Wioleta Jesionek

Thin-Layer Chromatography - Direct Bioautography for the Screening of Antimicrobial Properties of Plant Extracts

The main volatile compounds from three medicinal plants belonging to Lamiaceae family were screened for their biological properties. The plants were Salvia officinalis, Thymus vulgaris, and Mentha × piperita containing as the main volatile constituents thujone, thymol, and menthol, respectively. The applied chromatographic system was silica gel developed with toluene-ethyl acetate (93:7). Thin-layer chromatography — direct bioautography (TLC-DB) against Escherichia coli and Bacillus subtilis was used for detection of antibacterial activity of the plant extracts and essential oils. The bioautographic fingerprints were compared with the fingerprints obtained after derivatization with anisaldehyde.

Separation, Identification, and Investigation of Antioxidant Ability of Plant Extract Components Using TLC, LC–MS, and TLC–DPPH•

Ten common phenolic compounds from the extracts of five plant species, that is, Hypericum perforatum L., Matricaria recutita L., Achillea millefolium L., Thymus vulgaris L., and Salvia officinalis L., were separated using optimized thin-layer chromatography (TLC) conditions at normal phase mode and were visualized using Natural Product/Polyethylene Glycol Reagent. Antioxidant properties of components of the extracts were assessed using TLC–DPPH• directly on the TLC plate. Nine out of ten analyzed polyphenols were proved to be radical scavengers. The presence and identity of target substances in plant samples were confirmed by the liquid chromatography–mass spectrometry technique.

Effects-Directed Biological Detection: Bioautography

Effects-directed biological detection facilitates the identification of targeted substances in complicated matrices by biological detection after separation by planar chromatography. The term “bioautography” is used predominantly when antimicrobial or antifungal properties of the analytes are measured on high-performance thin-layer chromatography plates. However, other types of biological effects can be applied, for example, bioluminescence, antioxidant, enzymatic, or antiestrogenic assays. Increasingly, several modes of biological detection in parallel followed by chemical and/or spectroscopic detection are employed. Such hyphenations give complete information for the compounds of interest. They can be isolated, even from very complicated matrices, identified, and quantified. Additionally, their biological relevance and impact on the environment and human health can be assessed. In this chapter the principles of thin-layer chromatography–bioautography are presented, with special emphasis on direct bioautography. Various applications are described and new trends presented.Abstract Effects-directed biological detection facilitates the identification of targeted substances in complicated matrices by biological detection after separation by planar chromatography. The term “bioautography” is used predominantly when antimicrobial or antifungal properties of the analytes are measured on high-performance thin-layer chromatography plates. However, other types of biological effects can be applied, for example, bioluminescence, antioxidant, enzymatic, or antiestrogenic assays. Increasingly, several modes of biological detection in parallel followed by chemical and/or spectroscopic detection are employed. Such hyphenations give complete information for the compounds of interest. They can be isolated, even from very complicated matrices, identified, and quantified. Additionally, their biological relevance and impact on the environment and human health can be assessed. In this chapter the principles of thin-layer chromatography–bioautography are presented, with special emphasis on direct bioautography. Various applications are described and new trends presented.

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.

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.

SCREENING BACTERIAL AND RADICAL SCAVENGING PROPERTIES OF CHOSEN PLANT EXTRACTS USING THIN-LAYER CHROMATOGRAPHY–DIRECT BIOAUTOGRAPHY

Effect directed analysis method is proposed for searching compounds with antibacterial and radical scavenging properties in herbs. The method is based on thin-layer chromatography–direct bioautography of plant extracts and chemical screening using derivatization reagents. Bacillus subtilis and Escherichia coli were used as test bacteria for searching antibacterial properties of plant constituents and DPPH as a reagent for testing radical scavenging properties directly on earlier developed TLC plates. Anisaldehyde-sulfuric acid and natural product/polyethylene glycol 4000 reagent were used for derivatization of terpenoids and polyphenols, respectively.

