ChemiCal analysis and antimiCrobial aCtivities of the essential oils of Satureja thymbra l . and thymbra Spicata l . and their main Components

This work covers the chemical composition and antimicrobial activities of the essential oils isolated from Satureja thymbra and Thymbra spicata analyzed using gC/MS. The main components of S. thymbra oil were thymol (33.8 %), γ-terpinene (30.8 %) and p-cymene (11.8 %). The main components in T. spicata oil were carvacrol (74.5 %) and γ-terpinene (11.2 %). The oils and their main components, thymol and carvacrol, were assayed by applying the microdilution method for antibacterial and antifungal activity against food poisoning, plant, animal and human pathogenic microorganisms. The oil of T. spicata and carvacrol showed the highest antimicrobial activity.


InTrODUCTIOn
From ancient times, in addition to spices and their derivatives being used for flavoring foods and beverages and for medication, essential oils have also been highly valued for their use as antimicrobials (Akin and Oguz, 2010).
We focused our investigation on the antimicrobial activities of the essential oils of Satureja thymbra and Thymbra spicata from the Lamiaceae family, which has a wide distribution in greece and is of economic importance, and also of their components carvacrol, thymol, camphor, and 1,8-cineole, against soil-borne pathogens, food storage fungi, mycotoxic species, phytopathogens and human pathogens.This is the first report on the essential oil composition and antimicrobial activity of Thymbra spicata growing in greece.
Thymbra spicata L. is used in the traditional medicinal system of Turks, greeks, egyptians and romans to treat asthma and bronchitis as well as being used in the food industry for flavor, aroma and preservation (Daneshvar-royandezagh et al., 2009).The leaves have recently gained much popularity as a remedy to combat hypercholesterolemia (Akkol et al., 2009).The essential oils of this plant have wide industrial applications, from the flavoring of foods, liqueur production, perfumery and antiseptic used as antimicrobial agents (Sarac et al., 2009).Besides, the dried plant, softened in boiled water used to be applied to wounds as a drug (Akin and Oguz, 2010).Satureja thymbra L. has a concurrent warming, circulatory-stimulating action that makes it an excellent addition (at low levels) to massage blends for arthritis and rheumatism.It can also soothe painful joints and muscles (Simon et al., 1984).The acaricidal activity of the essential oil of S. thymbra and its major constituents, carvacrol and γ-terpinene, was also observed (Cetin et al., 2009).

Plant material
The plant material (aerial parts of S. thymbra and T. spicata) was collected at full blooming stage from experimental plots at 31' n,longitude 22°,58' e).The cultivated plants were primarily originated from seeds native to greece's S. thymbra and T. spicata populations.

Essential oil isolation procedure
The percentage oil content (as ml/100g of plant material) was determined using the european Pharmacopoeia apparatus (Clevenger-type).Dried inflorescences and the upper leaves (50 g) were chopped and distilled for 2 h.The obtained essential oils were dried over anhydrous sodium sulphate and stored in the refrigerator (-18 ° C) until used.

Identification and quantification of the oil components
The identification of the constituents was based on comparison of their kovats indices (rI) relative to n-alkanes with corresponding literature data (Adams, 1995), as far as matching a) their spectra with those from MS libraries (nIST 98, Adams 1995), and b) the rT of co-eluting reference compounds -peak enrichment technique (authentic samples by roth and Sigma Aldrich).
The bioassay was carried out by the microdilution method (Hanel and raether, 1988;espinel-Ingroff, 1989;Daouk et al., 1995).The bacterial cell 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 microplates were incubated for 24 h and 48 h at 37˚C.The lowest concentrations without visible growth (by the binocular microscope) were defined as MICs.The minimum bactericidal (MBCs) were determined by the serial subcultivation of 2 μl into microtiter plates containing 100 μl of broth per well and further incubation for 24 h and 48 h at 37˚C.
The lowest concentration with no visible growth was defined as MFC respectively indicating = 99.5% killing of the original inoculum.each experiment was repeated in triplicate.Streptomycin was used as positive controls (0.6-25 μg/ml).
The bioassay was carried out by the microdilution method (Hanel and raether, 1988;espinel-Ingroff, 1989;Daouk et al., 1995).The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v).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.Minimum inhibitory concentration (MIC) determinations were performed by a serial dilution technique using 96-well microtiter plates.The microplates were incubated for 72 h at 28 °C.The lowest concentrations without visible growth (by the binocular microscope) were defined as MICs.The fungicidal concentrations (MFCs) were determined by serial subcultivation of 2 µl into microtiter plates containing 100 µl of broth per well and further incubation 72 h at 28 °C.The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum.each experiment was repeated in triplicate.The commercial fungicide, ketoconazole, was used as positive control (0.6 -25 µg/ml).

