Comparison of the Chemical Composition and Antimicrobial Activity of Thymus serpyllum Essential Oils

The chemical composition of the essential oils obtained by hydrodistillation from the aerial parts of Thymus serpyllum and Thymus serpyllum ‘Aureus’ has been investigated by gas chromatography-mass spectrometry (GC-MS). Forty-seven compounds (99.67% of the total oil) were identified in the essential oil of T. serpyllum. The main components found in the oil were carvacrol (37.49%), γ-terpinene (10.79%), βcaryophyllene (6.51%), p-cymene (6.06%), (E)-β-ocimene (4.63%) and β-bisabolene (4.51%). Similarly, carvacrol (44.93%), γ-terpinene (10.08%), p-cymene (7.39%) and β-caryophyllene (6.77%) dominated in the oil of T. serpyllum ‘Aureus’. A total of forty three compounds were identified in this oil, representing 99.49% of the total oil content. On the basis of the obtained data it was proved that the content of 1-octen-3-ol, eucalyptol, (Z)-β-ocimene, (E)-β-ocimene, γ-terpinene, carvacrol methyl ether, germacrene D and β-bisabolene was significantly higher for T. serpyllum while T. serpyllum ‘Aureus’ was characterized by a significantly higher content of 3-octanone, 3-octanol, p-cymene, borneol and carvacrol. The isolated essential oils were evaluated for their antimicrobial activity against nine reference strains (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Enterococcus faecalis, Bacillus cereus, Micrococcus luteus, Proteus vulgaris and Candida albicans) by the microdilution technique. Based on this test, the minimum inhibitory concentrations (MIC) of essential oil were calculated. The volatile oil obtained from T. serpyllum showed the highest antimicrobial activity relative to the strain of E. coli (MIC=0.025 μL/mL) and to the yeast C. albicans (MIC=0.05 μL/mL). Similarly, a significant antimicrobial activity exhibited T. serpyllum ‘Aureus’ essential oil, although the MIC values obtained in that case for E. coli and C. albicans strains were twice as high and were respectively 0.05 μL/mL and 0.1 μL/mL.

Thymus oils and extracts have found wide applications in cosmetic and perfume industry as well as flavourings and preservative agents for different food products (Guseinov et al., 1987). Due to their antiseptic, antispasmodic and antimicrobial properties, they are also used for medicinal purposes (Jirovetz et al., 2007).
In traditional medicine, the flowering parts and leaves of Thymus species plants are mainly used as herbal tea, flavouring agents (condiment and spice), for treating colds, coughs, sore throat and indigestion (Zargari, 1990;Morales 2002;Amin, 2005).
Wild thyme is well-known for its cough-suppressant, antiseptic and spasmolytic properties (Zarzuelo and Crespo, 2002). Especially, a strong decoction, sweetened with honey is recommended for easing spasms of whooping cough (Aziz and Rehman, 2008). The plant can be applied for preparation of herbal tea, herbal baths and herbal pillows (Zarzuelo and Crespo, 2002). bottomed flask along with 500 mL distilled water was subjected to hydrodistillation for 2 hours using Clevenger apparatus according to the method recommended by European Pharmacopoeia (2010). The obtained essential oils were separated from water, then dried over anhydrous sodium sulphate, filtered and stored in dark sealed vial at low temperature (4 °C) prior to GC-MS analysis.

Gas Chromatography/Mass Spectrometry (GC/MS) analyses of essential oils
The qualitative analysis was conducted using HP 6890 gas chromatograph coupled with HP 5973 Mass Selective Detector operating at 70 eV mode. The essential oil samples (30 mg) were dissolved in dichloromethane (1.5 mL) and 2 µL of each solution were injected in a split mode at a ratio of 5:1. Compounds were separated on 30 m long capillary column (HP-5MS), 0.25 mm in diameter and with 0.25 µm thick stationary phase film ((5% phenyl)-methylpolysiloxane).
The flow rate of helium through the column was kept at 1.2 mL min -1 . The initial temperature of the column was 40 °C for 5 minutes, then increased to 60 °C at a rate of 30 °C min -1 , next to 230 °C at a rate of 6 °C min -1 (kept constant for 10 min), and then increased to a final temperature of 280 °C at a rate of 30 °C min -1 . The oven was held at this temperature for 5 minutes. The injector and the transfer line were kept at 280 °C . The ion source temperature was 230 °C . The solvent delay was 4 min. The scan range of the MSD was set from 40 to 550 m/z. The total running time for a sample was about 51 minutes.
The relative percentage of the essential oil constituents was evaluated from the total peak area (TIC) by apparatus software.
Essential oil constituents were identified by comparison of their retention indices (relative to n-alkanes C7-C40 on HP-5MS column) with those reported in NIST Chemistry WebBook (http://webbook.nist.gov/chemistry) and the literature (Adams, 2007).
Further identification was made by comparison of their mass spectra with those stored in the Wiley NBS75K.L and NIST/EPA/NIH (2002 version) mass spectral libraries using different search engines (PBM, Nist02) or with mass spectra of authentic compounds available in our laboratory (thymol, carvacrol and p-cymene), purchased from Fluka and Sigma-Aldrich.

