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1 Department of Microbiology/CEQOFF, Faculty of Pharmacy, University of Porto, 4050-Porto, Portugal
2 Department of Microbiology, Faculty of Medicine, University of Porto, 4200-Porto, Portugal
3 IPATIMUP – Institute of Pathology and Molecular Immunology, University of Porto, 4200-Porto, Portugal
4 Department of Pharmacognosy, Faculty of Pharmacy/CEF, University of Coimbra, 3000-Coimbra, Portugal
5 Department of Obstetrics/Gynaecology, Faculty of Medicine, University of Beira-Interior, 6200-Covilhã, Portugal
Correspondence
Eugénia Pinto
epinto{at}ff.up.pt
Received 30 November 2005
Accepted 15 June 2006
Abbreviations: MLC, minimal lethal concentration; PI, propidium iodide.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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In spite of the introduction of new antifungal drugs, they are limited in number. The increase of fungal resistance to classical drugs, the treatment costs, and the fact that most available antifungal drugs have only fungistatic activity, justify the search for new strategies (Rapp, 2004).
Aromatic plants have been widely used in folk medicine. It is known that most of their properties are due to their volatile oils. Essential oils from many plants are known to possess antifungal activity (Kalemba & Kunicka, 2003), but only limited information exists about activity toward human fungal pathogens. They have been empirically used as antimicrobial agents, but the mechanisms of action are still unknown.
According to our preliminary results (Pina-Vaz et al., 2004; Salgueiro et al., 2003, 2004), some essential oils show an important antifungal activity against yeasts, dermatophyte fungi and Aspergillus strains, which could predict therapeutic benefits, mainly for diseases with mucosal, cutaneous and respiratory tract involvement.
Several studies have shown that thyme oils, particularly those of Thymus vulgaris and Thymus zygis (Bruneton, 1999; Pina-Vaz et al., 2004; Stahl-Biskup & Sáez, 2002), possess antimicrobial activity, those of the phenol type being the most active. The limited occurrence of these phenols in nature is one of the reasons why Thymus oils containing thymol and carvacrol have been of great interest for some time.
Thymus pulegioides is widely distributed on the European continent south of the Mediterranean isles. In Portugal, it grows in the northeast, and it is locally used as an antiseptic. Previous results have demonstrated that this species is polymorphic (Salgueiro, 1994; Stahl-Biskup & Sáez, 2002), and that the thymol/carvacrol chemotype is one of the most abundant in Portugal.
The objective of our present research was to evaluate the antifungal activity and investigate the mechanism of action of this specific chemotype and of its main components.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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C. parapsilosis ATCC 90018 and C. krusei ATCC 6258 were used as controls. The fungal isolates were identified by standard microbiology methods and stored in Sabouraud dextrose broth with glycerol at –70 °C.
Plant material and chemicals. Aerial parts of the plants were collected at the flowering stage from Moimenta, Trás-os-Montes (north of Portugal). A voucher specimen was deposited at the Herbarium of the Instituto Botânico of the University of Coimbra (COI).
Thymol (99.5 %) was purchased from DBH, and carvacrol, 
-terpinene and p-cymene (all 99.5 %) from Fluka.
Essential oil analysis. Essential oil was isolated by water distillation for 3 h from air-dried material, using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia (Council of Europe, 1997).
Analysis of volatile oil was carried out by GC and GC-MS. Analytical GC was carried out in a Hewlett Packard 6890 gas chromatograph (Agilent Technologies) with a Hewlett Packard GC ChemStation Rev. A.05.04 data-handling system, equipped with a single injector and two flame-ionization detectors (FIDs). A graphpak divider (Agilent Technologies, part no. 5021-7148) was used for simultaneous sampling to two Supelco fused silica capillary columns with different stationary phases: SPB-1 (polydimethylsiloxane, 30 mx0.20 mm i.d., film thickness 0.20 µm) and SupelcoWax 10 (polyethyleneglycol, 30 mx0.20 mm i.d., film thickness 0.20 µm). The oven temperature programme was 70–220 °C (3 °C min–1), 220 °C (15 min); the injector temperature was 250 °C; the carrier gas helium, adjusted to a linear velocity of 30 cm s–1; the splitting ratio 1 : 40; and the detector temperature 250 °C.
