Synthetic polyploidization induces enhanced phytochemical profile and biological activities in Thymus vulgaris L. essential oil.

Essential oil from Thymus vulgaris L. has valuable therapeutic potential that is highly desired in pharmaceutical, food, and cosmetic industries. Considering these advantages and the rising market demand, induced polyploids were obtained using oryzalin to enhance essential oil yield. However, their therapeutic values were unexplored. So, this study aims to assess the phytochemical content, and antimicrobial, antioxidant, and anti-inflammatory activities of tetraploid and diploid thyme essential oils. Induced tetraploids had 41.11% higher essential oil yield with enhanced thymol and γ-terpinene content than diploid. Tetraploids exhibited higher antibacterial activity against all tested microorganisms. Similarly, in DPPH radical scavenging assay tetraploid essential oil was more potent with half-maximal inhibitory doses (IC50) of 180.03 µg/mL (40.05 µg TE/mg) than diploid with IC50 > 512 µg/mL (12.68 µg TE/mg). Tetraploids exhibited more effective inhibition of in vitro catalytic activity of pro-inflammatory enzyme cyclooxygenase-2 (COX-2) than diploids at 50 µg/mL concentration. Furthermore, molecular docking revealed higher binding affinity of thymol and γ-terpinene towards tested protein receptors, which explained enhanced bioactivity of tetraploid essential oil. In conclusion, these results suggest that synthetic polyploidization using oryzalin could effectively enhance the quality and quantity of secondary metabolites and can develop more efficient essential oil-based commercial products using this induced genotype.

Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens.

Essential oils (EOs) have great potential in inhalation therapy for the treatment of respiratory infections. However, innovative methods for evaluation of antimicrobial activity of their vapors are still needed. The current study reports validation of the broth macrodilution volatilization method for assessment of the antibacterial properties of EOs and shows the growth-inhibitory effect of Indian medicinal plants against pneumonia-causing bacteria in liquid and vapor phase. Among all samples tested, Trachyspermum ammi EO exhibits the strongest antibacterial effect against Haemophilus influenzae, with minimum inhibitory concentrations of 128 and 256 µg/mL in the liquid and vapor phases, respectively. Furthermore, Cyperus scariosus EO is found to be nontoxic to normal lung fibroblasts assessed by modified thiazolyl blue tetrazolium bromide assay. Chemical analysis performed using gas chromatography-mass spectrometry identified α-citral, cyperotundone, and thymol as the main constituents of Cymbopogon citratus, C. scariosus, and T. ammi EOs, respectively. In addition, ß-cymene is identified as the major compound of T. ammi EO vapors when analyzed using solid-phase microextraction and gas-tight syringe sampling techniques. This study demonstrates the validity of the broth macrodilution volatilization method for antimicrobial screening of volatile compounds in the vapor phase and suggests the therapeutic potential of Indian medicinal plants in inhalation therapy.

In Vitro Growth-Inhibitory Synergistic Effect of Zinc Pyrithione in Combination with Gentamicin against Bacterial Skin Pathogens of Livestock.

Bacterial skin diseases of livestock could be a serious global threat, especially in association with overcoming bacterial resistance. Combinatory action of antimicrobial agents proves to be an effective strategy to overcome the problem of increasing antibiotic resistance of microorganisms. In this study, the in vitro combined effect of zinc pyrithione with gentamicin against bacterial skin pathogens of livestock (Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae) was evaluated according to the sum of fractional inhibitory concentration indices (FICI) obtained by checkerboard method. The results showed that a combination of zinc pyrithione with gentamicin produced a strong synergistic effect (p < 0.001) against all tested streptococcal strains (with FICI values ranging from 0.20 to 0.42). Compared to that, only three out of eight S. aureus strains were highly susceptible to the combination of antimicrobial agents at single concentration (0.25 µg/mL) of zinc pyrithione with range of FICI 0.35−0.43. These findings suggest that interference between agents tested in this study can be used for the development of future veterinary pharmaceutical preparations for the treatment of bacterial skin infections of livestock.

