Peer Reviewed Articles About Essential Oils Antifungal Properties

Abstract

Vi essential oils (from oregano, thyme, clove, lavender, clary sage, and arborvitae) exhibited different antibacterial and antifungal properties. Antimicrobial activity was shown confronting pathogenic (Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis) and environmental bacteria (Bacillus cereus, Arthrobacter protophormiae, Pseudomonas fragi) and fungi (Chaetomium globosum, Penicillium chrysogenum, Cladosporium cladosporoides, Alternaria alternata, and Aspergillus fumigatus). Oregano, thyme, clove and arborvitae showed very strong antibacterial activeness confronting all tested strains at both total strength and reduced concentrations. These essential oils showed dissimilar fungistatic and fungicidal activities when tested by direct application and in the vapor phase. The genotoxic furnishings of these oils on HEL 12469 homo embryo lung cells were evaluated using an alkaline comet assay for the commencement fourth dimension, revealing that none of the oils induced meaning DNA damage in vitro after 24 h. This study provides novel approaches for assessing the antimicrobial potential of essential oils in both direct contact and the vapor stage and as well demonstrates the valuable properties of the phenol-free arborvitae oil. These results suggest that all the tested essential oils might be used as wide-spectrum anti-microbial agents for decontaminating an indoor environment.

Introduction

Essential oils (EOs) are products derived from effluvious plants which incorporate around 20–threescore components at quite unlike concentrations¹. Their about common constituents are terpenes, aromatic and aliphatic compounds (especially alcohols, esters, ethers, aldehydes, ketones, lactones, phenols and phenol ethers)^ane.

EOs from Origanum vulgare L., Thymus vulgaris Fifty., Salvia sclarea 50., and Lavandula angustifolia Mill. belonging to the Lamiaceae family have been used for their medicinal properties¹ for centuries; they possess antibacterial, antifungal^2,3,iv,5, antioxidant, anti-inflammatory^{vi, 7} and analgesic properties⁷. Clove EO from Eugenia caryophyllata L. (Myrtaceae) has shown antibacterial, antifungal, anti-oxidant^viii and anti-inflammatory furnishings^nine. EO from Thuja plicata (Cupressaceae) has been tested for antimicrobial^{10, 11} and insecticidal activity¹².

The antibacterial properties of EOs have been observed in several studies^{1, thirteen, 14}. Most of the studies have examined the direct issue of EOs on a range of microorganisms. For example several Gram-negative and Gram-positive leaner are sensitive to various EOs^{2, 3, 14,15,16}, showing clear zones on agar assays in which the tested EO inhibits the growth of a particular microorganism. Some studies as well adamant the minimal inhibition and minimal bactericidal concentrations in liquid medium^{11, 17}.

Nevertheless, EOs can too exist in a potentially highly bioactive vapor stage, and some EOs take shown antimicrobial activity that does not require direct contact with the EO^18,19,20,21. The vapor phase seems especially constructive against fungi, and a number of studies take shown that EOs are more than constructive antifungals in the vapor country than in the liquid^20,21,22. Ane possible caption for this behavior is that the lipophilic molecules responsible for at least part of the activity might acquaintance in the aqueous phase to form micelles, thereby suppressing their zipper to the organism, whereas the vapor phase allows free attachment²². In this state of affairs, the observed antimicrobial activity arising from the easily volatilized components would issue from a combination of the straight exposure to the vapor and the indirect exposure mediated by agar medium which captivated the vapor²³. Moreover, fungal strains tend to grow more on the agar surface than bacteria, and therefore would be more exposed to the vapor while the leaner would exist more strongly afflicted by the EO components that accumulated in the substrate.

EOs with biocidal activity were used to develop alternative disinfection strategies for indoor environments or in the nutrient industry, on contaminated surfaces and equipment in nutrient processing environments^{fifteen, 24,25,26}. The ability of some EOs to foreclose the germination of Listeria monocytogenes ¹⁵ and Salmonella enterica ²⁶ biofilm on stainless steel surfaces has previously been demonstrated.

Although EOs were applied in the past to successfully treat a diverseness of diseases and to preserve health, they have been used more oftentimes for a greater variety of applications in recent years, including drugs, crop protectants, nutrient additives, aromatherapy, and others. The resulting increase in homo exposure as a consequence of this expanded usage therefore requires a careful re-assessment of their toxicity and genotoxicity on the level of mammalian cells²⁷. The potential toxic effects of plant extracts, including EOs, on humans should not be underestimated. The mutagenicity of many plant extracts and their possible genotoxicity^{28,29,xxx,31,32} have been evaluated previously. There are several studies examining the genotoxic properties of EOs^{29, 33,34,35}, only there is non well-nigh enough data about the potential risk of sensitization when using EOs.

The purpose of this study was to determine the antimicrobial backdrop of six EOs (O. vulgare, T. vulgaris, S. sclarea, Fifty. angustifolia, Due east. caryophyllata and T. plicata) confronting clinical and food-borne bacterial pathogens and as well every bit several environmental bacterial and fungal strains. The antifungal properties of the vapor stage of these EOs were also investigated. Our in vitro trials determined the concentrations of EOs needed to reliably forestall the growth of pathogenic and environmental microorganisms. Finally, in this paper nosotros besides written report the showtime in vitro results on the cytotoxic and genotoxic activities of these EOs in human embryo lung cells (HEL 12469).

Results

Antibacterial activity of essential oils

The in vitro antibacterial activeness of six EOs against bacterial strains from both clinical and environmental origins (both Gram-positive and Gram-negative bacteria) was assayed using the disc diffusion method by measuring inhibition zone diameters (Fig. 1). All EOs tested showed antibacterial effects based on these inhibition zones (*p < 0.05; **p < 0.01; ***p < 0.001). Origanum vulgare (OR) and Thymus vulgaris (TY) EOs were extremely constructive on all tested leaner, with inhibition zones ranging from 26–54 mm. The differences in the measured inhibition halos of OR (p = 0.000457), TY (p = 0.000457) and Lavandula angustifolia (LA; p = 0.0117) on Staphylococus aureus were statistically different from the control. Interestingly, OR and TY produced inhibition halos much larger than those of chloramphenicol, suggesting that they are more than agile than this antibiotic. Eugenia caryophyllata (clove; CL) and Thuja plicata (arborvitae; AR) EOs exhibited a lower degree of bacterial growth inhibition than OR and TY, while the greatest inhibition observed was caused by AR confronting Yersinia enterocolitica (p < 0.01). Environmental bacterial strains were much more than sensitive to chloramphenicol than clinical strains; no meaning departure in susceptibility was found betwixt Gram-negative and Gram-positive bacteria. LA and Salvia sclarea (SA) EOs were both less agile against all leaner, with inhibition zones ranging from eight–14 mm.

