Evaluation of Thymus vulgaris and Thymbra spicata essential oils and plant extracts for chemical composition, antioxidant, and antimicrobial properties

Abstract The objectives of this study were (a) to obtain the essential oils (by hydrodistillation [HD] and microwave‐assisted extraction [MAE] methods) to determine the effect of the oil extraction method on the chemical composition, oil yield (%), free radical scavenging activity (IC50), ferric reducing antioxidant power (FRAP) value, and antimicrobial properties of Thymus vulgaris (thyme) and Thymbra spicata (zahter); and (b) to determine the effect of different solvents (methanol [80%] and ethanol [80%]) on extraction by means of the phenolic acid composition, total phenolic content, total flavonoid content, IC50, and FRAP value of thyme and zahter. Gas chromatography–mass spectrometry analysis showed that the amount of thymol (55.35%; 50.53%) and p‐cymene (11.2%; 11.79%) was found to be highest in thyme, when using HD and MAE, respectively. However, the highest amounts of carvacrol (68.20%; 66.91%) and γ‐terpinene (13.25%; 13.94%) were found in zahter, when using HD and MAE, respectively. Thyme essential oil had higher antioxidant capacity for both HD and MAE in comparison with zahter essential oil. Methanol extracts of both thyme and zahter had higher phenolic composition in comparison with their ethanol extracts. Extracts of both plants did not show any antimicrobial properties. However, essential oils of both thyme and zahter showed antimicrobial activity against chosen bacteria. Highest inhibition zone (radius) was shown against Staphylococcus aureus ATCC 9144 by the essential oils.

hydroxyanisole (BHA), from food formulations and replacing them with natural preservatives have gained significant importance. Many plants have been investigated for wide variety of active components for antimicrobial and antioxidant properties. Thyme and zahter could be useful plants due to their essential oils being high in thymol and carvacrol, respectively (Lee et al., 2005;Uysal et al., 2015), and also their extracts being rich in phenolic acids (Erturk et al., 2017;Roby, Sarhan, Selim, & Khalel, 2013). Phenolic acids, such as rosmarinic acid, have been studied for their antioxidant properties (Kostic et al., 2015;Skendi et al., 2017). Development of food additives made from essential oils and/or extracts with antioxidant and antimicrobial properties is a crucial step toward clean labeling and the production of healthier food products. and Thymbra spicata. This study was designed to compare hydrophobic and hydrophilic extracts of both species. Hydrophobic fractions (essential oils) were obtained by hydrodistillation (HD) and microwave-assisted extraction (MAE). MAE is fast and extracts essential oils quickly, so research is needed to determine whether oil compositions are affected by this extraction method. For the same reason, hydrophilic nonvolatile fractions will be extracted with two common solvents, methanol and ethanol. Therefore, the objectives of this study were (a) to obtain the essential oils by hydrodistillation (HD) and microwave-assisted extraction (MAE) and to determine the effect of the oil extraction method on the chemical composition, oil yield (%), free radical scavenging activity (IC 50

| Microwave extraction
Air-dried plants of cultivated Thymus vulgaris and Thymbra spicata were obtained from Diyarbakir Research Institute (July 2017), Turkey. A NEOS microwave extraction system (MA 125 Milestone, Italy) was used for this study. Seventy-five grams of dry plant material was weighed into a beaker. Then, 150 ml of distilled water was added (1:2, w/v). After 30 min of resting, extraction was performed at 550 W power for 30 min. Then, the essential oil was collected and placed into vial with anhydrous sodium sulfate to remove any water.
Then, vials were stored at 4°C for later analysis.

| Hydrodistillation with Clevenger apparatus
Hydrodistillation was conducted using an Electromantle™ (EM2000 CE, Electrothermal Engineering Ltd., UK, 500 W). Seventy-five grams of air-dried plant material was weighed into a round flask.
Then, 750 ml of distilled water (1:10, w/v) was added. Extraction was performed for three hours. Next, the essential oil was collected and placed into a vial with anhydrous sodium sulfate to remove any water. Then, vials were stored at 4°C for later analysis.

| Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC-MS) analysis of the essential oil was performed on a GC-MS QP2020 equipped with an Rxi-5Sil MS column (5% diphenyl-95% dimethylpolysiloxane 30 m × 0.25 mm i.d., df = 0.25 µm; RESTEK GC Columns, USA). The injector and detector temperatures were set at 250°C. Temperature programming of the oven included an initial hold at 40°C for 2 min and a rise to 240°C at 4°C/min and held for 53 min. Helium was the carrier gas, with a linear velocity of 43.4 cm/s. The samples were diluted with n-hexane (1:10, v/v), and a volume of 1.0 µl was injected into the GC with the injector in the split mode (split ratio: 1:25). The ionization voltage applied was 70 eV (electron volt), with a mass range m/z (mass-to-charge ratio) of 40-400 amu (atomic mass unit).
The National Institute of Standards and Technology (NIST), WILEY 7 mass spectral library data, and authentic standards were applied to match the separated components. Peak area integration was used for the determination of percentage of combination. This analysis was carried out in duplicate.

| Antioxidant assays
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging capacity was measured according to Cuendet, Hostettmann, and Potterat (1997). Next, 50 µl of either essential oil or plant extract (at different concentrations in methanol) was mixed with 5 ml of a 0.004% (w/v) DPPH methanolic solution. The reaction was allowed to stand at room temperature for 30 min, and absorbance was read against a blank at 517 nm. The inhibitions of the DPPH radical in percent were calculated as follows: where A blank is the absorbance of the control reaction (containing all reagents except the test sample), and A sample is the absorbance value of the essential oil or the extract. Extract or oil concentration providing 50% inhibition (IC 50 ) was calculated using the graph-plotted inhibition percentage against the extract or oil concentration.
Tests were conducted in duplicate, with gallic acid used as a positive control.
After incubation at 37°C for 15 min, the absorbance was measured at 595 nm, using the FRAP solution as a blank. The antioxidant capacity of plant extracts or oil was determined from a standard curve plotted using the FeSO 4 ·7H 2 O linear regression. Results were expressed as µM of Fe +2 /g of essential oil or dry extract. Tests were conducted in duplicate.

| Plant extraction and lyophilizing
Two solvents were used to extract polar fractions of the Thymus vulgaris and Thymbra spicata. Ten grams of sample was weighed into a beaker. Then, either 100 ml of 80% aqueous methanol (1:10 ratio of w/v) or 100 ml of 80% aqueous ethanol (1:10 ratio of w/v) was measured and placed into the beaker and shaken for 30 min to provide even mixing. Next, it was sonicated for 30 min, after which the slurry was filtered using a Buchner funnel, vacuum flask, and Whatman ® no. 1 filter paper. With the filtered solution in the dark and under refrigeration, the sonication procedure was repeated two more times.
The collected filtrates were combined, and the liquid extract was placed into a 500-ml round-bottom flask. The aqueous methanol or aqueous ethanol solution was completely evaporated using a rotary evaporator at 50°C. Then, extracts were frozen at −80°C and freezedried. The dried extracts were stored in the dark for later analysis.
The proper amount of lyophilized extracts was dissolved in a 50% methanolic solution and filtered with a 0.45-µm filter before use in the HPLC procedure.

| Standard preparation
A stock solution of standards was prepared at 1 mg/ml (1,000 ppm) concentration from a 50% methanolic solution. Working solutions of the standards were prepared at 1, 5, 25, 50, 75, and 100 ppm, and a 6-point standard curve was prepared for each standard, based on the UV signal.

| HPLC conditions
A Waters model W2690/5 autosampler equipped with Waters 2,695 pumps and a Waters 2,489 UV detector was used for this study.
The column temperature was fixed at 25°C, and the injection volume was 20 µl. The flow rate was kept constant at 1.2 ml/min. A gradient program with the following proportions of solvents was used: We started the flow of mobile phase A as 95% and B as 5% to 5 min; this gradually increased to 20%, 22%, 25%, 27%, 40%, 45%, and 65% at 5,8,10,17,19,30,35, and 40 min, respectively, for mobile phase B, while mobile phase A decreased. Then, mobile phase B was reduced to 10% at 45 min, while mobile phase C increased to 90%. During the next 5 min, mobile phase C was 100%, followed by a return to the initial conditions for 10 min. The chromatographic peaks were identified based on their retention times and compared with the retention times of the authentic standards. Phenolic compounds found in the plants were calculated based on the external standard curve of the standard compounds. This analysis was carried out in duplicate.