Thin-layer chromatography: Direct bioautography as a method of examination of antimicrobial activity of selected Potentilla species

ABSTRACT Simple method for preparing biologically active material from plant extract may consist of four steps: obtaining an extract from selected plant, analytical chromatography with biological detection and transition to preparative scale, preparative isolation of biologically identified bands/groups of bands from chromatogram, rechromatography of isolated fractions. The next step of proceeding may be a quality analysis of separated material by, e.g., thin-layer chromatography (TLC)-MS Interface or biological studies after isolation. Extracts obtained from four Potentilla species: P. erecta, P. collina, P. megalantha, and P. crantzi were separated on silica plates, developed with the nonaqueous eluent. Immersion—direct bioautography was chosen as a method of detection of bands’ activity for their selection for isolated bands. Antibacterial/bacteriostatic properties of marked areas was the preliminary criterion of choice the samples to the further examinations. Preparative thin layer chromatography allowed isolation of various number of fractions of each extracts. Obtained fractions were examined in various TLC systems (silica/nonaqueous mixture of solvents) with the various modes of development to obtain the best separation effect. The next step was a once more bioautographic detection of separated fractions on the TLC plates. The found chromatographic systems will be used to mass spectrometric determination of biologically active compounds contained in examined extracts. Bioautographic indication will be the method of choice for subsequently isolated compounds or groups of compounds.

Investigation of different extraction methods on the content and biological activity of the main components in Coffea arabica L. extracts

Coffee, due to its common consumption, is one of the main sources of polyphenols in human diet. Coffee species and coffee-related products differ in composition and content of main components, such as chlorogenic acid and caffeine. Chemical and biological fingerprints of various Coffea arabica L. extracts were obtained in order to check and compare their antibacterial and antioxidant properties. The antibacterial activity of green and roasted coffee seeds and pomace was evaluated against Bacillus subtilis using thin-layer chromatography (TLC)-direct bioautography. TLC-2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used to determine antioxidant properties of the afore-mentioned extracts. Furthermore, different solvents and several extraction methods such as simple maceration, maceration under stirring, and ultrasonic accelerated extraction were tested. The most efficient method of extraction of caffeine and chlorogenic acid was chosen based on quantitative TLC analysis. Additionally, these two main components of coffee were quantitatively determined in commercial products of green coffee.

Screening of antibacterial compounds in thymus vulgaris l. tincture using thin-layer chromatography-direct bioautography and liquid chromatography-tandem mass spectrometry techniques

In this study, thin-layer chromatography—direct bioautography (TLC—DB) was used for guiding the isolation and identification of antibacterial constituents of Thymus vulgaris L. ethanol extract. This TLC—bioassay method enables the separation and detection of active components directly on the surface of chromatographic plates. They can be identified by comparison with reference substances or using physicochemical methods, preferably spectroscopic ones (liquid chromatography—tandem mass spectrometry [LC—MS/MS], in the presented paper). The described method belongs to the effect-directed analyses (EDA). Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, including methicillin-resistant Staphylococcus aureus as well as luminescent bacteria like Aliivibrio fischeri. Five fractions with the widest antimicrobial spectra were detected using TLC—DB, isolated by semi-preparative TLC and subjected to LC—MS/MS analyses. Finally, two bioactive components were tentatively identified, basing on their fragmentation pattern, as eriodictyol and 4,4′-dihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-bifenylodion.

TLC–direct bioautography as a method for evaluation of antibacterial properties of Thymus vulgaris L. and Salvia officinalis L. essential oils of different origin

ABSTRACT The dot-blot bioautography was used to evaluate the antibacterial properties of Thymus vulgaris L. and Salvia officinalis L. essential oils (EOs) produced by three different manufacturers. The whole samples were applied at three concentrations on thin-layer chromatography (TLC) plates which were then subjected to bioautography against Bacillus subtilis. The samples of the highest activity were found. Then, they were separated using TLC and once again subjected to bioautography against B. subtilis. As was proved, only the essential oils of T. vulgaris L. possessed strong antibacterial properties for which mostly thymol and carvacrol were responsible. Their contents were calculated using TLC–UV densitometry. The highest contents were found in the essential oils of the highest antibacterial activity revealed in the dot-blot test. It means that a dot-blot test can be used for simple and fast evaluation of antibacterial properties of essential oils. GRAPHICAL ABSTRACT