Disparate Effects of Two Clerodane Diterpenes of Giant Goldenrod (Solidago gigantea Ait.) on Bacillus spizizenii.

New substances with antimicrobial properties are needed to successfully treat emerging human, animal, or plant pathogens. Seven clerodane diterpenes, previously isolated from giant goldenrod (Solidago gigantea) root, were tested against Gram-positive Bacillus subtilis, Bacillus spizizenii and Rhodococcus fascians by measuring minimal bactericidal concentration (MBC), minimal inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50). Two of them, Sg3a (a dialdehyde) and Sg6 (solidagoic acid B), were proved to be the most effective and were selected for further study. Bacillus spizizenii was incubated with the two diterpenes for shorter (1 h) or longer (5 h) periods and then subjected to genome-wide transcriptional analyses. Only a limited number of common genes (28 genes) were differentially regulated after each treatment, and these were mainly related to the restoration of cell membrane integrity and to membrane-related transports. Changes in gene activity indicated that, among other things, K+ and Na+ homeostasis, pH and membrane electron transport processes may have been affected. Activated export systems can be involved in the removal of harmful molecules from the bacterial cells. Inhibition of bacterial chemotaxis and flagellar assembly, as well as activation of genes for the biosynthesis of secondary metabolites, were observed as a general response. Depending on the diterpenes and the duration of the treatments, down-regulation of the protein synthesis-related, oxidative phosphorylation, signal transduction and transcription factor genes was found. In other cases, up-regulation of the genes of oxidation-reduction processes, sporulation and cell wall modification could be detected. Comparison of the effect of diterpenes with the changes induced by different environmental and nutritional conditions revealed several overlapping processes with stress responses. For example, the Sg6 treatment seems to have caused a starvation-like condition. In summary, there were both common and diterpene-specific changes in the transcriptome, and these changes were also dependent on the length of treatments. The results also indicated that Sg6 exerted its effect more slowly than Sg3a, but ultimately its effect was greater.

Antimicrobial Diterpenes from Rough Goldenrod (Solidago rugosa Mill.).

Solidago rugosa is one of the goldenrod species native to North America but has sporadically naturalized as an alien plant in Europe. The investigation of the root and leaf ethanol extracts of the plant using a bioassay-guided process with an anti-Bacillus assay resulted in the isolation of two antimicrobial components. Structure elucidation was performed based on high-resolution tandem mass spectrometric and one- and two-dimensional NMR spectroscopic analyses that revealed (-)-hardwickiic acid (Compound 1) and (-)-abietic acid (Compound 2). The isolates were evaluated for their antimicrobial properties against several plant pathogenic bacterial and fungal strains. Both compounds demonstrated an antibacterial effect, especially against Gram-positive bacterial strains (Bacillus spizizenii, Clavibacter michiganensis subsp. michiganensis, and Curtobacterium flaccumfaciens pv. flaccumfaciens) with half maximal inhibitory concentration (IC50) between 1 and 5.1 µg/mL (5-20 times higher than that of the positive control gentamicin). In the used concentrations, minimal bactericidal concentration (MBC) was reached only against the non-pathogen B. spizizenii. Besides their activity against Fusarium avenaceum, the highest antifungal activity was observed for Compound 1 against Bipolaris sorokiniana with an IC50 of 3.8 µg/mL.

Nine-dimensional bioprofiles of Tunisian sages (Salvia officinalis, S. aegyptiaca and S. verbenaca) by high-performance thin-layer chromatography - 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•), and enzyme inhibitory (α-glucosidase, acetylcholinesterase, and lipase) ones. The screening, using 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 α-glucosidase, and only c inhibited F. avenaceum. Compounds in these zones were identified by HPLC-high resolution tandem MS (LC-HRMS/MS) as rosmanol/epi-rosmanol 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 α-glucosidase enzyme. Linoleic and linolenic acids were detected in zone d, which showed strong lipase inhibition in all three sage species.

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)-direct 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 (HPTLC-EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC-mass 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 - ethyl acetate - methanol 15:3:2, V/V/V) was changed (to n-hexane - isopropyl acetate - methanol - acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1-2 and 3-4). 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 32.3-64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.

Separation and detection of apricot leaf triterpenes by high-performance thin-layer chromatography combined with direct bioautography and mass spectrometry.

Prunus armeniaca leaf extract was screened for antibacterial compounds by high-performance thin-layer chromatography (HPTLC)-direct bioautography using a Gram-positive Bacillus subtilis bacterium. Six chromatographic zones exhibited characteristic bioactivity. Five of them also appeared after derivatization with vanillin-sulfuric acid reagent and could be characterized with HPTLC-electrospray ionization (ESI)-mass spectrometry (MS), suggesting the presence of triterpenoids and the fatty acids linolenic and palmitic acid. To confirm the identification of triterpenoids an HPTLC method using in situ pre-chromatographic derivatization with iodine was developed to separate the closely related triterpenoids. After development, the iodine could be eliminated from the chromatogram (verified by HPTLC-MS), making it suitable for the B. subtilis assay. Ursolic acid, oleanolic acid, betulinic acid, corosolic acid, and maslinic acid were discovered for the first time as antibacterial components of P. armeniaca leaves. Their presence was proved also by 2D-HPTLC combined with intermediate in situ derivatization by iodine.

