Antifungal and anti-inflammatory potential of Lavandula stoechas and Thymus herba-barona essential oils

Industrial Crops and Products 44 (2013) 97–103

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Antifungal and anti-inflammatory potential of Lavandula stoechas and Thymus herba-barona essential oils M. Zuzarte a , M.J. Gonc¸alves a , C. Cavaleiro a , M.T. Cruz b , A. Benzarti c , B. Marongiu d , A. Maxia e , A. Piras d , L. Salgueiro a,∗ a

Center of Pharmaceutical Studies, Faculty of Pharmacy, Health Science Campus, University of Coimbra, Azinhaga de S. Comba 3000-354, Coimbra, Portugal Center for Neuroscience and Cell Biology and Faculty of Pharmacy, Coimbra, Portugal c Faculty of Science, University of Monastir, Monastir, Tunisia d Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Cagliari, Cittadella Universitaria di Monserrato, SS 554, Km 4.500, 09042 Cagliari, Italy e Dipartimento della Scienza della Vita e dell’Ambiente-Macrosezione di Botanica e Orto Botanico, Università degli Studi di Cagliari, Viale Sant’ Ignazio, I-09123 Cagliari, Italy b

a r t i c l e

i n f o

Article history: Received 20 July 2012 Received in revised form 2 November 2012 Accepted 2 November 2012 Keywords: Lavandula stoechas Thymus herba-barona Essential oil Antifungal Anti-inflammatory Nutraceutical

a b s t r a c t Lavandula L. and Thymus L. comprise several relevant species for the food, cosmetic, perfumery and pharmaceutical industries. Considering the traditional medicinal use of L. stoechas and T. herba-barona and the lack of scientific studies on their biological activities, the present study was designed to elucidate the composition and antifungal activity of their essential oils against fungi responsible for human infections as well as the anti-inflammatory potential and their cytotoxicity on a macrophage cell line. Moreover, the antifungal activity against fungi responsible for food contamination is also reported. Flowering parts of the plants were submitted to hydrodistillation in a Clevenger-type apparatus and the oils were analysed by GC and GC–MS. The minimal inhibitory and minimal lethal concentrations of the oils against fungi strains were determined using a macrodilution broth method. For the anti-inflammatory activity, an in vitro model of lipopolysaccharide-stimulated macrophages was used and the inhibition of nitric oxide production quantified. Assessment of the oils cytotoxicity was performed using the MTT reduction assay. L. stoechas essential oil was rich in fenchone (37.0%) and camphor (27.3%) while T. herba-barona oil showed high amounts of two phenols, carvacrol (54.0%) and thymol (30.2%). The latter was the most active oil against the tested fungi but evidenced high cytotoxicity on macrophages. L. stoechas was active against dermatophyte strains and showed potential anti-inflammatory activity at concentrations without affecting cell viability. These results support the use of L. stoechas in the development of phytopharmaceuticals or food supplements/nutraceuticals for the management of dermatophytosis and/or inflammatory-related diseases. Regarding T. herba-barona, it can be used as a preservative in storage products, due to its ability to inhibit Aspergilllus growth. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The incidence of fungal infections and inflammatory-related diseases has increased during the last decades. The difficulties encountered in their treatment, increase in drug resistance, sideeffects of conventional medication, and treatment costs justify the development of new effective, less toxic and cheaper drugs. Also fungi contamination in stored products is responsible for modifications in the appearance, flavour, and reduction of nutritional value of these products, as well as occurrence of allergies and mycotoxin intoxications (Magro et al., 2006). To avoid

∗ Corresponding author. Tel.: +351 239488400; fax: +351 239488503. E-mail address: [email protected] (L. Salgueiro). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.11.002

post-harvest losses, synthetic fungicides are currently used. However, the increasing development of resistance, the presence of chemical residues in the food chain, as well as limitations in the use of effective fungicides, justify the search for natural alternatives. As disappointments with conventional drugs and synthetic fungicides increase, the public acceptance and effectiveness of plant-based alternatives become more evident. Aromatic plants and their essential oils have long been used as preservatives and for medicinal purposes (Edris, 2007). Over the last years, different studies have confirmed the huge potential of these secondary metabolites, with several reviews pointing out their antimicrobial, virucidal, antiparasitical, insecticidal, and antioxidant properties (e.g. Bakkali et al., 2008; Cavanagh and Wilkinson, 2002; Edris, 2007; Miguel, 2010). Lavandula L. and Thymus L. are two important genera of the Lamiaceae family that comprise essential oil producing plants very

