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Antibacterial properties and chemical characterization of the essential oils from summer savory extracted by microwave-assisted hydrodistillation

Antibacterial properties and chemical characterization of the essential oils from summer savory (Satureja hortensis) extracted by microwave-assisted hydrodistillation (MAHD) were compared with those of the essential oils extracted using the traditional hydrodistillation (HD) method. While MAHD at 660 W required half as much time as HD needed, similar antibacterial efficacies were found from the essential oils obtained by the two extraction methods on two food pathogens (Staphylococcus aureus, a gram positive bacterium, and Escherchia coli, a gram negative bacterium). Also, as it was the case with the essential oils extracted by HD, that of MAHD indicated greater influence on S. aureus than on E. coli. The compositions of the extracted essential oils were also studied using GC-MS analysis. The same components with negligible differences in their quantities were found in the extracted essential oils using the two methods outlined above. Overall, to reduce the extraction time, MAHD can be applied at higher microwave levels without any compromise in the antibacterial properties of the essential oils extracted.


INTRODUCTION
Essential oils, which are the secondary metabolites produced within the various tissues of medicinal plants/herbs, are complex mixtures of volatile compounds such as terpenes (mostly monoterpenes and sesquiterpenes), phenolics and alcohols (17). It has been found that essential oils from spices, medicinal plants/herbs can possess certain levels of antimicrobial activities and as a result they can also be considered as potential antimicrobial agents against food pathogens (7). Satureja hortensis L., known as summer savory, from Labiatae family has been traditionally used as a stimulant, stomachic, carminative, expectorant, antidiarrheic, and aphrodisiac herb (23). Various biologically-active constituents from triterpene and flavonoid categories consisting of many components such as carvacrol, thymol, δ-terpinene, ρ-cymene, and α-and β-pinene (20) have been found in summer savory.
Carvacrol, which is a major component of savory essential oils with a concentration of 25-50 g kg -1 (20,22), has been known as a growth inhibitor against different bacteria and fungi (20,22). Effectiveness of the essential oils in preventing the growth of bacteria encourages research on the extraction of these oils The conventional method for the extraction of essential oils is hydrodistillation (HD), in which the essential oils are evaporated by heating a mixture of water and plant materials followed by the liquefaction of the vapors in a condenser.
Reportedly, this method suffers from several disadvantages including losses in the volatile compounds, long extraction time and degradation of some components through thermal and/or hydrolytic effects (14,17 Therefore, the objectives of this study were to focus only on summer savory and to quantitatively compare the components in the extracted essential oils when using MAHD and HD as extraction methods. Also, possible differences in the antibacterial activities of the extracted essential oils against a Gram-positive bacterium, Staphylococcus aureus, and a Gramnegative bacterium, Escherchia coli, were investigated.

Plant materials
Dried aerial parts of S. hortensis were provided by

Hydrodistillation
Thirty g of dried S. hortensis along with 600 mL distilled water was subjected to HD for 4 h using a Clevenger-type apparatus. The essential oils obtained this way was separated from water (due to its immiscibility with water and also as a result of a difference in its density level) and then dried over anhydrous sodium sulfate and stored in vials at 4 ºC. The extraction was repeated 3 times. with those of the literature.

Preparation of bacterial strains
The bacterial strains used as test microorganisms were E. h to allow the growing of the microorganism. Using sterile distilled water, serial dilutions were carried out to obtain a bacterial population of 10 6 -10 7 CFU/mL using the standard procedure of PCA (2).

Determination of antibacterial activity
The antibacterial activities of the essential oils were determined according to the disc diffusion method used by

Extraction yield
Data showing the lengths of times needed by MAHD and HD to reach the extraction temperature (so-called induction Antibacterial properties of the essential oils from summer savory times) and those indicating the total recoveries of essential oils at different power levels were published elsewhere (21). For MAHD at 660 W, the extraction started much earlier than that for HD. However, with the microwave power reduced to 220 W, the induction time with MAHD was longer than that with HD. Both MAHD and HD resulted in the same yield (3.1%, w/w) after the full extraction of essential oils. In Fig. 1, total times needed to fully recover the essential oils from summer savory at different power levels of microwave are compared against that with the traditional HD. No more essential oils were observed beyond these times. Total extraction time needed for HD to recover all the essential oils was 240 min.
However, MAHD needed much shorter times than did HD.
When microwave power was increased to higher levels, the extraction time was also reduced accordingly. According to Chen and Spiro (6) and Lucchesi et al. (17), at higher power levels, the microwaves (i.e., the radiations) can be absorbed more intensively resulting in a shorter extraction times.
Golmkani and Rezaei (11,12) investigated the effect of microwaves at higher power levels on the extraction of essential oils from Thymus vulgaris L. and Zataria multiflora Bois. They found no adverse effects from the microwaves on the molecular structures of the extracted essential oils in either case.  Kováts Retention Index (IK) relative to C9-C17 n-alkanes on the HP-5MS column. Antibacterial properties of the essential oils from summer savory

Structural image analysis of the leaves after the extraction
In order to investigate the changes in the cell during each extraction method, savory leaves were examined using a scanning electron microscope. Fig. 2a is a micrograph of the untreated leaf, which can be compared with those of the samples undergone 4 h of HD (Fig. 2b) and 2 h of MAHD at 660 W (Fig. 2c). The treated leaves indicated substantial differences compared to those untreated. Both HD and MAHD caused the burst of the glands and as a result the oil diffusion to the medium. These results are in good agreement with those of Golmakani and Rezaei (11,12) in the extraction of essential oils from Thymus vulgaris L. and Zataria multiflora Boiss.  Our results proved the antibacterial activities of essential oils from summer savory against two popular food pathogens, E. coli and S. aureus. Furthermore, use of microwave irradiation in MAHD for the extraction of essential oils did not disturb such activities. Therefore, essential oils obtained by such method can be used in many foods as natural preservatives as are the cases with the use of marjoram essential oils in sausage and thyme essential oils for beef (26).
Antibacterial properties of the essential oils from summer savory