Antimicrobial screening
Minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC) concentrations were determined by microdilution method in 96-well microtitre plates described by Wiegand et al. (2008) as well as Levic et al. T. serpyllum is also an important source of substances with antioxidant, antimicrobial and antitumor properties (Jaric et al., 2015). Recent studies have shown that Serpylli aetheroleum strongly act on fungi and bacteria (Farrukh et al., 2012;Sokolic-Mihalak et al., 2012;Nikolic et al., 2014). The oil is used medicinally as well as in the manufacturing of toothpastes, mouthwashes and gargles (Ahmad et al., 2006). According to Aziz and Rehman (2008), it can relieve rheumatism and may be used in hair loss treatments.
The high quality of essential oil is one of the main requirements of pharmaceutical and food industry. The essential oils derived from various Thymus species may differ in chemical composition and biological properties. Their market value may also be different.
Our previous studies concerned the influence of distillation time and distillation apparatus on the composition and quality of wild thyme oil (Wesołowska et al., 2012;Wesołowska et al., 2014).
The medicinal importance and biological activity of T. serpyllum volatile oil prompted us to investigate its cultivar 'Aureus'. The present study aimed to compare the chemical composition and antimicrobial activity of the oils extracted from T. serpyllum and T. serpyllum 'Aureus'. To the best of our knowledge, there are no scientific reports concerning this topic. Similarly, there is lack of publications on the composition and biological activity of oil obtained from T. serpyllum 'Aureus'.

Plant material
The studied biological material used in the current research consisted of wild thyme (Thymus serpyllum L.) and wild thyme 'Aureus' (Thymus serpyllum 'Aureus') from the Lamiaceae family. All the plants were grown in experimental plots with an area of 1.44 m 2 , in four replications at the Horticultural Experimental Station near Szczecin (North-Western Poland), which belongs to the West Pomeranian University of Technology Szczecin.
The seedlings, obtained from older plants, after rooting in horticultural substrate, were planted into the open field in the second half of May 2012, with spacing of 20 x 20 cm. For laboratory analyses, an herb from two-year-old plants was harvested at the flowering stage (harvest date: July 8, 2014). The field was prepared according to agrotechnique proper for thyme cultivation. Mineral fertilization was quantified according to the results of the chemical analysis of the soil samples and supplemented to those recommended for thyme level. In the first year of the experiment only nitrogen (60 kg N ha -1 ) and potassium (60 kg K2O ha -1 ) fertilization was applied, while in the second year -potassium (60 kg K2O ha -1 ) and phosphorous (60 kg P2O5 ha -1 ) fertilization.
The experiment was performed on sandy clay soil, which is characterized by low water-holding capacity. During the growing season manual weeding and irrigation were performed.
After the harvest, plant material was dried in a shady and well ventilated place at room temperature (drying room). Dry herb was cut into small pieces and stored (in paper bags in a dry and cool place) until chemical analyses were performed.

