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Chemical Composition of Essential Oils of Thymus and Mentha Species and Their Antifungal Activities

The potential antifungal effects of Thymus vulgaris L., Thymus tosevii L., Mentha spicata L., and Mentha piperita L. (Labiatae) essential oils and their components against 17 micromycetal food poisoning, plant, animal and human pathogens are presented. The essential oils were obtained by hydrodestillation of dried plant material. Their composition was determined by GC-MS. Identification of individual constituents was made by comparison with analytical standards, and by computer matching mass spectral data with those of the Wiley/NBS Library of Mass Spectra. MIC’s and MFC’s of the oils and their components were determined by dilution assays. Thymol (48.9%) and p-cymene (19.0%) were the main components of T. vulgaris, while carvacrol (12.8%), α-terpinyl acetate (12.3%), cis-myrtanol (11.2%) and thymol (10.4%) were dominant in T. tosevii. Both Thymus species showed very strong antifungal activities. In M. piperita oil menthol (37.4%), menthyl acetate (17.4%) and menthone (12.7%) were the main components, whereas those of M. spicata oil were carvone (69.5%) and menthone (21.9%). Mentha sp. showed strong antifungal activities, however lower than Thymus sp. The commercial fungicide, bifonazole, used as a control, had much lower antifungal activity than the oils and components investigated. It is concluded that essential oils of Thymus and Mentha species possess great antifungal potential and could be used as natural preservatives and fungicides.


Introduction
Moulds have various health effects. Excessive mould growth in the human environment needs to be taken care of, regardless of the species, as it may lead to increased allergies, toxicity, and house/building structural problems. Some Aspergillus species are able to produce mycotoxins such as aflatoxins [1] that are potent hepatocarcinogens in animals and humans [2]. Therefore, the presence of toxigenic fungi in foods and grains presents a potential hazard to human and animal health. Superficial fungal infections, dermatomycoses, are probably the most common communicable fungal disease affecting humans. They have become a serious problem in immunocompromised patients [3]. Fungal infections remain a therapeutic problem despite the availability of a number of treatments. Being largely synthetic and non-biodegradable, these agents can cause adverse effects and may have residual toxicity. As the number of reported cases of food-associated infections continues to increase, food safety is a fundamental concern of both consumers and the food industry. As aromatic plants, herbs and spices have been used for ages both as flavouring agents and as preservatives of food, they may be effective sources of biodegradable fungitoxicants without harmful side effects. In the present study, a search was made to find essential oils that could safely be used as natural alternatives for chemical fungicides/fungistatics.
Various Mentha and Thymus species have been credited with a long list of pharmacological properties. They have been used for their flavours in cooking, in folk medicine as antiseptic and as antimicrobial agents [4]. We chose these species for investigation of the antifungal activities of their oil. The essential oil of T. vulgaris is a known antiseptic, antiviral, antimicrobial agent [4][5][6]. The essential oil of T. tosevii contains a high amount of thymol and carvacrol and is also used in ethnobotany [7]. Its pharmacological activities are unknown. The present study reports the antifungal activities of the essential oils of two Thymus and two Mentha species and their main components against soil-borne pathogens, food storage fungi, mycotoxicogenic species, phytopathogens and opportunistic human pathogens.

Results and Discussion
The composition of Thymus sp. essential oils and their antifungal effects We have identified 27 components, accounting for total 97.2% of the oil, in the essential oil of T. vulgaris. The main components were thymol (48.9%) and p-cymene (19.0%) ( Table 1). The essential oil of T. vulgaris, tested by macrodilution method, showed very strong antifungal activity. A concentration of 0.25 μL/mL of oil inhibited Alternaria alternata, Fusarium tricinctum, all Aspergillus species and dermatomycetes. Phomopsis helianthi and Cladosporium cladosporioides were inhibited at lower concentrations (0.125 μL/mL). The essential oil dissolved in Tween ® showed better antifungal potential, with MIC 0.05 μL/mL. Penicillium species and Trichoderma viride were the most resistant fungi towards this oil; MIC in ethanol dissolved oil was 0.5 μL/mL, and 0.25 μL/mL when the oil was dissolved in Tween ® . MICs of T. vulgaris oil in ethanol were 0.125-0.5 μL/mL, and 0.05-0.25 μL/mL in Tween ® . Thymol showed the same antifungal potential as the oil, while carvacrol exhibited a slightly better effect, with MICs of 0.05-0.25 μL/mL in ethanol and 0.02-0.125 μL/mL in Tween ® , (Figure 1). In agreement with earlier publications [8][9][10][11][12][13] the essential oil of T. vulgaris showed very strong antifungal activity at low concentrations, 0.05-1.0 μL/mL. Strong antifungal activity of thymol and carvacrol were also reported in the literature [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Previous investigations of Thyme oil by our group [19] showed very high activity against the three major pathogens of the button mushroom, Agaricus bisporus, i.e. the fungi Verticillium fungicola and Trichoderma harzianum and the bacterium Pseudomonas tolaasii. This oil also showed very strong antibacterial activity against food spoilage bacteria [20]. From the essential oil of T. tosevii we identified 42 components, 97.3% of the total amount. Carvacrol (12.8%), α-terpinyl acetate (12.3%), cis-myrtanol (11.2%) and thymol (10.4%) were the dominant components (  The essential oil of T. tosevii possessed slightly lower antifungal potential than T. vulgaris oil. MICs and MFCs of this oil dissolved in ethanol were 0.25-1.0 μL/mL, and 0.125-0.5 μL/mL in Tween. Phomopsis helianthi was the most susceptible fungus, while Trichoderma viride was the most resistant species for this oil ( Figure 1). There is no published data about the antifungal activity of T. tosevii oil.
MICs of these oils were the same in macro-and microdilution methods, and the same as MFCs obtained by microdilution method, which means that concentrations that stopped fungal growth also killed the fungi. The commercial fungicide bifonazole, which was used as a control, possessed much higher MICs (8.0-15.0 μL/mL) than the essential oils and components investigated ( Figure 1). The lower antifungal effect of T. tosevii oil in comparison with T. vulgaris oil can be explained by its lower amount of thymol and its precursors (p-cymene and γ-terpinene) and by the higher percentage of acetates (α-terpinyl acetate and geranyl acetate) which may lead to lower antifungal potential [18]. Comparing the previous data with the chemical composition of the oils, it becomes evident that there is a relationship between the high activity of the Thymus type oils and the presence of phenol components, such as thymol and carvacrol. The high antifungal activity of these essential oils could be explained by the high percentage of phenol components. It seems possible that phenol components may interfere with cell wall enzymes like chitin synthase/chitinase as well as with the αand βglucanases of the fungus [21]. Consequently, the high content of phenol components may account for the high antifungal activity of oils [16]. From our results it can be seen that essential oils of Thymus species and carvacrol and thymol have very high antifungal activities, even higher than the commercial fungicide bifonazole.