Preliminary screening revealed that the OR, TY, CL, and AR EOs were the most constructive confronting all tested leaner; therefore, additional the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed with these four EOs. MIC and MBC assays were performed using a broth microdilution method in 96-well strip tubes covered with strip-caps. The results obtained from these assays are shown in Table ane. These antibacterial assays revealed that OR has a very strong activity (MIC 0.025%, MBC 0.025–0.05%) together with TY (MIC 0.025–0.125%, MBC 0.05–0.125%) while the CL and AR EOs had less antibacterial action (MIC 0.05–0.125%, MBC 0.125–0.five%). All iv EOs inhibited the growth of both clinical and environmental Gram-positive (South. aureus, L. monocytogenes, East. faecalis, B. cereus, and A. protophormiae) and Gram-negative bacteria (E. coli, Southward. typhimurium, Y. enterocolitica, and P. fragi).

Tabular array 1 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) (% w/v) of oregano, thyme, clove, and arborvitae EOs against tested bacteria.

Total size table

Antifungal activity of essential oils

A disc improvidence analysis was performed to make up one's mind the sensitivity of 5 fungal strains to the six EOs past measuring the inhibition zone diameters (in mm). Our goal was to determine whether the dissimilar EOs had similar inhibition effects on several different fungal strains (Cladosporium cladosporoides, Alternaria alternata, Aspergillus fumigatus, Chaetomium globosum and Penicillium chrysogenum). All tested EOs at concentrations of 75, 50, 25, ten and five% (w/v) showed antifungal activity, inhibiting the mycelial growth (Figs 2 and 3). The LA and SA EOs exhibited a lower level of inhibition.

The MIC and minimal fungicidal concentrations (MFC) of the OR, TY, CL, and AR EOs against Ch. globosum, P. chrysogenum, C. cladosporoides, A. alternata, and A. fumigatus are summarized in Table 2. The greatest antifungal action against all tested strains was exhibited by OR, which had MICs of 0.01% and 0.025% and MFCs of 0.025%, 0.05% and 0.075%. TY EO, despite being efficient confronting all tested fungal strains, appeared to have no fungicidal activity confronting P. chrysogenum, C. cladosporoides and A. fumigatus (Tabular array 2). CL also had no fungicidal activity against A. alternata and P. chrysogenum. Overall, OR, AR, TY and CL were effective as fungicidal agents but their efficiency varied from strain to strain (Table ii). The fungicidal effect was confirmed when sub-culturing the tested fungi from the agar dilution assays into fresh malt excerpt broth (MEB) without EO resulted in no further mycelial growth or resumption of spore germination. LA and SA EOs had no antifungal activity against any tested fungal strain.

Table ii Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) (% w/v) of oregano, thyme, clove, and arborvitae EOs against the tested fungal strains.

Full size table

Volatile vapor of essential oils

The efficacy of OR, TY, CL, AR, LA, and SA EOs in the vapor phase confronting Ch. globosum, P. chrysogenum, C. cladosporoides, A. alternata, and A. fumigatus was investigated. The volatile vapor of 0.005% EOs exhibited only a fungistatic effect on the tested fungi while the volatile vapor of 0.075% OR, TY, CL and AR completely inhibited the mycelial growth of all tested fungal strains (Fig. four) and were also revealed to accept a fungicidal effect after the re-inoculation of inhibited fungal mycelial plugs into fresh malt extract agar (MEA) and fresh MEB. Exceptionally, notwithstanding, P. chrysogenum and A. fumigatus treated with CL volatile vapor (0.075%) continued to grow in fresh MEB after re-inoculation, meaning that CL had only a fungistatic issue on these strains.

The volatile vapor of LA and SA at 0.075% concentration completely inhibited the growth of all tested fungi except A. alternata, but had no fungicidal properties against any of them. The vapor stage of TY and AR were more effective against P. chrysogenum, C. cladosporoides and A. fumigatus than in the liquid stage.

Cytotoxic and DNA-damaging furnishings of essential oils

The MTT (3-(4,five-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis was used to decide the cytotoxic effects on HEL 12469 cells of a 24 h exposure of different concentrations of EOs (0.0025–1.0 µL/mL). Figure 5 summarizes the results: IC_fifty values (the median inhibitory concentrations that cause approximately 50% cell death) were 0.058 µL/mL for OR, 0.fifteen µL/mL for AR and TY, 0.23 µL/mL for CL, 0.28 µL/mL for LA, and 0.45 µL/mL for SA; IC_twenty values (the median inhibitory concentrations that cause approximately 20% cell decease) were 0.026 µL/mL for OR, 0.10 µL/mL for AR, 0.085 µL/mL for TY, 0.13 µL/mL for CL, 0.23 µL/mL for LA, and 0.41 µL/mL for SA.

Further studies examined the genotoxic effects of these EOs, which were assessed at IC~_{10–twoscore}. Single-jail cell gel electrophoresis (SCGE; too known as comet analysis) was used to determine the level of DNA single-strand breaks in HEL 12469 cells. Simply one of the EOs, 0.2 µl/ml of AR, induced a significantly different level of Deoxyribonucleic acid breaks than those observed in the untreated command cells, (**p < 0.01) (Fig. vi).

Discussion

The antimicrobial efficacy of a given EO depends on its chemic composition, perhaps particularly its phenolic components^{1, fourteen}. In our report, therefore, we selected the EOs which are well-known for its high content phenols (OR, TY, CL), EOs with lower phenol content (LA and SA), and a phenol-free EO (AR)^{11, 36, 37}. This last was chosen based on previous studies on the susceptibility of diverse leaner and fungi to cedar leafage EO³⁶ and on our own preliminary experiments (data non shown). This item AR EO was obtained from heart-forest and independent mainly tropolones³⁷.

The pathogenic bacteria selected for this study were chosen based on previous findings on the ability of some EOs to inhibit some man pathogens^{2, 4, 17, 38}. The environmental bacterial and fungal strains were chosen to be representative of airborne contaminants which our group has isolated from indoor environments (unpublished information).

