Essential oils against Candida spp: in vitro antifungal activity of Origanum vulgare

1 Department of Pharmacology, Faculty of Veterinary, UFRGS-Federal University of Rio Grande do Sul Porto Alegre, RS, Brazil. 2 Department of Preventive Veterinary, Faculty of Veterinary, Federal University of Pelotas Campus Universitário, s/n° Caixa Postal 354, CEP 96010-900, Pelotas, RS, Brazil. 3 Department of Organic Chemistry, Chemistry Institute, Federal University of Pelotas, Campus Universitário, s/n° Caixa Postal 354, CEP 96010-900, Pelotas, RS, Brazil. 4 Universidad Autónoma de Nuevo León. UANL. Facultad de Medicina Veterinaria y Zootecnia, Av. Universidad S/N, Ciudad Universitaria. San Nicolás de los Garza, Nuevo León. C.P. 66451 México.


INTRODUCTION
Knowledge about medicinal plants was for a long time the only therapeutic resource of many communities and ethnic groups.The use of plants as treatment to cure illnesses has been since early times (Simões et al., 2003).Still today in poorer areas of the country and even in big Brazilian cities, medicinal plants are comercialized in popular markets and found in backyards of residences (Simões et al., 2003;Sartoratto et al., 2004).Regarding antimicrobial activity, plants of Lamiaceae family have been of interest due to their potential (Arango et al., 2004;Giordani et al., 2004;Rehder et al., 2004).Many species of this family, introduced in Brazil, are medicinal plants; they produce essential oils and are also used as seasoning or as ornamental flowers.Among them, Origanum vulgare stands out (Arcila-Lozano et al., 2004;Rehder et al., 2004;Rodrigues et al., 2004;Cleff et al., 2008a).Most important representatives of essential oils of this plant are terpenes, and among them monoterpenes and sesquiterpenes (Lambert et al., 2001;Arcila-Lozano et al., 2004;Rehder et al., 2004;Rodrigues et al., 2004;Cleff et al., 2008b).Studies with purpose of determining activity of O. vulgare essential oils have shown and demonstrated bacterial and fungal activity against different pathogens and this is attributed to the content of carvacrol and thymol that are major compounds in some oils (Lambert et al., 2001;Arango et al., 2004;Arcila-Lozano et al., 2004;Chami et al., 2004;Nostro et al., 2004;Rehder et al., 2004;Busatta et al., 2007Busatta et al., , 2008;;Cleff et al., 2008b).
Nowadays, due to higher frequency of patients susceptible to fungal infections, a larger number of reports of isolates resistant to antifungal have been observed as well as a higher rate of therapeutic failure, mainly in candidiasis (Chami et al., 2004;Santos Jr. et al., 2005;Magill et al., 2006).This mycosis has frequently been associated with hospital infections in immunesuppressed subjects, and in veterinary practice their importance is growing due to several reports of illness in animals (Mueller et al., 2002;Moretti et al., 2004;Magill et al., 2006;Cleff et al., 2007Cleff et al., , 2008a)).
The aim of this study was to evaluate in vitro antifungal activity and determine chemical composition of eight samples of O. vulgare essential oils, mainly in relation to thymol/carvacrol rate.

Chromatographic analyses
Essential oils were analyzed by gas chromatography with flame ionization detector (GC/FID -Schimadzu 17A) to identify main compounds.Chromatographic analyses were carried out in a chromatograph equipped with a DB-5 silica capillary column (methyl siloxane with 5% phenyl groups -30 m x 0.25 mm , film thickness 0.25 m).Nitrogen was used as a carrier gas with a flow rate of 1.0 mL min -1 and split ratio of 1:50.Injector and detector temperatures were both set at 280°C.Column temperature was programmed to 40°C, gradually increased to 145°C at 2°C min -1 and then increased to 280°C at 10°C min -1 , which was held for 10 min.Solution of each essential oil (5.000 µg mL -1 ) and chromatographic standards (40 µg mL -1 ) were prepared and 1 µL was injected at the same conditions.The compounds in the oregano samples were identified by comparison with retention time of standards and literature data (Rodrigues et al., 2004).