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relevant to the food, cosmetic, perfumery and pharmaceutical industries (Cavanagh and Wilkinson, 2002; Upson and Andrews, 2004). Several lavender and thyme species are long used as culinary herbs and their essential oils as flavouring ingredients in food, beverages and confectionary products, as well as aromas in soaps and perfumes. Also traditional remedies and food supplements include these plants based on their biological activities. Our team has performed several investigations on lavender and thyme essential oils, mainly on their antifungal, antioxidant, and anti-inflammatory properties (e.g. Figueiredo et al., 2008; Pina-Vaz et al., 2004; ValeSilva et al., 2010; Zuzarte et al., 2011). As part of our ongoing research on the valorization of aromatic plants for industrial purposes, we now focus the present work on two species, Lavandula stoechas and Thymus herba-barona. L. stoechas is widely distributed in the Mediterranean region. Traditional medicine uses this plant for its expectorant, antispasmodic, carminative properties and for wound healing. Also the essential oils are recognized as effective against colic and chest affections and are used to relieve nervous headaches. This species is one of the most explored lavenders in the world. Some studies have considered the antibacterial (Benabdelkader et al., 2011; Dadalio˘glu and Evrendilek, 2004), antifungal (Angioni et al., 2006; Benabdelkader et al., 2011), and antioxidant (Messaoud et al., 2012; Benabdelkader et al., 2011) properties of the oils. However, the antiinflammatory potential of the oils has not been evaluated before. T. herba-barona is an endemic species from Corsica and Sardinia. Traditionally it is known as a diaphoretic, sedative, antiseptic and antimycotic and its essential oils are used for their expectorant and antiseptic properties. Regarding its biological activities only two studies have been reported and both focus on the antimicrobial activity of the oils (Consentino et al., 1999; Juliano et al., 2000). We now report the chemical composition, antifungal and anti-inflammatory properties of the essential oils of L. stoechas and T. herba-barona from Sardinia. Also the effect of the oils on macrophages viability is reported in order to assess their potential use for health and/or cosmetic purposes. As far as we know, this is the first report on the antifungal activity of T. herba-barona essential oils against dermatophyte and Aspergillus strains. Also the cytotoxicity and the anti-inflammatory potential of both oils have not been assessed before. 2. Materials and methods 2.1. Plant material Aerial parts of L. stoechas and T. herba-barona were collected in Sardinia Island (Italy) and air-dried at 40 ◦ C with forced ventilation for two days. Before utilization, matter was ground with a Malavasi mill (Bologna, Italy) taking care to avoid overheating and then it was subjected to hydrodistillation. Vouchers specimens were deposited at the Herbarium of the Dipartimento de Scienze Botaniche e Orto Botanico, Università degli Studi di Cagliari (CAG n. 1067 for L. stoechas and 1065 for T. herba-barona). 2.2. Reference compounds Fluconazole was kindly provided by Pfizer (pure powder) and amphotericin B by Sigma (80.0% purity).