The measured inhibition halos of OR, TY, CL, AR, LA, SA indicated that all of EOs are constructive against bacteria. The OR and TY used in this study were fifty-fifty more effective than the antibody chloramphenicol. Our results are in accordance with a previous study showing that the inhibitory halos produced by the EOs of Eugenia caryophyllata, Origanum vulgare and Thymus vulgaris were larger than those produced by ciprofloxacin³⁸.

The low antibacterial activity of the LA and SA EOs may be due to the relatively depression phenol content of these EOs: their main components are, respectively, alcohols³⁹ and esters⁴⁰. This is consistent with another report on the antimicrobial efficiency of the EO^sixteen from Salvia officinalis, which reported a very depression antibacterial activity for 1,viii-cineole against S. aureus, B. subtilis, and E. coli. In the nowadays study, CL EO, which is known for its high eugenol⁴¹ content (a phenolic compound) was not found to be the most active EO against the microorganisms tested in the disc-improvidence analysis. It is possible that the sample we used had a lower concentration of the relevant compounds; it has previously been shown that the EOs of plants belonging to the same species, merely collected from unlike places can exhibit unlike antimicrobial activeness⁴².

It should be noted that the disc-diffusion method is limited by the hydrophobic nature of nigh EOs, which prevents their uniform improvidence through the agar medium. Therefore, most researchers prefer liquid medium methods⁴³. The EOs of OR, TY, CL and AR exhibited strong antimicrobial action against all microorganisms in liquid medium, every bit it has been previously described^{3, 16}. It is too quite interesting that no bacterial strain tested was resistant to any of the EOs studied.

Some studies accept reported that EOs tend to human action more than strongly on Gram-positive than Gram-negative leaner^{2,3,iv, 44}, presumably due to differences in cell wall composition⁴⁴. At that place is no general rule with respect to Gram sensitivity: the literature reports many conflicting studies showing that some Gram-negative strains are more sensitive than some Gram-positive ones to certain EOs^{21, 36, 38}. For case, Preuss et al.⁴⁵ constitute that origanum EO is lethal to East. coli and Klebsiella pneumoniae. Origanum syriacum L., Thymus syriacus Boiss. and Syzygium aromaticum L. EOs were constructive against the Gram-negative leaner Due east. coli O157:H7, Y. enterocolitica O9, Proteus spp., and Thou. pneumoniae ⁴⁶. Our results take also shown that some Gram-negative leaner (E. coli, Due south. typhimurium, Y. enterocolitica, and P. fragi) are sensitive to OR, TY, CL, AR.

Antifungal action of EOs was adamant past direct contact assay and also we tested their antifungal properties in the vapor stage. In a previous study, MICs of thyme red, clove, sage and lavender, for aspergilli and penicillin, ranged from 0.125% to one% afterwards 3 and 7 days^nineteen. Our results revealed that all tested fungal strains showed college susceptibility (MICs of OR, TY, CL and AR EOs ranged from 0.01% - 0.075%). The volatile components of OR, TY, CL and AR showed fungicidal activity while the LA and SA vapors demonstrated fungistatic activity. These results are very different from the straight contact assay, where LA and SA were unsuccessful in inhibiting the growth of the tested strains. At that place is growing testify that EOs in the vapor phase are more effective against fungi than in the liquid phase^{18,19,20,21,22, 47}. Thyme EOs vapours have been shown to be constructive against Aspergillus sp. and Penicillium sp.¹⁹. The high antifungal activity of vapour evidenced in our study is in accordance with previous finding⁴⁸, which showed that thyme and clove oils were more effective in vapour land against A. flavus.

AR vapors displayed comparable results to OR, TY and CL in spite of its lack of phenolic compounds and its loftier concentration of monoterpens, which are normally considered to be less constructive antimicrobial substances^xiv. According to¹⁴, antimicrobial activity can generally exist classified in the following order: phenols > aldehydes > ketones > alcohols > esters > hydrocarbons. The link between the well-nigh abundant constituent blazon and the antimicrobial activity is somewhat variable; for example, Inouye et al.²² reported that booze-containing EOs are more than active than ketone-containing EOs against Trichophyton mentagrophytes.

Most EOs are safe and free of adverse side effects when used properly⁴⁹. The most important safety factor for EOs is their dosage. EOs have shown antitumor activity both in vitro and in vivo and depression toxicity^{l, 51}. On the other hand, it has also been shown that high concentrations of some EOs contributed to harmful changes in the body⁵².

We have demonstrated the EOs do have cytotoxic outcome, but only at college concentrations on HEL 12469 human embryonic lung cells under in vitro conditions. The result of a 24 h treatment with a given EO on the viability of HEL cells was dependent on its concentration. IC₅₀ values declined in the order SA > LA > AR = TY > CL > OR. In other cell lines (human hepatocarcinoma cell line HepG2, human being keratinocytes HaCaT, man melanoma jail cell line HMB-2), the cytotoxic effect of LA EO was detected at a slightly college concentration ~ IC₅₀-0.4 µL/mL, which might be explained past differences in cell sensitivity⁵³. Finally, LA at loftier concentration was genotoxic to peripheral human lymphocytes⁵⁴ and to human monocyte THP-1 cells⁵⁵.

In our experiments the EC₅₀ value for all EOs was >20 µg/mL (for case: IC_fifty SA - 0.45 µl / ml = 0.351 mg/ml) indicating that none of them were cytotoxic based on the criteria gear up past the National Cancer Institute⁵⁶, which land that only natural substances with EC_l < 20 µg/mL are considered cytotoxic.

The level of Deoxyribonucleic acid single-strand breaks induced in HEL 12469 cells by these EOs was adamant using a Comet analysis. Handling with most EOs alone did not induce whatever significant increase in DNA strand breaks over the untreated control cells; the single exception was the highest concentration of AR EO examined (0.2 µL/mL). Similarly, it was recently shown that establish extracts of S. officinalis and T. vulgaris did non induce Dna damage in HepG2 cells or master rat hepatocytes^{57, 58}.

Conclusions

This study provides a wide range of data about the biological activities of EOs. It determined the biocidal efficiency of 6 EOs (from OR, TY, CL, AR, LA and SA) against five different fungal and nine unlike bacterial strains. In order to verify the potential take chances of EOs to human cells, the cytotoxicity and genotoxicity of each of these EOs on human HEL lung cells was assessed for the first time. Of the 6 EOs studied, OR, TY, CL, and AR were highly effective against all bacterial strains tested. LA and SA exhibited no antifungal activity by directly contact, merely did evidence a fungistatic event in the vapor phase. OR, TY, and AR exhibited important fungicidal activeness against all strains tested; CL showed fungicidal activity confronting most strains, merely just a fungistatic effect on P. chrysogenum and A. fumigatus.