Essential oil analyses
The results of the chemical analysis of the oils from S. thymbra and T. spicata are presented in Table 1.We identified 24 compounds in S. thymbra oil, rep-resenting 98.70% of the total amounts, and 20 compounds in T. spicata oil (99.00%).It can be seen that thymol (33.8%), γ-terpinene (30.8%) and p-cymene (11.8%) were the main components of S. thymbra oil.Carvacrol (74.5%) was the main component of the T. spicata oil, while the biosynthetically related monoterpene hydrocarbons γ-terpinene and p-cymene amounted to the 8.12 % and 5.62% of the oil, respectively.
Previous studies on the essential oil of Satureja thymbra have shown that it contains carvacrol and thymol as major components.From the analysis of S. thymbra oil, carvacrol was found to be the most abundant component (48.5%); these results are similar to those reported by Lagouri et al. (1993) for the same greek plant species.The biosynthetically related γ-terpinene and p-cymene were the other major components in the oils, representing 23.2% and 9.6%, respectively (Soković et al., 2002).The chemical composition of S. thymbra oil from Turkey showed that carvacrol (55.8%), γ-terpinene (21.7%) and p-cymene (8.7%) were the dominant components (Ugur et al., 2009) The chemical composition of the essential oils from S. thymbra species restricted to greece and the eastern Mediterranean region, showed that the main components were thymol (41%), γ-terpinene (22.2%) and p-cymene (11.8%) (Chorianopoulos et al., 2008).kokkini and vokou (1998) showed that mostly thymol containing chemotypes of S. thymbra can be found in greece but also in Sardinia.
It can be noticed that the oil examined in this paper is a thymol-type oil from the Satureja species.
The results of the antibacterial and antifungal activity of the essential oils tested are given in Table 2 and Table 3, respectively.Fungi proved to be more sensitive than bacteria to the effect of both essential oils.The oil of S. thymbra had minimal inhibitory concentration (MIC) in the range of 0.6-5.0μg/ml, and minimal bactericidal concentration (MBC) in the range of 2.5-10.0μg/ml.The most sensitive bac- teria was B. cereus with MIC of 0.6 μg/ml and MBC of 2.5 μg/ml, while the most resistant bacterial species were L. monocytogenes, E. faecalis and E. coli with MIC of 5 μg/ml and MBC of 10 μg/ml.In the cases of B. cereus and L. monocytogenes, the essential oil of S. thymbra was more effective than the commercial drug streptomycin (Table 2).The essential oil of T. spicata showed very strong antibacterial activity with MIC of 0.1-1.25 μg/ml and MBC of 0.6-2.5 μg/ml.This oil showed the best antibacterial activity against the gram-negative bacteria, P. aeruginosa, with very low MIC and MBC of 0.1 μg/ml and 0.6 μg/ml, respectively.The most resistant bacteria proved to be L. monocytogenes with MIC of 1.25 μg/ml and MBC of 2.5 μg/ml.This oil showed a higher antibacterial potential than streptomycin in all the cases, except for M. flavus and E. faecalis where the activity was the same (Table 2).It is obvious that T. spicata oil showed a higher antibacterial activity than the S. thymbra oil.
Thymol showed very strong antibacterial activity with MIC at 0.25-1.0µg/ml and 0.5-3.0µg/ml, respectively, while a bactericidal effect was achieved at 0.5-1.5 µg/ml.Carvacrol showed a stronger antibacterial activity with MIC at 0.02-0.5 µg/ml and MBC at 0.05-1.0µg/ml.Thymol and carvacrol showed higher antibacterial activity than streptomycin (MIC 0.6-12.5 µg/ml and MBC 1.2-25.0µg/ml) (Table 2).The essential oils of both tested species showed considerable antifungal activity against all the fungi tested.The oil of S. thymbra showed an inhibitory effect on the tested fungi in the range of 1.25-2.5 μg/ ml, and fungicidal activity in the range of 2.5-5.0 μg/ ml. A. fumigatus and T. viride were the most sensitive fungi to this oil, while A. ochraceus, A. niger and P. funiculosum showed themselves to be more resistant to this oil.ketoconazole showed a lower antifungal effect than S. thymbra oil in cases of almost all the fungi except for A. versicolor, where the activity was the same (Table 3).
The oil of T. spicata possessed a higher antifungal potential than S. thymbra oil.The MIC was 0.3-0.6 μg/ml and MFC in the range of 0.6-2.5 μg/ml.A. versicolor and A. fumigatus were the most sensitive, while P. ochrochloron was the most resistant to this oil.This oil showed much greater antifungal activity than the commercial antifungal agent ketoconazole (Table 3).
Thymol showed a great antifungal potential with an MIC of 0.125-0.2μg/ml and MBC at 0.25-0.5 μg/ ml.The Penicillium species and Trichoderma viride were more resistant than the Aspergillus species to thymol.Thymol showed a high antifungal potential with very low MIC values.Carvacrol exhibited very high antifungal activity, with MIC/MFC at 0.05-0.25 µg/ml.Both phenolic compounds tested showed much greater antifungal potential than commercial agents, even 10-500 times higher (Table 3).
