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Industrial Crops and Products 39 (2012) 204– 209

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h o me pag e: www.elsev ier .com/ locate / indcrop

omposition, antifungal activity and cytotoxicity of the essential oils ofeseli tortuosum L. and Seseli montanum subsp. peixotoanum (Samp.). Laínz from Portugal

aria José Gonc alvesa, Ana Cristina Tavaresa, Carlos Cavaleiroa, Maria Teresa Cruzb,aria Celeste Lopesb, Jorge Canhotoc, Lígia Salgueiroa,∗

Centro de Estudos Farmacêuticos, Faculdade de Farmácia, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Universidade de Coimbra, 3000-548 Coimbra, PortugalFaculdade de Farmácia Centro de Neurociências e Biologia Celular de Coimbra, Universidade de Coimbra, 3004-517 Coimbra, PortugalCentro de Ecologia Funcional, Departamento de Ciências da Vida, Universidade de Coimbra, Ap. 3046, 3001-401 Coimbra, Portugal

r t i c l e i n f o

rticle history:eceived 6 October 2011eceived in revised form 24 January 2012ccepted 18 February 2012

eywords:eseli tortuosumeseli montanum subsp. peixotoanumssential oilsntifungal activity

n vitro cytotoxicity

a b s t r a c t

Seseli L. is an important genus with high number of aromatic species used in traditional medicine dueto recognized therapeutic properties of their essential oils, namely antimicrobial activity. However, andin order to explore its potential utilization for industrial and commercial purposes, scientific studiesaddressing the chemical composition, the action mechanism and the toxicological safety of the oils aremissing. The present study was therefore designed to elucidate the chemical and biological effects of theessential oils of Seseli tortuosum and of Seseli montanum subsp. peixotoanum. Given that essential oils arecurrently used in topical applications we assessed its cytotoxicity in human keratinocytes. The oils wereinvestigated by GC and GC–MS and the antifungal activity (MIC and MLC) was evaluated against yeasts,dermatophyte and Aspergillus strains. Assessment of keratinocytes viability after essential oils treatmentwas made through the colorimetric assay MTT.

TT It is possible to distinguish the oils of the two taxa through differences in the contents of Z-�-ocimene,limonene and �-elemene. The results also demonstrated that the oil of S. tortuosum has higher anti-fungal activity than S. montanum oil. Furthermore, it was also observed that S. tortuosum essential oil hascytotoxic properties to human cells when used in concentrations higher than 0.64 �L/mL.

In conclusion, it is possible to find appropriate doses of S. tortuosum oil showing both antifungal activityand very low detrimental effect in human keratinocytes.

. Introduction

For a long time, plants from the Apiaceae family have beensed as spices or drugs, particularly due to their essential oils.ccordingly, the potential use of Apiaceae essential oils as antimi-

robial agents has been described previously (Demirci et al., 2007;i Pasqua et al., 2005; Kosalec et al., 2005; Oroojalian et al., 2010;avares et al., 2008, 2010).

Abbreviations: A., Aspergillus; ATCC, American Type Culture Collection; C., Can-ida; CECT, Colección Espanola de Cultivos Tipo; COI, Herbarium of the Botanicnstitute of Coimbra University; DMSO, dimethyl sulfoxide; FID, flame ionizationetector; GC, gas chromatography; GC–MS, gas chromatography–mass spectrome-ry; LPS, lipopolysaccharide; MIC, minimal inhibitory concentration; MLC, minimalethal concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumromide; NCCLS, National Committee for Clinical Laboratory Standards; NO, nitricxide; S.E.M., standard error of the mean.∗ Corresponding author. Tel.: +351 239 859995; fax: +351 239 827126.

E-mail address: ligia@ff.uc.pt (L. Salgueiro).

926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2012.02.025

© 2012 Elsevier B.V. All rights reserved.

Seseli is an old Greek name that was used by Hippocrates forcertain members of the Apiaceae family with finely divided leaves(Tosun et al., 2006). Numerous species of this genus have been usedin folk medicine since ancient times (Dioscorides, 2002). Tradi-tionally, the fruits of Seseli tortuosum are used as emmenagogueand in digestive diseases (Carretero Accame et al., 2011; Kupeliet al., 2006). On the other hand, the essential oils of Seseli indicum,Seseli libanotis and Seseli montanum subsp. tommasinii proved to beeffective against human pathogenic microorganisms (Adams et al.,2009; Ozturk and Ercisli, 2006; Siljegovic et al., 2011; Singh et al.,2002).