Essential oil extraction
Twenty grams of the whole dried aerial parts of T. serpyllum and T. serpyllum 'Aureus' (separately) in a 1000 mL round-(2011) with slight modifications (in case of yeast, the time of SDA plates incubation at 37 °C was prolonged to 48 h). The bacterial/fungal inoculates were prepared using fresh overnight cultures and suspensions was adjusted to 0.5 McFarland standard turbidity using turbidimeter (Biosan). Essential oils were diluted in propylene-glycol (2-(2-hydroxypropoxy)-1propanol) to the test concentration ranging from 500 to 2 µL/mL. The optical density of tested microorganisms cells (OD, λ=600 nm) under exposure to tested oils were used to quantify of results. The optical density of tested microorganisms cells was measured at the wavelength of 600 nm in 96-well microtitre plates with 200 µL of each sample using Infinite 200 PRO NanoQuant microplate reader (Tecan, Männedorf, Switzerland).
All tests were performed in Mueller Hinton Broth (MHB, Emapol, Poland), in a volume of 200 µL. The aliquots 20 µL bacteria (or yeast) suspensions and 20 µL essential oil in geometric dilutions (ranging from 500 µL to 0.125 µL) were added into each well of 96-well microtitre plate. Then, aliquots of 160 µL of MHB were added. The final essential oils concentrations were 50 to 0.0125 µL/mL. Simultaneously sterility control (MHB + tested oils) and control of toxicity of propylene glycol (MHB + tested microorganisms + propylene glycol) were performed. As the controls of all tests, the same tested microorganisms, incubated under the same conditions but without exposure to tested oils were used.
The microplates were incubated for 24 h at 37 °C for bacteria and 48 h at 37 °C for yeast. The MIC was defined as the lowest concentration of essential oil at which microorganism shows no visible growth. Then, the 5 µL solution from each well was transferred to BHIA plates (Emapol) and incubated for 24 h at 37 °C for bacteria. For yeast SDA plates (Emapol) were used and incubated for 48 h at 37 °C. The lowest concentration of the essential oil at which 99.5% inoculated microorganisms were killed was defined as MBC for bacteria and MFC for yeast. All tests with the controls were repeated three times.

Statistical analysis
In order to observe the differences in the composition of T. serpyllum and T. serpyllum 'Aureus' oils, seventeen constituents of the content greater than 1% of the oil were selected for statistical analysis. An analysis of variance was performed using AWAR software made by Department of Applied Informatics, Institute of Soil Science and Plant Cultivation in Puławy. The means were separated by the Tukey's test at p=0.05.
The data gathered in Table 4 are presented as the means ± standard deviations (mean ± SD) calculated for the three repetitions of the experiment using Statistica 9.0 (StatSoft, Poland).

Chemical composition of essential oils
The essential oils isolated by hydrodistillation from the aerial parts of T. serpyllum and T. serpyllum 'Aureus' were found to be yellow liquids and were obtained with a yield of 1.50% (v/w) and 1.45% (v/w), based on the dry weight of plant material. The present results are in conformity with the European Pharmacopoeia (2010) standard for T. serpyllum herb (a yield of at least 0.3%).
According to literature data, the content of essential oil in T. serpyllum is variable and depends mainly on the origin of the plants, but usually is between 0.1 and 1% (Raal et al., 2004). In Poland, the amounts of essential oil among wild growing populations vary from 0.21 to 0.60% (Pióro-Jabrucka and Osińska, 2003). However, plants grown in different regions of Jordan contain from 2.5 to 5.6% of oil (Abu-Darwish et al., 2009).
The relative percentage composition of essential oils and retention indices calculated for individual components of the oils are given in Table 1. All the constituents are listed in order of their elution from HP-5MS column. A total of 47 compounds were identified in the oil of T. serpyllum representing 99.67% of the total oil, and 43 compounds were identified in the oil of Thymus serpyllum 'Aureus', representing 99.49% of the total oil (Table 1).
For most of the analysed essential oil constituents there were significant differences found between their content in Thymus   both analysed essential oils (Tables 1 and 2). Soković et al. (2010) have proved that this compound had the greatest antimicrobial properties among all tested ingredients, including: camphor, 1,8cineole, linalool, linalyl acetate, limonene, menthol, α -pinene, βpinene and thymol. The similar activity of essential oils containing large amount of carvacrol has been pointed out by other researchers (Rasooli and Mirmostafa, 2002;Abed et al. 2014). Varga et al. (2015) additionally emphasize that essential oils of Thymus species, whose one of the most important ingredients is carvacrol, show a very wide spectrum of activity both against Gram-positive and Gram-negative bacteria and against yeast. Thus, these biologically active substances may play a significant role in the prevention of serious bacterial and fungal infections as pharmaceutical formulations or natural food preservatives (Hammer et al., 1999;Lambert et al., 2001).
The biological activity of essential oils depends mainly on the chemical structure of their components and their concentrations. Generally, phenolic compounds, terpenes, aliphatic alcohols, aldehydes, ketones, acids, and flavonoids are recognized as major components with antimicrobial activity, which can be found in plants. Among them, thymol, carvacrol, pcymene and γ-terpinene are considered as the most potent (Shelef, 1983;Farag et al., 1989;Davidson, 1993;Santoyo et al., 2006). Sikkema et al. (1995) reported that these compounds act on microbial cells and cause structural and functional damage of their membranes, resulting in increased permeability.

