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.

The Arabidopsis thaliana non-specific phospholipase C2 is involved in the response to Pseudomonas syringae attack.

Background and Aims: The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. Methods: The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. Key Results: The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. Conclusions: This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.

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.

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.

TLC-Direct Bioautography and LC/MS as Complementary Methods in Identification of Antibacterial Agents in Plant Tinctures from the Asteraceae Family.

Matricaria recutita L. (chamomile) and Achillea millefolium L. (yarrow) are very common herbs growing in meadows, pathways, crop fields, and home gardens. Preparations from these plants, e.g., infusions or alcohol extracts, are widely used as remedies. Both chamomile and yarrow have anti-inflammatory, analgesic, antimicrobial, and antioxidant properties. Most microbiological assays used today give information only on activity of whole extracts and do not provide information on the composition and activity of individual components. This problem can be solved by using TLC with direct microbiological detection, i.e., TLC-direct bioautography (TLC-DB), followed by LC/MS of active fractions. The aim of our study was chemical and microbiological screening of plant components of chamomile and yarrow tinctures using derivatization reagents and TLC-DB against eight bacterial strains: Staphylococcus epidermidis, S. aureus, methicillin-resistant S. aureus, Escherichia coli, Pseudomonas syringae pv. maculicola, Xanthomonas campestis pv. vesicatoria, Aliivibrio fischeri, and Bacillus subtilis. The identity of compounds exhibiting the widest range of activity (apigenin and α-linolenic acid) was confirmed by LC/MS.

TLC-Direct Bioautography as a Bioassay Guided Method for Investigation of Antibacterial Compounds in Hypericum perforatum L.

Fast high-throughput TLC-direct bioautography (DB) is an effect-directed analysis method that enables searching for biologically active (e.g., antimicrobial) substances in complex mixtures like plant extracts. The principle of the method is that separation and detection of biological properties of given mixture components is performed directly on a TLC plate. In searching for antibacterial activity, the developed plate is immersed in a bacterial broth, and bacteria grow directly on its layer during a proper incubation time. Inhibition zones are formed in places where antimicrobial components are located. The active compounds can be further identified using spectroscopic techniques. The aim of our study was investigation of plant components of Hypericum perforatum L. tincture by TLC-DB using nine bacterial strains: Micrococcus luteus, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus, S. epidermidis, Pseudomonas syringae pv. maculicola, Xanthomonas campestris pv. vesicatoria, and Aliivibrio fischeri. Compounds showing the widest range of antimicrobial activity were isolated using semipreparative TLC and identified as apigenin, 3,8'-biapigenin, quercetin, kaempferol, and linolenic acid by TLC, HPLC-diode array detection, and HPLC/MS/MS techniques.

BioArena system for knowing and understanding the biological world: a review with new experimental results.

A simple observation is the basis of the development of BioArena system: according to the first observations during the biological incubation after inoculation there is formaldehyde (HCHO) emission from the chromatographic spots; in this emission process, the level of HCHO molecules decreases time dependently. In fact, the antibiotic effect of an antibiotic-like compound decreases in parallel with the HCHO emission. The investigations demonstrated clearly a unique function and role of endogenous HCHO and its one main reaction product, ozone (O3), in the antiproliferative (e.g., antimicrobial) effect of different molecules with diverse chemical structures. The results in BioArena can be extended for in vivo conditions (e.g., greenhouse experiments), as well. For the pretreatment with different doses of inducers (immunostimulation-inducing molecules) there are always four bioequivalent immunostimulating response ranges (quadruple bioequivalent immune response system) in plants. The inducers (e.g., N-methylated basic amino acids, salicylic acid, cinnamic acid, and trace elements) do not participate directly in the induction of the immunostimulating effect. These new findings support a statement that HCHO and its reaction products (mainly O3), as bioreactive small molecules, are responsible for the immunostimulating activity (in vivo conditions), as well.

Characterization of volatile constituents from Origanum onites and their antifungal and antibacterial activity.