In Portugal there are 2 taxa usually used in traditional medicine:S. tortuosum L., widespread in several provinces and S. mon-tanum subsp. peixotoanum (Samp.) M. Laínz restricted to someultrabasic habitats in Trás-os-Montes province (north of Portugal)

(Castroviejo et al., 2003; Proenc a da Cunha et al., 2007). The last oneis an Iberian endemism; however, and to the best of our knowl-edge, its chemical composition and biological activities have notbeen studied yet. Our previous works with Apiaceae, widely used in

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raditional medicine in Portugal, have suggested that some essen-ial oils encompass therapeutic potential for fungal diseasesnvolving mucosal, cutaneous and respiratory tracts (Gonc alvest al., 2012; Tavares et al., 2008, 2010). Indeed, dermatomycoses areommon infections caused by dermatophytes and by some yeastshat can be severe in immunocompromised patients, due to theevelopment of opportunistic and superficial mycoses. However,he use of essential oils in therapeutic approaches may requireigh doses, and the exposure of humans to these products is aatter of concern. In this context, the analysis of the toxicologi-

al properties of the essential oils is of great relevance, being anmportant prerequisite for their application in cosmetic and healthare products.

The present study was undertaken to investigate the phy-ochemical composition and the antifungal activity of the oilsf the Portuguese Seseli spp. against yeasts, dermatophytes andspergillus strains. Since essential oils are currently used in topicalpplications we also assessed the cytotoxicity of the oils of Seselipp. in human keratinocytes.

. Materials and methods

.1. Plant material

Ripe umbels with mature seeds of S. montanum subsp. peixo-oanum and of S. tortuosum were collected in the north of Portugal

Trás-os-Montes (Samil – sample 1 and Alimonte – sample 2) andn the central part of Portugal (Vieira de Leiria – sample 3 and Baleal

sample 4), respectively.After harvesting, the umbels were air-dried in the shade. Plant

axonomy was confirmed and voucher specimens are depositedn the Herbariums of the Department of Life Sciences of the Uni-ersity of Coimbra (Alimonde-Carrazede, ACTavares 108 (COI) andamil-Braganc a, ACTavares 109 (COI)) and in the Laboratory of Phar-acognosy – Faculty of Pharmacy of the University of Coimbra.

.2. Essential oil isolation

Essential oils were isolated by water distillation for 3 h from airried material, using a Clevenger-type apparatus, according to therocedure described in the European Pharmacopoeia (Council ofurope, 1997).

.3. Gas chromatography (GC)

Analytical GC was carried out using a Hewlett Packard 6890Agilent Technologies, Palo Alto, CA, USA) gas chromatograph withP GC ChemStation Rev. A.05.04 data handling system, equippedith a single injector and two flame ionization detectors (FID). A

raphpak divider (Agilent Technologies, Part Number 5021-7148)as used for simultaneous sampling in two Supelco (Supelco Inc.,ellefonte, PA, USA) fused silica capillary columns with differenttationary phases: SPB-1 (polydimethylsiloxane 30 m × 0.20 mm,lm thickness 0.20 �m), and SupelcoWax 10 (polyethyleneglycol0 m × 0.20 mm, film thickness 0.20 �m). Oven temperature pro-ram: 70–220 ◦C (3 ◦C/min), 220 ◦C (15 min); injector temperature:50 ◦C; carrier gas: helium, adjusted to a linear velocity of 30 cm/s;plitting ratio 1:40; detectors temperature: 250 ◦C.

.4. Gas chromatography–mass spectrometry (GC–MS)

Analyses were carried out using a Hewlett Packard 6890

as chromatograph fitted with a HP1 fused silica columnpolydimethylsiloxane 30 m × 0.25 mm, film thickness 0.25 �m),nterfaced with an Hewlett Packard mass selective detector 5973Agilent Technologies, Palo Alto, CA, USA) operated by HP Enhanced

nd Products 39 (2012) 204– 209 205

ChemStation software, version A.03.00. GC parameters as above;interface temperature: 250 ◦C; MS source temperature: 230 ◦C; MSquadrupole temperature: 150 ◦C; ionization energy: 70 eV; ioniza-tion current: 60 �A; scan range: 35–350 u; scans/s: 4.51.

