Chemical Composition and Cytotoxic and Antioxidant Activities of Satureja montana L. Essential Oil and Its Antibacterial Potential against Salmonella Spp. Strains

1 Laboratory of Analysis, Treatment and Valorisation of Environmental Pollution and Products, Faculty of Pharmacy, Tunisia City Avicenne, 5000 Monastir, Tunisia 2 UR Study & Management of Urban and Coastal Environments, LARSEN, National Engineering School, BP 1173, 3038 Sfax, Tunisia 3 Laboratory of Biochemistry, Faculty of Medicine, 5019 Monastir, Tunisia 4 Range Ecology Laboratory, Arid Land Institute of Medenine, 4119 Medenine, Tunisia


Introduction
Salmonella species are responsible for causing foodborne bacterial illnesses.Human salmonellosis is generally increasing worldwide because of contaminated foods consumption, especially those of animal origin.Foodborne salmonellosis is characterized by gastrointestinal disorders manifested predominantly by diarrhea, fever, and abdominal cramps.Since the disease has not only an economic impact on individuals and countries but also affects people's health and well-being, many efforts have been spent to find out approaches reducing or eliminating Salmonella that contaminates foods [1].In this context, food safety has become a complex problem related to food products frequently introduced into the market.Indeed, these require generally a longer shelf life and a greater safety assurance, that is, lack of foodborne pathogenic microorganisms [2,3].Alternative preservation techniques based on the use of naturally derived ingredients are under investigation for their application in food products.Because of negative consumer perceptions of chemical preservatives, attention is shifting toward natural products used as alternatives, especially plant extracts, including the essential oils (EOs) and essences of plant extracts [4].However, plants EOs are a source of bioactive molecules and have been widely traditionally used and commercialized to increase the shelf life and food safety [5,6]; these are gaining significance for their potential as preservative [4,7].EOs are volatile, natural and complex compounds characterized by a strong odor which is the result of plants aromatic secondary metabolites [8].In addition, demand is growing for natural and highquality products as well, and are used in food preservation and in aromatherapy, the EOs have multiple antimicrobial, that is, antifungal and antiviral [8][9][10][11], as well as anticancer effects [8].
Satureja montana L., commonly known as winter savory or mountain savory, belongs to the Lamiaceae family, Nepetoideae subfamily, and Mentheae tribe and is a perennial semishrub (20-30 cm) that inhabits arid, sunny, and rocky regions.S. montana L. is native to the Mediterranean and is found throughout Europe, Russia, and Turkey [4].S. montana L. is a well-known aromatic plant, frequently used as traditional medicinal herb [12] and spice for food and teas.It is used in Mediterranean cooking, mainly as a seasoning for meats and fish and a flavoring agent for soups, sausages, canned meats, and spicy sauces [4,13].The EOs derived from plants of Satureja species possess strong antibacterial activities of different extents against organisms of importance to food spoilage and/or poisoning, as well as to those of interest to the medical field such as Salmonella, Listeria, and Staphylococcus.In view of their broad activity, these EOs may find industrial applications as natural preservatives and conservation agents in the cosmetic and/or food industries and as active ingredients in medical preparations [3].In this context, S. montana L. has biological properties that are related to the presence of its major EO chemical compounds which are carvacrol and p-cymene [12].
The aims of the present study were to assess the chemical composition and antioxidant and cytotoxicity activities of Satureja montana L. EO and to evaluate the antimicrobial effect of this winter savory against several foodborne pathogens especially the most common causative agent of foodborne salmonellosis.

Plant Material and Essential Oil Extraction. S. montana
L. plants were freshly collected in 2011 during the period of full flowering on the mountain in the south of France (Mediterranean climate country and mountainous region).The species was identified according to the forester flora of France [14].Aerial parts of the winter savory were dried at room temperature.Then EO was extracted by hydrodistillation (HD) for 3 h with 500 mL distilled water using a Clevenger-type apparatus according to the European Pharmacopoeia [15].The EO was collected and dried over anhydrous sodium sulfate and then stored in sealed glass vials in a refrigerator at 4 ∘ C prior to analysis.EOs yield was calculated based on dry weight.

Gas Chromatography (GC). An Agilent Technologies
6890N GC equipped with HP-5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 m; Hewlett-Packard) and connected to a FID was used.The column temperature was programmed at 50 ∘ C for 1 min, then 7 ∘ C/min to 250 ∘ C, and finally left at 250 ∘ C for 5 min.The injection port temperature was 240 ∘ C, while that of the detector was 250 ∘ C (split ratio: 1/60).
The carrier gas was helium (99.995% purity) with a flow rate of 1.2 mL/min.The analysed EO volume was 2 L.Percentages of the constituents were calculated by electronic integration of FID peak areas, without the use of response factor correction. Mean percentage of S. montana L. volatiles compounds represented the average calculated on three individuals.Retention indices (RIs) were calculated for separate compounds relative to C 8 -C 26 n-alkanes mixture (Aldrich Library of Chemicals Standards) [16].