Acetylcholinesterase inhibitors in the giant goldenrod root.

Eight bioactive clerodane diterpenes from the root extract of Solidago gigantea Ait. (giant goldenrod) were quantified by high-performance thin-layer chromatography (HPTLC) and two newly developed hyphenated methods. One uses vanillin sulphuric acid derivatization and densitometry, and the other an inhibition assay of acetylcholinesterase (AChE) and video densitometry. Both methods gave figures of merit for quantification including 5.8-33.9 ng and 175.5-448.7 ng LOQs and 2.7-6.9 RSD% and 8.8-13.9 RSD% inter-day precisions, respectively. Based on the diterpenes' content of 14 root samples collected over a year from the same plant population, the fully flowering plant is suggested to collect the root as a source of these compounds. Excepting one diterpene (with the lowest retardation factor), the quantitative results for the richest sample obtained by the two methods were in harmony. The difference could be due to a matrix effect.

Goldenrod Root Compounds Active against Crop Pathogenic Fungi.

Root extracts of three goldenrods were screened for antimicrobial compounds. 2Z,8Z- and 2E,8Z-matricaria esters from European goldenrod (Solidago virgaurea) and E- and Z-dehydromatricaria esters from grass-leaved goldenrod (Solidago graminifolia) and first from showy goldenrod (Solidago speciosa) were identified by high-performance thin-layer chromatography combined with effect-directed analysis and high-resolution mass spectrometry or nuclear magnetic resonance spectroscopy after liquid chromatographic fractionation and isolation. Next to their antibacterial effects (against Bacillus subtilis, Aliivibrio fischeri, and Pseudomonas syringae pv. maculicola), they inhibited the crop pathogenic fungi Fusarium avenaceum and Bipolaris sorokiniana with half maximal inhibitory concentrations (IC50) between 31 and 107 µg/mL. Benzyl 2-hydroxy-6-methoxybenzoate, for the first time found in showy goldenrod root, showed the strongest antifungal effect, with IC50 of 25-26 µg/mL for both fungal strains.

Bioactive clerodane diterpenes of giant goldenrod (Solidago gigantea Ait.) root extract.

Giant goldenrod (Solidago gigantea Ait.) root extract was screened for bioactive compounds by high-performance thin-layer chromatography (HPTLC), coupled with effect-directed analysis including antibacterial (Bacillus subtilis F1276, B. subtilis subsp. spizizenii, Aliivibrio fischeri and Xanthomonas euvesicatoria), antifungal (Fusarium avenaceum) and enzyme inhibition (acetyl- and butyrylcholinesterases, α- and ß-glucosidases and α-amylase) assays. Compounds of six multipotent zones (Sg1-Sg6) were characterized by HPTLC-heated electrospray ionization-high-resolution mass spectrometry (HRMS) and HPTLC-Direct Analysis in Real Time-HRMS. Apart from zone Sg3, containing three compounds, a single characteristic compound was detectable in each bioactive zone. The bioassay-guided isolation using preparative-scale flash chromatography and high-performance liquid chromatography provided eight compounds that were identified by NMR spectroscopy as clerodane diterpenes. All isolates possessed inhibiting activity against at least one of the tested microorganisms.

High-performance thin-layer chromatography hyphenated to high-performance liquid chromatography-diode array detection-mass spectrometry for characterization of coeluting isomers.

The effect-directed analysis on a planar chromatogram allows for fast non-target screening, multi-imaging detection of effects (bioprofiling) and highly targeted characterization and isolation of bioactive compounds. For direct characterization by high-resolution mass spectrometry (HRMS), however, the orthogonal hyphenation of two different liquid chromatographic techniques (planar and column chromatography) is still underexplored. In particular, it can be helpful in case of coeluting compounds. Exemplarily, lemon balm (Melissa officinalis L.) leaf extract was analysed by high-performance thin-layer chromatography in combination with bioactivity assays for antibacterial (against the Gram-positive Bacillus subtilis and the Gram-negative Aliivibrio fischeri) and α-glucosidase-inhibitory compounds (HPTLC-UV/Vis/FLD-EDA). High-resolution mass spectra of two bioactive compound zones were directly recorded via an elution head-based interface. By HPTLC-HESI-HRMS, the compound in zone a inhibited A. fischeri and was identified as linolenic acid, whereas the two closely related constitutional isomers oleanolic acid and ursolic acid were present in zone b. This was proven by two-dimensional liquid chromatography. Heart-cutting HPTLC-UV/Vis/FLD-HPLC-DAD-MS allowed the separation of the two isomers and proved both to be present in the bioactive zone with ursolic acid at a much higher abundance.

