Microbiological Studies on the Influence of Essential Oils from Several Origanum Species on Respiratory Pathogens.

Essential oils (EOs) with established and well-known activities against human pathogens might become new therapeutics in multidrug-resistant bacterial infections, including respiratory tract infections. The aim of this study was to evaluate the antimicrobial activity of EOs obtained from several samples of Origanum vulgare, O. syriacum, and O. majorana cultivated in Poland. EOs were analyzed by GC-MS and tested against four bacterial strains: Staphylococcus aureus (MRSA), Haemophilus influenzae, Haemophilus parainfluenzae, and Pseudomonas aeruginosa. Chemical analyses showed that the Eos were characterized by a high diversity in composition. Based on the chemical data, four chemotypes of Origanum EOs were confirmed. These were carvacrol, terpineol/sabinene hydrate, caryophyllene oxide, and thymol chemotypes. Thin-layer chromatography-bioautography confirmed the presence of biologically active antibacterial components in all tested EOs. The highest number of active spots were found among EOs with cis-sabinene hydrate as the major compound. On the other hand, the largest spots of inhibition were characteristic to EOs of the carvacrol chemotype. Minimal inhibitory concentrations (MICs) were evaluated for the most active EOs: O. vulgare 'Hirtum', O. vulgare 'Margarita', O. vulgare 'Hot & Spicy', O. majorana, and O. syriacum (I) and (II); it was shown that both Haemophilus strains were the most sensitive with an MIC value of 0.15 mg/mL for all EOs. O. majorana EO was also the most active in the MIC assay and had the highest inhibitory rate in the anti-biofilm assay against all strains. The most characteristic components present in this EO were the trans-sabinene hydrate and terpinen-4-ol. The strain with the least sensitivity was the MRSA with an MIC of 0.6 mg/mL for all EOs except for O. majorana, where the MIC value reached 0.3 mg/mL. Scanning electron microscopy performed on the Haemophilus influenzae and Haemophilus parainfluenzae biofilms showed a visible decrease in the appearance of bacterial clusters under the influence of O. majorana EO.

Pinus sylvestris L. and Syzygium aromaticum (L.) Merr. & L. M. Perry Essential Oils Inhibit Endotoxin-Induced Airway Hyperreactivity despite Aggravated Inflammatory Mechanisms in Mice.

Scots pine (SO) and clove (CO) essential oils (EOs) are commonly used by inhalation, and their main components are shown to reduce inflammatory mediator production. The aim of our research was to investigate the chemical composition of commercially available SO and CO by gas chromatography-mass spectrometry and study their effects on airway functions and inflammation in an acute pneumonitis mouse model. Inflammation was evoked by intratracheal endotoxin and EOs were inhaled three times during the 24 h experimental period. Respiratory function was analyzed by unrestrained whole-body plethysmography, lung inflammation by semiquantitative histopathological scoring, myeloperoxidase (MPO) activity and cytokine measurements. α-Pinene (39.4%) was the main component in SO, and eugenol (88.6%) in CO. Both SO and CO significantly reduced airway hyperresponsiveness, and prevented peak expiratory flow, tidal volume increases and perivascular edema formation. Meanwhile, inflammatory cell infiltration was not remarkably affected. In contrast, MPO activity and several inflammatory cytokines (IL-1ß, KC, MCP-1, MIP-2, TNF-α) were aggravated by both EOs. This is the first evidence that SO and CO inhalation improve airway function, but enhance certain inflammatory parameters. These results suggest that these EOs should be used with caution in cases of inflammation-associated respiratory diseases.

Immortelle (Helichrysum italicum (Roth) G. Don) Essential Oil Showed Antibacterial and Biofilm Inhibitory Activity against Respiratory Tract Pathogens.