Candida spp. isolates
Twelve (12) isolates were used for in vitro tests: nine C. albicans (five of mucous membrane and cutaneous tegument of dogs and four standard strains -ATCC 44858, 4053, 18804 and IOC 3691), and three were non-albicans species: C. parapsilosis (ATCC 22019), C. lusitanie (ATCC 34449) and C. krusei (ATCC 34135).Regarding Candida spp.isolates, four isolates of C. albicans were from vaginal mucous membrane of canine and one isolate of cutaneous candidiasis was from a dog, from Pelotas, Rio Grande do Sul, Brazil.Isolated yeasts were maintained in subcultures in Sabouraud dextrose agar (SDA) and stored at 5°C, in Mycology Laboratory, Veterinary College, UFPel; Standard strains were kindly supplied by Osvaldo Cruz Foundation (Fiocruz, INCQS), Rio de Janeiro, Brazil.

Inoculums preparation
Yeast inoculum was prepared by growing Candida isolates on SDA for 24 h at 35°C and then suspended in 5 mL of sterile physiological saline solution and homogenized.The inoculum suspension had its turbidity adjusted according to 0.5 McFarland standards which corresponds to an initial inoculum with approximately 5 x 10 6 CFU mL -1 .Then, 1:50 dilution in sterile physiological saline solution was prepared, and another 1:20 dilution in RPMI Media 1640 was prepared in 96-well microtiter plate (Becton Dickinson Labware, Franklin Lakes, NJ, USA).Adjusted inoculum suspension (100 μL) was dispensed in each well, resulting in desired final drug concentration and inoculum size between 1 and 5 x 10 3 CFU mL -1 .

Antifungal activity
Microdilution in broth technique was used to evaluate susceptibility of Candida spp.against oils, according to National Committee for Clinical Laboratory Standards, NCCLS, M 27-A2 (CLSI) with few modifications.A 1% solution of Tween 80 as dispersant of essential oil in RPMI Media was used, avoiding micellas formation and favoring dilution.Ten (10) serial dilutions from stock solutions of essential oil of Origanum were prepared in RPMI medium with L-glutamine without bicarbonate and buffered with MOPS at pH 7.0.For yeasts, minimum inhibitory concentrations (MICs) were determined by subculturing 100 µL of those concentrations from each well (1 to 10) of microtiter plate.Positive control (inoculum/ media) and negative control (essential oil/media) were placed into wells 11 and 12. Plates were incubated aerobically at 35°C for 48 h with shaking.Susceptibility was expressed as MIC, defined as lowest concentration required to delay growth of fungi by 24 h of incubation in relation to the positive control.Also, minimum concentration were determined in μg/ml, capable to inhibit 50 and 90% from isolated, calculated as MIC 50 and MIC 90.  1, other important compounds such as 4-terpineol, -terpinene, p-cymene, -terpinene and terpineol can be observed.Figure 1 presents a graph with concentration variations (%) of p-cymene, 4-terpineol, thymol and carvacrol, in different essential oil samples of O. vulgare (origanum), where it was possible to observe great variation in tymol/carvacrol rate.

