Studies on Chemical Composition, Antimicrobial and Antioxidant Activities of Five Thymus vulgaris L. Essential Oils

This study is aimed at assessing the essential oil composition, total phenolic content, antimicrobial and antioxidant activities of Thymus vulgaris collected in five different area of the Campania Region, Southern Italy. The chemical composition of the essential oils was studied by GC-flame ionization detector (FID) and GC/MS; the biological activities were evaluated through determination of MIC and minimum bactericidal concentration (MBC) and evaluation of antioxidant activity. In total, 134 compounds were identified. The oils were mainly composed of phenolic compounds, and all oils belonged to the chemotype thymol. The antimicrobial activity of the five oils was assayed against ten bacterial strains. The oils showed different inhibitory activity against some Gram-positive pathogens. The total phenol content in the essential oils ranged from 77.6–165.1 mg gallic acid equivalents (GAE)/g. The results reported here may help to shed light on the complex chemotaxonomy of the genus Thymus. These oils could be used in many fields as natural preservatives of food and as nutraceuticals.

. Chemical composition of the essential oils isolated from the aerial part of Thymus vulgaris collected at the campus of the University of Salerno (S), Frigento (F), Contrada la Francesca (LF), Morigerati (M) and Zungoli (Z).

Minimum Inhibitory Concentrations
The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values of the five essential oils against ten selected microorganisms are reported in Table 2. The five essential oils showed different inhibitory activity against the Gram-positive pathogens. Among the Gram-negative bacteria, E. coli was affected by the oil of Frigento (F). Staphylococcus epidermidis was the more sensitive bacterial strain.

Total Phenolic Content
The concentration of total phenols was determined in the five essential oils of T. vulgaris plants. In Figure 1, the results of the colorimetric analysis are given; they were derived from the absorbance values of the oil solutions compared to the standard solutions of gallic acid equivalents (standard curve equation: y = 0.00119x − 0.00532, r 2 = 0.9996). The total phenol content of the five oils ranged from 77.6-165.1 mg gallic acid equivalents (GAE)/g of sample (essential oil). The essential oil from Zungoli contained significantly higher total phenols (165.1 mg GAE/g) than the other oils.

Free Radical-Scavenging Capacity
The antioxidant activity of T. vulgaris essential oils was assessed by DPPH assay, evaluating the H-donating or radical scavenging ability of the oils using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a reagent. Table 3 shows the concentrations that led to 50% inhibition (IC50) for three of the studied thyme oils (data for essential oils from Zungoli and Morigerati are unavailable). Ascorbic acid was used as a standard antioxidant. In this study, the IC50 values of the studied oils were less than the value of the reference antioxidant ascorbic acid (IC50 values of 3.10 ± 1.13 μg/mL) [7]. The essential oil composition of the five T. vulgaris populations appeared similar, and the oils belonged to the same chemotype. Indeed, the five oils were characterized by high percentages of phenols and can be classified as oils belonging to the thymol chemotype. The variations between the main compounds of thyme essential oil can be explained by the biosynthetic relationship between the two phenols.
The metabolic pathway for the carvacrol and thymol formation begins with the autoxidation of γ-terpinene to p-cymene and the subsequent hydroxylation to thymol [8]. In the literature, it was reported that Thymus vulgaris has a chemical polymorphism with six different chemotypes that show spatial segregation in nature: phenolic chemotypes (thymol and carvacrol) and non-phenolic chemotypes (geraniol, α-terpineol, linalool and trans-thujan-4-ol/terpinen-4-ol) [9].
The different antimicrobial activity of these oils might be due to the little variation in their chemical profile. In the literature, it was reported that various chemical compounds have direct activity against many species of bacteria, such as terpenes and a variety of aliphatic hydrocarbons (alcohols, aldehydes and ketones). The lipophilic character of their hydrocarbon skeleton and the hydrophilic character of their functional groups are of main importance in the antimicrobial action of essential oils components, and the importance of the hydroxyl group of phenolic structures has been confirmed.
Moreover, the aldehyde group conjugated to a carbon-to-carbon double bond is a highly electronegative arrangement, which may explain their activity, suggesting a proportional increase of the antibacterial activity with electronegativity. The activity increased with the length of the carbon chain. Secondly, there is some evidence that minor components have a critical part to play in antibacterial activity, possibly by producing a synergistic effect between other components. This has been found for sage, some species of Thymus and oregano [10]. The appreciable total phenol contents of the five essential oils can also contribute to the antimicrobial activity. Ahmad and coworkers [3] reported that synergistic and additive interactions occur between the major and minor constituents present in the essential oil of Thymus vulgaris, and in this way, the antimicrobial efficacy of the essential oil could be enhanced.
Our data concerning total phenolic content are in line with previous research [11,12], which reports that the phenolic compounds are the main compounds in the thyme essential oil. The variation of the total phenolic content may be due to environmental conditions, such as soil composition and nitrogen content, which can modify the constituents of the plant [13,14].
The moderate antioxidant activity of the essential oil from the campus of the University of Salerno is probably due to the high amount of oxygenated compounds (phenolic compounds, 75.5%; oxygenated monoterpenes, 6.4%; oxygenated sesquiterpenes, 7.4%) and to the total phenolic content (112.3 mg GAE/g of sample). Our results are in agreement with previous studies, which showed that greater antioxidant potential of several Thymus species' essential oils could be related to the nature of the phenolic compounds and their hydrogen ability. Besides, such activity could be ascribable to the oxygenated compounds, such as carvacrol and thymol. Moreover, the activities of essential oils of Thymus species depend on several structural features of the molecules and are primarily attributed to the high reactivity of the hydroxyl group substituent [15]. Moreover, the essential oils that contain oxygenated monoterpenes and/or sesquiterpenes have been reported for their greater antioxidative properties [1].

