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Journal of Medical Microbiology (2011), 60, 612–618 Chemical composition and antifungal activity of theessential oils of Lavandula viridis L'He´r.
Mo´nica Zuzarte,1,2 Maria Jose´ Gonc¸alves,1 Carlos Cavaleiro,1Jorge Canhoto,2 Luı´s Vale-Silva,3 Maria Joa˜o Silva,3 Euge´nia Pinto3and Lı´gia Salgueiro1 1Center of Pharmaceutical Studies, Faculty of Pharmacy, Health Science Campus, University of Lı´gia Salgueiro Coimbra, Azinhaga de S. Comba, 3000-354 Coimbra, Portugal 2Center of Pharmaceutical Studies, Department of Life Sciences, University of Coimbra, Ap. 3046, 3001-401 Coimbra, Portugal 3Microbiology Service/CEQUIMED, Faculty of Pharmacy, University of Porto, Rua Anı´bal Cunha 164, 4050-047 Porto, Portugal In the present work we report for what we believe to be the first time the antifungal activity andmechanism of action of the essential oils of Lavandula viridis from Portugal. The essential oilswere isolated by hydrodistillation and analysed by GC and GC/MS. The MIC and the minimallethal concentration (MLC) of the essential oil and its major compounds were determined againstseveral pathogenic fungi. The influence of subinhibitory concentrations of the essential oil on thedimorphic transition in Candida albicans was also studied, as well as propidium iodide and FUN-1staining of Candida albicans cells by flow cytometry following short treatments with the essentialoil. The oils were characterized by a high content of oxygen-containing monoterpenes, with1,8-cineole being the main constituent. Monoterpene hydrocarbons were present at lowerconcentrations. According to the determined MIC and MLC values, the dermatophytes andCryptococcus neoformans were the most sensitive fungi (MIC and MLC values ranging from 0.32to 0.64 ml ml"1), followed by Candida species (at 0.64–2.5 ml ml"1). For most of these strains,MICs were equivalent to MLCs, indicating a fungicidal effect of the essential oil. The oil wasfurther shown to completely inhibit filamentation in Candida albicans at concentrations well belowthe respective MICs (as low as MIC/16). Flow cytometry results suggested a mechanism of actionultimately leading to cytoplasmic membrane disruption and cell death. Our results show thatL. viridis essential oils may be useful in the clinical treatment of fungal diseases, particularly Received 31 October 2010 dermatophytosis and candidosis, although clinical trials are required to evaluate the practical Accepted 19 January 2011 relevance of our in vitro research.
In recent years, research on aromatic plants, andparticularly their essential oils, has attracted many Over the last few decades, there has been an increase in the investigators. Essential oils have traditionally been used number of serious human infections in immunocompro- for centuries for their antifungal properties mised patients caused by fungi The More recently, several studies have confirmed the range of severity of these infections is a consequence of the huge potential of these natural products as antifungal host reaction to the metabolic products produced by fungi, the virulence of the infecting strain, the site of infection and also environmental factors Nowadays, Therefore, it is not surprising that essential oils are one of the increasing impact of these infections, the limitations the most promising groups of natural products for the encountered in their treatment (e.g. resistance, side-effects development of broad-spectrum, safer and cheaper anti- and high toxicity) and the rising overprescription and fungal agents.
overuse of conventional antifungals all stimulate a search for The genus Lavandula provides valuable essential oils alternative natural drugs.
mainly for the food (flavouring), perfumery and cosmeticindustries, and is also very popular in aromatherapy.
Abbreviations: MLC, minimal lethal concentration; PI, propidium iodide.
However, many other applications can be foreseen, as 027748 G 2011 SGM Printed in Great Britain Antifungal activity of Lavandula viridis oils suggested in several reports on the biological activity of this strains from the American Type Culture Collection (Candida albicans genus. Lavandula oils have been reported to have sedative ATCC 10231, Candida tropicalis ATCC 13803 and Candida and antispasmodic properties parapsilopsis ATCC 90018); one Cryptococcus neoformans type strainfrom the Coleccio´n Espano˜la de Cultivos Tipo (Cryptococcus neofor- as well as acaricidal ), mans CECT 1078); one Aspergillus clinical strain isolated from antibacterial (e.g. bronchial secretions (Aspergillus flavus F44) and two Aspergillus type ), antifungal (e.g. strains from the American Type Culture Collection (Aspergillus niger ) and antioxidant activities.