gas chromatography–mass spectrometry (GC–MS). Analytical GC was carried out in a gas chromatograph (Agilent, Model 7890A, Palo Alto, CA), equipped with a flame ionization detector (FID), an autosampler (Agilent, Model 7683B), Agilent HP5 fused silica column (5% phenyl-methylpolysiloxane), 30 m × 0.25 mm i.d., film thickness 0.25 ␮m, and a Agilent ChemStation software system. Oven temperature was settled at 60 ◦ C, raising at 3 ◦ C/min to 250 ◦ C and then held 20 min at 250 ◦ C; injector temperature: 250 ◦ C; carrier gas: helium at 1.0 ml min−1 ; splitting ratio 1:10; detectors temperature: 300 ◦ C. GC–MS analyses were carried out in a gas chromatograph (Agilent, Model 6890N, Palo Alto, CA) equipped with a splitsplitless injector, an autosampler Agilent model 7683 and an Agilent HP5 fused silica column; 5% phenyl-methylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.25 ␮m. GC conditions used were: programmed heating from 60 to 250 ◦ C at 3 ◦ C/min followed by 20 min under isothermal conditions. The injector was maintained at 250 ◦ C. Helium was the carrier gas at 1.0 ml min−1 ; the sample (1 ␮l) was injected in the split mode (1:10). The GC was fitted with a quadrupole mass spectrometer, MS, Agilent model 5973 detector. MS conditions were as follows: ionization energy 70 eV, electronic impact ion source temperature 200 ◦ C, quadrupole temperature 150 ◦ C, scan rate 3.2 scan s−1 , mass range 30–480 u. Software adopted to handle mass spectra and chromatograms was a ChemStation. NIST 02 and LIBR (TP) Mass Spectra Libraries were used as references. Samples were run in chloroform with a dilution ratio of 1:100. Compounds were identified by matching their mass spectra and retention indices with those reported in the literature. Moreover, whenever possible, identification was confirmed by injection of pure compounds. Percentage of individual components was calculated based on GC peak areas without FID response factor correction. 2.4. Fungal strains Antifungal activity of the essential oils and their main compounds (fenchone, camphor, carvacrol and thymol) was evaluated against yeasts, dermatophyte and Aspergillus strains: two clinical Candida strains isolated from recurrent cases of vulvovaginal and oral candidosis (Candida krusei H9 and Candida guillermondii MAT23); three Candida type strains from the American Type Culture Collection (Candida albicans ATCC 10231, Candida tropicalis ATCC 13803, and Candida parapsilopsis ATCC 90018); one Cryptococcus neoformans type strain from the Colección Espanõla de Cultivos Tipo (Cryptococcus neoformans CECT 1078); one Aspergillus clinical strain isolated from bronchial secretions (Aspergillus flavus F44) and two Aspergillus type strains from the American Type Culture Collection (Aspergillus niger ATCC 16404 and Aspergillus fumigatus ATCC 46645); three dermatophyte clinical strains isolated from nails and skin (Epidermophyton floccosum FF9, Microsporum canis FF1, and Trichophyton mentagrophytes FF7), and four dermatophyte type strains from the Colección Espanõla de Cultivos Tipo (Microsporum gypseum CECT 2908, Trichophyton mentagrophytes var. interdigitale CECT 2958, Thrichophyton rubrum CECT 2794, Trichophyton verrucosum CECT 2992). The fungal isolates were identified by standard microbiology methods and stored on Sabouraud broth with glycerol at −70 ◦ C. Prior to antifungal susceptibility testing, each isolate was inoculated on Sabouraud agar (SDA) or Potato dextrose agar (PDA) to ensure optimal growth characteristics and purity.

2.3. Essential oil isolation and analysis 2.5. Antifungal activity Hydrodistillation was performed for 4 h in a circulatory Clevenger-type apparatus up to exhaustion of the oil contained in the matrix, according to the procedure described in the European Pharmacopoeia (Council of Europe, 1997). Analysis of the volatile extracts was carried out by gas chromatography (GC) and by

A macrodilution broth method was used to determine the Minimal Inhibitory Concentrations (MICs) and Minimal Lethal Concentrations (MLCs), according to Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) references documents M27-A3