Antibacterial Activity of Thymus vulgaris L. Essential Oil Vapours and Their GC/MS Analysis Using Solid-Phase Microextraction and Syringe Headspace Sampling Techniques.

While the inhalation of Thymus vulgaris L. essential oil (EO) is commonly approved for the treatment of mild respiratory infections, there is still a lack of data regarding the antimicrobial activity and chemical composition of its vapours. The antibacterial activity of the three T. vulgaris EOs against respiratory pathogens, including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes, was assessed in both liquid and vapour phases using the broth microdilution volatilisation (BMV) method. With the aim of optimising a protocol for the characterisation of EO vapours, their chemical profiles were determined using two headspace sampling techniques coupled with GC/MS: solid-phase microextraction (HS-SPME) and syringe headspace sampling technique (HS-GTS). All EO sample vapours exhibited antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 512 to 1024 µg/mL. According to the sampling technique used, results showed a different distribution of volatile compounds. Notably, thymol was found in lower amounts in the headspace-peak percentage areas below 5.27% (HS-SPME) and 0.60% (HS-GTS)-than in EOs (max. 48.65%), suggesting that its antimicrobial effect is higher in vapour. Furthermore, both headspace sampling techniques were proved to be complementary for the analysis of EO vapours, whereas HS-SPME yielded more accurate qualitative results and HS-GTS proved a better technique for quantitative analysis.

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro.

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (ß-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

Validation of Qualitative Broth Volatilization Checkerboard Method for Testing of Essential Oils: Dual-Column GC-FID/MS Analysis and In Vitro Combinatory Antimicrobial Effect of Origanum vulgare and Thymus vulgaris against Staphylococcus aureus in Liquid and Vapor Phases.

Combinatory action of antimicrobial agents such as essential oils (EOs) show to be an effective strategy to overcome the problem with increasing antibiotic resistance of microorganisms, including Staphylococcus aureus. The objective of this study was to evaluate in vitro antimicrobial interactions between Origanum vulgare and Thymus vulgaris EOs against various S.aureus strains in both liquid and vapor phases using the broth volatilization checkerboard method. Fractional inhibitory concentrations (FICs) were determined for both liquid and vapor phases, and the composition of EOs was analyzed by gas chromatography-mass spectrometry using dual-column/dual-detector gas chromatograph. Results of oregano and thyme EOs combination showed additive effects against all S. aureus strains in both phases. In several cases, sums of FICs were lower than 0.6, which can be considered a strong additive interaction. The lowest FICs obtained were 0.53 in the liquid phase and 0.59 in the gaseous phase. Chemical analysis showed that both EOs were composed of many compounds, including carvacrol, thymol, γ-terpinene, and p-cymene. This is the first report on oregano and thyme EOs interactions against S. aureus in the vapor phase. It also confirms the accuracy of the broth volatilization checkerboard method for the evaluation of combinatory antimicrobial effects of EOs in the vapor phase.

Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays.

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated.

Chemical Composition and Determination of the Antibacterial Activity of Essential Oils in Liquid and Vapor Phases Extracted from Two Different Southeast Asian Herbs-Houttuynia cordata (Saururaceae) and Persicaria odorata (Polygonaceae).

Essential oils obtained via the hydrodistillation of two Asian herbs (Houttuynia cordata and Persicaria odorata) were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Additionally, both the liquid and vapor phase of essential oil were tested on antimicrobial activity using the broth microdilution volatilization method. Antimicrobial activity was tested on Gram-negative and Gram-positive bacteria-Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes, Klebsiella pneumoniae, Seratia marcescense and Bacillus subtilis. Hydrodistillation produced a yield of 0.34% (Houttuynia cordata) and 0.40% (Persicaria odorata). 41 compounds were identified in both essential oils. Essential oils contained monoterpenes and their oxidized forms, sesquiterpenes and their oxidized forms, oxidized diterpenes, derivates of phenylpropene and other groups, such as, for example, aldehydes, alcohols or fatty acids. Both essential oils were antimicrobial active in both vapor and liquid phases at least in case of one bacterium. They expressed various antimicrobial activity in the range of 128-1024 µg∙mL-1, 512-1024 µg∙mL-1 in broth and 1024 µg∙mL-1, 512-1024 µg∙mL-1 in agar, respectively. Research showed new interesting information about P. odorata and H. cordata essential oils and demonstrated that both essential oils could be possibly used in the field of natural medicine or natural food preservation.