ATCC 16404 and Aspergillus fumigatus ATCC 46645); three More recently, application as a biopesticide has also been (Epidermophyton floccosum FF9, Trichophyton mentagrophytes FF7and Microsporum canis FF1) and four dermatophyte type strains from Lavandula viridis L'He´r. is a highly aromatic shrub the Coleccio´n Espano˜la de Cultivos Tipo (Trichophyton mentagro- endemic to the south Iberian Peninsula. It is commonly phytes var. interdigitale CECT 2958, Trichophyton rubrum CECT 2794, known as green or white lavender due to its white flowers Trichophyton verrucosum CECT 2992 and Microsporum gypseumCECT 2908). All strains were stored in Sabouraud dextrose broth with and green floral bracts, which are very distinct from those 20 % glycerol at 280 uC and subcultured on Sabouraud dextrose agar of the other lavenders. Dried leaves of L. viridis are used (SDA) or potato dextrose agar (PDA) before each test, to ensure with medical applications in Madeira, Portugal optimal growth conditions and purity.
Antifungal activity. Broth macrodilution methods based on the As part of our ongoing study on the valorization of Clinical and Laboratory Standards Institute (CLSI) reference proto- Portuguese lavenders, we now report the chemical cols M27-A3 and M38-A2 for yeasts and composition, antifungal activity and mechanism of action filamentous fungi, respectively, were used to determine MICs of the of L. viridis essential oils. As far as we know, this is the first essential oils and their major constituents. A macrodilution rather report on the antifungal activity of this species.
than a microdilution design was used to allow the use of glass testtubes, thus avoiding the interaction of the essential oil with the plasticpolymer material of the 96-well microtitre plates. Briefly, inoculumsuspensions were prepared at appropriate densities in RPMI 1640 broth (with L-glutamine, without bicarbonate, and with the pHindicator phenol red) from SDA or PDA cultures and distributed into Plant material. Aerial parts of two samples of L. viridis were 12675 mm glass test tubes. Inoculum densities were confirmed by collected from field-growing plants in the flowering stage in the south viability counts on SDA. Serial twofold dilutions of the oil were of Portugal (A, Barranco do Velho region; B, Salir region). Voucher prepared in DMSO and added to the cell suspensions in order to specimens were deposited at the herbarium of the Department of Life obtain test concentrations ranging from 0.08 to 20.0 ml ml21 (final Sciences of the University of Coimbra (COI).
DMSO concentrations never exceeded 2 %, v/v). Oil-free growthcontrols, as well as sterility and DMSO control tubes, were also Essential oil isolation and analysis. The essential oils from air- included. The test tubes were incubated aerobically at 35 uC for 48 h/ dried plant material were isolated by hydrodistillation for 3 h, using a 72 h (Candida and Aspergillus species/Cryptococcus neoformans) and Clevenger-type apparatus according to the European Pharmacopoeia at 30 uC for 7 days (dermatophytes). MIC values were determined as The oils were preserved in a sealed vial at the lowest concentration of the oil causing full growth inhibition.
4 uC. Oil analyses were carried out by both GC and GC/MS using Quality control was performed by testing fluconazole and amphoter- fused silica capillary columns with two different stationary phases icin B with the reference strains Candida parapsilopsis ATCC 22019 (SPB-1 and SupelcoWax-10) as previously reported and Candida krusei ATCC 6258 and the results were within the predetermined limits. To measure minimal lethal concentrations The volatile compounds were identified by both their retention (MLCs), 20 ml samples were taken from each negative tube plus the indices and their mass spectra. Retention indices, calculated by linear first tube showing growth (to serve as a growth control) after MIC interpolation relative to retention times of a series of n-alkanes, were reading to SDA plates and incubated at 35 uC for 48 h/72 h (Candida compared with those of authenticated samples from the database of and Aspergillus species/Cryptococcus neoformans) or at 30 uC for the Laboratory of Pharmacognosy, Faculty of Pharmacy, University of 7 days (dermatophytes). MLC values were determined as the lowest Coimbra. Mass spectra were compared with reference spectra from a concentration of the oil causing fungal death. All experiments were home-made library or from literature data performed in triplicate and repeated whenever the results of each Relative amounts of individual components were triplicate did not agree. A range of values is presented when different calculated based on GC peak areas without flame ionization detector results were obtained.