M. Zuzarte et al. / Industrial Crops and Products 44 (2013) 97–103

(CLSI, 2008a) and M38-A2 (CLSI, 2008b) for yeasts and filamentous fungi, respectively. The serial doubling dilution of each oil or isolated compound was prepared in dimethyl sulphoxide (DMSO), with concentrations ranging from 0.02 to 20 ␮l ml−1 . Final concentration of DMSO never exceeded 2%. Recent cultures of each strain were used to prepare the cell suspension adjusted to 1–2 × 103 cells per ml for yeasts and 1–2 × 104 cells per ml for filamentous fungi. The concentration of cells was confirmed by viable count on Sabouraud agar. The test tubes were incubated aerobically at 35 ◦ C for 48 h/72 h (Candida spp. and Aspergillus spp./C. neoformans) and at 30 ◦ C for 7 days (dermatophytes) and MICs were determined. To evaluate MLCs, aliquots (20 ␮l) of broth were taken from each negative tube after MICs reading, and cultured in Sabouraud dextrose agar plates. Plates were then incubated for 48 h at 35 ◦ C (Candida spp. and Aspergillus spp.), 72 h for C. neoformans and 7 days at 30 ◦ C (dermatophytes). In addition, two reference antifungal compounds, amphotericin B (Fluka) and fluconazole (Pfizer) were used to control the sensitivity of tested microorganisms. All tests were performed in RPMI medium. For each strain tested, the growing conditions and the sterility of the medium were checked in two control tubes. The innocuity of the DMSO was also checked at the highest tested concentration. All experiments were performed in triplicate and repeated if the results differed.

99

2.8. Anti-inflammatory activity To evaluate the anti-inflammatory potential of the oils, the in vitro model of lipopolysaccharide (LPS)-stimulated macrophages was used. The production of nitrite oxide (NO) was measured by the accumulation of nitrites in the culture medium, using the colorimetric Griess reaction. Briefly, 170 ␮l of culture supernatants were added to an equal volume of Griess reagent [0.1% (w/v) N-(1-naphthyl)-ethylenediamine dihydrochloride and 1% (w/v) sulphanilamide containing 5% (w/v) H3 PO4 ] and maintained during 30 min, in the dark. Absorbance was measured using an ELISA automatic microplate reader (SLT, Austria) at 550 nm and the nitrite concentration determined from a regression analysis prepared with serial dilutions of sodium nitrite. All the experiments were performed in duplicate, being the results expressed as mean ± SEM of the indicated number of experiments. Statistical analysis comparing LPS-stimulated cells to control was performed using two-sided unpaired t-test. To compare the effect of different concentrations of the essential oils with LPS-stimulated cells, one-way ANOVA followed by a Dunnett’s multiple comparison test was used. The statistical tests were applied using GraphPad Prism, version 5.02 (GraphPad Software, San Diego, CA, USA). 3. Results

2.6. Cell culture

3.1. Essential oil composition

The mouse macrophage cell line, Raw 264.7 (ATCC number: TIB-71) was kindly supplied by Dr. Otília Vieira (Center for Neuroscience and Cell Biology, University of Coimbra, Portugal). The cells were cultured on endotoxin-free Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) non inactivated foetal bovine serum, 3.02 g l−1 sodium bicarbonate, 100 ␮g ml−1 streptomycin and 100 U ml−1 penicillin at 37 ◦ C in a humidified atmosphere with 5% CO2 . Morphological appearance of macrophages was microscopically monitored during the assays.

The essential oils were obtained in yields of 0.7% (v/w) for L. stoechas and 1.0% for T. herba-barona (v/w). The qualitative and quantitative composition of the essential oils of L. stoechas and T. herba-barona are represented in Table 1. Both oils were characterized by high contents of oxygenated monoterpenes. For L. stoechas oil the main compounds identified were fenchone (37.0%), camphor (27.3%), bornyl acetate (6.2%), 1,8-cineole (6.0%), thymol (3.1%) and carvacrol (3.4%) while for T. herba-barona essential oil high amounts of phenolic compounds, namely thymol (30.2%) and carvacrol (54.0%) were found. 3.2. Antifungal activity

2.7. Cell viability Assessment of cell respiration, an indicator of cell viability, was performed using a colorimetric assay with 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT), as described by Mosmann (1983). The cells (0.6 × 106 cells/well) were cultured in a 48-well microplate and left to stabilize for 12 h. The cells were then incubated for 24 h in the culture medium alone (control) or with different concentrations of the essential oils. After cells treatments, 43 ␮l of MTT solution (5 mg ml−1 in phosphate buffered saline) were added to each well and the microplates were further incubated at 37 ◦ C for 15 min, in a humidified atmosphere with 5% CO2 . Supernatants were centrifuged (1000 × g during 5 min) to recover viable cells. To dissolve formazan crystals formed in adherent cells in the microplates, 300 ␮l of acidified isopropanol (0.04 N HCl in isopropanol) were added to each cell and recovered to the respective Eppendorf containing the pellet formed after centrifugation. Quantification of formazan was performed using an ELISA automatic microplate reader (SLT, Austria) at 570 nm, with a wavelength of 620 nm. All the experiments were performed in duplicate, being the results expressed as mean ± SEM of three independent experiments. The means were statistically compared using one-way ANOVA, with a Dunnett’s multiple comparison test. The statistical tests were applied using GraphPad Prism, version 5.02 (GraphPad Software, San Diego, CA, USA).

