Thin-layer Chromatography Direct Bioautography Research Articles

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Thin layer chromatography-direct bioautography guided isolation of β-sitosterol from the leaves of Capparis fascicularis

The aim of the study was to isolate and characterise antimicrobial agents from the leaves of Capparis fascicularis using thin-layer chromatography-direct bioautography (TLC-DB). Capparis fascicularis is a medicinal plant used traditionally to treat various ailments. Previous studies have shown that species of the genus Capparis, contain several classes of secondary metabolites, including sterols. In this study, the leaves of C. fascicularis were extracted with 80% aqueous methanol, and the extract’s fractions were screened for antimicrobial activity against Staphylococcus aureus ATCC 25923 using a broth micro-dilution assay. The hexane fraction was the most active, with a minimum inhibitory concentration (MIC) of 512 µg/mL. TLC-DB and flash column chromatography of the hexane fraction resulted in the isolation of β-Sitosterol for the first time from C. fascicularis. The compound was characterised using NMR and HRMS.

Gram-negative rough mutants used as test bacteria can increase sensitivity of direct bioautography

Currently, the antimicrobial activity of essential oils (EOs) is an outstanding research field due to antibiotic resistance of microorganisms. Thin-layer chromatography‒direct bioautography (TLC‒DB) is an effective, fast method to find components with antimicrobial activity in a mixture of plant compounds, e.g., in EOs. The volatility and hydrophobic characters of EOs require special experimental conditions, and disc diffusion assay is not appropriate to explore the antimicrobial activity of them. The aim of this study was to use “R” mutants, which are more sensitive to synthetic antimicrobial drugs, in DB to increase the sensitivity of this method. Our hypothesis was that these mutants show sensitivity to some EOs (thyme, clove, and peppermint) as well. The chemical composition of our tested EOs was measured with gas chromatography‒mass spectrometry (GC‒MS). The main compounds (39.8% thymol, 78.8% eugenol, and 50.4% menthol) of EOs showed notable antibacterial activity in TLC‒DB. Based on our results, we suggest to use Salmonella minnesota Re595 rough strain as test bacterium in bioautography, because it showed the highest sensitivity to the tested antibiotics (gentamicin and cephalexin) and EOs. Furthermore, this rough mutant could make TLC‒DB more faster, because only 4 h incubation time was enough to detect the inhibition zones of the active compounds used in this study.

Screening and identification of antibacterial components in Artemisia argyi essential oil by TLC–direct bioautography combined with comprehensive 2D GC × GC-TOFMS

One of the primary components that contribute to Artemisia argyi 's effectiveness is essential oil, which has an exceptional antibacterial effect that has been well documented. The actual cause of its antibacterial activity and associated mechanism, however, are still not completely understood. For the first time, comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (2D GC × GC-TOFMS) and thin-layer chromatography-direct bioautography (TLC-DB) were employed to investigate its antibacterial components. The antibacterial properties of A. argyi essential oil were investigated, and the antibacterial activity of six compounds was evaluated, using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coil) as test microorganisms. TLC-direct bioautography was used to screen two bioactive clusters. Following 2D GC × GC-TOFMS identification of bioactive clusters, six compounds were evaluated for in vitro antibacterial activity verification. All the components tested displayed antibacterial action. Results showed that α-terpineol and eugenol had high potent antibacterial activity (MIC＜0.62 mg/mL, IC50＜2.00 mg/mL). For complex essential oils from traditional Chinese medicine, this method is efficient for quick screening and identifying antibacterial compounds.