Essential oils obtained by hydrodistillation (HD) and microwave-assisted HD (MWHD) of Origanum onites aerial parts were analyzed by GC and GCIMS. Thirty-one constituents representing 98.6% of the water-distilled oil and 52 constituents representing 99.6% of the microwave-distilled oil were identified. Carvacrol (76.8% HD and 79.2% MWHD) and thymol (4.7% HD and 4.4% MWHD) were characterized as major constituents in both essential oils. Separation of carvacrol and thymol was achieved by overpressured layer chromatography. HPTLC and TLC separations were also compared. Essential oils were evaluated for antifungal activity against the strawberry anthracnose-causing fungal plant pathogens Colletotrichum acutatum, C. fragariae, and C. gloeosporioides using a direct overlay bioautography assay. Furthermore, main oil components carvacrol and thymol were then evaluated for antifungal activity; only carvacrol demonstrated nonselective antifungal activity against the three Colletotrichum species. Thymol and carvacrol were subsequently evaluated in a 96-well microdilution broth assay against Phomopsis obscurans, Fusarium oxysporum, three Colletotrichum species, and Botrytis cinerea. No activity was observed against any of the three Colletotrichum species at or below 30 pM. However, thymol demonstrated antifungal activity and produced 31.7% growth inhibition of P. obscurans at 120 h and 0.3 pM, whereas carvacrol appeared inactive. Thymol and carvacrol at 30 pM showed 51.5 and 36.9% growth inhibition of B. cinerea at 72 h. The mechanism of antibacterial activity was studied in a bioautography-based BioArena system. Thymol and carvacrol showed similar inhibition/killing effect against Bacillus subtilis soil bacteria; the action could be enhanced by the formaldehyde generator and transporter copper (II) ions and could be decreased in the presence of L-arginine, a formaldehyde capturer. Results indicated that Origanum essential oils and its major components thymol and carvacrol appear to generate antimicrobial activity through a mechanism of action where formaldehyde and its reaction products are produced.

Applicability of preparative overpressured layer chromatography and direct bioautography in search of antibacterial chamomile compounds.

In situ sample preparation and preparative overpressured layer chromatography (OPLC) fractionation on a 0.5 mm thick adsorbent layer of chamomile flower methanol extract prepurified by conventional gravitation accelerated column chromatography were applied in searching for bioactive components. Sample cleanup in situ on the adsorbent layer subsequent to sample application was performed using mobile phase flow in the opposite direction (the input and output of the eluent was exchanged). The antibacterial effect of the fractions obtained from the stepwise gradient OPLC separation with the flow in the normal direction was evaluated by direct bioautography against two Gram-negative bacteria: the luminescence gene tagged plant pathogenic Pseudomonas syringae pv. maculicola, and the naturally luminescent marine bacterium Vibrio fischeri. The fractions having strong activity were analyzed by SPME-GC/MS and HPLC/MS/MS. Mainly essential oil components, coumarins, flavonoids, phenolic acids, and fatty acids were tentatively identified in the fractions.

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.

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.

Two Non-Necrotic Disease Resistance Types Distinctly Affect the Expression of Key Pathogenic Determinants of Xanthomonas euvesicatoria in Pepper.

Pepper (Capsicum annuum L.) carrying the gds (corresponding to bs5) gene can prevent the development of bacterial leaf spot disease without HR. However, little is known regarding the development of the resistance mechanism encoded by gds, especially its influence on the bacterium. Here, the effect of gds was compared with pattern-triggered immunity (PTI), another form of asymptomatic resistance, to reveal the interactions and differences between these two defense mechanisms. The level of resistance was examined by its effect on the bacterial growth and in planta expression of the stress and pathogenicity genes of Xanthomonas euvesicatoria. PTI, which was activated with a Pseudomonas syringae hrcC mutant pretreatment, inhibited the growth of Xanthomonas euvesicatoria to a greater extent than gds, and the effect was additive when PTI was activated in gds plants. The stronger influence of PTI was further supported by the expression pattern of the dpsA bacterial stress gene, which reached its highest expression level in PTI-induced plants. PTI inhibited the hrp/hrc expression, but unexpectedly, in gds plant leaves, the hrp/hrc genes were generally expressed at a higher level than in the susceptible one. These results imply that different mechanisms underlie the gds and PTI to perform the symptomless defense reaction.

Cacopsyllapruni (Hemiptera, Psyllidae) in an apricot orchard is more attracted to white sticky traps dependent on host phenology.

The colour preference of the plum psyllid, Cacopsyllapruni (Hemiptera, Psyllidae), is yet poorly studied. This species is the only known vector of the 'Candidatus Phytoplasma prunorum', the agent of European stone fruit yellows (ESFY), a devastating disease of several cultivated Prunus species in Europe. As ESFY is still uncurable, vector control, thus vector monitoring, is pivotal to protect these trees. Cacopsyllapruni is a univoltine, host-shelter-alternating species; overwintered adults migrate from conifer to wild or cultivated Prunus species (family Rosaceae) in late winter-early spring. To select the most effective colour indicating the arrivals of the immigrants, yellow, fluorescent yellow, white, red and transparent sticky traps were deployed in an apricot orchard in Hungary. The two most abundant species in sticky traps were C.pruni and C.melanoneura. Catches of white traps were significantly biased towards C.pruni as compared to C.melanoneura specimens. Moreover, white sticky traps were better at catching plum psyllids than the other colours. Attraction to white was strongest when immigrants from shelter plants kept arriving in the orchard, coinciding with the blooming principal phenophase of apricot trees. When the host flowering growth stage was over, catches of C.pruni in white traps declined sharply to the level of yellow traps that was highest during this post-blooming period. We recommended white sticky traps for promptly monitoring C.pruni in apricot orchards because it is more potent and more selective than yellow ones during the critically important early flowering interval.

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.

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.