval development had higher bufadienolide content at metamorphosis than non-exposed tadpoles, whereas exposure for 9 days to the same concentration or to 2 mg a.e./L throughout larval development or for 9 days had no detectable effect. In outdoor mesocosms, tadpoles from 16 populations exhibited elevated bufadienolide content after three-weeks exposure to both concentrations of the herbicide. These results show that pesticide exposure can have unexpected effects on non-target organisms, with potential consequences for the conservation management of toxin-producing species and their predators.

Separation and Detection of Bioactive Essential Oil Components by Overpressured Layer Chromatography Coupled with Bioactivity Tests

Overpressured layer chromatography (OPLC), ensuring pumpforced constant mobile phase flow and the possibility of overrun, offers the expanded exploitation of fine-particle adsorbent layers for a longer development distance. Using an infusion—transfusion OPLC method with a 26-cm long development, the separation of clove, rosemary, eucalyptus, tea tree, spearmint, thyme, and cinnamon bark essential oil components was achieved with good resolutions. In the combination of OPLC and Aliivibrio fischeri assay, the main essential oil components eugenol, borneol, (−)-R-carvone, thymol, and trans-cinnamaldehyde exhibited antibacterial effect. The OPLC—2,2-diphenyl-1-picrylhydrazyl (DPPH*) test showed two antioxidant components: eugenol and thymol.

Investigation of antibacterial components of button mushroom (Agaricus bisporus) by direct bioautography and HPLC–DAD–MS

ABSTRACT Agaricus bisporus (button mushroom) is one of the most consumed edible mushrooms in the world. The components of its methanol and ethyl acetate extracts were tested for their ability to inhibit the growth of a soil bacterium Bacillus subtilis utilizing direct bioautography (thin-layer chromatography (TLC) coupled with bioassay). The active components separated were also characterized by different chemical reagents. The major antibacterial compound was eluted from the layer and further investigated by the use of high-performance liquid chromatography coupled with photodiode array detector and mass spectrometer (HPLC-DAD-ESI/MS) and identified as linoleic acid by comparison of retention time and mass spectra with authentic standards.

Rapid, Bioassay-Guided Process for the Detection and Identification of Antibacterial Neem Oil Compounds

Bioassay guidance was used along the whole process including method development, isolation and identification of antibacterial neem (Azadirachta indica) oil compounds. The biomonitoring was performed by direct bioautography (DB), a combination of thin-layer chromatography (TLC) and antimicrobial detection. DB of neem oil showed one antibacterial zone that was not UV-active; therefore, the TLC separation was improved under DB control. The chromatographic zone that exhibited activity against Bacillus subtilis, Xanthomonas euvesicatoria, Aliivibrio fischeri, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus was characterized by TLC reagents, indicating a lipophilic, fatty acid-like chemical feature. Two compounds were found and identified in the active zone by high-performance liquid chromatography-electrospray ionization mass spectrometry as linoleic and oleic acids. Both fatty acids inhibited B. subtilis, but A. fischeri was sensitive only against linoleic acid.

Bioassay-guided detection and identification of an antibacterial compound from greater burdock

In this study, the antibacterial profiling of the ethanolic leaf extract of greater burdock (Arctium lappa L.) is demonstrated, applying thin-layer chromatography (TLC) coupled bioassays against the Gram-positive soil bacterium Bacillus subtilis and the Gram-negative pepper pathogen Pseudomonas syringae pv. maculicola. The main active component was isolated by eluting from the adsorbent bed and subjected to a targeted characterization by high-performance liquid chromatography—diode array detection—electrospray ionisation—mass spectrometry. The identification of the germacranolide sesquiterpene lactone onopordopicrin was based on its retardation factor, bioactivity in TLC-based methods, and retention tim as well as ultraviolet (UV) and mass spectra, compared to those of the reference substance isolated earlier in our laboratory from Onopordum acanthium leaf.

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.