Thin layer chromatography-direct bioautography for investigation of antibacterial activities of Artemisia sieversiana Ehrhart ex Willd. essential oil

The aim of the study was to characterise the chemical composition of the essential oil extracted from a Chinese traditional aromatic herb, Artemisia sieversiana Ehrhart ex Willd and investigate its antimicrobial activities against Staphylococcus aureus, the test bacterium, using thin-layer chromatography-direct bioautography (TLC-DB). Gas chromatography-mass spectrometry analysis showed that the essential oil was dominated by chamazulene (44.44%). Undeveloped TLC-DB enabled the measurement of zone of inhibition as valid as and more sensitive than the traditional agar diffusion method. The overall antimicrobial effect was weak at the tested concentration range and the antimicrobial strength did not exhibit concentration dependence. At high essential oil concentration (>1000μg/ml), size of zone of inhibition was all <7mm. Developed TLC-DB separated the components and visualised the inhibition of bacterial growth on the plate surface where the active antimicrobials were determined to be the minor components, hydroxylated terpenes, rather than the dominant component, chamazulene.

Chemical Composition, Antioxidant Properties, and Antibacterial Activity of Essential Oils of Satureja macrostema (Moc. and Sessé ex Benth.) Briq.

Satureja macrostema is a plant that is located in various regions of Mexico and is used in a traditional way against illness. Essential oils (EOs) were obtained from leaves Satureja macrostema and the chemical composition was evaluated by gas chromatography-mass spectrometry (GC-MS). The antioxidant effect of the oil was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and by Trolox Equivalent Antioxidant Capacity (TEAC). In vitro antibacterial activity against Escherichia coli and Staphylococcus aureus was determined using a broth microdilution assay and thin layer chromatography-direct bioautography (TLC-DB) to identify active antibacterial compounds. The EOs analysis showed 21 compounds, 99% terpenes, and 96% oxygenated monoterpenes, with trans-piperitone epoxide (46%), cis-piperitone epoxide (22%), and piperitenone oxide (11%) as more abundant compounds. Likewise, S. macrostema EOs showed an antioxidant activity of DPPH = 82%, with 50% free radical scavenging (IC50) = 7 mg/mL and TEAC = 0.005, an antibacterial effect against E. coli of 73% inhibition, and 81% over S. aureus at dose of 100 µL of undiluted crude oil. The TLC-DB assay showed that the most active compounds were derived from piperitone. The comparison with other studies on S. macrostema shows variability in the compounds and their abundances, which can be attributed to climatic factors and the maturity of plants with similar antioxidant and antibacterial activities.

Flowering phenophases influence the antibacterial and anti-biofilm effects of Thymus vulgaris L. essential oil

BackgroundEssential oils are becoming increasingly popular in medicinal applications because of their antimicrobial effect. Thymus vulgaris L. (Lamiaceae) is a well-known and widely cultivated medicinal plant, which is used as a remedy for cold, cough and gastrointestinal symptoms. Essential oil content of thyme is responsible for its antimicrobial activity, however, it has been reported that the chemical composition of essential oils influences its biological activity. In order to explore flowering phenophases influence on the chemical composition of thyme essential oil and its antibacterial and anti-biofilm activity, plant materials were collected at the beginning of flowering, in full bloom and at the end of flowering periods in 2019.MethodsEssential oils from fresh and dried plant materials were distilled and analyzed with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). The antibacterial activity was performed by broth microdilution and thin layer chromatography-direct bioautography (TLC-DB) assays and the anti-biofilm effect by crystal violet assay, respectively. Scanning electron microscopy was applied to illustrate the cellular changes of bacterial cells after essential oil treatment.ResultsThymol (52.33–62.46%) was the main component in the thyme essential oils. Thyme oil distilled from fresh plant material and collected at the beginning of flowering period exerted the highest antibacterial and anti-biofilm activity against Haemophilus influenzae, H. parainfluenzae and Pseudomonas aeruginosa.ConclusionThe different flowering periods of Thymus vulgaris influence the antibacterial and anti-biofilm activity of its essential oils, therefore, the collection time has to be taken into consideration and not only the full bloom, but the beginning of flowering period may provide biological active thyme essential oil.