The composition of Mentha sp. essential oils and their antifungal effects
In M. piperita essential oil 26 components were detected and identified (97.7%). Menthol (37.4%), menthyl acetate (17.4%) and menthone (12.7%) were the main components in this oil (Table 1). In essential oil of M. spicata 27 components were identified (92.4%). The main components were carvone (49.5%) and menthone (21.9%) ( Table 1). The results of antifungal activities of both essential oils and components are presented in Figure 2. The essential oil of M. spicata showed the same fungistatic activity in macro-and microdilution method with MICs of 1.0-2.5 μL/mL in ethanol and 0.5-1.5 μL/mL in Tween ® . MICs of M. piperita essential oil were higher, 1.5-3.0 μL/mL in ethanol and 1.0-2.5 μL/mL in Tween ® . Figure 2   Carvone showed higher antifungal activities than previous components. MICs and MFCs of this component were 0.25-1.0 μL/mL in ethanol and 0.05-0.5 μL/mL in Tween ® . There is no effect of ethanol and Tween against tested micromycetes. The commercial fungicide (bifonazole), which was used as a control showed lower antifungal potential than the essential oils investigated (Figure 2).
The differences between antifungal activities of these two essential oils could be due to different chemical composition of essential oils. The greater antifungal potential of M. spicata essential oil could be explained by the presence of carvone, which possesses very strong antifungal activity [14,16]. The essential oil of M. piperita possesses menthol and 1,8-cineole as main components, which also exhibited very good antifungal properties but lower than carvone [18]. Carvone has better antifungal properties because of its high water solubility. One of the reasons for lower antifungal activity of M. piperita essential oil could be the large amount of menthyl acetate, which causes a decrease of antifungal properties [18].
Earlier investigations of the antimicrobial activity of Mentha spicata and M. piperita oils showed strong activity against the three major pathogens of the button mushroom, Agaricus bisporus, i.e. the fungi Verticillium fungicola and Trichoderma harzianum and the bacterium Pseudomonas tolaasii [19]. Both Mentha oils tested inhere showed strong antibacterial activity against a variety of bacteria [20]. Our results are in accordance with previous investigations of antifungal activities of Mentha essential oils. Previous results showed a fungicidal effect of M. spicata essential oil against human pathogens in concentration of 0.25 -2.0 μL/mL, while this oil possessed lower antifungal potential against phytopathogenic species [9,16,22]. The results obtained by both methods suggested that carvone and menthol possessed greater antifungal activities than other compounds investigated, while limonene showed the lowest antifungal activity. Previous results of investigation of antifungal properties had shown that hydrocarbon monoterpenes had the lowest antifungal activity, larger antifungal potential could be due to the presence of oxygenated terpenes or of those with phenolic structures [9,14,16,29]. The hydrocarbons tend to be relatively inactive regardless of their structural type, and this inactivity is closely related to their limited hydrogen binding capacity and water solubility [17]. Ketones, aldehydes and alcohols showed activity but with differing specificity and levels of activity, which is in connection with the functional groups present but also associated with hydrogen-binding parameters in all cases.
From our results it can be seen that MICs and MFCs are generally lower for all the essential oils and components investigated in microdilution method. The low water solubility of the oil and its components limit their diffusion through the agar medium. Only the more water-soluble components, such as carvone or 1,8-cineole diffuse into the agar. The hydrocarbon components either remain on the surface of the medium or evaporate [18]. That could be the reason for better results obtained by the microdilution method. Broth method, carried out in microtitre trays, has the advantage of lower workloads for a larger number of replicates and the use of small volumes of the test substance and growth medium. In this method dilution of the oil is better and there is no agar in the medium, which both enable better diffusion through the liquid medium.

