Bioactivity Profiles on 15 Different Effect Mechanisms for 15 Golden Root Products via High-Performance Thin-Layer Chromatography, Planar Assays, and High-Resolution Mass Spectrometry.

Planar chromatography has recently been combined with six different effect-directed assays for three golden root (Rhodiola rosea L.) samples. However, the profiles obtained showed an intense tailing, making zone differentiation impossible. The profiling was therefore improved to allow for the detection of individual bioactive compounds, and the range of samples was extended to 15 commercial golden root products. Further effect-directed assays were studied providing information on 15 different effect mechanisms, i.e., (1) tyrosinase, (2) acetylcholinesterase, (3) butyrylcholinesterase, (4) ß-glucuronidase, and (5) α-amylase inhibition, as well as endocrine activity via the triplex planar yeast antagonist-verified (6-8) estrogen or (9-11) androgen screen, (12) genotoxicity via the planar SOS-Umu-C bioassay, antimicrobial activity against (13) Gram-negative Aliivibrio fischeri and (14) Gram-positive Bacillus subtilis bacteria, and (15) antioxidative activity (DPPH• radical scavengers). Most of the golden root profiles obtained were characteristic, but some samples differed substantially. The United States Pharmacopeia reference product showed medium activity in most of the assays. The six most active compound zones were further characterized using high-resolution mass spectrometry, and the mass signals obtained were tentatively assigned to molecular formulae. In addition to confirming the known activities, this study is the first to report that golden root constituents inhibit butyrylcholinesterase (rosin was tentatively assigned), ß-glucuronidase (rosavin, rosarin, rosiridin, viridoside, and salidroside were tentatively assigned), and α-amylase (stearic acid and palmitic acid were tentatively assigned) and that they are genotoxic (hydroquinone was tentatively assigned) and are both agonistic and antagonistic endocrine active.

Effect-Directed Profiling of Akebia quinata and Clitoria ternatea via High-Performance Thin-Layer Chromatography, Planar Assays and High-Resolution Mass Spectrometry.

Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, ß-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.

Qualitative and Quantitative Evaluation of Rosavin, Salidroside, and p-Tyrosol in Artic Root Products via TLC-Screening, HPLC-DAD, and NMR Spectroscopy.

Artic root is a well-known plant adaptogen with multipotential pharmacological properties. Thin-layer chromatography (TLC)-screening followed by diode-array high-performance liquid chromatography and nuclear magnetic resonance spectroscopy proved to be a reliable and convenient method for the simultaneous determination of the quality of various herbal raw materials and supplements. This combination allowed for comparing and differentiating arctic root samples as well as defining their authenticity. The study provided information on the chemical and biological properties of the seven chosen samples as well as qualitative and quantitative evaluation of the quality markers: rosavin, salidroside, and p-tyrosol. The absence of rosavin, salidroside, and p-tyrosol in three samples was detected using TLC screening and confirmed by HPLC-DAD and NMR. The paper highlighted the importance of quality control and strict regulation for herbal medicine supplements and preparations.

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.

A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-positive bacteria, Bacillus subtilis.

A TLC-direct bioautography (DB) assay using Bacillus subtilis as test bacteria was developed. Various factors affecting the microorganism's viability on the TLC plates were studied and verified for the flumequine standards. The Dhenasar's method called "direct sample determination" was used for TLC; the antibiotic samples were spotted on the TLC plates and subjected to bioautography without developing with a mobile phase. The best preincubation and incubation times of bacterial broth were found to be 1 h at 37 degrees C and 6 h at 37 degrees C. The optimal viscosity of broth was obtained by the addition of agarose to obtain a 0.05% solution in the Mueller-Hinton broth. The best incubation time of seeded TLC plates was 17 h at 37 degrees C. The plates were visualized by spraying with 0.2% aqueous 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide solution and incubated again for 0.5 h at 37 degrees C. The method was validated by determination of linearity, interday and intraday precision, LOD, and LOQ. The calibration curves showed good linearity in the range 0.005-0.5 microg (0.5-50.0 microg/mL). The regression coefficients were 0.9970 and 0.9955 for intraday and interday plots, respectively. The LOD of flumequine equalled 0.5 microg/mL, i.e., 5 ng of the antibiotic in the spot. The sensitivity of the developed TLC-DB test was compared with that of the two most commonly used standard antimicrobial susceptibility assays: agar disc diffusion and agar cylinder diffusion. The obtained minimum inhibitory concentration values clearly indicate much higher sensitivity of the TLC-DB method compared to the standard antimicrobial susceptibility assays.