2.5. Qualitative and quantitative analyses

The identity of the compounds was achieved from their reten-tion indices on SPB-1 and SupelcoWax 10 columns and fromtheir mass spectra. Retention indices, calculated by linear inter-polation relative to retention times of C8–C22 n-alkanes, werecompared with those of authentic samples included in our ownlaboratory database. Acquired mass spectra were compared withcorresponding data of components of reference oils and com-mercial available standards from a home-made library or fromliterature data (Adams, 2004; Joulain and Konig, 1998). Relativeamount of individual components was calculated based on GC peakareas without FID response factor correction. The relative percent-age of components which co-elute on apolar column was calculatedon SupelcoWax 10 column.

2.6. Antifungal strains

Antifungal activity of the oils was evaluated against yeasts,Aspergillus and dermatophyte strains: two clinical Candida strainsisolated from recurrent cases of vulvovaginal candidosis (C. kruseiH9 and C. guillermondii MAT23), three type strains from the Ameri-can Type Culture Collection (Candida albicans ATCC 10231, Candidatropicalis ATCC 13803, and Candida parapsilosis ATCC 90018) andone type strain from the Colección Espanola de Cultivos Tipo(Cryptococcus neoformans CECT 1078); three dermatophyte clini-cal strains isolated from nails and skin (Epidermophyton floccosumFF9, Trichophyton mentagrophytes FF7 and Microsporum canis FF1)and two type strains from the Colección Espanola de Cultivos Tipo(Trichophyton rubrum CECT 2794 and Microsporum gypseum CECT2908); and one Aspergillus clinical strain isolated from bronchialsecretions (A. flavus F44) and two type strains from the Amer-ican Type Culture Collection (Aspergillus niger ATCC 16404 andAspergillus fumigatus ATCC 46645). Antifungal activity of the maincompounds of the essential oils (�-pinene, �-pinene and �-ocimene), was also evaluated against the same strains.

The fungal isolates were identified by standard microbiologymethods and stored on Sabouraud broth with glycerol at −70 ◦C.Prior to antifungal susceptibility testing, each isolate was inocu-lated on Sabouraud agar to ensure optimal growth characteristicsand purity.

2.7. Antifungal activity

A macrodilution broth method was used to determine theminimal inhibitory concentrations (MICs) and minimal lethal con-centrations (MLCs), according to Clinical and Laboratory StandardsInstitute (CLSI, formerly NCCLS) reference documents M27-A3(CLSI, 2008), M27-S3 (CLSI, 2008) and M38-A2 (CLSI, 2008) foryeasts and filamentous fungi, respectively.

The serial doubling dilution of the essential oil and its majorcompound was prepared in dimethyl sulfoxide (DMSO), with con-centrations ranging from 0.08 to 20 �L/mL. Final concentration ofDMSO never exceeded 2%. Recent cultures of each strain were usedto prepare the cell suspension adjusted to 1–2 × 103 cells per mLfor yeasts and 1–2 × 104 cells per mL for filamentous fungi. Theconcentration 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 7days (dermatophytes) and MICs were determined. To evaluate MLC,aliquots (20 �L) of broth were taken from each negative tube after

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specific effects of the oils on MTT (data not shown). Taken together,these results demonstrate that S. tortuosum essential oil has cyto-toxic properties to human cells when used in concentrations above0.64 �L/mL.

Fig. 1. Effect of S. tortuosum essential oil on cell viability (MTT assay). The HaCaT

06 M.J. Gonc alves et al. / Industrial C

IC reading, and cultured in Sabouraud dextrose agar plates. Platesere then incubated for 48 h at 35 ◦C (Candida spp. and Aspergillus

pp.), 72 h for C. neoformans and 7 days at 30 ◦C (dermatophytes).n addition, two reference antifungal compounds, amphotericin BFluka) and fluconazole (Pfizer) were used to control the sensitiv-ty of tested microorganisms. All tests were performed in RPMI

edium. For each strain tested, the growth conditions and theterility of the medium were checked in two control tubes. Thennocuity of the DMSO was also checked at the highest testedoncentration. All experiments were performed in triplicate andepeated if the results differed.

.8. Cell culture and materials

The fetal calf serum was from Biochrom KG (Berlin, Germany)nd trypsin from Gibco (Paisley, UK). 3-(4,5-Dimethylthiazol-2-yl)-,5-diphenyltetrazolium bromide (MTT), LPS from Escherichia coliserotype 026:B6), and all the other reagents were from Sigmahemical Co. The human keratinocyte cell line HaCaT, obtainedrom DKFZ (Heidelberg), was kindly supplied by Dr. Eugénia Car-alho (Centro de Neurociências e Biologia Celular, Universidade deoimbra, Coimbra, Portugal). Keratinocytes were cultured in Dul-ecco’s Modified Eagle Medium (high glucose) supplemented with

mM glutamine, 10% heat inactivated fetal bovine serum, 100 U/mLenicillin, and 100 �g/mL streptomycin at 37 ◦C in a humidifiedtmosphere of 95% air and 5% CO2. Along the experiments, cellsere monitored by microscope observation in order to detect anyorphological change.

