Essential oils obtained by hydrodistillation (HD) and microwave-assisted HD (MWHD) of Origanum onites aerial parts were analyzed by GC and GC/MS. 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 μM. However, thymol demonstrated antifungal activity and produced 31.7% growth inhibition of P. obscurans at 120 h and 0.3 μM, whereas carvacrol appeared inactive. Thymol and carvacrol at 30 μM 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.

Components of 50% aqueous ethanol chamomile (Matricaria recutica L.) flower extract, previously found antibacterial in a TLC-bioautographic study, were separated and isolated by the use of on-line overpressured layer chromatography (OPLC). This system consisted of an OPLC 50 BS system, an on-line coupled flow-through UV detector, and a manual fraction collector. The collected fractions were investigated by GC-MS analysis and by TLC re-chromatography with subsequent visualization, performed after use of the vanillin-sulphuric acid reagent, or under UV illumination, or applying bioautographic detection. The main compounds of the collected 11 fractions were identified by GC-MS. The results showed that the antibacterial effect of 50% aqueous ethanol extract of chamomile is ascribable to cis-, trans-spiroethers, and the coumarins like herniarin and umbelliferone.

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.