Thus, it seems likely that the inhibitory effects of the examined essential oils are due to their major components.These inhibitory effects are interesting in connection with the presentation of mycotoxin contamination in many foods.
As a consequence, the chemical composition of the essential oils and their biological activities have been extensively studied, indicating that the essential oils possesses significant antimicrobial activities and may be used as effective agents that does not exhibit any genotoxic activities.Moreover, many reports concern the biological activities (antioxidant, antifungal, anti-inflammatory etc.) of the essential oils of Satureja and Thymus species (Chorianopoulos et al., 2004).The in vitro efficacy of T. spicata essential oil against 21 bacteria and seven Candida species was examined using disc diffusion and minimum inhibitory concentration (MIC) methods.The essential oil demonstrated strong anti-microbial activity in a wide spectrum against most microorganisms, particularly the yeasts tested.That is the first report on the anticandidal properties of the essential oil of T. spicata (Sarac et al., 2009).In previous studies, Yegen et al., (1992) and Müller-riebau et al., (1995) have reported that the essential oils of T. spicata and S. thymbra, and their main phenolic constituents, carvacrol and thymol, show a remarkable antifungal activity by inhibition of the mycelial growth of the soil-borne phytopathogenic fungi Fusarium moniliforme, Rhizocto-nia solani, Sclerotinia sclerotiorum, and Phytophthora capsici, one of the most important plant pathogens of pepper growing in the east Mediterranean region of Turkey.These essential oils were effective against gram-positive and gram-negative bacteria, which included multiple antibiotic resistant strains.However, Pseudomonas aeruginosa ATCC 27853 and Pseudomonas fluorescens MU 87 were resistant to these oils.The essential oils were very effective against Candida albicans.The antimicrobial activity of the essential oils showed some variations depending on the localities from which they were collected.The essential oil of the S. thymbra was especially very effective against the resistant strains such as Stenotrophomonas maltophilia MU 64, S. maltophilia MU 99 and Chryseomonas luteola MU 65 (Sarac and Ugur, 2008).The essential oil of S. thymbra (1% v/v) growing in greece, and the decoction and hydrosol fraction of the essential oil were tested against biofilms formed by five bacterial species, either as monospecies, or as a mixedculture of all species.The tested bacterial species were Staphylococcus simulans and Lactobacillus fermentum (useful technological bacteria), Pseudomonas putida (spoilage bacterium), Salmonella enterica and Listeria monocytogenes (pathogenic bacteria).Both methods revealed that the essential oil and the hydrosol of S. thymbra exhibited a strong antimicrobial action against both monospecies and mixed-culture biofilms (Chorianopoulos et al., 2008).The essential oil of S. thymbra was found to be active against the bacteria E. coli, P. aeruginosa, S. typhimurium, S. sonnei and S. aureus and the yeast C. albicans (gören et al., 2004).S. thymbra essential oil originating from greece possessed very good antifungal properties with low MIC (0.1-1.0 μl/ml) and MFC (0.2-2.0 μl/ml) values (Soković et al., 2002).The results of the antifungal activity of S. thymbra oil against M. perniciosa, a contaminator of Agaricus bisporus, obtained by the micro atmosphere method, showed an MIQ of 0.001-0.05μl/ml and MFQ of 0.1-0.25 μl/ml (glamočlija et al., 2006).Previous investigation confirms the high antifungal activity of thy-mol against food storage, phytopathogenic fungi (Müller-riebau et al., 1995) and dermatomycetes (Adam et al., 1998).

DISCUSSIOn
After comparison of the presented data with the chemical composition of the oil, it is evident that there is a relationship between the high activity of Satureja and Thymus oils and their high phenolic content.Although this study has mainly identified compounds, thymol and carvacrol, which appear to contribute significantly to the antifungal activity of oils, the possibility remains that other minor components contribute to the antimicrobial activity.
The results showed that S. thymbra and T. spicata essential oils and their components (thymol and carvacrol) are potent bacterial and mould inhibitors, confirming the possibility of using them in food preservation.The result of this study supports the traditional use of these plants.

table 1 .
Chemical composition of S. thymbra and T. spicata essential oils.
*rI, calculated retention indices on the DB 5 column **Identification method: a) rI b) Mass spectra c) authentic samples(co-elution, rT)

table 2 .
Antibacterial activity of S. thymbra and T. spicata essential oils and streptomycin (MICs and MBCs in μg/ml).

table 3 .
Antifungal activity of S. thymbra and T. spicata essential oils and ketoconazol (MICs and MFCs in μg/ml).