MIC and MLC values of L. stoechas and T. herba-barona essential oils and their main compounds are shown in Tables 2 and 3, respectively. The essential oil of L. stoechas was active against C. neoformans and dermatophytes with MIC values ranging from 0.32 to 0.64 ␮l ml−1 (Table 2). For Candida spp., MIC values varied from 1.25 to 2.5 ␮l ml−1 while for Aspergillus strains MIC values reached up to 5 ␮l ml−1 (Table 2). The antifungal activity of the oil seems to result from a synergistic effect of several compounds since the major compounds, fenchone and camphor, tested alone showed very low antifungal activity against the referred strains. The essential oil of T. herba-barona showed a wide-spectrum antifungal activity, including Candida spp., C. neoformans, dermatophytes and Aspergillus strains, with MIC values ranging from 0.16 to 0.32 ␮l ml−1 (Table 3). For most Candida spp. and dermatophytes, MIC was equivalent to MLC suggesting a fungicidal effect of the oil. The essential oil’s major compounds confirm this fungicidal activity, with carvacrol and thymol proving to be more active than the essential oil, particularly the former with MIC values as low as 0.04 ␮l ml−1 . 3.3. Cell viability The cytotoxicity of the oils was evaluated in order to assess their potential safety, a crucial step required for further development of

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Table 1 Composition of the essential oils of Lavandula stoechas and Thymus herba-barona from Sardinia. RI

Compound

L. stoechas (%)

T. herba-barona (%)

886 926 930 938 953 987 992 996 1019 1027 1032 1034 1062 1085 1090 1090 1100 1115 1147 1168 1169 1179 1196 1209 1237 1247 1287 1293 1301 1327 1418 1523 1581 1589 1601 1642

3-Heptanone Tricyclene ␣-Thujene ␣-Pinene Camphene 3-Octanone Myrcene 3-Octanol ␣-Terpinene p-Cymene Limonene 1,8-Cineole ␥-Terpinene Camphenilone Terpinolene Fenchone Linalool ␣-Fenchol Camphor Borneol p-Mentha-1,5-dien-8-ol Terpinen-4-ol Myrtenol Verbenone Thymol methyl ether Carvacrol methyl ether Bornyl acetate Thymol Carvacrol Myrtenyl acetate trans-␤-Caryophyllene ␦-Cadinene Caryophyllene oxide Viridiflorol Guaiol T-Muurolol

– 0.2 – 0.4 2.8 – – – – 0.4 0.2 6.0 – 0.3 – 37.0 – 0.4 27.3 0.8 0.4 – 0.5 0.3 – – 6.2 3.1 3.4 1.7 – 0.3 – 2.6 1.7 0.4

0.8 – 0.1 0.1 0.2 0.7 0.2 0.8 0.4 2.1 0.1 – 2.2 – 0.1 – 1.6 – – 3.2 – 0.7 – – 0.6 0.5 – 30.2 54.0 – 1.0 – 0.2 – – –