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.

Distinction and valorization of 30 root extracts of five goldenrod (Solidago) species.

A high-performance thin-layer chromatography (HPTLC) method was developed for rapid and easy-to-perform discrimination between five goldenrod species present in Europe: the native Solidago virgaurea and the four invasive aliens, S. canadensis, S. gigantea, S. rugosa and S. graminifolia. The chemotaxonomic distinction was based on the chemical profile of their root extracts, confirmed by principal component analysis. This allowed the distinction of the goldenrods in wintertime, when classical morphological methods are not applicable. Their enzyme inhibitory profiles were determined by HPTLC combined with α-glucosidase, ß-glucosidase, α-amylase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) assays. Two compounds of S. canadensis showed the most intense enzyme inhibition in all assays, having also antibacterial activity against Bacillus subtilis, Xanthomonas euvesicatoria and Aliivibrio fischeri strains. HPTLC-high-resolution mass spectrometry (HRMS), bioassay-guided isolation, NMR spectroscopy and literature data led to the characterization and identification of the labdane diterpenes solidagenone and presolidagenone as the active S. canadensis root components. The previously identified polyacetylenes (2Z,8Z and 2E,8Z matricaria esters) of S. virgaurea, also inhibited all enzymes. Except for the known anti-AChE effect of the 2Z,8Z-matricaria ester, this is the first report on the α-glucosidase, ß-glucosidase, α-amylase, AChE and BChE inhibitory activity of these potent compounds. The anti-hyperglycemic effects of the S. canadensis labdanoids were also observed for the first time. Combined with effect-directed assays and HRMS, hyphenated HPTLC allowed an effect-directed high-throughput screening and a fast characterization of multipotent compounds. The investigation of botanicals by fast, hyphenated, bioanalytical tools substantially increased the information gain with regard to active principles (bioprofiling) and efficiently pointed to potent candidates for drug development.

Isolation and structural elucidation of a novel brunnein-type antioxidant ß-carboline alkaloid from Cyclocybe cylindracea.

Effect-directed isolation of free radical scavengers from the methanol extract of the freeze-dried fruiting bodies of the cultivated basidiomycetous mushroom, black poplar (Cyclocybe cylindracea), yielded a ß-carboline alkaloid. Its structure was determined based on ESI-TOF-MS/MS, NMR and circular dichroism spectra by comparison with published data. The compound, identified as the C1-S diastereomer of brunnein B, exhibited explicit radical scavenging activity (EC50 = 119.1 ±â€¯1.2 µg/mL). The quantity of the active component was determined with HPLC-MS in the fruiting body (36.2 ±â€¯2.8 ng/g DW, dry weight) and its different tissues such as peel (94.7 ±â€¯1.9 ng/g DW), inner cap (90.5 ±â€¯1.3 ng/g DW), gills (71.5 ±â€¯0.6 ng/g DW), and stipe (162.2 ±â€¯1.7 ng/g DW). It is a ß-carboline alkaloid that was not reported previously.

Discovered acetylcholinesterase inhibition and antibacterial activity of polyacetylenes in tansy root extract via effect-directed chromatographic fingerprints.

The knowledge about the activity of polyacetylenes was extended by their new acetylcholinesterase inhibition and antibacterial activity against plant pathogenic bacteria. For this discovery, an utmost streamlined workflow, which we consider to be of high potential in the field of natural product or superfood search was developed. It demonstrates the combined power of biological, biochemical and chemical fingerprints. Bioactive components of tansy (Tanacetum vulgare L.) root extract were profiled and identified by high-performance thin-layer chromatography hyphenated with in situ effect-directed analysis, chemical derivatizations and high-resolution mass spectrometry (HPTLC-UV/Vis/FLD-EDA-HRMS). The effect-directed profiling was performed using four bacterial bioassays including two plant pathogens, an antioxidant assay and acetyl- and butyrylcholinesterase inhibitory assays. The chromatographic, spectral and powerful mass spectrometric study of zones that exerted substantial antibacterial and/or antioxidant and/or acetylcholinesterase inhibitory effects allowed these multi-potent zones to be identified as polyacetylenes. Five polyacetylene compounds were assigned to be 2-non-1-ene-3,5,7-triynyl-3-vinyl-oxirane, 2-(2,4-hexadiynylidene)-3,4-epoxy-1,6-dioxaspiro[4.5]decane, trans- and cis-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.5]dec-3-ene and tetradeca-2,4,6-triine-8-en-12-one. This study clearly showed the advantage of the combined use of different ionization sources, i.e. electrospray ionization via an elution-head based interface and also the Direct Analysis in Real Time interface, for HRMS analysis of compounds from the same class with very similar chromatographic behavior and polarity.