GC-MS analyses were carried out in a Hewlett Packard 6890 gas chromatograph fitted with an HP1 fused silica column (polydimethylsiloxane, 30 mx0.25 mm i.d., film thickness 0.25 µm), interfaced with a Hewlett Packard mass selective detector 5973 (Agilent Technologies) operated by Hewlett Packard Enhanced ChemStation software, version A.03.00. GC parameters were as above, and other parameters were as follows: interface temperature, 250 °C; MS source temperature, 230 °C; MS quadrupole temperature, 150 °C; ionization energy, 70 eV; ionization current, 60 µA; scan range, 35–350 u; scans per second, 4.51.
The identity of the components was ascertained based on their retention indices, calculated by linear interpolation relative to retention times of a series of n-alkanes, and their mass spectra, which were compared with those from our own library and literature data (Adams, 1995; Joulain & Konig, 1998). Relative amounts of individual components were calculated based on GC peak areas without FID response factor correction.
Antifungal activity. MICs, determined by the macrodilution broth method, and minimal lethal concentrations (MLCs) were performed according to reference documents M27-A and M38-A (National Committee for Clinical Laboratory Standards, 1997, 2002) for yeasts and filamentous fungi, respectively.
Serial twofold dilutions in DMSO, ranging from 0.02 to 20 µl ml–1, were tested for essential oil and its main components (thymol, carvacrol, p-cymene and -terpinene). In addition, the reference antifungal compounds, fluconazole (Pfizer) for yeasts and dermatophytes, or amphotericin B (Sigma) for Aspergillus, were used as standard antifungal drugs. Twofold serial dilutions ranging from 0.25 to 128 µg ml–1 for fluconazole and 0.016 to 16 µg ml–1 for amphotericin B were used.
Quality control determinations of the MICs of fluconazole and amphotericin B were performed by testing C. parapsilosis ATCC 90018 and C. krusei ATCC 6258. The results obtained were within the recommended limits.
These experiments performed in duplicate were repeated independently three times and yielded essentially the same results. A range of values is presented where different results were obtained. Two growth controls, RPMI medium and RPMI with 2.0 % (v/v) DMSO, were included for each strain.
Mechanism of activity
Lesion of cytoplasmic membrane.
Flow cytometry analysis using propidium iodide (PI; Sigma) was performed. PI is a fluorescent probe used to study the effect of drugs on membranes. It only penetrates cells with severe membrane lesions, showing increased red fluorescence (Pina-Vaz et al., 2001). Cells (106 ml–1) were incubated with serial concentrations of the oil and its main components (0.32–1.25 µl ml–1) for 1 h (Candida) or 7 h (Aspergillus), and then stained with 1 µg PI ml–1 for 30 min. Cells were also incubated with the same compounds, at MLC values, for 5, 10, 15 and 30 min at 30 °C for C. albicans, representing yeasts, and 3, 5, 7 and 16 h for A. fumigatus and A. niger, representing moulds. Scattergram analysis was performed to evaluate morphological changes (size and complexity). The percentage of stained cells at FL3 (620 nm, red), representing dead cells with severe lesions of the membrane, was quantified.