The assayed EOs are non considered cytotoxic as judged by the criteria set by the National Cancer Institute and appeared non to damage the Deoxyribonucleic acid of HEL cells.

The information reported in this report evidence that EOs might provide an alternative way to fight microbial contagion and that they can exist considered condom for humans at relatively low concentrations. Generally, it is possible to recommend the use of EOs for various environmental disinfection strategies, merely only later accurate in vitro trials, such as those described in this investigation.

Materials and Methods

Essential oils

The commercially available EOs used in this work were OR from O. vulgare L., TY from T. vulgaris L., CL from Eastward. caryophyllata 50., LA from L. angustifolia Mill., SA from S. sclarea L., and AR from T. plicata Donn. (all from doTERRA, Pleasant Grove, USA). A GC/MS assay was provided past the producer, who guaranteed the chemical composition of each EO. The EOs were stored in amber glass vials and sampled using sterile pipet tips to minimize contamination and oxygen exposure. Since the EOs varied in density, each of the EOs was weighed to make up one's mind the book that comprised 10 mg. This amount was used in testing every bit the total-force (100%) concentration and was then serially diluted in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., United states of america).

Microbial strains and growth atmospheric condition

The EO antimicrobial activities were investigated against different clinical and nutrient-borne bacterial pathogens: S. aureus (FRIC 418), L. monocytogenes (FRIC 270), E. faecalis (FRIC 282); E. coli (FRIC 375), S. typhimurium (FRIC 305), and Y. enterocolitica (FRIC thirty); environmental bacterial strains from our own drove were too examined, including B. cereus, P. fragi, and A. protophormiae.

The fungal strains used in this study (Ch. globosum, P. chrysogenum, C. cladosporioides, A. alternata, A. fumigatus) were air-borne isolates from our laboratory drove. The bacterial strains were kept frozen in stock cultures at −80 °C in cryovials, and the fungal cultures were stored at iv °C and subcultured once a month. Prior to the inoculation of the strains with EOs, the leaner were grown at 28 or 37 °C (depending on the type of microorganism) for 12–18 h on Luria–Bertani agar (LBA) for environmental bacteria or Mueller Hinton agar (MHA) for pathogenic bacteria. The fungal strains were grown at 26 °C on Malt Extract Agar (MEA).

Cell civilisation

HEL 12469 human embryo lung cells (Human embryonic lung fibroblast; ECACC 94101201), were cultivated in Hawkeye's Minimum Essential Medium (MEM) supplemented with 10% fetal dogie serum (FCS), 1% non-essential amino acids and antibiotics (streptomycin l μg/mL and penicillin 50 U/mL)^l. Cell lines were cultured in a humidified atmosphere of five% CO_two at 37 °C. The chemicals and media used for cell cultivation were purchased from Gibco BRL (Paisley, Uk).

Screening for antibacterial activity

A disc-diffusion assay was used to make up one's mind the growth inhibition of bacteria by EOs. A single colony from an overnight bacterial culture plate was seeded into 5 mL of an appropriate pre-warmed growth medium goop (LBB or MHB). Culture tubes were shaken at 300 rpm and 37 °C until the 600 nm absorbance of the growth solution was greater than 1.0. Using a sterile swab, cultures were spread evenly onto pre-warmed 37 °C agar plates. Sterile filter paper discs (half-dozen mm Ø Whatman No.i) were gently pressed onto the surface of the agar plates, and EOs were so pipetted onto the discs. Each EO was tested at 100% strength, and at various dilutions (5, 10, 25, 50, and 75%) in DMSO. A pure DMSO control was included with each test to ensure that microbial growth was not inhibited by DMSO itself. Chloramphenicol (30 μg/disc; Sigma-Aldrich, U.s.a.) was used equally a positive command. Plates were then inverted and incubated for approximately 24 h at 37 °C and the diameter of the inhibition zones was measured in mm, including the diameter of the disc. The sensitivity was classified according to Ponce et al.⁵⁹ every bit follows: not sensitive for a bore less than 8 mm, sensitive for a diameter of ix–14 mm, very sensitive for a diameter of 15–19 mm, and extremely sensitive for a diameter larger than 20 mm. Each exam was performed in iii replicates.

Evaluation of MIC and MBC in liquid medium

The MIC and MBC of each EO was determined using a broth microdilution method in 96-well strip tubes with transparent strip-caps according to Poaty et al.¹⁷ with modification. Bacterial suspensions were adjusted to a final concentration of 10⁶ CFU/mL in MHB. One hundred microliters of MHB containing 5% DMSO was distributed into the wells of the micro titer plates. EOs (10 µL) were added to these wells at a range of concentrations, from stock solutions 0.05, 0.10, 0.50, one.0, 2.5, five.0, and 10% (w/v). For each dilution, the same book every bit the full-strength sample was added. One hundred microliters of bacterial suspension was finally added to each. The plates were incubated at 37 °C for 24 h. MIC was adamant after calculation 40 µL of 0.2 mg/mL ρ-iodonitrotetrazolium violet (INT; Sigma-Aldrich, United states), followed by incubation at 37 °C for thirty min. MIC was determined every bit the everyman concentration of EO that inhibited visible growth of the tested microorganism. Growth of bacterial cells in each of the wells was verified by color change. When bacterial growth occurred (absence of inhibition), the INT changed from articulate to purple. Wells with DMSO solitary were used as controls. MBC is the everyman concentration of EO that results in microbial expiry. It was determined by subculturing from wells that exhibited no color change to sterile MHA plates that exercise not contain the test EO. The plates were then incubated at 37 °C for 24 h.