.9. MTT assay for cell viability

Since the oil from S. montanum revealed little anti-fungal activ-ty, viability assays were only performed for S. tortuosum essentialil. Assessment of cell viability was made through a colorimetricssay, using MTT, as previously reported at our lab (Tavares et al.,008, 2010). The cells (0.1 × 106 cells/well) were cultured in 48-ell microplates, in a final volume of 600 �L, allowed to stabilize

or 3 h, and then incubated for 24 h with culture medium alonecontrol), or stimulated with varying concentrations of the essen-ial oil (0.32 �L/ml, 0.64 �L/mL and 1.25 �L/mL) diluted in culture

edium. 60 �L of MTT solution (5 mg/mL in PBS) were added toach well. The microplates were further incubated at 37 ◦C for0 min, in a humidified atmosphere of 95% air/5% CO2. Supernatantsere then discarded and 300 �L of acidified isopropanol (0.04 N HCl

n isopropanol) were added to the cultures and mixed thoroughlyo dissolve the dark blue crystals of formazan. Formazan quantifica-ion was performed using an automatic plate reader (SLT, Austria)t 570 nm, with a reference wavelength of 620 nm.

.10. Data analysis

All the experiments were performed in duplicate. Results areresented as mean ± standard error of the mean (S.E.M.) of the 3–8xperiments and the means were statistically compared using thene-way ANOVA test, with a Dunnett’s post-test. The significanceevel was *p < 0.05 and ***p < 0.001.

. Results

.1. Essential oil analysis

The average yields of the essential oils of S. tortuosum and S.

ontanum subsp. peixotoanum were 1.3% and 0.5% (v/w), respec-

ively.The oils were analyzed by GC and GC–MS and the qualitative

nd quantitative compositions are presented in Table 1.

nd Products 39 (2012) 204– 209

Monoterpene hydrocarbons were shown to be the main group ofconstituents in all samples (79.2–85.4%). �-Pinene (24.8–24.9%), �-pinene (23.5–23.9%) and Z-�-ocimene (13.3–16.0%) are importantconstituents in the essential oils of S. tortuosum. On the other handthe main constituents of the oils of S. montanum subsp. peixotoanumare �-pinene (36.0–37.1%), �-pinene (22.5–23.6%) and limonene(7.7–8.8%). Z-�-ocimene is a minor compound in S. montanumsubsp. peixotoanum oils. Among the sesquiterpenes, �-elemene isthe major one (5.2–5.8%).

3.2. Antifungal activity

Evaluation of MIC and MLC of the oils showed a variability ofinhibition among all the fungi tested (Table 2). C. neoformans anddermatophyte strains showed more sensibility to these oils whencompared with Candida and Aspergillus ssp. S. tortuosum oil provedto be more active with MIC and MLC values ranging from 0.64 to1.25 �L/mL (Table 2). The fungicidal activity of the main compoundsof these oils, �-pinene, �-pinene and Z-�-ocimene, was also eval-uated. �-Pinene showed to be the most active compound with MICand MLC ranging from 0.08 to 2.5 �L/mL, whereas �-pinene showedthe weakest activity (Table 2).

3.3. Effect of the essential oils on keratinocytes viability

To evaluate the potential cytotoxic activity of the oil in humancells, a keratinocyte cell line was used for the cellular viabilityassays, through the MTT assay. After 24 h of cells exposure, theconcentrations of the oil 1.25 �L/mL and 0.64 �L/mL decreased MTTreduction by keratinocytes to 4.3 ± 2.3% (p < 0.001) and 60.6 ± 18.2%(p < 0.05) of the control, respectively (Fig. 1). The lower concen-trations of the oil slightly decreased the reduction of MTT bykeratinocytes; however this effect was not statistically significantwhen compared to cells incubated with culture medium alone(control). A cell-free control was performed in order to exclude non-

cells were exposed to different concentrations of the essential oil (0.32–1.25 �L/mL),for 24 h. Results are expressed as percentage of MTT reduction by control cells main-tained in culture medium alone. Each value represents the mean ± S.E.M. from threeto six experiments, performed in duplicate (*p < 0.05, ***p < 0.0001, compared tocontrol).