Antibacterial potential of the Cistus incanus L. phenolics as studied with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis.

The main aim of this study was to detect and identify antibacterial components of fraction I derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer chromatography - direct bioautography (TLC-DB) using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid glycosides and then separate and visualize the liberated glucose and some other building blocks of the zones' components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid) to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts produced through fractionation.

Effect-directed analysis via hyphenated high-performance thin-layer chromatography for bioanalytical profiling of sunflower leaves.

High-performance thin-layer chromatography (HPTLC) coupled with effect-directed analysis was used for non-targeted screening of sunflower leaf extract for components exhibiting antioxidant, antibacterial and/or cholinesterase enzyme inhibitory effects. The active compounds were characterized by HPTLC-electrospray ionization-high resolution mass spectrometry (ESI-HRMS) and HPTLC-Direct Analysis in Real Time (DART)-MS/MS. The latter ambient ionization technique (less soft than ESI) resulted in oxidation and fragmentation products and characteristic fragment ions. NMR spectroscopy after targeted isolation via preparative normal phase flash chromatography and semi-preparative reversed phase high-performance liquid chromatography supported the identification of two diterpenes to be (-)-kaur-16-en-19-oic acid and 15-α-angeloyloxy-ent-kaur-16-en-19-oic acid. Both compounds found to be multi-potent as they inhibited acetylcholinesterase and butyrylcholinesterase and showed antibacterial effects against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri bacteria. Kaurenoic acid was also active against the Gram-negative pepper pathogenic Xanthomonas euvesicatoria bacteria.

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.

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.

Detection and Identification of Antibacterial and Antioxidant Components of Essential Oils by TLC-Biodetection and GC-MS.

Components of cinnamon bark, rosemary, clove and thyme essential oils were screened for antioxidant and antibacterial activity utilizing thin-layer chromatography (TLC) coupled with the DPPH(.) test and direct bioautography using Bacillus subtilis cells. The compounds in the active chromatographic zones were identified by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) after their elution. Seven antibacterial components were found: cinnamaldehyde and eugenol in cinnamon bark oil, 1,8-cineole, camphor, borneol and α-terpineol in rosemary oil, eugenol in clove oil and thymol in thyme oil. Only two of them, thymol and eugenol displayed a free radical scavenging effect.

Tracking and identification of antibacterial components in the essential oil of Tanacetum vulgare L. by the combination of high-performance thin-layer chromatography with direct bioautography and mass spectrometry.

Two tansy (Tanacetum vulgare L.) essential oils were obtained by steam distillation of the capitula with subsequent liquid-liquid extraction (oil 1) or with use of an auxiliary phase for the trapping of the steam components (oil 2). These oils were investigated against Bacillus subtilis F1276, B. subtilis spizizenii (DSM 618), Xanthomonas euvesicatoria, Pseudomonas syringae pv. maculicola, Ralstonia solanacearum strain GMI1000 and Aliivibrio fischeri, using the coupling of high-performance thin-layer chromatography to direct bioautography (HPTLC-DB). Using this method with the potato and tomato pathogen R. solanacearum is shown for the first time. Due to the advanced extraction process, oil 2 was richer in components and provided more inhibition zones. The main bioactive components were identified by scanning HPTLC-Direct Analysis in Real Time mass spectrometry (HPTLC-DART-MS) and solid-phase microextraction gas chromatography electron impact MS (SPME-GC-EI-MS) as cis- and trans-chrysanthenol as well as trans-chrysanthenyl acetate. cis-Chrysanthenol exhibited antibacterial effects against all tested bacteria, whereas trans-chrysanthenol inhibited B. subtilis, R. solanacearum and A. fischeri. trans-Chrysanthenyl acetate was an inhibitor for X. euvesicatoria, R. solanacearum and A. fischeri. Although HPTLC-DART-MS resulted in a comparable fragmentation, the ionization characteristics and the recorded mass spectra clearly showed that DART is a softer ionization technique than EI. It is also more affected by ambient conditions and thus prone to additional oxidation products.

Direct Bioautography as a High-Throughput Screening Method for the Detection of Antibacterial Components from Plant Sources.

The applicability of direct bioautography, the combination of planar chromatography with antimicrobial assay, is demonstrated with special emphasis on its recent developments such as BioArena and the use of genetically modified luminescent bacteria. Its methodological advancement is put into a historical perspective. In comparison with other commonly used antimicrobial susceptibility tests, the main advantage of direct bioautography resides in its simplicity, rapidity, and ability to detect separated individual matrix components exhibiting antimicrobial activity in situ. It is confirmed with examples that high-throughput direct bioautography is suitable as a biomonitoring-screening system for bioassay-guided isolation.