Screening for antifungal activeness

Fungal suspensions were prepared according to De Lira Mota et al.^lx by washing the surface of the MEA slant civilization with 5 mL of sterile saline and shaking the suspensions for 5 min. The resulting mixture of sporangiospores and hyphal fragments was withdrawn and transferred to a sterile tube. After heavy particles were allowed to settle for 3–five min, the upper intermission was nerveless and vortexed for 15 south. Final conidia suspensions were adjusted using a Neubauer's chamber to 10^six conidia per mL. 300 µL of each fungal suspension were applied to MEA plates. Filter paper discs (half-dozen mm Ø Whatman No.i) were placed on the agar surface of the Petri dishes and each EO, dissolved in DMSO at different concentrations (75, 50, 25, 10, five%) was individually added. For each dilution, the aforementioned book equally the full-force sample was placed on the sterile disc. Discs impregnated with 10 µL of DMSO, nystatin (50 µg/mL) and cycloheximide (50 µg/mL) (all Sigma-Aldrich, USA) were used equally controls. Petri dishes were incubated at 26 °C for 5 days. Inhibition zone diameters were measured in mm. An inhibition zone larger than 1 mm was taken to indicate a positive upshot.

Determination of fungistatic and fungicidal activities

The procedure reported by Thompson⁶¹ was used to determine whether a given EO possessed only a fungistatic effect or if it as well had fungicidal action. Different concentrations (10 µL) of each stock EO solution, ranging from 0.50, 1.0, ii.5, 5.0, 10, 25, 50 and 75% (w/v), were prepared by mixing various quantities of a given EO in DMSO (v/v) with x mL molten MEA; this mixture was then poured into sterile Petri dishes. The center of each solidified medium was inoculated upside down with 6-mm foursquare mycelial plugs cut from the periphery of 7-day-erstwhile cultures. Positive controls were simultaneously run with DMSO and without EO. Later incubation of the plates for 7 days at 26 °C, those fungal plugs that did not prove whatever growth were transferred to fresh MEA plates without EO for an additional 7 days at 26 °C to determine which concentration of each EO had a fungicidal consequence. The lowest concentration of each EO that completely prevented visible fungal growth and allowed a revival of fungal growth during the transfer experiment was considered the MIC for that EO. This effect was identified every bit fungistatic. The concentration unfavorable for growth revival during the transfer experiment was taken as the MFC and this effect was identified every bit fungicidal. Seven days after reinoculation, the inhibited fungal mycelial plugs were once once again reinoculated into fresh MEB without EO to see if their growth revived. Microscopic observations were carried out to investigate fungal jail cell growth after five days incubation at 26 °C. No growth was taken to confirm again the fungicidal activity and also to suggest a possible sporocidal effect.

Antifungal activity of vapor phase of essential oils

In order to determine the fungistatic or fungicidal activeness of volatized EOs, 6 mm squares of growing fungal mycelia were taken from the margin of the active growth area of fungal colonies and placed onto MEA plates. EOs from stock solutions (v% and 75%) at dose levels of 1 µL/1 mL air space were placed on the inner surface of the Petri dish lid; controls with DMSO and without EO were too prepared. The plates were sealed with parafilm to prevent vapor leakage and were incubated inverted for vii days at 26 °C. The radial mycelial growth of the fungus was then checked.

Transfer experiments for determining the fungistatic or fungicidal action of EO vapors were carried out by replacing the Petri dish hat with a new, untreated ane and incubating in an inverted orientation for an additional 7 days at 26 °C. The upshot was identified as fungistatic if growth was observed after the new incubation menses, and fungicidal if no growth was observed⁶². The effect was also confirmed by reinoculating the inhibited fungal mycelial plugs into fresh MEB without EO.

Cytotoxicity of essential oils

Exponentially growing HEL 12469 cells cultured in complete MEM were seeded onto 96 well plates (density of 2 × x^iv cells/well) and subsequently incubated in the presence or absence (negative command) of 0.0025–i.0 µL/mL EO for 24 h to test for cytotoxicity using the MTT assay⁵⁸. The MTT test is a colorimetric method for measuring the activity of the mitochondrial enzymes that reduce MTT, a yellow tetrazole, to purple formazan. This reduction takes place simply when reductase enzymes are active, and therefore conversion is often used equally a measure of viable (living) cells. In our experiments, later incubation with EO, HEL 12469 cells were washed with fresh MEM and 100 μL of complete MEM medium and 50 μL of one mg/mL MTT solution was added followed by a iv h incubation. The MTT solution was then replaced with 100 μL of DMSO and the plates were placed on an orbital shaker for 30 min to completely deliquesce the formazan crystals. At least 4 parallel wells were used for each sample. Absorbance at 540 nm was measured using an xMark™ Microplate Spectrophotometer (Bio-Rad Laboratories, Inc.) and the background absorbance at 690 nm was subtracted.

Genotoxicity of essential oils

HEL 12469 cells were seeded into a series of Petri dishes (1 × 10⁶ cells, Ø = 60 mm) and cultured in MEM. Cells were and so exposed to different EO concentrations (0.0025–1.0 µL/mL) for 24 h; cells with no treatment were used every bit an intact control. Afterwards the treatment, the cells were washed, trypsinized, re-suspended in a fresh civilisation medium and the level of DNA lesions was detected using the single prison cell gel electrophoresis (SCGE), also known as comet analysis (element of group i). The procedure of Singh et al.⁶³ was used with pocket-size modifications suggested by Slameňová et al.⁶⁴. In cursory, 2 × 10⁴ treated and control HEL 12469 cells were embedded in 0.75% low melting point (LMP) agarose. This jail cell suspension was spread as a single layer on a base layer of one% normal melting point (NMP) agarose in PBS on microscopic slides and covered with encompass slips. As positive command, hydrogen peroxide (300 μM) was used. After solidification of the gel, the cover slips were removed and placed in lysis solution (2.5 1000 NaCl, 100 mM Na₂EDTA, 10 mM Tris-HCl, pH 10 and one% Triton X-100, at four °C) for 1 h to remove cellular proteins and membranes. The slides were transferred to an electrophoresis box and immersed in an alkaline solution (300 mM NaOH, i mM Na_iiEDTA, pH > thirteen). After xl min unwinding fourth dimension, a voltage of 25 Five (0.6 V/cm) was applied for thirty min at iv °C. The slides were then neutralized with iii × v min washes with Tris-HCl (0.four M, pH seven.four), and stained with ethidium bromide (EtBr, five µg/mL; Sigma Chemical Company, St. Louis, MO). EtBr-stained nucleoides were examined with a Zeiss Imager Z2 fluorescence microscope with computerized image analysis (Metafer 3.half dozen, Meta Systems GmbH, Altlussheim, Deutschland). The percentage of DNA in the tail was used every bit a parameter for estimating the number of Dna strand breaks. One hundred comets were scored for each sample in one electrophoresis run.