High-performance thin-layer chromatography–direct bioautography combined with chemometrics for the distinction of goldenrod species

Thirteen root extract samples of four goldenrod (Solidago) species present in Europe were investigated by hyphenated high-performance thin-layer chromatography (HPTLC). Only S. virgaurea is native, whereas S. gigantea, S. canadensis, and S. graminifolia have been introduced from North America. The bioactive zones in the Aliivibrio fischeri bioautogram were identified as polyacetylenes, labdane diterpenes, or clerodane diterpenes by HPTLC coupled to high-resolution mass spectrometry, exploiting the two interfaces, heated electrospray ionization, and direct analysis in real time. Principal component analysis of the obtained bioprofiles enabled the discrimination of the Solidago species. Furthermore, chemometrics pointed to the discriminative components, the main bioactive markers of the species: Z,Z-matricaria ester from S. virgaurea, solidagenone from S. canadensis, solidagoic acid A, and a dialdehyde clerodane diterpene from S. gigantea, and Z-dehydromatricaria ester from S. graminifolia.

Exploring the Antifungal Activity and Action of Saussurea costus Root Extracts against Candida albicans and Non-albicans Species.

The isolation and assessment of the active constituents in polar and non-polar crude extracts of Saussurea costus roots as antifungal agents, against Candida albicans and non-C. albicans (NAC) species, was the aim of this current investigation. The SEM “Scanning electron microscopy” imaging provided potential action modes of n-hexane extract (nhhE) toward Candida spp., whereas the TLC-DB “Thin layer chromatography-direct bioautography” was employed for detecting the anticandidal compounds. nhhE had the greatest biocidal activity against all strains and clinical isolates of Candida spp. with maximum zones of inhibition. SEM revealed the occurrence of irregular, dense inclusions of C. albicans cell walls after treatment with nhhE for 12 h. Complete morphological distortions with lysed membranes and deterioration signs appeared in most treated cells of C. parapsilosis. The most effectual compound with anticandidal activity was isolated using TLC-BD and identified as sesquiterpene by GC/MS analysis. The infra-red analysis revealed the presence of lactone ring stretching vibrations at 1766.72 cm−1. The anticandidal activity of nhhE of S. costus roots was confirmed from the results, and the treated cotton fabrics with nhhE of S. costus possessed observable activity against C. albicans. Data could recommend the practical usage of S. costus extracts, particularly nhhE, as influential natural bioactive sources for combating pathogenic Candida spp.

Combination of Analytical and Statistical Methods in Order to Optimize Antibacterial Activity of Clary Sage Supercritical Fluid Extracts.

The extraction of clary sage (Salvia sclarea L.) using supercritical carbon dioxide (SC-CO2) was systematically studied by using thin layer chromatography-direct bioautography (TLC-DB) and response surface methodology (RSM). The three parameters temperature, pressure, and cosolvent ratio were optimized for the maximum antibacterial activity of clary sage extracts against Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA). The highest inhibition zone was 7.51 mm for P. aeruginosa and 7.57 mm for MRSA. According to RSM analysis, the predicted optimum extraction parameters are 18.6 MPa pressure, 40 °C temperature, and 2% ethanol (EtOH) ratio. The combination of this analytical and statistical method allows saving time, money, and instrument runtime in the optimization of essential oil composition, which is tailored to a specific task and could be useful on any kind of herbs in a wide range of use from perfume manufacturing to the food industry.