The biofilm formation of bacteria in different parts of the human body can influence the success of antibiotic therapy. Essential oils (EOs) and their components are becoming increasingly popular in point of view of medicinal applications, because of their antibacterial efficacy. The immortelle EO has been used traditionally as an expectorant; however, there are no studies summarizing its antibacterial effect against respiratory tract bacteria. Our aim was to investigate the antibacterial and biofilm inhibitory activity of immortelle (Helichrysum italicum) EO against respiratory tract pathogens such as Haemophilus influenzae, H. parainfluenzae, Pseudomonas aeruginosa and Streptococcus pneumoniae. In order to prove the antibacterial effect of the immortelle EO, broth microdilution and biofilm inhibition tests, and membrane damage assay were investigated. Scanning electron microscopy was used to identify the structural modifications in bacterial cells. Our results showed that immortelle EO has antibacterial and anti-biofilm effects against respiratory tract bacteria used in this study. H. parainfluenzae was the most sensitive to each treatment, however, P. aeruginosa was the most resistant bacteria. In conclusion, the studied EO may have a role in the treatment of respiratory tract infections due to their antibacterial and anti-biofilm activity.

Antibacterial Effect of Lemongrass (Cymbopogoncitratus) against the Aetiological Agents of Pitted Keratolyis.

Pitted keratolysis (PK) is a bacterial skin infection mostly affecting the pressure-bearing areas of the soles, causing unpleasant symptoms. Antibiotics are used for therapy, but the emergence of antiobiotic resistance, makes the application of novel topical therapeutic agents necessary. The antibacterial effects of 12 EOs were compared in the first part of this study against the three known aetiological agents of PK (Kytococcus sedentarius, Dermatophilus congolensis and Bacillus thuringiensis). The results of the minimal inhibitory concentration, minimal bactericidal concentration and spore-formation inhibition tests revealed that lemongrass was the most effective EO against all three bacterium species and was therefore chosen for further analysis. Seventeen compounds were identified with solid-phase microextraction followed by gas chromatography-mass spectrometry (HS-SPME/GC-MS) analysis while thin-layer chromatography combined with direct bioautography (TLC-BD) was used to detect the presence of antibacterially active compounds. Citral showed a characteristic spot at the Rf value of 0.47, while the HS-SPME/GC-MS analysis of an unknown spot with strong antibacterial activity revealed the presence of α-terpineol, γ-cadinene and calamenene. Of these, α-terpineol was confirmed to possess an antimicrobial effect on all three bacterium species associated with PK. Our study supports the hypothesis that, based on their spectrum, EO-based formulations have potent antibacterial effects against PK and warrant further investigation as topical therapeutics.

A Themed Issue in Honor of Professor K. Hüsnü Can Baser-Outstanding Contributions in the Fields of Pharmacognosy, Phytochemistry, Botany and Ethnopharmacology.

Dear Colleagues, [...].

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.

Treatment of Benign Prostatic Hyperplasia by Natural Drugs.

Benign prostatic hyperplasia (BPH) is one of the most common urinary diseases affecting men, generally after the age of 50. The prevalence of this multifactorial disease increases with age. With aging, the plasma level of testosterone decreases, as well as the testosterone/estrogen ratio, resulting in increased estrogen activity, which may facilitate the hyperplasia of the prostate cells. Another theory focuses on dihydrotestosterone (DHT) and the activity of the enzyme 5α-reductase, which converts testosterone to DHT. In older men, the activity of this enzyme increases, leading to a decreased testosterone/DHT ratio. DHT may promote prostate cell growth, resulting in hyperplasia. Some medicinal plants and their compounds act by modulating this enzyme, and have the above-mentioned targets. This review focuses on herbal drugs that are most widely used in the treatment of BPH, including pumpkin seed, willow herb, tomato, maritime pine bark, Pygeum africanum bark, rye pollen, saw palmetto fruit, and nettle root, highlighting the latest results of preclinical and clinical studies, as well as safety issues. In addition, the pharmaceutical care and other therapeutic options of BPH, including pharmacotherapy and surgical options, are discussed, summarizing and comparing the advantages and disadvantages of each therapy.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Effects of Thymus vulgaris L., Cinnamomum verum J.Presl and Cymbopogon nardus (L.) Rendle Essential Oils in the Endotoxin-induced Acute Airway Inflammation Mouse Model.