Volatile Antimicrobial Agents and In Vitro Methods for Evaluating Their Activity in the Vapour Phase: A Review.

This review summarizes data on the in vitro antimicrobial effectiveness of volatile agents of plant origin and in vitro methods for evaluating their activity in the vapour phase. As a result of literature analysis, the antimicrobial efficacy of vapours from 122 different plant species and 19 pure compounds examined in 61 studies using different in vitro tests against a broad spectrum of microorganisms was identified and summarized. In addition, 11 different techniques found in the literature are described in detail. An original classification of methods based on the solid and liquid matrix volatilization principle is proposed because carrier medium/matrix selection is crucial for the volatilization of any agents tested. This review should be useful for medicinal, pharmaceutical, food, and agricultural experts working in areas related to the management of infectious diseases (especially respiratory and skin infections), food preservation (active packaging), and protection of agriculture products (controlled atmosphere). It may also stimulate the interest of pharmaceutical, cosmetic, food, and agriculture industries in the research and development of new antimicrobial agents of natural origin. Since several original apparatuses previously developed for antimicrobial susceptibility testing in the vapour phase are described in this review, labware manufacturers may also be interested in this topic. The review also provides specific guidelines and recommendations for researchers studying the antimicrobial activity of volatile agents. The article will therefore appeal to communities of industrial stakeholders, pharmacists, physicians, food experts, agriculturists, and researchers in related areas such as pharmacology, medicinal chemistry, microbiology, natural product chemistry, food preservation and plant protection.

Evaluation of in vitro growth-inhibitory effect of carvacrol and thymol combination against Staphylococcus aureus in liquid and vapour phase using new broth volatilization chequerboard method.

Carvacrol and thymol, both plant-derived volatile compounds, have extensively been studied individually as well as in combination with other agents for their antimicrobial activity in liquid phase. However, in contrast to well-established assays for testing of antimicrobial combinatory effects in liquid media, there are no standardized methods for evaluation of interactions between volatile compounds in vapour phase. The objective of this study was to verify new broth volatilization chequerboard method by testing the combination of carvacrol and thymol and to determine in vitro inhibitory effect of these compounds in liquid and vapour phase against twelve Staphylococcus aureus strains. The new method, based on combination of standard microdilution chequerboard and new broth volatilization tests allowing calculation of fractional inhibitory concentrations (FICs), was used. Combination of carvacrol and thymol produced the additive antimicrobial effect against all strains tested. In several cases, they reached ΣFIC values lower than 0.6, which can be considered as a strong additive interaction. The best result was found in vapour phase against one standard strain at combination of 128 µg/mL of carvacrol and 16-256 µg/mL of thymol (ΣFIC = 0.51) and in liquid phase against one clinical isolate at combination of 256 µg/mL of carvacrol and 256 µg/mL of thymol (ΣFIC = 0.53). The study verified that the new technique is suitable for simple and rapid high-throughput combinatory antimicrobial screening of volatile compounds simultaneously in vapour and liquid phase and that it allows determination and comparison of MIC and FIC values in both, liquid and solid media.

Evaluation of antibacterial potential and toxicity of plant volatile compounds using new broth microdilution volatilization method and modified MTT assay.

With aim to develop effective proof-of-concept approach which can be used in a development of new preparations for the inhalation therapy, we designed a new screening method for simple and rapid simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmat™ as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. Antibacterial activity of carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone was determined against Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae using new broth microdilution volatilization method. The cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128µg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86-2.95µg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71µg/mL, however this compound produced only weak antibacterial potency with MICs 512-1024µg/mL. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents without need of special apparatus. In our opinion, this assay can also potentially be used for development of various medicinal, agricultural, and food applications that are based on volatile antimicrobials.