Gas Chromatography/Mass Spectrometry (GC/MS).
The volatile compounds isolated by HD were analysed by GC/MS, using an Agilent Technologies 6890N GC.The fused HP-5MS capillary column (the same as that used in the GC/FID analysis) was coupled to an Agilent Technologies 5973B MS (Hewlett-Packard, Palo Alto, CA, USA).The oven temperature was programmed as before (50 ∘ C for 1 min, then 7 ∘ C/min to 250 ∘ C, and then left at 250 ∘ C for 5 min).The injection port temperature was 250 ∘ C and that of the detector was 280 ∘ C (split ratio: 1/100).The carrier gas was helium (99.995% purity) with a flow rate of 1.2 mL/min.The MS conditions were as follows: ionization voltage, 70 eV; ion source temperature, 150 ∘ C; electron ionization mass spectra were acquired over the mass range 50 to 550 m/z.

Volatile Compounds Identification.
The volatile compounds of S. montana L. aerial parts were identified by comparing the mass spectra data with spectra available from the Wiley 275 mass spectra libraries (software, D.03.00).Further identification confirmations were made referring to RI data generated from a series of known standards of nalkanes mixture (C 8 to C 26 ) [16] and to those previously reported in the literature [17][18][19].

Antioxidant Activity
DPPH Radical Method.The free-radical scavenging activity of S. montana L. EOs was measured by 2,2-diphenyl-2picrylhydrazyl (DPPH, Sigma-Aldrich, France) using the method described by Hatano et al. [20].One milliliter of the EO at known concentrations was added to 0.25 mL of a DPPH methanolic solution.The mixture was shaken vigorously and left standing at room temperature for 30 min in the dark.The absorbance of the resulting solution was then measured at 517 nm and corresponded to the ability of the EO to reduce the stable radical DPPH to the yellow-colored diphenylpicrylhydrazine.The antiradical activity was expressed as IC 50 (g/mL), the extract dose required to cause a 50% inhibition.Absorption of a blank sample containing the same amount of methanol and DPPH solution acted as negative control.All determinations were performed in triplicate.The ability to scavenge the DPPH radical was calculated using the following equation: where  0 was the absorbance of the control at 30 min and  1 was the absorbance of the sample at 30 min.All samples were analyzed in triplicate.
2.4.Cytotoxic Activity.The S. montana L. EOs was screened for cytotoxic activity using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphynyltetrazolium bromide] colorimetric assay against the human lung adenocarcinoma epithelial cell line (A549) as described previously [21].Briefly, cells were treated with concentrations of EOs ranging from 12.5 to 800 g/mL and seeded in 96-well microplates.The EO was first dissolved in DMSO and then in RPMI 1640 supplemented with 2% foetal calf serum (FBS).The final DMSO concentrations in the test medium and controls were 1% (v/v).Each concentration was tested in quadruplicate together with that in the control and repeated two times in separate experiments.After incubation for 24, 48 and 72 hours, the medium in each well was collected and the cytotoxic effect was measured with the MTT colorimetric assay.To determine the cell viability, 20 L of MTT (5 mg/mL) was added to each well and cells were cultured in additional incubation for 4 h.After washing the supernatant out, the insoluble formazan product was dissolved in acidified isopropanol.Then, optical density (OD) of 96-well culture plates was measured using an enzyme-linked immunosorbent assay (ELISA) reader at 540 nm.The OD of formazan formed in untreated control cells was taken as 100% of viability.The antibacterial effect was also tested against 31 strains belonging to Salmonella genus, including 12 species of enteritidis responsible for collective food intoxication isolated in hospital Fattouma Bougruiba Monastir (Tunisia) in June 2000.These microorganisms were kindly provided by Prof. Rhim Amel from the Regional Laboratory of Public Health of Monastir (Tunisia), and the serotyping of the strains was performed at the Pasteur Institute, Tunisia.

Disc-Diffusion Assay. Antimicrobial activity testing was done according to the Clinical and Laboratory Standards
Institute [22] guidelines (CLSI, 2006).For the experiments, a loopful of the microorganisms working stocks were enriched on a tube containing 9 mL of Mueller-Hinton (MH) broth and then incubated at 37 ∘ C for 18-24 h.The overnight cultures were used for the S. montana L. EO antimicrobial activity test, and the optical density was adjusted to 0.5 McFarland turbidity standards with a DENSIMAT (Biomérieux).The inoculums were streaked onto MH agar plates at 37 ∘ C.
Sterile filter discs (diameter 6 mm, Whatman Paper no. 3) were impregnated with 10 L of EO placed on the MH agar mediums.The treated Petri dishes were placed at 4 ∘ C for 1-2 h and then incubated at 37 ∘ C for 18-24 h under anaerobic condition.The antibacterial activity was evaluated by measuring the growth inhibition diameter zone around the disk.Standard disks of the antibiotic ciprofloxacin (5 g) served as the positive antibacterial controls according to the committee of the French Society of Antimicrobial for all strains except L. monocytogenes which standard disks of the antibiotic gentamycin (10 g/disc), served as the positive antibacterial controls [23].Each experiment was carried out in triplicate and the mean diameter of the inhibition zone was recorded.