M.J. Gonc alves et al. / Industrial Crops and Products 39 (2012) 204– 209 207

Table 1Composition of the essential oils of Seseli montanum and Seseli tortuosum from Portugal.

RI SPB-1 RI SW 10 Compound S. montanum S. tortuosum

1 2 3 4

921 1028 Tricyclene t 0.1 t t922 1029 Thujene 0.5 0.4930 1029 �-Pinene 36.0 37.1 24.9 24.8943 1063 �-Fenchene 0.1 0.1943 1072 Camphene 3.1 2.9 2.8 2.7943 1127 Verbenene 0.1 0.2964 1123 Sabinene 0.8 0.7 t t969 1116 �-Pinene 22.5 23.6 23.9 23.5977 1161 Myrcene 6.5 7.0 4.9 5.3

1005 1152 �-Phellandrene 0.1 0.1 0.5 0.61012 1272 p-Cymene 0.1 0.2 1.6 1.61021 1201 Limonene 8.8 7.7 2.5 2.01021 1210 �-Phellandrene 0.6 0.5 t t1027 1233 Z-�-Ocimene 0.1 0.2 13.3 16.01035 1249 E-�-Ocimene 0.2 0.3 4.4 3.91047 1249 �-Terpinene t t 3.5 3.51076 1288 Terpinolene t 0.1 0.6 0.71081 1544 cis-Sabinene hydrate 0.1 0.11082 1542 Linalool 0.1 0.2 0.1 0.11117 1396 allo-Ocimene 0.2 0.31120 1639 Pinocarveol 0.2 0.2 0.1 0.11134 1556 Pinocarvone 0.1 0.21158 1595 Terpinen-4-ol 0.2 0.2 1.2 1.31164 1615 Myrtenal 0.1 0.11169 1689 �-Terpineol 0.3 0.2 0.4 0.41175 1842 Myrtenol 0.1 0.21223 1600 Carvacrol methyl ether 0.1 0.11369 2004 Methyleugenol 0.1 0.11377 1519 �-Bourbonene 0.1 t1382 1580 �-Elemene 5.8 5.2 0.1 t1408 1590 E-caryophyllene 0.2 0.3 0.6 0.71442 1662 �-Humulene 0.1 0.11467 1699 Germacrene D 0.8 0.8 2.5 2.21471 1707 �-Selinene 0.4 0.61481 1717 Bicyclogermacrene 0.9 1.0 0.5 0.41482 1701 �-Selinene 0.7 0.61490 1746 Germacrene A 1.6 1.81497 1746 �-Cadinene t t t 0.11506 1746 �-Cadinene 0.3 0.1 0.1 0.11540 1818 Germacrene B 0.3 0.41557 1964 Caryophyllene oxide 0.5 0.21577 2004 Carotol 0.2 0.21663 1709 �-Bisabolol 0.6 0.5

Total identified 91.5 92.8 91.0 92.5

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. Discussion

In this paper it was demonstrated that S. tortuosum essential oilas significant antifungal properties. However, we also detected aytotoxic effect on human keratinocytes, especially when used inoncentrations above 0.64 �L/mL, suggesting that attention muste paid in the application of the oil for health promoter activities,pecifically in the selection of concentrations with fungicidal activ-ty and without cytotoxicity. Indeed, very few papers addressed theoxicological safety of essential oils in human keratinocytes, besidests usual application in the cosmetic industry.

The oils of S. tortuosum and S. montanum were characterizedy high amounts of �-pinene and �-pinene (>45%). Neverthe-

ess, S. tortuosum oil has high amounts of Z-�-ocimene, whereas. montanum subsp. peixotoanum possesses important amounts ofimonene and �-elemene. More samples of both species should benalyzed to conclude that it is possible to distinguish these two taxa

ased on the composition of their essential oils.

Similarly, high amounts of pinenes were also observed in theils of S. tortuosum from Turkey (>42%) (Kaya et al., 2003), Iran>35%) (Zohreh et al., 2003) and Italy (>31%) (Bader et al., 2003).

tion indices relative to C9–C23 n-alkanes on the Supelco SPB-1 column; RI SW 10: (≤0.05%).

On the other hand, the oils of S. tortuosum from Portugal are quitedifferent to the Italian oil, which is characterized by high amountsof myrcene (29.2%) (Bader et al., 2003), and to the oil from Turkeythat has (E)-sesquilavandulol (37.0%) as major compound (Doganet al., 2006). This last compound was not detected in the Portugueseoils. High amounts of pinenes (>52%) were also observed in Seselicampestre from Anatolia (Kaya et al., 2010) and Seseli rigidum fromSerbia (Savikin-Fodulovic et al., 2006; Stojkovic et al., 2009).