Statistical assay

The data are given as means of 3 to 5 experiments ± one standard difference (SD). The differences betwixt the given groups were tested for statistical significance using Pupil'due south t-test (*p < 0.05; **p < 0.01; ***p < 0.001)⁵⁰. Because the antibacterial activity datasets were normally distributed, the independent samples t-test was performed to test for significant differences between groups. Differences between more than ii groups were assessed by one way analysis of variance (ANOVA) followed past the Bonferroni test if equal variances were assumed or Tamhane'due south test if equal variances were not causeless⁵⁰. Differences with p < 0.05 are considered to be statistically significant.

References

Bakkali, F., Averbeck, S., Averbeck, D. & Idaomar, M. Biological effects of essential oils - a review. Food Chem. Toxicol. 46, 446–475 (2008).

CAS Commodity PubMed Google Scholar
Fournomiti, M. et al. Antimicrobial activity of essential oils of cultivated oregano (Origanum vulgare), sage (Salvia officinalis),and thyme (Thymus vulgaris) confronting clinical isolates of Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae. Microb. Ecol. Health Dis. 26, 23289–23295 (2015).
Lambert, R. J. W., Skandamis, P. Northward., Coote, P. & Nychas, 1000. J. E. A study of the minimum inhibitory concentration and manner of action of oregano essential oil, thymol and carvacrol. J. Appl.Microbiol. 91, 453–462 (2001).

CAS Commodity PubMed Google Scholar
Mith, H. et al. Antimicrobial activities of commercial essential oils and their components against food-borne pathogens and food spoilage bacteria. Food Sci Nutr. 2(4), 403–416 (2014).

CAS Article PubMed PubMed Cardinal Google Scholar
Yuce, E., Yildirim, North., Yildirim, N. C., Paksoy, M. Y. & Bagci, E. Essential oil limerick, antioxidant and antifungal activities of Salvia sclarea L. from Munzur Valley in Tunceli, Turkey. Jail cell. Mol. Biol. 60(2), 1–5 (2014).

CAS PubMed Google Scholar
Cavanagh, H. M. A. & Wilkinson, J. M. Biological activities of lavender essential oil. Phytother. Res. 16(4), 301–308 (2002).

CAS Article PubMed Google Scholar
Hajhashemi, Five., Ghannadi, A. & Sharif, B. Anti-inflammatory and analgesic properties of the leaf extracts and essential oil of Lavandula angustifolia Mill. J. Ethnopharmacol. 89(1), 67–71 (2003).

Article PubMed Google Scholar
Vanin, A. B. et al. Antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced by enzymatic esterification. Applied biochemistry and biotechnology 174(4), 1286–1298 (2014).

CAS Article PubMed Google Scholar
Taher, Y. A. et al. Experimental evaluation of anti-inflammatory, antinociceptive and antipyretic activities of clove oil in mice.Libyan. J Med. ten(1), 28685 (2015).

Google Scholar
Johnston, W. H., Karchesy, J. J., Constantine, One thousand. H. & Craig, A. Thou. Antimicrobial action of some Pacific Northwest wood against anaerobic leaner and yeast. Phytotherapy Research. 15(7), 586–588 (2001).

CAS Commodity PubMed Google Scholar
Tsiri, D. et al. Chemosystematic value of essential oil compostion of Thuja species cultivated in Poland-Antimicrobial activity. Molecules fourteen, 4707–4715 (2009).

CAS Article PubMed Google Scholar
Pavela, R. Insecticidal activeness of some essential oils against larvae of Spodoptera littoralis. Fitoterapia. 76(7), 691–696 (2005).

CAS Article PubMed Google Scholar
Burt, S. Essential oils: their antibacterial properties and potential applications in foods a review. Int. J. Nutrient Microbiol. 94, 223–53 (2004).

CAS Article PubMed Google Scholar
Kalemba, D. & Kunicka, A. Antibacterial and antifungal properties of essential oils. Curr. Med. Chem. 10, 813–829 (2003).

CAS Article PubMed Google Scholar
De Oliveira, G. M. M., Brugnera, D. F., Cardoso, Chiliad. G., Alves, E. & Piccol, R. H. Disinfectant activeness of Cymbopogon sp. essential oils in different phases of biofilm formation past Listeria monocytogenes on stainless steel surface. Nutrient Control 21, 549–553 (2010).

Commodity Google Scholar
Mitić-Ćulafić, D., Vuković-Gačić, B., Knežević-Vukčević, J., Stanković, Due south. & Simić, D. Comparative study on the antibacterial activity of volatiles from sage (Salvia officinalis L.). Arch. Biol. Sci. Belgrade 57 , 173–178 (2005).
Poaty, B., Lahlah, J., Porqueres, F. & Bouafif, H. Composition, antimicrobial and antioxidant activities of seven essential oils from the North American boreal forest. World J. Microbiol. Biotechnol. 31, 907–919 (2015).

CAS Article PubMed Google Scholar
Inouye, S., Abe, South., Yamaguchi, H. & Asakura, M. Comparative report of antimicrobial and cytotoxic furnishings of selected essential oils by gaseous and solution contacts. Int. J. Aromather. thirteen, 33–41 (2003).

Commodity Google Scholar
Tullio, V. et al. Antifungal activity of essential oils confronting filamentous fungi determined by goop microdilution and vapour contact methods. J. Appl. Microbiol. 102, 1544–1550 (2007).

CAS Article PubMed Google Scholar
Tyagi, A. K. & Malik, A. Liquid and vapour-phase antifungal activities of selected essential oils against Candida albicans: microscopic observations and chemical label of Cymbopogon citratus. BMC Complement. Altern. Med. 10, ane–11 (2010).

Article Google Scholar
Tyagi, A. Thousand. & Malik, A. Antimicrobial potential and chemical composition of Eucalyptus globulus oil in liquid and vapour phase against nutrient spoilage microorganisms. Food Chem. 126, 228–235 (2011).

CAS Article Google Scholar
Inouye, S., Uchida, K. & Abe, S. Vapor activity of 72 essential oils against a Trichophyton mentagrophytes. J. Infect. Chemother. 12, 210–216 (2006).

CAS Article PubMed Google Scholar
Bergkvist, T.P. Antimicrobial activity of four volatile essential oils. Master thesis in Chemist's shop, Charles Sturt University, Goteborg, Sweden. (2007).
Chia, T. W. R., Goulter, R. 1000., McMeekin, T., Dykes, One thousand. A. & Fegan, Due north. Attachment of different Salmonella serovars to materials unremarkably used in a poultry processing plant. Food Microbiology 26, 853–859 (2009).