Thyme (TO), cinnamon (CO), and Ceylon type lemongrass (LO) essential oils (EOs) are commonly used for inhalation. However, their effects and mechanisms on inflammatory processes are not well-documented, and the number of in vivo data that would be important to determine their potential benefits or risks is low. Therefore, we analyzed the chemical composition and investigated the activity of TO, CO, and LO on airway functions and inflammatory parameters in an acute pneumonitis mouse model. The components of commercially available EOs were measured by gas chromatography-mass spectrometry. Airway inflammation was induced by intratracheal endotoxin administration in mice. EOs were inhaled during the experiments. Airway function and hyperresponsiveness were determined by unrestrained whole-body plethysmography on conscious animals. Myeloperoxidase (MPO) activity was measured by spectrophotometry from lung tissue homogenates, from which semiquantitative histopathological scores were assessed. The main components of TO, CO, and LO were thymol, cinnamaldehyde, and citronellal, respectively. We provide here the first evidence that TO and CO reduce inflammatory airway hyperresponsiveness and certain cellular inflammatory parameters, so they can potentially be considered as adjuvant treatments in respiratory inflammatory conditions. In contrast, Ceylon type LO inhalation might have an irritant effect (e.g., increased airway hyperresponsiveness and MPO activity) on the inflamed airways, and therefore should be avoided.

Antimicrobial Activity of Different Artemisia Essential Oil Formulations.

The extreme lipophilicity of essential oils (EOs) impedes the measurement of their biological actions in an aqueous environment. We formulated oil in water type Pickering Artemisia annua EO nanoemulsions (AEP) with surface-modified Stöber silica nanoparticles (20 nm) as the stabilizing agent. The antimicrobial activity of AEP and its effects on mature Candida biofilms were compared with those of Tween 80 stabilized emulsion (AET) and ethanolic solution (AEE) of the Artemisia EO. The antimicrobial activity was evaluated by using the minimum inhibitory concentrations (MIC90) and minimum effective concentrations (MEC10) of the compounds. On planktonic bacterial and fungal cells beside growth inhibition, colony formation (CFU/mL), metabolic activity, viability, intracellular ATP/total protein (ATP/TP), along with reactive oxygen species (ROS) were also studied. Artemisia annua EO nanoemulsion (AEP) showed significantly higher antimicrobial activity than AET and AEE. Artemisia annua EO nanoemulsions (AEP) generated superoxide anion and peroxides-related oxidative stress, which might be the underlying mode of action of the Artemisia EO. Unilamellar liposomes, as a cellular model, were used to examine the delivery efficacy of the EO of our tested formulations. We could demonstrate higher effectiveness of AEP in the EO components' donation compared to AET and AEE. Our data suggest the superiority of the AEP formulation against microbial infections.

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.

Antibacterial activity evaluation of selected essential oils in liquid and vapor phase on respiratory tract pathogens.