Bioactivities of plant extracts collected in Halmahera, Indonesia: A bioprospection study of underexplored plant species

The discovery of new antibiotics to overcome the growing resistance problem as well as the discovery of new natural, safe antioxidants to combat oxidative stress are still urgently needed. Medicinal plants are known to produce potential therapeutic substances which are more biologically selective than synthetic compounds. Therefore, we explored the bioactivities of 35 ethanolic extracts from 24 underexplored plant species collected in Halmahera, to find potential sources for antibacterial and antioxidant agents. &#x0D; Dried plant parts were extracted using ethanol 96%. Thin layer chromatography-direct-bioautography (TLC-DB) and minimum inhibitory concentration (MIC) determination were used to evaluate the antibacterial effect. Antioxidant activity was determined against DPPH using TLC-DB and microdilution assay. Total phenolic content (TPC) was determined using Folin-Ciocalteu’s method.&#x0D; The ethanolic extracts exhibited moderate to weak antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the leaf extract of Elaeocarpus dolichostylus, Elaeocarpus multiflorus, and Psychotria celebica as well as the stem bark extract of Elaeocarpus dolichostylus, Cinnamomum sintoc, and Garcinia latissima displayed very strong antioxidant activities against DPPH with AAI values between 4.60 to 13.42. A strong correlation between TPC and antioxidant activity with r = 0.8712 was observed.&#x0D; Despite the moderate to weak antibacterial activity, eight underexplored plant species exhibit strong antioxidant activities. A high correlation between TPC and antioxidant activity indicating a prominent role of phenolic compounds in the plants’ antioxidant properties. These findings indicate that collected plants from Halmahera are potential to be studied and developed further as the potential sources for novel antioxidants.

In Vitro Antibacterial and Antibiofilm Activity of Hungarian Honeys against Respiratory Tract Bacteria.

Honey is a rich source of carbohydrates, while minor compounds such as amino acids and polyphenols contribute to its health-promoting effects. Honey is one of the oldest traditional remedies applied for microbial infections, due to its antibacterial, anti-inflammatory, and antioxidant properties. The aim of this study was to investigate the antibacterial and antibiofilm effects of Hungarian black locust, linden, and sunflower honeys against the most common biofilm-forming respiratory tract pathogens Haemophilus spp., Pseudomonas aeruginosa, and Streptococcus pneumoniae. The unifloral character of all three honey types was confirmed by melissopalynological analysis. The antibacterial activity of each honey sample against each bacterium strain was proven with agar well diffusion assay and thin layer chromatography—direct bioautography. Kinetics and mechanisms of antibacterial action were clarified with time-kill assay and membrane degradation study. The anti-biofilm activity was evidenced using crystal violet assay. In each assay, linden honey was the most effective, followed by sunflower and black locust honey. In addition, each honey sample had greater potential to suppress respiratory tract bacteria, compared to major sugar components. In conclusion, honey in general and linden honey in particular, can have a role in the treatment of respiratory tract infections caused by biofilm-forming bacteria.

A new thin-layer chromatography–direct bioautography assay for the qualitative and quantitative determination of peroxidase inhibitors in plant extracts

Thin-layer chromatography (TLC) hyphenated to bioassays is a modern tool used for discovery of drugs with diverse biological activities from plant extracts which justifies the continuous need for developing such methods. A new TLC–bioautography assay for detecting peroxidase enzyme inhibitors was developed and validated in this study. Peroxidases are related to pathogenesis of many diseases such as neurodegenerative disorders and rheumatoid arthritis and are considered as inflammatory markers. The developed TLC–bioautography method was based on reaction of peroxidase with hydrogen peroxide and the subsequent formation of blue color by the reaction between the released oxygen and benzidine. Compounds with peroxidase inhibition activity are observed as yellowish spots against a blue background. The developed method was validated according to the ICH guidelines. Quantitative estimation of peroxidase inhibitors was achieved by applying image analyses techniques, showing good linearity (R2 = 0.998), accuracy (% recovery 96.6–99.7%), intra-day precisions (RSD% = 0.95–2.35) and inter-day precision (RSD% = 1.80–3.20) as well as low LOD (0.28 μg/spot) and LOQ (0.85 μg/spot). The developed method was applied to different Juniperus species fractions as a demonstrative illustration. Overall, the method proved to be efficient, simple and rapid for identification of peroxidase inhibitors in complex matrices and can be applied in high-throughput screening of plant extracts.