response factor correction.
Mechanism of action Pure and reference compounds. Authentic samples of 1,8-cineole Germ tube inhibition assay.
(Merck; 99.5 % purity), Cell suspensions from overnight SDA a-pinene (Fluka; 99.0 % purity), linalool (Aldrich; 99.0 % purity) and camphor (Extrasynthese) were used.
cultures of Candida albicans strains ATCC 10231, D5 and M1 wereprepared in NYP medium [N-acetylglucosamine (Sigma; 1023 mol Fluconazole was kindly provided by Pfizer as the pure powder and l21), Yeast Nitrogen Base (Difco; 3.35 g l21), proline (Fluka; 1023 amphotericin B was from Sigma (80.0 % purity).
mol l21) and NaCl (4.5 g l21), pH 6.7±0.1] and adjusted to obtain a density of 1.0±0.26106 c.f.u. ml21. The Fungal strains. The antifungal activity of the essential oil of sample essential oil was diluted in DMSO and added in 10 ml volumes to A was evaluated against yeasts and filamentous fungi: four clinical 990 ml of the yeast suspensions (final DMSO concentration of 1 %, v/ Candida strains isolated from recurrent cases of vulvovaginal and oral v), obtaining a series of subinhibitory concentrations (as low as 1/64 candidosis (Candida albicans D5, Candida albicans M1, Candida of the MIC). The samples were incubated for 3 h at 37 uC without krusei H9 and Candida guilliermondii MAT23); three Candida type agitation and 100 cells from each sample were then counted in a

M. Zuzarte and others haemocytometer. The percentage of germ tubes was determined as the comparison to the drug-free controls. Results are presented as number of cells showing hyphae at least as long as the diameter of the means±SD of at least three replicate experiments.
blastospore. Cells showing a constriction at the point of connection ofthe hypha to the mother cell, typical for pseudohyphae, were excluded.
The results are presented as means±standard deviation (SD) of three RESULTS AND DISCUSSION Flow cytometry. Yeast suspensions were prepared in PBS solution Chemical compositions of the essential oils with 2 % (w/v) D-glucose from overnight SDA cultures of Candida The essential oils were obtained in yields ranging from 0.7 albicans ATCC 10231 at 35 uC and adjusted, using a haemocytometer, to 1.2 % (v/w). A total of 51 compounds were identified, to a final density of 2.0±0.26106 c.f.u. ml21. Serial twofold dilutionsof the essential oil (final concentrations of 0.64–10.0 ml ml21) and a representing 93.2 % (sample A) and 95.3 % (sample B) of single solution of amphotericin B at 2 mg ml21 (four times the the total volatile oils. The oils were characterized by high respective MIC of 0.5 mg ml21) in PBS with 2 % (w/v) D-glucose were contents of oxygen-containing monoterpenes (69.5 and added to the cell suspensions and the mixtures were incubated at 75.7 %), followed by monoterpene hydrocarbons (17.1 and 35 uC in a humid atmosphere without agitation for 30 min, 4 h or 15.5 %). The main constituents of the oils were 1,8-cineole 24 h. Drug-free control tubes were included in each experiment. After (34.5 % and 42.2 %), camphor (13.4 %), a-pinene (9.0 %) this period, the cells were washed in PBS and resuspended in 500 ml and linalool (7.9 and 6.7 %). Sesquiterpenic compounds PBS with 2 % (w/v) D-glucose for FUN-1 (Invitrogen) staining andPBS for propidium iodide (PI; Sigma) staining. Five microlitres of the attained only 4.8 and 2.3 %.
