Essential oils (EOs) extracted from Lavandula luisieri and Cymbopogon citratus were tested for their antifungal activity against ten clinical isolates of dermatophytes isolated from cases of tinea pedis. Inhibition of conidial germination and antifungal drug/EO combination assay were tested on two ATCC reference strains of Trichophyton rubrum and Trichophyton mentagrophytes. EOs were characterised by high amount of oxygenated monoterpenes in their composition. Strong antifungal activity was observed for the majority of clinical strains, and fungicidal activity was demonstrated. Positive interaction between L. luisieri EO combined with terbinafine was observed against terbinafine-resistant strain (Tr ATCC MYA-4438). Significative reduction of the germination was observed above 100 g mL1. Both oils were safe to macrophage mammalian cells at tested concentration. This study describes the antifungal activity of L. luisieri and C. citratus EOs against dermatophytes, which could be useful in designing new formulations for topical treatments.This work was supported by Portuguese Foundation for Science and Technology (FCT) [grant number PEST-C/FIS/UI607/2013], [grant number UID/BIO/04469/2013], [grant number PEst-OE/EQB/LA0023/2013] and European Community fund FEDER [grant number FCOMP-01-0124-FEDER-007025], [grant number RECI project: FCOMP-01-0124-FEDER-027462].The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013. Nicolina Dias acknowledges the project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, Ref. FCOMP-01-0124-FEDER-027462

Cavaleiro, C.

Dias, M. C.

Dias, Nicolina

Lima, Nelson

Machado, M.

Sousa, M. C.

Natural Product Research

Essential oils (EOs) extracted from Lavandula luisieri and Cymbopogon citratus were tested for their antifungal activity against ten clinical isolates of dermatophytes isolated from cases of tinea pedis. Inhibition of conidial germination and antifungal drug/EO combination assay were tested on two ATCC reference strains of Trichophyton rubrum and Trichophyton mentagrophytes. EOs were characterised by high amount of oxygenated monoterpenes in their composition. Strong antifungal activity was observed for the majority of clinical strains, and fungicidal activity was demonstrated. Positive interaction between L. luisieri EO combined with terbinafine was observed against terbinafine-resistant strain (Tr ATCC MYA-4438). Significative reduction of the germination was observed above 100 μg mL−1. Both oils were safe to macrophage mammalian cells at tested concentration. This study describes the antifungal activity of L. luisieri and C. citratus EOs against dermatophytes, which could be useful in designing new formulations for topical treatments.</p

N. Dias (2832356)

M.C. Dias (2832362)

C. Cavaleiro (624003)

M.C. Sousa (2832365)

N. Lima (2832353)

M. Machado (2832359)

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Oxygenated monoterpenes-rich volatile oils as potential antifungal agents for dermatophytes