Total

86.1

99.8

essential oil-based products for pharmaceutical and/or cosmetics purposes. In vitro tests based on the MTT assay on a macrophage cell line were used, avoiding animal sacrifices at this stage. L. stoechas essential oil did not affect cell viability or showed very low detrimental effects at concentrations (<0.64 ␮l ml−1 ) with antifungal activity (Fig. 1a). On the other hand, T. herba-barona proved to be very toxic even at lower concentrations (0.04 ␮l ml−1 , Fig. 1b). 3.4. Anti-inflammatory activity The anti-inflammatory potential of the oils was investigated on macrophages that are able to produce NO, an inflammatory marker, after cells stimulation with LPS. The effect of the oils on NO production was analysed by measuring the accumulation of nitrites in the culture medium. As shown in Fig. 2, untreated cells produced very low quantities of nitrites (<1 ␮M), being nitrites production strongly increased in the presence of LPS. In the presence of L. stoechas essential oil the production of nitrites was inhibited at concentrations that did not affect cell viability (Fig. 1a), namely after cells treatment with 0.16 ␮l ml−1 and 0.32 ␮l ml−1 of essential oil. T. herba-barona did not show anti-inflammatory potential since it was not able to inhibit the production of NO at concentrations without cytotoxicity to the cells (Figs. 1b and 2b). 4. Discussion L. stoechas and T. herba-barona have been object of several phytochemical studies that have pointed out a high chemical variability

allowing the establishment of several chemotypes. L. stoechas oil is characterized by significant variations in the amounts of fenchone, camphor and 1,8-cineole, being the fenchone/camphor chemotype the most commonly identified (Angioni et al., 2006; Benabdelkader et al., 2011; Dadalio˘glu and Evrendilek, 2004; Dob et al., 2006; Messaoud et al., 2012). For T. herba-barona oil several studies report the presence of high amounts of phenolic compounds, with mainly one compound (carvacrol or thymol) occurring in very high quantities (Corticchiato et al., 1998; Juliano et al., 2000; Usai et al., 2003). Also, other chemotypes have been reported for this species, namely carvone, linalool, geraniol, ␣-terpinyl acetate, terpinen-4-ol, and cis-dihydrocarvone chemotypes (Corticchiato et al., 1998). In our study, the chemical composition of L. stoechas essential oil is in accordance to that reported previously, with high amounts of fenchone and camphor. Thymol (3.1%) and carvacrol (3.4%) are not usual in this specie. For Lavandula genus, only L. multifida has been reported as a phenolic-rich lavender (Zuzarte et al., 2012). However, the genus Thymus comprises species particularly rich in carvacrol and thymol, namely T. pulegioides, T. vulgaris and T. zygis (Gonc¸alves et al., 2010; Pinto et al., 2006; Stahl-Biskup, 2002). In the present study, T. herba-barona from Sardinia also showed high levels of carvacrol (54%) and thymol (30.2%), suggesting great potentialities of this species for industrial exploration, mainly as a antimicrobial agent (Ahmad et al., 2011). The antifungal activity of L. stoechas essential oil from different localities of Algeria was previously evaluated against C. albicans, A. niger and A. flavus (Benabdelkader et al., 2011). In our study other Candida species, C. neoformans, dermatophytes and A. fumigatus were considered. L. stoechas oil was more active against C. neoformans and dermatophytes strains, exhibiting a fungicidal effect against most of the tested fungi (MIC values equivalent to MLC). Furthermore, the antifungal activity of the oil was probably due to the activity of minor compounds or related with a synergistic effect between different compounds present in the oil since the main compounds tested alone showed low antifungal activity. T. herba-barona oil was more active against all the tested strains, being the activity related to the phenolic nature of its main compounds (carvacrol and thymol) (Table 3). Although L. stoechas essential oil showed lower antifungal activity than T. herba-barona, it did not affect cell viability when used at concentrations up to 0.32 ␮l ml−1 or showed very low detrimental effect at 0.64 ␮l ml−1 . On the other hand, T. herba-barona was toxic to the cells even at lower concentrations (0.04 ␮l ml−1 ). Although many studies focus on the biological activity of essential oils, cytotoxicity assays are scarce. However, for the development of bio-products for human or animal use, safety and tolerance assays are fundamental. In vitro tests on cell lines are useful in earlier stages of investigations, avoiding animal sacrifices. Our results prove the safety of L. stoechas essential oil for pharmaceutical and cosmetic purposes but alert for a more cautious use of T. herbabarona oil (without toxicity only up to 0.02 ␮l ml−1 ). Studies on the anti-inflammatory potential of lavender and thyme essential oils are very limited. Concerning Lavandula spp. only one study reported the ability of L. angustifolia essential oil to inhibit the carrageenan-induced oedema in rats paw at concentrations of 200 mg kg−1 , although the action mechanism was not addressed in this study (Hajhashemi et al., 2003). Regarding Thymus spp., the potential anti-inflammatory properties of T. vulgaris essential oil was demonstrated by its ability to repress the enzymatic activity of 5-lipoxygenase and to reduce the secretion of the pro-inflammatory cytokines TNF-␣, IL-1␤ and IL-8 in THP-1 cells (Tsai et al., 2011). In our study a well-established in vitro assay was used to evaluate the capacity of the oils to inhibit NO production, an important inflammatory marker. L. stoechas showed very promising results,