FUN-1 and PI solutions in DMSO and PBS, respectively, were added In a previous study carried out by to the cell suspensions to obtain final concentrations of 0.5 mM FUN- some individual samples of L. viridis from the south of 1 and 1.0 mg PI ml21. FUN-1-stained cells were incubated for a Portugal and Spain were analysed. The chemical composi- further 20 min at 35 uC, away from incident light, while PI-stainedsamples were read after about 10 min at room temperature.
tion of these samples was very similar to that of our Unstained cell suspensions were included as autofluorescence collective samples, 1,8-cineole being the major component controls. Flow cytometry was performed using a FACSCalibur in all samples. This fact points to a very high homogeneity (Becton Dickinson Biosciences) flow cytometer with a 488 nm blue in the composition of the essential oils of L. viridis from argon laser emitting at 15 mW and the results were analysed using Portugal and Spain.
CellQuest Pro Software (Becton Dickinson). Intrinsic parameters(forward and side scatter, for relative cell size and complexityanalysis) and fluorescence in the FL2 channel (log yellow/orange Antifungal activity of the essential oils fluorescence, bandpass filter 585/42 nm) for FUN-1 and the FL3channel (log red fluorescence, longpass filter .650 nm) for PI were The essential oil was used to evaluate the antifungal activity acquired and recorded for a minimum of 7500 events per sample against several pathogenic strains involved in human using logarithmic scales. Markers (M1) were adjusted to include a diseases. Various degrees of inhibition were registered maximum of 5 % of the cells in monoparametric histograms of the against all the fungi tested fluorescence intensity of control samples (see for examples) andkept unchanged through the analysis of the remaining samples to The highest antifungal activity was observed against quantify the percentages of cells showing altered fluorescence in dermatophyte strains and Cryptococcus neoformans, with Fig. 1. Flow cytometry histograms showing fluorescence intensity versus number of events (Candida albicans ATCC 10231cells) in relative units. (a) Orange fluorescence (FL2 channel) intensity of samples stained with FUN-1. (b) Red fluorescence(FL3 channel) intensity of samples stained with PI. Af, Autofluorescence of unstained cells; control, untreated cells; AmB, cellstreated with amphotericin B at 2.0 mg ml"1; EO, cells treated with the essential oil of L. viridis at 10.0 ml ml"1.
Journal of Medical Microbiology 60 Table 1. Antifungal activity (MIC and MLC) of the essential oil of Lavandula viridis (sample A) for Candida, dermatophyte and Aspergillus strains Results were obtained from three independent experiments performed in duplicate. When different MIC values were obtained, a range of values is presented. NT, Not tested.
Candida albicans ATCC 10231 Candida albicans D5 Candida albicans M1 Candida tropicalis ATCC 13803 Candida krusei H9 Candida guilliermondii MAT23 Candida parapsilopsis ATCC Cryptococcus neoformans CECT var. interdigitale CECT 2958 Trichophyton rubrum CECT Trichophyton verrucosum CECT Microsporum canis FF1 Microsporum gypseum CECT Epidermophyton floccosum FF9 Aspergillus niger ATCC16404 Aspergillus fumigatus ATCC Aspergillus flavus F44 *MIC and MLC were determined by a macrodilution method and expressed in ml ml21 (v/v).
DMIC and MLC were determined by a macrodilution method and expressed in mg ml21 (w/v).
