Essential oils (EOs) have been gaining popularity in the past decades among researchers due to their potential to replace conventional chemicals used in the fight against pests, pathogenic and spoilage microbes, and oxidation processes. EOs are complex mixtures with many chemical components, the content of which depends on many factors-not just the plant genus, species, or subspecies, but also chemotype, locality, climatic conditions, phase of vegetation, method of extraction, and others. Due to this fact, there is still much to study, with antimicrobial effect being one of the key properties of EOs. There are many methods that have been frequently used by researchers for in vitro evaluation; however, although the research has been going on for decades, an internationally accepted standard is still missing. Most of methods are based on time-proven standards used for the testing of antibiotics. Due to the specific properties of EOs and their components, such as volatility and hydrophobicity, many modifications of these standard procedures have been adopted. The aim of this review is to describe the most common methods and their modifications for the testing of antimicrobial properties of EOs and to point out the most controversial variables which can potentially affect results of the assays.

• Klíčová slova
• agar diffusion, agar dilution, antibacterial, biofilm, broth dilution, plant extracts, vapor phase,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

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, β-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.

• Klíčová slova
• Cymbopogon citratus, Cyperus scariosus, GC/MS, MTT assay, Trachyspermum ammi, antimicrobial activity, headspace analysis, macrodilution, respiratory infections, vapor phase, volatiles,
• MeSH
• antibakteriální látky chemie   MeSH
• antiinfekční látky * analýza   MeSH
• léčivé rostliny * MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• oleje prchavé * farmakologie   chemie   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• pneumonie * MeSH
• volatilizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• antiinfekční látky * MeSH
• oleje prchavé * MeSH

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.

• Klíčová slova
• antimicrobial activity, broth microdilution, headspace analysis, respiratory infections, thyme, vapour phase,
• MeSH
• antibakteriální látky chemie   izolace a purifikace   farmakologie   MeSH
• Haemophilus influenzae účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• mikroextrakce na pevné fázi * MeSH
• oleje prchavé chemie   izolace a purifikace   farmakologie   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• Staphylococcus aureus účinky léků   MeSH
• Streptococcus pyogenes účinky léků   MeSH
• Thymus (rostlina) chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• oleje prchavé MeSH

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 (β-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.

• Klíčová slova
• antimicrobial, cytotoxicity, macrodilution method, respiratory infections, thymohydroquinone, thymoquinone, vapor phase, volatile compound, β-thujaplicin,
• MeSH
• antibakteriální látky analýza   chemie   farmakologie   MeSH
• aplikace inhalační MeSH
• Bacteria účinky léků   MeSH
• benzochinony aplikace a dávkování   farmakologie   MeSH
• Haemophilus influenzae účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• monoterpeny aplikace a dávkování   farmakologie   MeSH
• oleje prchavé chemie   farmakologie   MeSH
• Staphylococcus aureus účinky léků   MeSH
• Streptococcus pneumoniae účinky léků   MeSH
• Streptococcus pyogenes účinky léků   MeSH
• těkavé organické sloučeniny chemie   farmakologie   MeSH
• thymol aplikace a dávkování   analogy a deriváty   farmakologie   MeSH
• tropolon aplikace a dávkování   analogy a deriváty   farmakologie   MeSH
• volatilizace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• benzochinony MeSH
• beta-thujaplicin MeSH Prohlížeč
• monoterpeny MeSH
• oleje prchavé MeSH
• těkavé organické sloučeniny MeSH
• thymohydroquinone MeSH Prohlížeč
• thymol MeSH
• thymoquinone MeSH Prohlížeč
• tropolon MeSH

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.

• Klíčová slova
• GC/MS, antimicrobial interactions, broth volatilization chequerboard method, chemical composition, fractional inhibitory concentration, gaseous phase, oregano, thyme, volatile compound,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• DPPH, MTT, bioassay, broth microdilution, essential oil, microtiter plate, plant compounds, supercritical CO2 extract, volatilization,
• MeSH
• antiinfekční látky chemie   izolace a purifikace   farmakologie   MeSH
• antioxidancia chemie   izolace a purifikace   farmakologie   MeSH
• bifenylové sloučeniny antagonisté a inhibitory   MeSH
• biologické přípravky chemie   izolace a purifikace   farmakologie   MeSH
• Candida albicans účinky léků   MeSH
• galgán chemie   MeSH
• mikrobiální testy citlivosti MeSH
• Myrtaceae chemie   MeSH
• Nigella chemie   MeSH
• oleje prchavé chemie   izolace a purifikace   farmakologie   MeSH
• pikráty antagonisté a inhibitory   MeSH
• rostlinné extrakty chemie   izolace a purifikace   farmakologie   MeSH
• skořicovník chemie   MeSH
• Staphylococcus aureus účinky léků   MeSH
• těkavé organické sloučeniny chemie   izolace a purifikace   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 1,1-diphenyl-2-picrylhydrazyl MeSH Prohlížeč
• antiinfekční látky MeSH
• antioxidancia MeSH
• bifenylové sloučeniny MeSH
• biologické přípravky MeSH
• oleje prchavé MeSH
• pikráty MeSH
• rostlinné extrakty MeSH
• těkavé organické sloučeniny MeSH