Study of ergosterol amount. For determination of the amount of ergosterol, the strains were incubated in RPMI medium (Sigma) supplemented with 2 % glucose (Difco) for 48 h at 35 °C (yeasts), 3 days at 25 °C (Aspergillus) or 5 days at 25 °C (dermatophytes), while shaking. A quantification of ergosterol amount was performed after incubation with and without the essential oil, its main components, or fluconazole as control, at both MIC and subinhibitory concentrations. Ergosterol was isolated from fungal cells by saponification, and the non-saponifiable lipids were extracted with heptane. Ergosterol was identified by its spectrophotometric absorbance profile (230–300 nm) (Arthington-Skaggs et al., 1999).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Fifty-seven components representing 98.5 % of the volatile oil were identified. The oil was characterized by high amounts of thymol (26.0 %), and of carvacrol (21.0 %) and its biogenetic precursors, -terpinene (8.8 %) and p-cymene (7.8 %) (thymol/carvacrol chemotype).
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). T. pulegioides essential oil exhibited significant antifungal activity. MIC values ranged from 0.16 to 0.32 µl ml–1 against dermatophyte and Aspergillus strains. Candida showed the highest MIC values, ranging from 0.32 to 0.64 µl ml–1. For Candida and most dermatophyte strains, MIC and MLC values were similar, ranging from 0.16 to 0.64 µl ml–1 (Table 2). It is difficult to attribute the activity of a complex mixture to particular constituents. Nevertheless, it is reasonable to speculate that the activity of this oil can be related to the presence of carvacrol and thymol. These compounds were found to be the most active constituents (Table 2) of T. pulegioides oil, with MIC values ranging from 0.04 to 0.32 µl ml–1 and 0.08 to 0.32 µl ml–1, respectively. The importance of the phenolic hydroxyl groups for the antimicrobial activity of the monoterpenoids has previously been reported (Adam et al., 1998; Aligiannis et al., 2001; Dorman & Deans, 2000; Nostro et al., 2004; Sivropoulou et al., 1996). Other species of the genus Thymus, such us T. zygis and T. vulgaris, with high amounts of phenols, also show a broad spectrum of activity against a variety of pathogenic yeasts and filamentous fungi, including fungi with decreased susceptibility to fluconazole (Dorman & Deans, 2000; Nguefack et al., 2004; Pina-Vaz et al., 2004). Nevertheless, carvacrol proved to be more active against dermatophyte strains, in a similar manner to the essential oil. MIC and MLC values were very similar and the fungistatic and fungicidal properties of the oil were suspected to be associated with high carvacrol and thymol content.
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). Previous work carried out with essential oils has revealed anti-Candida activity (Pina-Vaz et al., 2004; Salgueiro et al., 2003, 2004). Incubating Aspergillus with essential oil of T. pulegioides, PI began to stain the cells after 7 h incubation. At MLC values, 
40 % of A. fumigatus cells and 20 % of A. niger cells were stained, the effect being dose dependent (Fig. 2). Thymol and carvacrol gave identical cytometric results to T. pulegioides essential oil against Candida and Aspergillus. To understand the mechanism of action on moulds, Aspergillus species were studied as typical. Aspergillus begins its germination at 7 h incubation, so it is understandable that it was only after this period that the essential oil was effective and PI could enter the cells. This effect is superior to the effect of most antifungals, as most of them are fungistatic.
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The large spectrum of activity of this essential oil acting on Candida, Aspergillus and dermatophytes agrees with the mechanism of action proposed: cytoplasmic membrane lesion.
In conclusion, the findings of the present study indicate that T. pulegioides essential oil has potential as a topical antifungal agent against fungi that are pathogenic to humans. This essential oil is a broad-spectrum agent that inhibites not only dermatophytes, Aspergillus and Candida species (such as C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii), but also fluconazole-resistant C. albicans isolates, and C. krusei and C. glabrata, which are intrinsically resistant to fluconazole or whose resistance is easily inducible.
Given the results described above, particularly the possible mechanisms of action, which might induce side-effects in humans, these antifungals require further investigation.
The results presented should stimulate studies on toxicity, improved formulations and the determination of optimal concentrations for clinical applications, as well as comparative studies alongside currently used drugs of the therapeutic efficacy of essential oils to control mucocutaneous infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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