Applications of novel direct bioautography tests for analysis of antimicrobials: a review.

TLC coupled to direct bioautography detection can be applied to the analysis of various antimicrobial agents in complex matrixes. Because of the lack of commercially available microbiological detection methods, two direct bioautography tests were developed in our laboratory to be used after TLC separation. One method was based on Gram-positive bacteria, Bacillus subtilis, and the other on Gram-negative bacteria, Escherichia coli. These tests can be used for detection and determination of wide spectrum of antimicrobials as well as for other, nontypical purposes, such as choosing the best sample preparation method before the analysis. Some of the more interesting applications of the newly developed tests are described in this paper.

Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-negative bacteria, Escherichia coli.

With the aim of developing a TLC-direct bioautography assay using Escherichia coli as test bacteria, various parameters influencing the viability of microorganisms on TLC plates were examined and checked for flumequine standards. The optimal times for preincubation and incubation of bacterial broth were 20 h at 37 degrees C and 2 h at 37 degrees C, respectively. The optimal viscosity of the broth was obtained for 0.05% agarose solution in Mueller-Hinton broth. Various incubation times of the seeded TLC plates were also tested (5 h proved to be optimal). After incubation, the plates were sprayed with 0.2% aqueous [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide] (MTT) solution and incubated for 0.5 h at 37 degrees C. The precision of the method was evaluated by the repeatability (intraday assay) and intermediate precision (interday assay). The regression coefficients were 0.9977 and 0.9968, respectively, for intraday and interday curves. The calibration curves show good linearity in the range of 0.005-0.50 microg (0.5-50.0 microg/mL). The established LOD of flumequine equaled 0.5 microg/mL, i.e., 5 ng flumequine in the spot. The developed direct bioautography test significantly enhances the sensitivity of the TLC method.

Investigation of the composition and antibacterial activity of Ukrain™ drug using liquid chromatography techniques.

The greater celandine (Chelidonium majus L.) has been known for the centuries as a medicinal plant. One of the therapeutic agents based on C. majus is anticancer drug Ukrain™ known as a semi-synthetic C. majus alkaloid derivative. Although there are no doubts about antitumor properties of the drug, there is still controversy about its composition. In this study, Ukrain™ was subjected to TLC and LC-MS/MS analyses to compare it with C. majus alkaloid root extract and to determine its composition. Moreover, microbiological activity of both Ukrain™ and the alkaloid extract were tested against Bacillus subtilis strains using TLC-direct bioautography. Sanguinarine, chelidonine, α-homochelidonie and chelerythrine were found to have antibacterial properties. Besides chelidonine, sanguinarine, chelerythrine, protopine, allocryptopine, homochelidonie, berberine and coptisine reported earlier in literature, the presence of stylopine, norchelidonine, dihydrochelidonine and hydroberberine in Ukrain™ was detected, and here they have been reported for the first time.

Comparison of deproteinization methods used before TLC-DB and HPLC analysis of flumequine residues in milk.

Seventeen various extraction procedures based on precipitation of proteins in milk samples spiked with flumequine were tested. Several criteria were taken into account, when choosing the most effective. The supernatants were analyzed by thin-layer chromatography direct bioautography (TLC-DB) and high performance liquid chromatography (HPLC-UV). The results obtained from both methods indicate as the best the same deproteinization procedure. The addition of acetonitrile to milk in 1:1 volume proportions gave the highest concentration of flumequine in supernatant and prompt coagulation of proteins in milk samples.

Bioautography detection in thin-layer chromatography.

Bioautography is a microbial detection method hyphenated with planar chromatography techniques. It is based mainly on antimicrobial or antifungal properties of analyzed substances. The review discusses three versions of bioautography, i.e. contact, immersion and direct bioautography. The more concern is given to the last one. Many applications are quoted, not only for testing various groups of compounds, but also for investigating biochemical processes and factors influencing bacterial growth. Additionally, related methods, which can be included into direct bioautography, are discussed. The most promising among them seems to be TLC-bioluminescence screening.