Universidade do Minho: RepositoriUM

Natural Product research, 2017Vol. 31, No. 4, 460–464http://dx.doi.org/10.1080/14786419.2016.1195379SHORT COMMUNICATIONOxygenated monoterpenes-rich volatile oils as potential antifungal agents for dermatophytesN. Diasa, M.C. Diasb, C. Cavaleiroc, M.C. Sousac,d, N. Limaa and M. Machadob,c,eaceB – centre of Biological engineering, university of Minho, campus Gualtar, Braga, Portugal; bcesPu – Instituto de Investigação e Formação avançada em ciências e tecnologias da saúde, Gandra, Portugal; cceF – centro de estudos Farmacêuticos/Faculty of Pharmacy, university of coimbra, coimbra, Portugal; dcNc – center for Neuroscience and cell Biology, university of coimbra, coimbra, Portugal; ecIBIo – research centre in Biodiversity and Genetic resources, InBIo, universidade do Porto, campus agrário de Vairão, Vila do conde, PortugalABSTRACTEssential oils (EOs) extracted from Lavandula luisieri and Cymbopogon citratus were tested for their antifungal activity against ten clinical isolates of dermatophytes isolated from cases of tinea pedis. Inhibition of conidial germination and antifungal drug/EO combination assay were tested on two ATCC reference strains of Trichophyton rubrum and Trichophyton mentagrophytes. EOs were characterised by high amount of oxygenated monoterpenes in their composition. Strong antifungal activity was observed for the majority of clinical strains, and fungicidal activity was demonstrated. Positive interaction between L. luisieri EO combined with terbinafine was observed against terbinafine-resistant strain (Tr ATCC MYA-4438). Significative reduction of the germination was observed above 100 μg mL−1. Both oils were safe to macrophage mammalian cells at tested concentration. This study describes the antifungal activity of L. luisieri and C. citratus EOs against dermatophytes, which could be useful in designing new formulations for topical treatments. © 2016 Informa uK limited, trading as taylor & Francis GroupKEYWORDSantifungals; dermatophytes; pathogenic fungi; skin infectionsARTICLE HISTORYreceived 2 February 2015 accepted 5 May 2016CONTACT N. dias nidias@deb.uminho.pt experimental details relating to this article are available online at http://dx.doi.org/10.1080/14786419.2016.1195379.NATURAL PRODUCT RESEARCH 4611. IntroductionDermatophytoses are among the most prevalent infectious diseases worldwide, which inci-dence has increased, mainly on urban environments. Occasionally, it can spread to other parts of the body, and it is highly contagious. High rate of recurrence following antifungal treatment limited use in some patients due to side effect, and medication interactions are common problems found when using conventional drugs (Vander Straten et al. 2003). Thus, clinical isolates of drug-resistant Trichophyton rubrum exhibiting high-level primary resistance to terbinafine have been reported (Favre et al. 2004). Medicinal plants are the richest natural source of bioactive phytochemicals and antioxidants. Among those, monoterpenes are main constituents of essential oils (EOs). Furthermore, EOs containing high amounts of oxygenated monoterpenes (OM) are related to broad spectrum antibacterial activities (Messaoud et al. 2012; Ait Said et al. 2015; Carrasco et al. 2016).In this study, Lavandula luisieri and Cymbopogon citratus, which are known for their high content of OM, were tested for their potential use as topical therapeutic agents against dermatophyte strains involved in foot dermatophytoses.2. Results and discussion2.1. Analysis of oils, antifungal activities and viability assayMajor compounds are listed in Table S1. L. luisieri EO was predominantly composed by oxy-genated necrodane derivatives, followed by 1,8-cineol as main compounds. The presence of necrodane derivatives has been reported as the characteristic and dominant compounds in L. luisieri EO, which could be used as a chemotaxonomic marker of this species (Zuzarte et al. 2012). Both EOs were characterised by high amounts of oxygen-containing monoter-penes (OM > 75%) totalizing 80.4% and 97.9% of monoterpenic compounds (MH and OM) for L. luisieri and C. citratus, respectively. In Table S2, a strong antifungal activity and a fun-gicidal mode of action with MFC = 200 μg mL−1 were observed for both EOs. Antifungal effect of both EOs was dose dependent for all dermatophytes (Figure S1), although C. citratus showed a mild effect on clinical strains of T. mentagrophytes. Strong antifungal activity has been positively correlated with the presence of OM (Mesa-Arango et al. 2009; Prusinowska et al. 2016). Besides, L. luisieri EO with the highest amount of necrodane compounds shown to be the most active against dermatophyte strains (Zuzarte et al. 2012).2.2. In vitro antifungal drug/EO combination assayThe combination between EOs and conventional antimicrobial drugs has been referred as a strategy to bring about the maximum of therapeutic efficacy by additive or synergistic effect (Wagner & Ulrich-Merzenich 2009). Although combination therapy has been already recommended to improve monotherapy results, few studies (Shin & Lim 2004; Khan & Ahmad 2011) have reported on the antifungal effect against dermatophytes. Table S3 shows a pos-itive interaction of L. luisieri EO with terbinafine (TER) against the terbinafine-resistant Tr ATCC MYA-4438 in all fixed-ratio concentrations suggesting a synergistic activity between the drug and EO, as the effect of the combined substances is greater than the sum of the individual effects (Burt 2004). Consequently, a minor dose of terbinafine would be necessary to obtain the same antifungal effect when used in combination with L. luisieri EO. In this 462 N. DIAS ET AL.study, the best antifungal effect was obtained for the ratios 1:1 and 3:1. At TER/EO ratio 1:3, combined activity was apparently additive. In this perspective, combined therapy of EO with conventional drugs may lead to a reduction of drug doses by decreasing possible adverse side effects or increasing therapeutic effects (Khan & Ahmad 2011).2.3. Effect of EOs on conidial germination and morphologyBoth oils proved to be effective in delaying germination of conidia on reference strains. Results on the percentage of conidia germinated after 24 h of treatment with L. luisieri and C. citratus EOs are displayed in Figure 1. Significant reduction of the germination (p < 0.001) was observed for strains treated with EO concentrations above 100 μg mL−1, and most conidia were non-germinated at 200 μg mL−1. The effect of L. luisieri EO on the morphology of conidia and hyphae after 24 and 48 h of treatment, respectively, is shown in Figure S2. L. luisieri EO induced morphological alterations on T. rubrum conidia. Germination alterations were clearly visible after 24 h of exposure at sub-MIC concentrations (A–D). Thus, growth of the germinative tube was severely impaired at 50 μg mL−1. At 100 μg mL−1, alterations on the morphology of conidia were clearly observed. After 48 h of incubation (E–H), the myce-lium structure became irregular with the loss of ramifications. No mycelium growth was observed for most cells, at 200 μg mL−1. Inhibition of DNA synthesis or disruption of mem-branes is mechanism of phytotoxicity found in OM, which lead to anatomical and physio-logical changes (De Martino et al. 2010). Therefore, it was expected that impaired germ tubes originated distorted hyphae compromising cell morphogenesis and further growth. In a previous work, Zuzarte et al. (2012) reported on the capacity of inhibition of the germ tube of Candida albicans by L. luisieri at sub-MIC values.2.4. Cytotoxicity of EOsCareful use of lavender oil in highly diluted forms, when applied to the skin, has been rec-ommended by Prashar et al. (2004). In our study, EOs did not cause a significant alteration Figure 1.  effect of L. luisieri and C. citratus on the germination of conidia of reference strains of dermatophytes T. rubrum atcc MYa-4438 (a) and T. mentagrophytes atcc MYa-4439 (b). Fungal cultures were treated for 24 h with different concentrations of the oils ranging from 0 to 200 μg ml−1. results are expressed as percentage of germinated conidia on untreated and treated cultures of dermatophytes. Values are expressed as mean  ±  seM from three independent experiments. ***p  <  0.001 significant statistical differences compared to control culture. n.s. = not significative.NATURAL PRODUCT RESEARCH 463on the viability of treated mouse macrophage cells when compared to control cells (Figure S3). No induction of alterations on the morphology of cells was observed under light micros-copy (data not shown). In addition, a very low detrimental effect was found on mouse mac-rophage cells at concentrations of L. luisieri EO up to 320 μg mL−1 (Zuzarte et al. 2012).3. ConclusionThe chemical composition of the tested EOs, mainly the OM fraction, suggests an association with the activity profile of EOs against dermatophytes. In addition to fungistatic and fungi-cidal activity, the EOs revealed an important inhibitory effect on germination of conidia at sub-MIC concentrations, thus reinforcing their potential use in topical therapy. This study also proposes L. luisieri as a suitable oil for combined treatment against terbinafine-resistant fungal strains, although further in vivo experiments are needed to evaluate the therapeutic efficacy and safety of the EO in combination with antifungal drugs.AcknowledgementsThe authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013. Nicolina Dias acknowledges the project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, Ref. FCOMP-01-0124-FEDER-027462.Disclosure statementNo potential conflict of interest was reported by the authors.FundingThis work was supported by Portuguese Foundation for Science and Technology (FCT) [grant num-ber PEST-C/FIS/UI607/2013], [grant number UID/BIO/04469/2013], [grant number PEst-OE/EQB/LA0023/2013] and European Community fund FEDER [grant number FCOMP-01-0124-FEDER-007025], [grant number RECI project: FCOMP-01-0124-FEDER-027462].ReferencesAit Said L, Zahlane K, Ghalbane I, El Messoussi S, Romane A, Cavaleiro C, Salgueiro L. 2015. Chemical composition and antibacterial activity of Lavandula coronopifolia essential oil against antibiotic-resistant bacteria. Nat Prod Res. 29:582–585.Burt S. 2004. Essential oils: their antibacterial properties and potential applications in foods – a review. Int J Food Microbiol. 94:223–253.Carrasco, A, Martinez-Gutierrez, M, Tomasc, V, Tudela, J. (2015). Lavandin (Lavandula × intermedia Emeric ex Loiseleur) essential oil from Spain: determination of aromatic profile by gas chromatography–mass spectrometry, antioxidant and lipoxygenase inhibitory bioactivities. Nat Prod Res. 30:1123–1130.Martino L, Mancini E, Almeida LF, Feo V. 2010. The antigerminative activity of twenty-seven monoterpenes. Molecules. 15:6630–6637.Favre B, Ghannoum MA, Ryder NS. 2004. Biochemical characterization of terbinafine-resistant Trichophyton rubrum isolates. Med Mycol. 42:525–529.Khan MSA, Ahmad I. 2011. Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum. Appl Microbiol Biotechnol. 90:1083–1094.464 N. DIAS ET AL.Mesa-Arango AC, Montiel-Ramos J, Zapata B, Durán C, Betancur-Galvis L, Stashenko E. 2009. Citral and carvone chemotypes from the essential oils of Colombian Lippia alba (Mill.) N.E. Brown: composition, cytotoxicity and antifungal activity. Mem Inst Oswaldo Cruz. 104:878–884.Messaoud C, Chograni H, Boussaid M. 2012. Chemical composition and antioxidant activities of essential oils and methanol extracts of three wild Lavandula L. species. Nat Prod Res. 26:1976–1984.Prashar A, Locke IC, Evans CS. 2004. Cytotoxicity of lavender oil and its major components to human skin cells. Cell Prolif. 37:221–229.Prusinowska R, Śmigielski K, Stobiecka A, Kunicka-Styczyńska A. 2016. Hydrolates from lavender (Lavandula angustifolia) – chemical composition, antimicrobial and antioxidant properties. Nat Prod Res. 30:386–393.Shin S, Lim S. 2004. Antifungal effects of herbal essential oils alone and in combination with ketoconazole against Trichophyton spp. J Appl Microbiol. 97:1289–1296.Vander Straten MR, Hossain MA, Ghannoum MA. 2003. Cutaneous infections. Infect Dis Clin North Am. 17:87–112.Wagner H, Ulrich-Merzenich G. 2009. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine. 16:97–110.Zuzarte M, Gonçalves MJ, Cruz MT, Cavaleiro C, Canhoto J, Vaz S, Pinto E, Salgueiro L. 2012. Lavandula luisieri essential oil as a source of antifungal drugs. Food Chem. 135:1505–1510.

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Oxygenated monoterpenes-rich volatile oils as potential antifungal agents for dermatophytes

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