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Table 2 Antimicrobial activity (MIC and MLC) of Lavandula stoechas essential oil and its main compounds against yeasts, dermatophyte and Aspergillus strains. Strains

Candida albicans ATCC 10231 Candida tropicalis ATCC 13803 Candida krusei H9 Candida guillermondii MAT23 Candida parapsilosis ATCC 90018 Cryptococcus neoformans CECT 1078 Epidermophyton floccosum FF9 Microsporum canis FF1 Microsporum. gypseum CECT 2908 Trichophyton mentagrophytes FF7 Trichophyton mentagrophytes var. interdigitale CECT 2958 Trichophyton rubrum CECT 2794 T. verrucosum CECT 2992 Aspergillus fumigatus ATCC 46645 Aspergillus flavus F44 Aspergillus niger ATCC 16404

Lavandula stoechas

Fenchone

MICa

MICa

MLCa

MICa

MLCa

MICb

MLCb

MICb

MLCb

MLCa

Camphor

Fluconazole

Amphotericin B

2.5

2.5

5

5

>20

>20

1

>128

N.T.c

N.T.

2.5

2.5

5

5

>20

>20

4

>128

N.T.

N.T.

2.5 1.25

2.5 1.25

2.5 2.5

2.5 2.5

>20 >20

>20 >20

64 8

64–128 8

N.T. N.T.

N.T. N.T.

2.5

2.5

5

5–10

>20

>20

<1

<1

N.T.

N.T.

0.64

0.64

2.5

2.5

>20

>20

16

128

N.T.

N.T.

0.32

0.64

1.25–2.5

1.25–2.5

>20

>20

16

16

N.T.

N.T.

0.64 0.64

0.64 0.64–1.25

2.5–5 2.5

2.5–5 2.5–5

>20 >20

>20 >20

128 128

128 >128

N.T. N.T.

N.T. N.T.

0.64

0.64

2.5–5

2.5–5

>20

>20

16–32

32–64

N.T.

N.T.

0.64

1.25

>20

>20

128

≥128

N.T.

N.T.

0.64

1.25

1.25–2.5

>20

>20

16

64

N.T.

N.T.

5

>20

>20

>128

>128

N.T.

N.T.

>20

>20

N.T.

N.T.

2

4

>20 >20

>20 >20

N.T. N.T.

N.T. N.T.

2 1–2

8 4

0.64

0.64

10

10

1.25–2.5 5

1.25

>20

10

5 2.5

>20 >20

20 10

10–20 20 >20

Results were obtained from 3 independent experiments performed in duplicate. a MIC and MLC were determined by a macrodilution method and expressed in ␮l ml−1 (v/v). b MIC and MLC were determined by a macrodilution method and expressed in ␮g ml−1 (w/v). c Not tested.

Table 3 Antimicrobial activity (MIC and MLC) of Thymus herba-barona essential oil and its main compounds against yeasts, dermatophyte and Aspergillus strains. Strains

Thymus herba barona MIC

a

MLC

a

Carvacrol MIC

a

Timol a

MLC

MIC

a

Fluconazole MLC

a

MIC

b

Amphotericin B MLC

b

MICb

MLCb

Candida albicans ATCC 10231 Candida tropicalis ATCC 13803 Candida krusei H9 Candida guillermondii MAT23 Candida parapsilosis ATCC 90018 Cryptococcus neoformans CECT 1078

0.32 0.32

0.32 0.32

0.16 0.16

0.16–0.32 0.16–0.32

0.16 0.16–0.32

0.32 0.32

1 4

>128 >128

N.T.c N.T.

N.T. N.T.