CAS Article PubMed Google Scholar
Jun, W. et al. Microbial biofilm detection on food contact surfaces by macro-scale fluorescence imaging. J. Food Eng. 99, 314–322 (2010).

Article Google Scholar
Valeriano, C. et al. The sanitizing action of essential oil-based solutions against Salmonella enterica serotype Enteritidis S64 biofilm germination on AISI 304 stainless steel. Food Control 25, 673–677 (2012).

CAS Article Google Scholar
Slamenova, D., Horvathova, E., Kovacikova, Z., Kozics, Thousand. & Hunakova, 50. Essential rosemary oil protects testicular cells against DNA-damaging effects of H₂O_ii and DMNQ. Food Chem. 129, 64–lxx (2011).

CAS Article Google Scholar
Basaran, A. A., Yu, T., Plewa, M. J. & Anderson, D. An investigation of some Turkish herbal medicines in Salmonella typhimurium and in the Comet assay in human lymphocytes. Teratog. Carcinog. Mutagen. 16, 125–138 (1996).

CAS Article PubMed Google Scholar
Llana-Ruiz-Cabello, M. et al. In vitro toxicological evaluation of essential oils and their chief compounds used in active food packaging: a review. Food Chem. Toxicol. 81, 9–27 (2015).

CAS Article PubMed Google Scholar
Ruiz-Pérez, N. J. et al. Antimycotic Activity and Genotoxic Evaluation of Citrus sinensis and Citrus latifolia Essential Oils. Sci. Reports half dozen, 253–271 (2016).

Google Scholar
Schimmer, O., Kruger, A., Paulini, H. & Haefele, F. An evaluation of 55 commercial found extracts in the Ames mutagenicity test. Pharmazie 49, 448–451 (1994).

CAS PubMed Google Scholar
Sturchio, Eastward. et al. Molecular and structural changes induced past essential oils treatments in Vicia faba roots detected by genotoxicity testing. J. Toxicol. Environ. Health A. 79(iv), 143–152 (2016).

CAS Article PubMed Google Scholar
Ortiz, C., Morales, L., Sastre, Chiliad., Haskins, W. E. & Matta, J. Cytotoxicity and genotoxicity assessment of Sandalwood essential oil in human breast cell lines MCF-7 and MCF-10A. Evid. Based Complement. Alternat. Med. (2016).
Navarra, Yard. et al. Furnishings of bergamot essential oil and its extractive fractions on SH‐SY5Y human being neuroblastoma jail cell growth. J. Pharm. Pharmacol. 67, 1042–1053 (2015).

CAS Commodity PubMed Google Scholar
Sinha, Southward., Jothiramajayam, Grand., Ghosh, M. & Mukherjee, A. Evaluation of toxicity of essential oils palmarosa, citronella, lemongrass and vetiver in man lymphocytes. Food Chem. Toxicol. 68, 71–77 (2014).

CAS Article PubMed Google Scholar
Hudson, J., Kuo, Thousand. & Vimalanathan, S. The Antimicrobial Properties of Cedar Leaf (Thuja plicata) Oil; A Safe and Efficient Decontamination Agent for Buildings. Int. J. Environ. Res. Public Health 8, 4477–4487 (2011).

CAS Article PubMed PubMed Cardinal Google Scholar
Daniels, C. R. & Russell, J. H. Analysis of western redcedar (Thuja plicata Donn) heartwood components by HPLC as a possible screening tool for trees with enhanced natural immovability. J. Chromatogr. Sci. 45(five), 281–285 (2007).

CAS Commodity PubMed Google Scholar
Maida, I. et al. Exploring the anti-Burkholderia cepacia circuitous activity of essential oils: a preliminary analysis. Evid. Based Complement. Altern. Med. vol. 2014, pp. 10 (2014).
Shellie, R., Mondello, 50., Marriott, P. & Dugo, G. Characterisation of lavender essential oils past using gas chromatography–mass spectrometry with correlation of linear retention indices and comparison with comprehensive 2-dimensional gas chromatography. J. Chromatogr. A 970(i), 225–234 (2002).

CAS Article PubMed Google Scholar
Cai, J., Lin, P., Zhu, X. & Su, Q. Comparative assay of clary sage (S. sclarea L.) oil volatiles by GC–FTIR and GC–MS. Food chem. 99(2), 401–407 (2006).

CAS Article Google Scholar
Politeo, O., Jukic, M. & Milos, M. Comparing of chemic composition and antioxidant activity of glycosidically bound and gratuitous volatiles from clove (Eugenia caryophyllata Thunb.). J. food biochem. 34(1), 129–141 (2010).

CAS Article Google Scholar
Sarac, Northward. & Ugur, A. Antimicrobial activities and usage in folkloric medicine of some Lamiaceae species growing in Mugla, Turkey. EurAsia J. BioSci. 4, 28–37 (2007).

Google Scholar
Bilia, A.R., Santomauro, F., Sacco, C., Bergonzi, Grand.C. & Donato, R. Essential Oil of Artemisia annua L. An Extraordinary Component with Numerous Antimicrobial Properties. Evid. Based Complement. Altern. Med. Vol 2014, seven (2014).
Ratledge, C. & Wilkinson, S.G. An overview of microbial lipids in Microbial Lipids (eds. Ratledge, C. & Wilkinson South.G.) vol. one. 3–22 (Academic Press, 1988).
Preuss, H. One thousand., Echard, B., Enig, M., Brook, I. & Elliott, T. B. Minimum inhibitory concentrations of herbal essential oils and monolaurin for gram-positive and gram-negative leaner. Mol. Cell Biochem. 272, 29–34 (2005).

CAS Article PubMed Google Scholar
Al-Mariri, A. & Safi, M. In vitro antibacterial action of several constitute extracts and oils against some gram-negative bacteria. Iran. J. Med. Sci. 39(i), 36–43 (2014).

PubMed PubMed Central Google Scholar
Lopez, P., Sanchez, C., Batile, R. & Nerin, C. Solid- and vapour-stage antimicrobial activities of half-dozen essential oils: susceptibility of selected foodborne bacterial and fungal strains. J. Agric. Nutrient Chem. 53, 6939–6946 (2005).

CAS Commodity PubMed Google Scholar
Suhr, K. I. & Nielsen, P. V. Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi. J. Appl. Microbiol. 94(four), 665–674 (2003).