| Total phenolic and flavonoid content
To determine the total polyphenol content, 0.5 ml of the sample extract was mixed with 2 ml of Folin-Ciocalteu's reagent. After 5 min, 2.5 ml of 7.5% Na 2 CO 3 solution was added, and the mixture was incubated for 90 min in the dark. The reaction mixture absorbance was measured at 760 nm, and the reaction mixture without the sample was used as a blank. Gallic acid was chosen as a standard, and a 6point standard curve was prepared (0-50 mg/L). The TPC of the plant extract was expressed as gallic acid equivalents (mg GA/g) for dry powder (Riahi et al., 2013;Singleton & Rosi, 1965). All samples were analyzed in duplicate.
To determine the TFC, 1 ml of diluted plant extract was mixed with 1 ml of 2% AlCl 3 methanolic solution. After incubation at room temperature for 15 min, the absorbance of the reaction mixture was measured at 430 nm. Quercetin was chosen as a standard, and a standard curve was prepared (0-50 mg/L). The TFC was expressed as mg quercetin equivalents/g for dry weight (Djeridane et al., 2006;Riahi et al., 2013). All samples were analyzed in duplicate.

| Disk diffusion assay
A disk diffusion assay was used to determine the antibacterial properties. Essential oils of hydrodistillation (HD) and microwave-assisted extraction (MAE) along with methanol and ethanol extracts of Thymus vulgaris and Thymbra spicata were individually tested against six bacteria. All the bacterial species were first inoculated into tryptic soy agar and incubated overnight at 37°C. After checking for purity, the bacteria were suspended in a 0.9% NaCl solution using a densitometer to adjust the final cell concentration to a 0.5 McFarland number (1 × 10 8 cfu/ml). Then, 100 µl of the bacterial suspensions was spread on Mueller-Hinton agar. The 6-mm-diameter, sterile, empty disks were either impregnated with 10 µl essential oils, or 20 µl of extracts was placed on the inoculated agar. Empty standard antibiotic disks were used as a control. The inoculated plates were incubated at 37°C for 24 hr. Antibacterial activity was determined by measuring the zone of inhibition in mm without including the radius of the disk.

| Statistical analysis
All determinations were conducted in duplicate, and results for each parameter were expressed as the mean ± standard deviation.
Data were evaluated by an analysis of variance (ANOVA) procedure.
Means were separated by the least significant difference (LSD) when significant (p < 0.05) treatment effects were found.

| Essential oil yields
The total yields of volatile chemicals from Thymus vulgaris (thyme) and Thymbra spicata (zahter) are given in Table 1. Results showed that microwave-assisted extraction (MAE) of zahter had a significantly (p < 0.05) higher oil yield (2.16% ± 0.16%) in comparison with its hydrodistillation (HD; 1.59% ± 0.06%). However, the distillation method did not affect the oil yield (%) for thyme (1.8 ± 0.14 for HD; 1.77 ± 0.05 for MAE). The essential oils of both thyme and zahter obtained with MAE were darker in color. Golmakani and Rezaei (2008) reported similar results for the essential oil of Thymus vulgaris: 2.52% for microwave-assisted extraction, with the exception of a longer extraction time (2 hr) with a higher power wattage (990 W) and 2.39% for hydrodistillation (4 hr). Ozel, Gogus, and Lewis (2003) used supercritical water extraction for Thymbra spicata essential oil, finding that an increase in the extraction temperature also increased oil yield, but they found a comparable extraction efficiency of 2% at 100°C. Similar results were reported by Uysal et al. (2015) for the essential oil of Thymbra spicata using solvent-free microwave extraction (2.5%) and hydrodistillation (2.4%). In another study, ultrasound-assisted ohmic heating provided much higher oil yield (%) for Thymus daenensis in comparison with hydrodistillation (Tavakolpour et al., 2017). Microwave extraction provides comparable yields with other methods and uses less time, energy, and only a small amount of water to hydrate the dry plants.