Isolation of the Volatile Oils
One hundred grams of fresh aerial parts of each sample were ground in a Waring blender and then subjected to hydrodistillation for 3 h according to the standard procedure described in the European Pharmacopoeia [16]. The oils were solubilized in n-hexane, dried over anhydrous sodium sulfate and stored under N2 at +4 °C in the dark until tested and analyzed. The calculated essential oil yield was expressed in % (v/w), based on the weight of the fresh plant material. All extractions were done in triplicate.

GC-FID Analysis
The gas chromatography-flame ionization detector (GC-FID) analysis was carried out on a Perkin-Elmer Sigma-115 gas chromatograph equipped with a flame ionization detector (FID) and a data handling processor. The separation was achieved using an apolar HP-5 MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25-μm film thickness); column temperature: 40 °C, with 5 min initial hold, then to 270 °C at 2 °C/min and, finally, at 270 °C for 20 min; injection mode: splitless (1 μL of a 1:1000 n-pentane solution). Injector and detector temperatures were 250 °C and 290 °C, respectively. Analysis was also run by using a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm i.d., 0.25-μm film thickness). In both cases, helium was used as the carrier gas (1.0 mL/min). The relative essential oil contents of the components were obtained by peak area normalization, without calculating response factors.

GC/MS Analysis
The gas chromatography-mass spectroscopy (GC/MS) analysis was performed with an Agilent 6850 Ser. II apparatus, fitted with a fused silica DB-5 capillary column (30 m × 0.25 mm i.d., 0.33-μm film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization energy voltage: 70 eV; electron multiplier voltage energy: 2000 V. Mass spectra were scanned in the range 40-500 amu, with a scan time of 5 scans/s. The gas chromatographic conditions were as reported in the previous paragraph; transfer line temperature: 295 °C.

Identification of the Essential Oil Components
The identification of the essential oil constituents was based on the comparison of their Kovats retention indices (RIs), determined relative to the tR values of n-alkanes (C10-C35) on both capillary columns with those in literature [17][18][19][20] and their mass spectra with those of authentic compounds available in our laboratories or those listed in the NIST 02 and Wiley 275 mass spectral libraries [21]. For some compounds, the identification was confirmed by coinjection with an authentic sample (Table 1).

Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
The antibacterial activity was evaluated by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) using the broth dilution method [22]. . The strains were maintained on Tryptone Soya agar (Oxoid, Milan, Italy); for the antimicrobial tests, Tryptone Soya broth (Oxoid, Milan, Italy) was used. In order to facilitate the dispersion of the oil in the aqueous nutrient medium, it was diluted with Tween 20, at a ratio of 10%. Each strain was tested with sample that was serially diluted in broth to obtain concentrations ranging from 100 μg/mL down to 0.8 μg/mL. The sample was previously sterilized with a Millipore filter of 0.20 μm. The samples were stirred, inoculated with 50 μL of physiological solution containing 5 × 10 6 microbial cells, and incubated for 24 h at 37 °C. The MIC value was determined as the lowest concentration of the sample that did not permit any visible growth of the tested microorganism after incubation. The control containing only Tween 20 was not toxic to the microorganisms. As positive controls, cultures containing only sterile physiological solution Tris buffer were used. MBC was determined by subculture of the tubes with inhibition in 5 mL of sterile nutrient broth. After incubation at 37 °C, the tubes were observed. When no growth was observed, the sample denoted a bactericidal action. The oil sample was tested in triplicate. Chloramphenicol was used as the standard antibacterial agent.

Determination of Total Phenolics
The total phenolic content was determined following the microscale protocol for Folin-Ciocalteu colorimetry, an alternative protocol for small sample volumes [23]. Each oil sample (20 μL, dissolved in ethanol, to obtain a final concentration of 50 mg/5 mL), a gallic acid calibration standard (50 mg/mL; 100 mg/mL; 250 mg/mL; 500 mg/mL) or blank (distilled water) was taken in a test cuvette. The absorbance was determined at room temperature at k = 765 nm using a Cary UV/Vis spectrophotometer (Varian Cary 50 MPR). The quantification was based on a standard curve generated with gallic acid; the results were expressed as mg gallic acid equivalents (GAE)/g of essential oil. A methanolic solution of gallic acid was tested in parallel as a reference compound.

Antioxidant Activity
The antiradical activity of the extracts under investigation was determined using the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), according to the method reported by Brand-Williams and coworkers [24] with some modifications to adapt the procedure using 96-well microplates [25]. In its radical form, DPPH has an absorption band at 517 nm, which disappears upon reduction by an antiradical compound. Briefly, an aliquot (7 μL) of the MeOH solution containing different amounts of the oils was added to 280 μL of DPPH solution (7.6 × 10 −5 M), prepared daily, kept in the dark when not used. An equal volume (7 μL) of the vehicle alone was added to control tubes. Absorbances at 517 nm were measured on a Multiskan Spectrum Microplate Spectrophotometer (Thermo Fischer Scientific, Vantaa, Finland) 0, 10, 20, 30, 40, 50 and 60 min after starting the reaction. For preparation of the standard curve, different concentrations of DPPH methanol solutions (5-40 μg/mL) were used. Moreover, the solution of ascorbic acid was used for a calibration curve of DPPH reduction and as a chemical reference in comparison to the antioxidant capacities of the oils. Ascorbic acid was obtained from Fluka (Buchs, Switzerland). Ascorbic acid is an effective antioxidant [26]. Ascorbic acid was solved in methanol to have the following final concentrations (5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.625 μg/mL, 0.3125 μg/mL). The DPPH concentration (μg/mL) in the reaction medium was calculated from the following calibration curve, determined by linear regression (r 2 : 0.9974): Absorbance (λ517) = 0.00186 + 0.0187 × [DPPH] The IC50 value was defined as the concentration of sample that reduced the initial DPPH concentration by 50%, as compared to the negative control.

Statistical Analysis
Data from the determination of total phenolics were analyzed in GraphPad Prism 6.0 for correlation and significance (one-way ANOVA and Dunnett's multiple comparison post-test). Data on antioxidant activity are expressed as the mean ± SD of five experiments.

Conclusions
The results reported here may help to shed light on the apparently complex chemotaxonomy of the genus Thymus. All five samples belong to the thymol chemotype, showing a homogeneity of prevalent monoterpenes in the oils. This finding seems to be related to the circum-Mediterranean distribution of this chemotype, which is the only one with the characteristic flavor and aroma of true thyme. Moreover, this study focused on the phenolic fraction and the effectiveness of T. vulgaris essential oils as an antimicrobial and antioxidant. Therefore, these oils could be used in many fields as natural preservatives of food and as nutraceuticals.

Conflicts of Interest
The authors declare no conflict of interest.