TLC-Direct Bioautography as a High Throughput Method for Detection of Antimicrobials in Plants

The richness of bioactive compounds in plant materials encourages continuous development of separation methods and bioassays for their isolation and identification. Thin-layer chromatography-direct bioautography links separation on the adsorbent layer with biological tests performed directly on it. Therefore, the method is very convenient for searching plant constituents with biological activity, such as antibiotics. Test bacteria grow directly on a plate surface excluding places where antibacterials are located. They can be detected with reagents converted by living bacteria. TLC-DB is a high throughput method enabling analyses of many samples in parallel and the comparison of their activity. Both screening and semi-quantitative analysis is possible. The targeted compounds can be identified using spectroscopic methods, mostly mass spectrometry, that can be performed directly on a TLC plate. This paper discusses all above mentioned aspects of TLC-DB, illustrating them with literature, schemes and our own results

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) β-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), β-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 <i>Akebia quinata</i> and <i>Clitoria ternatea</i> 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, β-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

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) β-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), β-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 Isolation and Identification of Antibacterial Chelidonium majus L. Alkaloids

The components of alkaloid, methanol and ethyl acetate extracts of Chelidonium majus L. root, leaf and stem were tested for antibacterial activity against Bacillus subti - lis and Escherichia coli by thin-layer chromatography cou - pled with direct bioautography (TLC-DB). Effect-directed analyses were performed to obtain the active compounds as follows: separation with preparative layer chromatogra - phy (PLC, 0.5-mm-thick silica layer, 20 × 10 cm); a part of the preparative layer (3 cm wide) was used for PLC-DB to detect the antibacterial components against B. subtilis ; elu - tion of the appropriate lanes as indicated by PLC-DB from the remaining 17-cm-wide layer; verification of the bioac - tivity of the eluted compounds by TLC-DB; further purifi - cation of the fractions was carried out with TLC, when it was necessary. The eluates of interest were analysed by LC-HRMS and LC-Q/TOF. The antibacterial effect against B. subtilis was mainly attributed to the isoquinoline alka - loid components, which were identified as chelidonine, sanguinarine, chelerythrine, berberine, coptisine, corydine, dihydrosanguinarine, dihydrochelerythrine, 6-acetonyl- dihydrosanguinarine, 6-acetonyl-dihydrochelerythrine and stylopine. Only chelidonine, sanguinarine and chelerythrine possessed inhibiting effect against E. coli