BACKGROUND: The increasing number of multidrug-resistant bacteria and the fact of antibiotic resistance is leading to a continuous need for discovering alternative treatments against infections, e.g. in the case of respiratory tract diseases. Essential oils (EOs), because of their volatility, can easily reach both the upper and lower parts of the respiratory tract via inhalation. Therefore, the aim of the present study was the antibacterial evaluation of clove, cinnamon bark, eucalyptus, thyme, scots pine, peppermint, and citronella EOs against respiratory tract pathogens such as Streptococcus pneumoniae, S. mutans, S. pyogenes, Haemophilus influenzae, H. parainfluenzae, and Moraxella catarrhalis. Furthermore, we wanted to compare the antibacterial effect of these EOs in two different test systems to provide data for the development of an appropriate product formulation. METHODS: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with in vitro vapor phase test (VPT) and broth macrodilution test (BDT). The chemical and percentage compositions of the EOs were determined by GC-MS and GC-FID analysis. RESULTS: Among the EOs, thyme was the most effective against S. mutans (MIC: 0.04 mg/mL in BDT, but cinnamon bark and clove oils also presented high inhibition in liquid medium with MIC values of 0.06 mg/mL and 0.1 mg/mL against S. pneumoniae and S. pyogenes, respectively. M. catarrhalis was the most sensitive to thyme EO (MIC: 0.09 mg/mL). Cinnamon bark EO was the most effective against Haemophilus spp. (MIC: 0.06 mg/mL). In the VPT, cinnamon bark was the most effective oil against all investigated pathogens with MIC values in the range of 15.62-90 µl/L. Surprisingly, the eucalyptus and scots pine showed weak activity against the test bacteria in both test systems. CONCLUSIONS: The EO of thyme, clove and cinnamon bark may provide promising antibacterial activity against respiratory tract pathogens either in liquid medium or in vapor phase. However, their effect is lower than that of the reference antibiotics. The combination of EOs and antibiotics may be beneficial in the alternative treatment of respiratory tract diseases. In vivo studies are necessary to calculate the effective dose of EOs in patients and determine their possible side effects and toxicity.

Antimicrobial and Virulence-Modulating Effects of Clove Essential Oil on the Foodborne Pathogen Campylobacter jejuni.

Our study investigated the antimicrobial action of clove (Syzygium aromaticum) essential oil (EO) on the zoonotic pathogen Campylobacter jejuni After confirming the clove essential oil's general antibacterial effect, we analyzed the reference strain Campylobacter jejuni NCTC 11168. Phenotypic, proteomic, and transcriptomic methods were used to reveal changes in cell morphology and functions when exposed to sublethal concentrations of clove EO. The normally curved cells showed markedly straightened and shrunken morphology on the scanning electron micrographs as a result of stress. Although, oxidative stress, as a generally accepted response to essential oils, was also present, the dominance of a general stress response was demonstrated by reverse transcription-PCR (RT-PCR). The results of RT-PCR and two-dimensional (2D) PAGE revealed that clove oil perturbs the expression of virulence-associated genes taking part in the synthesis of flagella, PEB1, PEB4, lipopolysaccharide (LPS), and serine protease. Loss of motility was also detected by a phenotypic test. Bioautographic analysis revealed that besides its major component, eugenol, at least four other spots of clove EO possessed bactericidal activity against C. jejuni Our findings show that clove EO has a marked antibacterial and potential virulence-modulating effect on C. jejuni IMPORTANCE: This study demonstrates that the components of clove essential oil influence not only the expression of general stress genes but also the expression of virulence-associated genes. Based on this finding, alternative strategies can be worked on to control this important foodborne pathogen.

Effects of essential oil combinations on pathogenic yeasts and moulds.

Essential oils (EOs) can be used as alternative or complementary antifungal agents against human pathogenic moulds and yeasts. To reduce the effective dose of antimicrobial agents, EOs are combined which can lead to synergistic or additive effect. In this study the anti-yeast and anti-mould activities of selected EOs were investigated, alone and in combinations, against clinical isolates of Candida albicans, C. parapsilosis, Aspergillus fumigatus, A. terreus, Rhizopus microsporus, Fusarium solani and Lichtheimia corymbifera. Minimum inhibitory concentrations (MICs) were determined for the EOs of cinnamon, citronella, clove, spearmint and thyme. To investigate the combination effect of the EOs, fractional inhibitory concentrations (FICs) were defined by the checkerboard method and the type of interaction was determined by the FIC index (FICI). FIC index below 0.5 was considered as synergism and between 0.5 and 1 as additive effect. Strongest antifungal activity was showed by thyme EO with MIC values below 1.0 mg/ml. Combination of EOs resulted in additive or indifferent effect, with occasional "borderline synergism". The best combination was cinnamon with clove leading to additive effect in all cases.

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.

Linalool and Geraniol Defend Neurons from Oxidative Stress, Inflammation, and Iron Accumulation in In Vitro Parkinson's Models.