Antifungal activity
After incubation period, the microdilution plates were observed, by the technique of microdilution in broth, to detect the sensitivity of isolates of Candida spp. to O. vulgare essential oils.Table 2 shows the results of the MIC of eight O. vulgare essential oils using this technique.In this table, the MIC did not demonstrate significant variation among different isolates; what is observed when pure antifungal substances are used.MIC 50 and MIC 90 are indicative of a less concentration capable of growth inhibition of 50 and 90% of the isolates.This data is shown in Table 3, with an average of MIC 50 of 0.62 µL/mL and the average of MIC 90 of 5.52 µL/mL.
Results demonstrate susceptibility differences among field isolates, standards and different species of Candida.It was observed that the most active essential oils were one and eight (EO1 and EO8) which presented lower MIC 90 values than other oils (EO2, EO3, EO4, EO5, EO6 and EO7).
These data are in accordance to what has been described in literature, where O. vulgare essential oil presents a great variation in composition, mainly in active compounds, where monoterpene phenols such as thymol and carvacrol reached 80.2 to 98% of total composition of this oil (Simões et al., 2003;Arcila-Lozano et al., 2004;Rehder et al., 2004;Rodrigues et al., 2004;Cleff et al., 2008).Geographic factors such as altitude, soil, climate, harvest, culture, drying process and storage conditions among others, have influence on composition, quality and amount of each compound present in essential oil from plants (Cox et al., 2000;Simões et al., 2003;Arango et al., 2004;Arcila-Lozano et al., 2004).
Chromatographic analyses of oregano essential oil samples (EO1 and EO8) showed better result in vitro (MIC 90= 3,54 and 3,87, respectively); presented mostly 4terpineol, thymol and carvacrol components who deserves attention to concentration balance between thymol (10.20 and 8.42) and carvacrol (12.67 and 9.44) associated to a large concentration of 4-terpineol (21.32 and 47.95), respectively.It was reported that the composition 4-terpineol acts be inducing deformations in the cell membrane, modifying consequently its permeability (Cox et al., 2000).Another factor to be considered is that the use of commercial oregano could have leaf mixtures, flowers and stems of O. vulgare in the sample, as well as the presence of different oregano genus in the same sample.Although all organs of a plant can accumulate volatile oils, its composition can change according to part or species studied, since just Origanum genus presents 39 different species (Simões et al., 2003).
Compounds without aromatic ring and/or hydroxyl groups, such as -terpinene, -terpinene and p-cymene, monoterpenes hydrocarbons, were found in significant amount in those samples.That could justify the MIC values achieved by these essential oils (Nostro et al., 2004).Presence of the p-cymene, has an antagonistic effect with carvacrol and thymol, which would explain lower antimicrobial activity of essential oils EO2 (20.38);EO4 (13.96) and EO6 (7.01).
High concentrations of these compounds and their proportion are of great importance for effectiveness of the product.However studies have demonstrated that these isolated compounds do not have same efficiency of essential oil.Bio-synthetic precursors -terpinene and pcymene, as well as -terpineol, 1.8-cineol and linalool, also showed antimicrobial activity, justifying higher efficiency  (Rodrigues et al., 2004;Busatta et al., 2008).
of essential oil when compared to isolated compounds (Simões et al., 2003).Action mechanism of essential oils is complex and it is not totally elucidated.Some studies suggested an alteration in activity of calcium canals, causing increase in permeability and liberation of vital intracellular constituents, promoting a decrease in intracellular ATP in cells.Simultaneously, it causes an increase in extracellular ATP, leading to rup-ture in cellular membrane of microorganism (Sikkema et al., 1994(Sikkema et al., , 1995;;Sartoratto et al., 2004).Antimicrobial activity can be due to damage caused to several enzymes, including those involved in production of energy and synthesis of structural components of microorganism (Lambert et al., 2001).
In the present work, samples analyzed, besides phenol compounds, 4-terpineol was sometimes in larger amount than thymol or carvacrol (phenolics compounds) and terpineol; both terpenes alcohol, monoterpenes with hydroxyl group (OH), were able to make polar hydrogen bonds, which justify the good result of MIC 90 .For  Several authors believe that aromatic ring with a polar functional group, is responsible for antimicrobial activity (Milos et al., 2000;Lambert et al., 2001).It is suggested that the presence of this group and hydroxyl group (-OH), would form hydrogen bond with the active microbial enzyme small sites.Therefore, it is possible to suppose that these groups (aromatic ring and hydroxyl group) are responsible for antimicrobial activity (Milos et al., 2000;Lambert et al., 2001;Ultee et al., 2002).

Conclusion
Results demonstrate that differences between concentrations of thymol and carvacrol influenced the MIC 90 of O. vulgare oils against yeasts studied.Balance of concentration of phenolic compounds, thymol and carvacrol, seems to have synergic and positive effect on growth inhibition of different species of Candida, however the eight oils analyzed presented antifungal activity in vitro.
Table1shows the results obtained from chromatographic analysis of eight O. vulgare essential oils where the difference between phenol constituent can be observed.

Table 1 .
Concentration (%) of compound in eight samples of O. vulgare essential oils analyzed by gas chromatography (GC/FID).

Table 3 .
MIC 50 and MIC 90 of eight oil extracts from O. vulgare against Candida spp isolates strains from regions of South Brazil and controls.