| Antioxidant activity of the essential oils
To measure antioxidant activity, two assays-the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and the FRAP TA B L E 1 Yield of free radical scavenging activity and FRAP value of essential oils obtained by hydrodistillation (HD) and microwaveassisted extraction (MAE) of Thymus vulgaris and Thymbra spicata (ferric reducing antioxidant power)-were used to determine the in vitro antioxidant activities of the essential oils of Thymus vulgaris and Thymbra spicata. The free radical scavenging activity is higher for lower IC 50 values. The amount of essential oil or extract needed to decrease the initial radical DPPH˙ concentration by 50% is used for the free radical scavenging activity and defined as IC 50 . Results of the antioxidant activity test are shown in Table 1. The free radical scavenging activity of thyme essential oil obtained with MAE (93.77 ± 13.0 µg/ml) was significantly (p < 0.05) higher than that found with HD (159.59 ± 12.79 µg/ml). When comparing the results with a corresponding commercial product (250 ± 0.01 µg/ml), the essential oil of thyme used in the current study had a much higher free radical scavenging activity (Teixeira et al., 2013). A similar pattern was observed for the free radical scavenging activity of the zahter essential oil. The MAE extraction (129.48 ± 11.58 µg/ ml) had a significantly (p < 0.05) higher free radical scavenging activity than the HD (181.56 ± 2.13 µg/ml). Earlier studies found that compounds such as carvacrol and thymol exist in high amounts in Lamiaceae family plants. Lee et al. (2005) found that volatile components such as carvacrol and thymol had antioxidant properties that hindered the hexanal inhibition by 95%-100% over a 30-day period at 5 µg/ml concentration. It was also reported that thymol and carvacrol were successfully used in active packaging as antioxidants (Ramos et al., 2014). The present study found that thyme had a higher antioxidant capacity than zahter. This could be due to the presence of a high thymol content in T. vulgaris. Previous studies found that thymol has a higher antioxidant activity due to the greater steric hindering effects of the phenolic group in thymol, which is greater than that of carvacrol (Yanishlieva, Marinova, Gordon, & Raneva, 1999), which corresponds with our results.
Our study is the first to report on the determination of the DPPH˙ free radical scavenging activity of zahter essential oil. In another study, the FRAP values of Thymus mastichina were studied for both essential oil and methanolic extracts, finding that due to the use of a phosphate buffer at pH 6.6, the essential oil underwent phase separation and did not produce proper results (Delgado et al., 2014

| Chemical composition of the essential oils
Gas chromatography-mass spectrometry analysis resulted in the identification of 25 compounds for Thymus vulgaris, as shown in Furthermore, Boruga et al. (2014) reported that the essential oil of thyme from Romania had lower thymol (47.59%) and p-cymene (8.41%) levels but a much higher γ-terpinene content (30.9%).

| Chemical compositions of the plant extracts
The chemical composition of the Thymus vulgaris and Thymbra spicata methanol and ethanol extracts is shown in 8.69%. High rosmarinic acid content has a great potential because of its possible antioxidant, antibacterial, and antiviral properties (Kostic et al., 2015). Our results agree with other studies reported for members of Lamiaceae family plants (Martins et al., 2015;Roby et al., 2013). Some researchers reported lower rosmarinic acid con- studied Ocimum bacillicum (sweet basil) and Thymbra spicata from Amasya, Turkey. They did not report rosmarinic acid content, but the highest percentages of phenolics were found to be trans-cinnamic acid (0.0545%) and syringic acid (0.0183%). We found a cinnamic acid content of 0.037% (methanol) and 0.021% (ethanol) for thyme and 0.026% (methanol) and 0.011% (ethanol) for zahter. Roby et al.
(2013) reported very high cinnamic acid content, with 28.54% for thyme. Many factors could influence the phenolic composition of the plants, such as the extraction solvent, methodology, location, soil composition, season, altitude (Kizil et al., 2015;Magwaza et al., 2016;Roby et al., 2013). Among the identified and quantified compounds, the second most frequently occurring compound after rosmarinic acid was benzoic acid. Its content ranged from 0.53% to 0.75% for zahter and 1.08% to 1.47% for thyme, when using ethanol and methanol extracts, respectively. Benzoic acid is an aromatic carboxylic acid naturally found in plant and animal tissues. It can be produced by the plant as protection against fungal attacks. Benzoic acid and its derivatives have been employed in the food industry as antifungal and antibacterial food additives (Olmo, Calzada, & Nunez, 2017). Based on all the available data, we found that Thymus vulgaris and Thymbra spicata provided a relatively high amount of rosmarinic acid and benzoic acid. Plant extracts with such high levels of rosmarinic acid and benzoic acid can be used as a natural food additive to improve the shelf life of food products.