Parkinson's disease is one of the most prevalent neurological disorders affecting millions of people worldwide. There is a growing demand for novel and natural substances as complementary therapies. Essential oils and their various compounds are highly investigated natural plant-based products as potential treatment options for common human diseases, such as microbial infections, chronic diseases, and neurodegenerative disorders. The present study focuses on the beneficial effects of linalool and geraniol, the major compounds of lavender (Lavandula angustifolia L.) and geranium (Pelargonium graveolens L'Hér. in Aiton) essential oils, on oxidative stress, inflammation, and iron metabolism of the rotenone and 6-hydroxydopamine-induced in vitro Parkinson's models. The experiments were carried out on all-trans retinoic acid differentiated SH-SY5Y cells. The effects of linalool and geraniol were compared to rasagiline, an MAO-B inhibitor. The results revealed that both essential oil compounds reduce the level of reactive oxygen species and alter the antioxidant capacity of the cells. They lower the secretion of IL-6, IL-8, and IL-1ß pro-inflammatory cytokines. Moreover, linalool and geraniol change the expression of iron-related genes, such as the iron importer transferrin receptor 1, heme-oxygenase-1, and ferroportin iron exporter, and influence the intracellular iron contents. In addition, it has been unveiled that iron availability is concatenated with the actions of the essential oil compounds. Based on the results, linalool and geraniol are vigorous candidates as an alternative therapy for Parkinson's disease.

TLC-direct bioautography for determination of antibacterial activity of Artemisia adamsii essential oil.

The aim of the present study was the chemical characterization of the essential oil of a Mongolian medicinal plant, Artemisia adamsii Besser, and the investigation of the antibacterial effect of its oil on different human pathogenic bacteria (Staphylococcus aureus, methicillin-resistant S. aureus, and S. epidermidis). The chemical composition of the oil was established by GC and GC/MS. Direct bioautography was used for detecting the antibacterial activity of the essential oil. The result of GC experiments showed that a-thujone was the main component (64.4%) of the oil, while the amount of beta-thujone was 7.1%. 1,8-Cineole seemed to be the other relevant component (15.2%). The antibacterial activity of the A. adamsii essential oil against all three investigated bacteria was observed in the bioautographic system, but this effect was not proportional to the concentrations of a- or beta-thujone; therefore, from a microbiological aspect, thujone content does not determine the medicinal value of this oil. On the whole, the combination of TLC separation with biological detection is an appropriate method for evaluating multicomponent and hydrophobic plant extracts, for instance, essential oils, and it provides more reliable results than traditional microbiological methods (e.g., disc diffusion and agar plate techniques).

Phenolic compounds in Hungarian acacia, linden, milkweed and goldenrod honeys.

Honey is a valuable source of nutrients, minerals and phenolic compounds. Phenolic acids and flavonoids are associated with health benefits of honey and can serve as markers for distinguishing honey types. This study aimed at determining the phenolic profile of four Hungarian unifloral honeys that were not analyzed previously. After verifying their botanical origin with melissopalynological analysis, total reducing capacity was determined with Folin-Ciocalteau method, and phenolic composition was analyzed with HPLC-DAD-MS. From the 25 phenolic substances examined, pinobanksin was the most abundant, followed by chrysin, p-hydroxybenzoic acid and galangin. Quercetin and p-syringaldehyde were detected only in acacia honey, which contained higher levels of chrysin and hesperetin compared to the other three honeys. Milkweed and linden honeys displayed higher levels of caffeic, chlorogenic, ferulic and p-coumaric acids compared to acacia and goldenrod honeys. Taxifolin may serve as a unique marker compound of milkweed honey. Goldenrod honey contained the highest level of syringic acid. Principal component analysis supported the indicator role of polyphenols in honey identification, discriminating clearly the four unifloral honeys. Our results suggest that phenolic profiles may be useful to find markers of honey's floral origin, but geographical origin can strongly influence the composition of characteristic compounds.

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

BACKGROUND: Essential 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. METHODS: Essential 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. RESULTS: Thymol (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. CONCLUSION: The 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.