Antimicrobial activity
The antimicrobial activity of T. serpyllum oils was evaluated against Gram-positive and Gram-negative bacterial strains as well as for yeast Candida albicans using the microdilution method. The obtained results are summarized in Table 4. It was demonstrated a significant, but varied for each of the analysed test strains the antimicrobial activity of essential oils. At the same time, based on conducted scientific controls, the sterility of tested essential oils was confirmed and also the toxicity of propylene glycol was excluded with respect to all of the tested microorganisms. Reported values of MIC and MBC/MFC were respectively in the case of the T. serpyllum L. essential oil 0.025-0.78 µL/mL and 0.1-1.56 µL/mL, whereas for the T. serpyllum 'Aureus' essential oil 0.05-0.39 µL/mL and 0.1-1.56 µL/mL. The T. serpyllum L. essential oil showed the highest antimicrobial activity relative to the strain of E. coli (MIC=0.025 µL/mL and MBC=0.1 µL/mL) and to C. albicans (MIC=0.05 µL/mL and MFC=0.1 µL/mL), however the lowest to the strain of S. epidermidis (MIC=0.78 µL/mL and MBC=1.56 µL/mL), and to M. luteus (MBC=1.56 µL/mL). Similarly, a significant antimicrobial activity exhibited the T. serpyllum 'Aureus' essential oil, although the MIC values obtained in that case for E. coli and C. albicans strains were twice as high and were respectively (0.05 µL/mL and 0.1 µL/mL). On the other hand, the lowest antimicrobial activity of the T. serpyllum 'Aureus' essential oil showed relative to the strain of P. vulgaris (MIC=0.39 µL/mL and MBC=0.78 µL/mL), and strains of S. epidermidis and E. faecalis, for which the observed for that oil MBC value was as high as 1.56 µL/mL.

Discussion
The present results are in agreement with the data provided by other authors who also showed significant antimicrobial effect after the application of O. vulgare, T. vulgaris, T. serpyllum, as well as T. algerientis essential oils (Lambert et al., 2001;Levic et al., 2011;Nikolic et al., 2014). Similarly to Hammer et al. (1999), the lowest values of minimum inhibitory concentrations (MIC) were shown for tested strains of E. coli and C. albicans. This may prove the particular sensitivity of these microorganisms to the essential oils. Significant antimicrobial activity of these oils is mainly connected with the destruction of the integrity and function of biological membranes of microbial cells exposed to their operation (Lambert et al., 2001). An essential role in this mechanism may be especially performed by carvacrol -an organic compound that constituted a dominant ingredient of from the territory of European North-East Russia and Ural (Alekseeva and Gruzdev, 2012). Uncharacteristic compounds, such as: 2,4,6-trimethylanisol (73.41%), 3,5-dimethyl benzoic acid (5.38%) and β-bisabolene (3.67%), were present in the essential oil obtained from plants growing in Turkey (Topal et al., 2008).
Comparing the content of active ingredients in both essential oils, it appears that T. serpullum 'Aureus' oil should be more effective against tested microbial strains. Surprisingly, slightly higher activity against E. coli and C. albicans was noted for T. serpyllum oil. Probably less abundant constituents of the oil are also responsible for the antimicrobial activity. They may be involved in some type of synergism with the other active compounds.

Conclusion
In all two Thymus essential oils, carvacrol, γ-terpinene, p-cymene and β-caryophyllene were identified as the major oil constituents. The investigated oils were the most active against Escherichia coli and yeast Candida albicans, and less potent against Staphylococcus epidermidis, Enterococcus faecalis, Micrococcus luteus and Proteus vulgaris. The current study suggest the potential use of T. serpyllum and T. serpyllum 'Aureus' oils as antimicrobials for food preservation as well as in pharmaceutical formulations for prevention of bacterial and fungal infections.