M. Zuzarte and others MIC and MLC values ranging from 0.32 to 0.64 ml ml21.
inhibition of cell metabolism after short incubation periods For Candida strains, MIC and MLC values ranged from with the oil at concentrations starting from the respective 0.64 to 2.5 ml ml21. The oil was less effective against MIC The dye FUN-1 freely permeates fungal Aspergillus strains The higher susceptibility of plasma membranes into the cell and is distributed in the dermatophytes has also been reported for other essential cytoplasm as a bright diffuse green/yellow stain. In normal fungal cells, the dye is metabolized into orange/red cylindrical intravacuolar structures. However, in cells withimpaired metabolism, this change does not occur and FUN-1 For most of the dermatophytes, Cryptococcus neoformans remains in the cytoplasm in a diffuse pattern, indicating a and Candida strains, the MIC was equivalent to the MLC, disorder in cell metabolism . This change indicating a clear fungicidal effect of L. viridis essential oil.
was detected by a reduction of orange fluorescence (FL2 The major constituents of the oil (1,8-cineole, camphor, a- channel) in cells exposed to the essential oil in comparison to pinene and linalool) were also assayed individually for their untreated controls (and . To observe PI staining of antifungal activity. 1,8-Cineole and camphor displayed the the test cells, on the other hand, a 4 h incubation with a lowest antifungal activity against all strains but a-pinene concentration of the oil at least two log2 dilutions above the proved to be a very active compound, particularly against MIC was required ). The nucleic acid binding dermatophyte strains Since the essential oils are fluorescent probe PI penetrates only dead cells showing complex mixtures of several compounds, it is difficult to severe membrane lesions . The attribute their biological activity to a particular constituent.
observed asymmetry between metabolic inhibition and cell Usually, major compounds are the ones responsible for the death shows that cells clearly become metabolically inactive antifungal activity of the essential oils. However, some in the presence of the essential oil of L. viridis before it leads studies show that minor components may have a crucial to cell death, thus appearing to exclude a potential role in the biological activity of the oils mechanism of antifungal action relying on primary leakage Our results seem to indicate that the activity of L.
of cytoplasmic contents due to direct damage to cell viridis essential oil is mainly due to the presence of a- membranes. It is worth pointing out that under the same pinene in the oil.
experimental conditions, the reference fungicidal drugamphotericin B tested at a concentration two log2 dilutionsabove the respective MIC did not lead to PI staining .
Mechanism of action of the essential oil After 24 h, however, over 90 % of the cells presented positive The essential oil was also found to inhibit filamentation in PI staining with amphotericin B treatment (data not shown).
the tested Candida albicans strains at concentrations of The mechanism of action of essential oils remains 0.08–0.16 ml ml21, well below the corresponding MICs somewhat controversial. While some studies suggest that This marked difference between MICs and the compounds may penetrate the micro-organism and filamentation-inhibiting concentrations seems to suggest react with active sites of enzymes and/or interfere with that different mechanisms may be responsible for these two cellular metabolism, most evidence supports direct disrup- biological activities. This finding is particularly relevant tion of cellular membranes and concentration-dependent considering the fact that filamentation has long been pro-oxidant cytotoxic effects shown to be essential for virulence in Candida albicans Concerning antifungal activity specifically, the mechanism In fact, inhibition of the dimorphic of action of the oils seems to involve penetration through transition alone has been suggested to be sufficient to treat cell walls and direct damage to both cytoplasmic and disseminated candidosis, thus proving to be a good target mitochondrial membranes This leads mechanism in the development of novel antifungal agents to changes in permeability leading to leakage and Additionally, flow cytometry analyses ultimately resulting in cell death after FUN-1 staining have revealed a dose-dependent Bearing this knowledge in mind, the present results for the Table 2. Percentage of germ tubes after treatment of three Candida albicans strains with subinhibitory concentrations of theessential oil of L. viridis for 3 h in a filamentation-inducing medium at 37 6C Results are presented as mean (±SD) values of three independent experiments. Concentration is in ml ml21 (v/v).
C. albicans ATCC 10231 47.5±10.6 (0.04) 42.7±10.5 (0.08) *Untreated samples including the solvent (1 % DMSO) only.
Journal of Medical Microbiology 60

Antifungal activity of Lavandula viridis oils Fig. 2. Percentage (and SD) of M1-gated Candida albicans ATCC 10231 cells, analysed by flow cytometry, after treatmentswith different concentrations of the essential oil of L. viridis in comparison with amphotericin B (AmB) and an untreated control.
Cells were treated with the compounds for 30 min for staining with FUN-1 and 4 h for staining with PI. *MIC of the essential oil.
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