| Total phenolic content and total flavonoid content of the extracts
The results of the TPC of the plant extracts are shown in Table 4.
The Thymus vulgaris methanol extract had significantly (p < 0.05) higher TPC than its 80% ethanol extract and also more than the  Table 4.
The TFC ranged from 3.24 to 7.285 mg QUE/g DW for all the extracts. Methanol extracts exhibited significantly (p < 0.05) higher amounts of TFC in comparison with ethanol extracts. Also, thyme extracts had a significantly (p < 0.05) higher amount of TFC than zahter extracts. Tohidi et al. (2017) reported between 1.89 and 8.55 mg QUE/g among the 14 Thymus species from Iran and found the highest TFC in T. vulgaris, with 8.55 mg QUE/g. However, in some other studies, the TFC was determined based on rutin equivalent (Fatma et al., 2014;Miliauskas, Venskutonis, & van Beek, 2004). Both the TPC and the TFC supplied information about the antioxidant capacity because higher phenolic content is associated with higher antioxidant activity.

| Antioxidant activity of the extracts
Reactive oxidative species, such as singlet oxygen, hydroxyl radical, peroxyl radical, and lipid hydrogen peroxide, cause oxidation of food products. Food oxidation can lead to the production of toxic compounds as well as a loss of nutrition. Also, oxidation TA B L E 4 Composition of phenolic compounds, total phenolic content (TPC), total flavonoid content (TFC), free radical scavenging activity, and FRAP value of Thymus vulgaris and Thymbra spicata obtained by methanolic and ethanolic extraction reduces the shelf life and consumer acceptability of food products.
Antioxidants scavenge radicals in food by donating hydrogen to these radicals and produce antioxidant radicals with low reduction potential. Also, due to the low reduction potential of these antioxidant radicals, they cannot cause oxidation of other molecules (Choe & Min, 2009

| Antibacterial activity of the extracts
Results of the antibacterial activity test based on disk diffusion are shown in  et al., 2004). This also aligns with our results. However, the fact that these major components occurred in a much higher quantity in hydrodistilled essential oil (Tables 1 and 2) indicates that minor components can also influence antibacterial properties. Similar results were reported earlier (Bounatirou et al., 2007;Rota et al., 2008). For Thymbra spicata, the microwave-assisted extraction of essential oil provided a larger inhibition zone (23-35 mm) for Gr (+) bacteria, while hydrodistilled oil provided a larger inhibition zone (14-19 mm) for Gr (−) bacteria. Overall, both essential oils showed a higher inhibition effect against Gr (+) bacteria in comparison with Gr (−) bacteria; this could be due to the difference in the wall type for Gr (+) and Gr (−) bacteria (Erturk et al., 2017;Teixeira et al., 2013). In addition to successful results obtained from in vitro 13.5a ± 0.87 14.5a ± 1.66 14a ± 1.42 11b ± 1.00 a Mean ± SD (n = 2). b Different letters in the same row denote a significant difference, LSD Fisher's test (p < 0.05).
TA B L E 5 Antibacterial activity of essential oils obtained by hydrodistillation (HD) and microwave-assisted extraction (MAE) of Thymus vulgaris and Thymbra spicata tested using the disk diffusion method (mm) antimicrobial studies, thyme essential oil or its bioactive components such as thymol and carvacrol have been successfully used as antimicrobial agents in surface washes on fresh produce and meat (Lu et al., 2014;Lu & Wu, 2010. The essential oil of both Thymus vulgaris and Thymbra spicata provided an antibacterial effect against these six bacteria. This is especially important because these bacteria are major foodborne pathogens (except E. coli ATCC 25922) associated with serious foodborne illnesses. Therefore, the essential oils of Thymus vulgaris and Thymbra spicata can be useful, natural antimicrobials for food preservation.

| CON CLUS ION
This study provided a comprehensive investigation of hydrophilic and hydrophobic fractures of Thymus vulgaris and Thymbra spicata for chemical composition, antioxidant activity, and antimicrobial activity. Concerning the extraction methodology for essential oils, microwave-assisted extraction provided results comparable to hydrodistillation for the GC-MS and antioxidant assays. To the best of our knowledge, the results of the FRAP assay for Thymbra spicata essential oil are the first to be reported. Bioactive compounds, such as rosmarinic acid, were high in both methanol extracts of the plants. Also, methanol extracts provided significantly (p < 0.05) higher bioactive compounds than ethanol extracts, which also re-

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.