CAS Article PubMed Google Scholar
Edris, A. E. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review. Phytother. Res. 21(4), 308–323 (2007).

CAS Article PubMed Google Scholar
Russo, A. et al. Comparative phytochemical profile and antiproliferative activity on homo melanoma cells of essential oils of three Lebanese Salvia species. Ind. Ingather. Prod. 83, 492–499 (2016).

CAS Article Google Scholar
Sertel, Due south., Eichhorn, T., Plinkert, P. K. & Efferth, T. Cytotoxicity of Thymus vulgaris essential oil towards human oral cavity squamous prison cell carcinoma. Anticancer Res. 31, 81–87 (2011).

CAS PubMed Google Scholar
Maistro, Due east. 50., Mota, S. F., Lima, Due east. B., Bernardes, B. M. & Goulart, F. C. Genotoxicity and mutagenicity of Rosmarinus officinalis (Labiatae) essential oil in mammalian cells in vivo. Genet. Mol. Res. 9(four), 2113–2122 (2010).

CAS Article PubMed Google Scholar
Kozics et al. Antioxidant potential of essential oil from Lavandula angustifolia in in vitro and ex vivo cultured liver cells. Neoplasma (doi:10.4149/neo_2017_401) in printing.
Di Sotto, A., Mazzanti, Grand., Carbone, F., Hrelia, P. & Maffei, F. Genotoxicity of lavender oil, linalyl acetate, and linalool on human lymphocytes in vitro. Environ. Mol. Res. 52(1), 69–71 (2011).

Google Scholar
Huang, M. Y., Liao, M. H., Wang, Y. K., Huang, Y. Southward. & Wen, H. C. Effect of essential oil on LPS-stimulated inflammation. Am. J. Chin. Med. 40(4), 845–859 (2012).

Article PubMed Google Scholar
Geran, R. I., Greenberg, N. H., Macdonald, M. M., Schumacher, A. Yard. & Abbott, B. J. Protocols for screening chemical agents and natural products confronting fauna tumours and other biological systems. Cancer Chemother. Rep. three, 59–61 (1972).

Google Scholar
Horvathova, East. et al. Enriching the drinking water of rats with extracts of Salvia officinalis and Thymus vulgaris increases their resistance to oxidative stress. Mutagenesis 31, 51–59 (2016).

CAS PubMed Google Scholar
Kozics, K. et al. Effects of Salvia officinalis and Thymus vulgaris on oxidant-induced DNA damage and antioxidant status in HepG2 cells. Food Chem. 141, 2198–2206 (2013).

CAS Article PubMed Google Scholar
Ponce, A. 1000., Fritz, R., Del Valle, C. E. & Roura, S. I. Antimicrobial action of essential oils on native microbial population of organic Swiss chard. Lebensm. Wiss. Technol. 36, 679–684 (2003).

CAS Article Google Scholar
De Lira Mota, K. S. et al. Antifungal Activity of Thymus vulgaris L. Essential Oil and Its Constituent Phytochemicals against Rhizopus oryzae: Interaction with Ergosterol. Molecules 17, 14418–14433 (2012).

Article PubMed Google Scholar
Thompson, D. P. Fungitoxic action of essential oil components on food storage fungi. Mycologia 81, 151–153 (1989).

CAS Article Google Scholar
Feng, West., Chen, J., Zheng, 10. & Liu, Q. Thyme oil to control Alternaria alternata In vitro and in vivo as fumigant and contact treatments. Nutrient Control 22, 78–81 (2011).

CAS Article Google Scholar
Singh, N. P., McCoy, One thousand. T., Tice, R. R. & Schneider, East. L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175(1), 184–191 (1988).

CAS Article PubMed Google Scholar
Slameňová, D. et al. Detection of MNNG-induced Deoxyribonucleic acid lesions in mammalian cells; validation of comet analysis confronting Dna unwinding technique, alkaline elution of DNA and chromosomal aberrations. Mutation Research. Deoxyribonucleic acid Repair. 383, 243–252 (1997).

Article PubMed Google Scholar

Download references

Acknowledgements

This study was funded by VEGA projects no. 2/0061/17 "Innovative disinfection strategies: the essential oils effect on microflora and materials of cultural heritage objects" and no. 2/0027/sixteen "Antioxidative, anticarcinogen and photoprotective effects of the essential oil from lavender in vitro". We are very grateful to Dr. Jacob Bauer for the English language revision of the text.

Author information

Affiliations

Institute of Molecular Biological science, Slovak Academy of Sciences, Dúbravská cesta 21, 84551, Bratislava, Slovakia

Andrea Puškárová, Mária Bučková, Lucia Kraková & Domenico Pangallo
Cancer Research Plant, Biomedical Research Heart, Slovak Academy of Sciences, Dúbravská cesta 9, 84505, Bratislava, Slovakia

Katarína Kozics

Contributions

A. Puškárová, M. Bučková and L. Kraková performed the antibacterial and antifungal assay. K. Kozics was responsible for the cytotoxicity and genotoxicity assays. D. Pangallo critically revised the manuscript. A. Puškárová wrote the article. A. Puškárová, M. Bučková, and D. Pangallo participated in drafting the article. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Andrea Puškárová.

Ethics declarations

Competing Interests

The authors declare that they have no competing interests.

Boosted information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This commodity is licensed under a Creative Eatables Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as y'all give appropriate credit to the original author(s) and the source, provide a link to the Creative Eatables license, and signal if changes were made. The images or other tertiary party material in this article are included in the commodity'southward Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/four.0/.

Reprints and Permissions

Virtually this article

Verify currency and authenticity via CrossMark

Cite this article

Puškárová, A., Bučková, Grand., Kraková, Fifty. et al. The antibacterial and antifungal activeness of 6 essential oils and their cyto/genotoxicity to human HEL 12469 cells. Sci Rep seven, 8211 (2017). https://doi.org/10.1038/s41598-017-08673-9

Download citation

Received: 29 November 2016
Accepted: 12 July 2017
Published: 15 August 2017
DOI : https://doi.org/10.1038/s41598-017-08673-9

Comments

By submitting a comment y'all agree to abide by our Terms and Customs Guidelines. If you observe something abusive or that does not comply with our terms or guidelines delight flag it as inappropriate.

fowlerwilcorts.blogspot.com

Source: https://www.nature.com/articles/s41598-017-08673-9