As far as we know there is no information about the essential oilof S. montanum subsp. peixotoanum. There is only one work report-ing the oil composition of S. montanum subsp. tommasinii, whichdemonstrates important amounts of �-pinene (30.2%), germacreneD (10.1%), sabinene (8.0%), �-pinene (7.2%) and limonene (6.6%)(Siljegovic et al., 2011). According to our results, the amounts ofgermacrene D and sabinene can help to distinguish this subspeciesfrom the subsp. peixotoanum.

Essential oils of some species of the genus Seseli are known to

possess antimicrobial activity against a wide spectrum of microor-ganisms (Adams et al., 2009; Milosavljevic et al., 2007; Ozturk andErcisli, 2006; Siljegovic et al., 2011; Stojkovic et al., 2009; Tosunet al., 2004). Our data demonstrated that S. tortuosum oil has higher

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.

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ider

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n

flocc

osum

FF9

0.64

0.64

0.64

–1.2

5

0.64

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5

0.16

0.16

5–10

5–10

0.64

0.64

16

16

N.T

.

N.T

.

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ergi

llus

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ATC

C

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>20

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10

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5

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5

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2

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s

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.

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Res

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from

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nd

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MIC

and

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det

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by

a

mac

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tion

met

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and

exp

ress

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in

�L/

mL

(v/v

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MIC

and

MLC

wer

e

det

erm

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a

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Not

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ed.

nd Products 39 (2012) 204– 209

activity than S. montanum oil. This essential oil showed effectiveantifungal activity against C. neoformans and dermatophyte strains,with MIC and MLC values ranging from 0.64 to 1.25 �L/mL. In mostcases the MIC was equivalent to the MLC, indicating a fungicidaleffect of the oil. The activity of the oil is mainly due to the contribu-tion of �-pinene, which accounts for the activity of other essentialoils, such as those of Juniperus sp. (Cavaleiro et al., 2006). Z-�-ocimene may also contribute for the S. tortuosum activity against C.neoformans and dermatophyte strains. These results highlight thepotential utilization of S. tortuosum oil in dermatomycoses, whichare common infections caused by filamentous fungi and by someyeasts that can be severe in immunocompromised patients. How-ever, S. tortuosum essential oil exhibited cytotoxicity against humankeratinocytes when used in concentrations above 0.64 �L/mL. Morestudies are imperative in order to explore its potential applicationfor cosmetic and pharmaceutical purposes. In fact, the cytotoxicactivity of S. tortuosum essential oil has not been studied so farand from data available in the literature we can hypothesize that�-pinene, �-pinene and ocimene may account for the cytotoxicactivity of the essential oil. Indeed, �-pinene was demonstratedto exhibit in vitro cytotoxicity against mouse lymphoblast cells andseveral cell lines (Beattie et al., 2011; Díaz et al., 2008), although nocytotoxic effects were reported in the human lung epithelial A549cells and in vero and RC-37 cells (Gminski et al., 2010; Loizzo et al.,2008; Schnitzler et al., 2008). Concerning ocimene, very few studieshave addressed its cytotoxic properties. For instance, the leaf essen-tial oil of Liriodendron tulipifera, dominated largely by Z-�-ocimene,in the later part of the growing season, exhibited in vitro antibacte-rial activity against Bacillus cereus and Staphylococcus aureus, as wellas cytotoxic activity on MDA-MB-231 and Hs 578T human breasttumor cells (Miller et al., 2009).

5. Conclusions

These results demonstrated that S. tortuosum essential oils havehigher biological activity than those of S. montanum, being a poten-tial source of bioactive compounds with beneficial anti-fungalproperties. Its essential yield is also compatible with an indus-trial use. More scientific studies are needed to assure the absenceof cytotoxicity at concentrations with strong biological activity, inorder to explore its potential utilization by the pharmaceutical andcosmetic industries.

Conflict of interest

There is no conflict of interest for any of the contributing authors.

Acknowledgments

Our thanks to the following institutions for supporting thiswork: Center of Pharmaceutical Studies of the University of Coim-bra (POCI2010FEDER) and EDP/Electricity Portuguese Foundation.To the Institute of Interdisciplinary Research of the University ofCoimbra our thanks for the Ph.D. grant – III/05/2007 – of AnaCristina Tavares.

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