Solvents affect the antifungal effects of Thymus and Mentha species essential oils
Also, it can be seen that the essential oils and components investigated showed greater antifungal activities when they are diluted in Tween ® . Non-ionic emulsifiers, such as Tween ® 20 or 80, are relatively inactive when tested alone and have been widely reported in the literature for their use as emulsifying agents [18]. It was achieved that dispersion of the oil and components is better than when ethanol was used as a solvent.
However, essential oils and all the components investigated showed inhibitory effects against all 17 fungi tested. The mycelial growth of test fungi responded differently to the investigated essential oil, which indicated that oils may have different modes of action or that the metabolism of some fungi was able to better overcome the effect of the oil or adapt to it. Phytopathogen Phomopsis helianthi was the most susceptible to the investigated essential oils, while Trichoderma viride and Penicillium species were the most resistant species (Figures 1 and 2).

Thymus and Mentha species essential oils can be used safely
In an earlier study of one of us [28], it was found that the LC50 value of orally applied Thyme oil in rat was 9543.5 μL kg -1 bodyweight. The LD 50 values of pyrethrin (350-500 mg kg -1 ) and carvone (1640 mg kg -1 ) are well below that of Thyme oil [29]. Mentha piperita and Thymus vulgaris as well as their oils and the major oil components are generally regarded as safe and full toxicology has been obtained [30].
Taken together, these results suggest that the essential oils investigated here could find practical application in the prevention and protection of fungal infections of plants, animals and humans. Essential oils could safely be used as preservative materials on foods for protection to fungal infection, since they are natural, and mostly non-toxic to humans. The selected plant species are popular culinary herbs, and their essential oils have been used extensively for many years in food products, perfumery, and dental and oral products.

Conclusions
The essential oils of T. vulgaris, T. tosevii and of M. piperita and M. spicata are highly active as fungitoxicants and could safely be used as natural preservatives to replace synthetic fungicides in the prevention and cure of some plant, human and animal fungal disease.  [26]. The cultures were stored at +4°C and subcultured once a month.

Instrumentation
The essential oils were investigated for their composition by the use of analytical GC/FID and GC/MS techniques. For this purpose a HP 5890 series II gas chromatograph, equipped with splitsplitless injector, fused silica capillary column (25 m x 0.32 mm), coated with cross-linked methyl silicone gum (0.5 μm film thickness), and FID was employed. Essential oil solutions in ethanol (1%) were injected in split mode (1:30). Injector was heated at 250 o C, FID at 300 °C, while column temperature was linearly programmed from 40-280 °C (4 °C /min). GC/MS analyses were carried out on a HP-GCD, equipped with split-splitless injector, fused silica capillary column (50 m x 0.2 mm) PONA, coated with cross-linked methyl silicone gum (0.5 μm film thickness). The chromatographic conditions were as above. Transfer line (MSD) was heated at 280 °C. EIMS spectra (70eV) were acquired in scan mode in m/e range 40-300. Identification of individual constituents was made by comparison of their retention times with those of analytical standards, and by computer searching, matching mass spectral data with those held in Wiley/NBS Library of Mass Spectra. For quantification purposes area percent reports obtained by FID were used.

Tests for antifungal activity
Macrodilution method: In order to investigate antifungal activity of essential oils and their components, the modified mycelial growth test with malt agar was used [27]. The minimum inhibitory concentration (MIC) of investigated components necessary for the complete inhibition of mycelial growth of the fungal strain was determined. Different concentrations of essential oils and components were diluted in Petri dishes with malt agar (MA). All fungal species were tested in triplicate. Petri dishes with ethanol and bifonazole were used as a control. Essential oil was added into molten malt agar (MA) and poured into Petri dishes. The tested fungi were inoculated at the center of the plates. Plates were incubated for three weeks at room temperature, after this period MIC was determined.
Microdilution method: The modified microdilution technique was used [26,27]. The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween ® 80 (vol/vol). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0 x 10 5 in a final volume of 100 μL per well. The inocula were stored at +4 °C for further use. Dilutions of the inocula were cultured on solid MA to verify the absence of contamination and to check the validity of the inoculum. Minimum inhibitory concentrations (MICs) determination was performed by a serial dilution technique using 96-well microtitre plates. Essential oils and compounds investigated were dissolved in malt medium broth with fungal inoculum. The microplates were incubated for 72 h at 28 °C. The lowest concentrations without visible growth (at the binocular microscope) were defined as concentrations, which completely inhibited fungal growth (MICs). The minimum fungicidal concentrations (MFCs) was determined by serial subcultivation of 2 μL into microtitre plates containing 100 μL of broth per well and further incubation for 72 h at 28 °C. The lowest concentration with no visible growth was defined as the MFC, indicating = 99.5 % killing of the original inoculum.