Anti-Haemophilus Activity of Selected Essential Oils Detected by TLC-Direct Bioautography and Biofilm Inhibition.

Essential oils (EOs) are becoming increasingly popular in medical applications because of their antimicrobial effect. Direct bioautography (DB) combined with thin layer chromatography (TLC) is a screening method for the detection of antimicrobial compounds in plant extracts, for example, in EOs. Due to their lipophilic character, the common microbiological assays (etc. disk diffusion) could not provide reliable results. The aim of this study was the evaluation of antibacterial and anti-biofilm properties of the EO of cinnamon bark, clove, peppermint, thyme, and their main components against Haemophilus influenzae and H. parainfluenzae. Oil in water (O/W) type Pickering nano-emulsions stabilized with silica nanoparticles from each oil were prepared to increase their water-solubility. Samples with Tween80 surfactant and absolute ethanol were also used. Results showed that H. influenzae was more sensitive to the EOs than H. parainfluenzae (except for cinnamon bark oil). In thin layer chromatography-direct bioautography (TLC-DB) the ethanolic solutions of thyme oil presented the best activity against H. influenzae, while cinnamon oil was the most active against H. parainfluenzae. Pickering nano-emulsion of cinnamon oil inhibited the biofilm formation of H. parainfluenzae (76.35%) more efficiently than samples with Tween80 surfactant or absolute ethanol. In conclusion, Pickering nano-emulsion of EOs could inhibit the biofilm production effectively.

Characterisation and screening of antimicrobial essential oil components against clinically important antibiotic-resistant bacteria using thin layer chromatography-direct bioautography hyphenated with GC-MS, LC-MS and NMR.

The antimicrobial activity of many essential oils (EOs) is well established, indicating that EOs may be a source of compounds for antimicrobial drug development. Thin layer chromatography-direct bioautography (TLC-DB) can quickly identify antimicrobial components in complex mixtures and can be applied to the screening of EOs for lead compounds. This study aimed to identify antimicrobial components of oregano, rosewood and cumin EOs against antibiotic-sensitive and -resistant bacteria using TLC-DB and a multi-faceted approach of GC-MS, LC-MS and NMR techniques to characterise bioactive compounds. The study also aimed to quantify the antimicrobial activity of bioactive compounds in order to evaluate their potential for the development of therapies against antibiotic-resistant bacteria. EOs were eluted on TLC plates and sprayed with a suspension of Staphylococcus aureus, Enterococcus faecium, Escherichia coli or Pseudomonas aeruginosa (antibiotic-sensitive and -resistant isolates). Zones of inhibition, visualised with iodonitrotetrazolium chloride, were subject to GC-MS, LC-MS and NMR to characterise the bioactive compounds. Seven compounds were identified from the three EOs using GC-MS, while LC-MS and NMR failed to detect the presence of any further non-volatile or heat labile compounds. Carvacrol was most antimicrobial compound identified, with minimum inhibitory concentrations ranging 0.99-31.62 mM. The identified antimicrobial compounds present in oregano, rosewood and cumin EOs including carvacrol may be candidates for the development of novel antimicrobial therapies against antibiotic-resistant bacteria.

Review of the Determination of the Antioxidant Activity of Foods, Food Ingredients, and Dietary Supplements by Thin Layer Chromatography-Direct Bioautography, Spectrometry, and the Dot-Blot Procedure.

This paper reviews a selection of the most important studies on antioxidants in foods (including beverages), food ingredients, and dietary supplements by effect-directed analysis using TLC with DPPH*, ABTS*+, and β-carotene direct bioautography. Total antioxidant activity by visible mode spectrometry (colorimetry) with TLC used offline to obtain additional analytical results, mostly for identification and quantification of phenolic compounds in samples, is also discussed. Finally, dot-blot assay for total antioxidant activity, carried out on a TLC plate without mobile-phase development, is reviewed as an alternative with possible advantages compared with spectrometry.