0.32 0.16 0.32

0.32 0.32 0.32

0.16 0.08–0.16 0.16

0.16–0.32 0.16 0.16–0.32

0.16–0.32 0.16 0.32

0.32 0.16 0.32

64 8 <1

64–128 8 <1

N.T. N.T. N.T.

N.T. N.T. N.T.

0.16

0.32

0.16

0.16

0.16

0.32

16

128

N.T.

N.T.

Epidermophyton floccosum FF9 Microsporum canis FF1 Microsporum gypseum CECT 2908 Trichophyton mentagrophytes FF7 T. mentagrophytes var. interdigitale CECT 2958 Trichophyton rubrum CECT 2794 Trichophyton verrucosum CECT 2992

0.16

0.16

0.08

0.08

0.16

0.16

16

16

N.T.

N.T.

0.16 0.16

0.16 0.16

0.04 0.04

0.08 0.08–0.16

0.08 0.16

0.16 0.32

128 128

128 >128

N.T. N.T.

N.T. N.T.

0.16

0.32

0.04

0.08

0.16

0.16–0.32

16–32

32–64

N.T.

N.T.

0.16

0.32

0.08

0.16

N.T.

N.T.

128

≥128

N.T.

N.T.

0.16

0.16

0.08

0.08

0.16

0.16

16

64

N.T.

N.T.

0.16

0.16

0.08

0.16

N.T.

N.T.

>128

>128

N.T.

N.T.

0.32 0.16

0.64 0.64

0.32 0.16

0.32 0.32

0.32 0.16

0.64 0.64

N.T. N.T.

N.T. N.T.

2 2

8 4

0.32

0.64

0.16

0.16–0.32

0.16

0.64

N.T.

N.T.

1–2

4

Aspergillus flavus F44 Aspergillus fumigatus ATCC 46645 Aspergillus niger ATCC 16404

Results were obtained from 3 independent experiments performed in duplicate. a MIC and MLC were determined by a macrodilution method and expressed in ␮l ml−1 (v/v). b MIC and MLC were determined by a macrodilution method and expressed in ␮g ml−1 (w/v). c Not tested.

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M. Zuzarte et al. / Industrial Crops and Products 44 (2013) 97–103

Fig. 1. Effect of Lavandula stoechas (a) and Thymus herba-barona (b) on macrophages viability using the MTT assay. Results are expressed as percentage of MTT reduction by control cells maintained in culture medium. Each value represents the mean ± SEM from 3 independent experiments performed in duplicate (**p < 0.01 and ***p < 0.001, compared to control cells).

Fig. 2. Effect of Lavandula stoechas (a) and Thymus herba-barona (b) essential oils on nitrites production by LPS-stimulated macrophages. Nitrite levels in culture supernatants were evaluated by Griess reaction. Results are expressed as concentration (␮M) of nitrites in culture medium. Each value represents the mean ± SEM from 3 independent experiments performed in duplicate (### p < 0.001, compared to control; **p < 0.01 and ***p < 0.001, compared to LPS).

since it inhibited NO production at concentrations that did not affect cells viability. Taken together our results provide evidence on the antifungal, anti-inflammatory and safety/cytotoxicity of L. stoechas and T. herba-barona essential oils. The results obtained with T. herbabarona pointed its use as a preservative in storage products, due to its ability to inhibit Aspergilllus growth. L. stoechas essential oil is more suitable as a potential natural source of antifungal and anti-inflammatory drugs, suggesting its application in food supplements/nutraceuticals or cosmeceuticals by the pharmaceutical, food and cosmetic industries. Nutraceuticals are gaining impact in the society due to their ability to provide medical or health benefits, including the prevention or treatment of diseases, and studies addressing its safety and mechanism of action are of great value, since an increasing number of people are more amenable to consume these products as alternatives/complements to conventional drugs. Acknowledgements The authors thank Dr. Otília Vieira (CNC, University of Coimbra, Portugal) for the kind gift of the mouse macrophage-like cell line Raw 264.7. This work was funded through national funds from FCT – Fundac¸ão para a Ciência e a Tecnologia under the project CEF/POCI2010/FEDER and by a Ph.D. fellowship to Mónica R. Zuzarte (SFRH/BD/40218/2007).

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