Investigation of antibacterial and cytotoxic potential of phenolics derived from Cistus incanus L. by means of thin-layer chromatography-direct bioautography and cytotoxicity assay

ABSTRACTCistus incanus L. (hairy rockrose) is a medicinal plant which belongs to the Cistaceae family and the Cistus genus, with a well established position in traditional medicine of the Mediterranean basin and the Middle East. It was the aim of this study to compare antibacterial activity of the phenolics derived from fourteen C. incanus samples of different origin (Turkey, Albania, Greece, and an unknown geographical location) obtained as herbal teas from a local market of diet supplements. This activity was assessed with the use of thin-layer chromatography–direct bioautography (TLC-DB) applied to crude extracts against the Gram negative naturally luminescent marine bacterium Aliivibrio fischeri and the Gram positive soil bacterium Bacillus subtilis as the test microorganisms. It was established that in spite of different origin of the investigated herbal samples, in qualitative terms their antibacterial activity was closely comparable and more strongly pronounced against the Gram positive than the Gram negative bacterium. Crude extract originating from one herbal specimen labelled A3 (sample no. 3 from Albania) underwent selective multi-step extraction of the phenolics, dividing them into six fractions (I to VI) that expectedly contain flavonoid aglycons, free phenolic acids, non-polar flavonoid glycosides, polar flavonoid glycosides, and phenolic acids obtained through the acidic and basic hydrolysis from the respective glycosides. Antibacterial activity of each A3 fraction was then assessed with the use of the same TLC-DB approach and it was established that the strongest effect was exerted by fractions I and II (flavonoid aglycons and free phenolic acids). Moreover, cytotoxic assay was performed for the crude C. incanus extracts against the human colon adenocarcinoma cells and a moderate yet well measurable cytotoxic effect was observed with all investigated C. incanus samples. In analogy to antibacterial activity, also in this case cytotoxic potential of all investigated crude C. incanus extracts was similar.

Antibacterial potential of the Cistus incanus L. phenolics as studied with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis

The main aim of this study was to detect and identify antibacterial components of fraction I derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer chromatography – direct bioautography (TLC-DB) using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid glycosides and then separate and visualize the liberated glucose and some other building blocks of the zones’ components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid) to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts produced through fractionation.

High-performance thin-layer chromatography-direct bioautography as a method of choice for alpha-amylase and antioxidant activity evaluation in marine algae

High-Performance Thin-layer chromatography (HPTLC) combined with DPPH free radical method and α-amylase bioassay was used to compare antioxidant and antidiabetic activities in ethanol and ethyl acetate extracts from 10 marine macroalgae species (3 Chlorophyta, 4 Phaeophyta and 3 Rhodophyta) from Blue Lagoon beach (Malaysia). Samples were also evaluated for their phenolic and stigmasterol content. On average, higher antioxidant activity was observed in the ethyl acetate extracts (55.1mg/100g gallic acid equivalents (GAE) compared to 35.0mg/100g GAE) while, as expected, phenolic content was higher in ethanol extracts (330.5mg/100g GAE compared to 289.5mg/100g GAE). Amounts of fucoxanthin, stigmasterol and α-amylase inhibitory activities were higher in ethyl acetate extracts. Higher enzyme inhibition is therefore related to higher concentrations of triterpenes and phytosterols (Note: these compounds are more soluble in ethyl acetate). Ethyl acetate extracts from Caulerpa racemosa and Padina minor, had the highest α-amylase inhibitory activity, and also showed moderately high antioxidant activities, stigmasterol content and polyphenolic content. Caulerpa racemose, being green algae, does not contain fucoxanthin, while Padina minor, being brown algae, contains high amounts of fucoxanthin. Therefore, it is very unlikely that fucoxanthin contributes to α-amylase inhibitory activity as previously reported.

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

ABSTRACTThe 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.