Microwell Determination of MIC and MBC.
The minimal inhibition concentration (MIC) and the minimal bactericidal concentration (MBC) values were determined for all bacterial strains used in this study as described by Güllüce et al. [24].The inoculums of the bacterial strains were prepared from 12 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity.The S. montana L. EOs dissolved in 10% dimethylsulfoxide (DMSO) were first diluted to the highest concentration (50 mg/mL) to be tested, and then serial twofold dilutions were made in a concentration range from 0.0488 to 50 mg/mL in 5 mL sterile test tubes containing nutrient broth.The 96-well plates were prepared by dispensing into each well 95 L of nutrient broth and 5 L of the inoculum.A 100 L aliquot from the stock solutions of each EO was added to the first wells.Then, 100 L from the serial dilutions was transferred into 100 L consecutive wells.The last well containing 195 L of nutrient broth without EO and 5 L of the inoculum on each strip was used as the negative control.The final volume in each well was 200 L.The plates were incubated at 37 ∘ C for 18-24 h.
After incubation, bacterial growth was evaluated by the presence of turbidity and a pellet on the well bottom.The MIC was defined as the lowest concentration of the compounds to inhibit the microorganism growth.The BMC values were interpreted as the highest dilution (lowest concentration) of the sample, which showed clear fluid with no turbidity development and without visible growth.All tests were performed in triplicate.

Statistical Analysis.
Values were expressed as means ± standard deviation.Analysis of variance was conducted, and differences between variables were tested for significance by one-way ANOVA with an SPSS 11 (Statistical Package for the Social Sciences) programme.Differences at  < 0.05 were considered statistically significant.

Results and Discussion
3.1.Essential Oil Composition.The S. montana L. EO chemical composition was investigated using both GC and GC/MS techniques.The volatile compounds composition was based on the chemical functions (acids, alcohols, aliphatics, aromatics, sulfurs, and terpenes).The oil yield of the French variety of the winter savory Satureja montana was 1.56% while that of the Croatian variety was 1.7% [12].The identified components, their percentages, their calculated RI, and their comparison according to the RI values previously published in the literature are listed in Table 1, considering the compounds elution order on the HP-5MS column.A total of 32 compounds were identified, accounting for 99.85% of the total oil content (Table 1).These compounds were divided into five classes that are monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and others.This oil was characterized by very high percentage of monoterpenes (92.31%) and especially the oxygenated ones (59.11%) which constituted the predominant class as was found for the majority of S. montana L. [12].Furthermore, S. montana L. EO is characterized by a high content of the phenolic carvacrol (53.35%).Other important compounds were -terpinene (13.54%) and the monoterpenic hydrocarbons p-cymene (13.03%).Moreover, low percentages of -caryophyllene (2.23%), linalool (1.84%), -terpinene (1.7%), myrcene (1.3%), -bisabolene (1.3%), and the oxygenated compounds borneol (1.14%) were evidenced.The EOs of the aerial parts of S. montana L. collected in Croatia contained an important percentage of carvacrol (45.7%) [12].Twenty-six chemical compounds were identified in the EO of winter savory spice (S. montana L.) originating from Albania [4], and the major constituents were thymol (28.99%), p-cymene (12.00%), linalool (11.00%), and carvacrol (10.71%).
In fact, the composition of the EO of S. montana L. depends on many factors, such as environmental conditions, harvest time, geographic origin, and storage conditions which seem to have a significant influence on the EOs relative compounds concentration in S. montana [25,26].Various isolates of winter savory from Croatia, Bosnia, and Herzegovina have carvacrol (up to 84.19%) as the main constituent [27], and a review of the published literature [25] reveals that the composition of S. montana oil shows large variations in the relative concentration of major components: carvacrol (5%-69%) was found to be the major component among the oxygenated monoterpenes, linalool (1%-62%), -terpinene (1%-31%), and p-cymene (3%-27%) arising from the existence of different chemotypes, and environmental conditions seem to have a significant influence on the relative amounts of EO components of S. montana [25,26].

Antioxidant Activity.
The antioxidant properties of S. montana L. bioactive extracts were evaluated by DPPH radical scavenging, one of the most used methods to evaluate evaluate EOs, and phenolic extracts.The antioxidant activity in this study showed that the IC 50 value was 410.5 ± 4.27 g/mL.
In this study, S. montana L. EO IC 50 was higher than that found by Güllüce et al. [24] for Satureja hortensis EO (350 ± 5 g/mL).In contrast, Cavar et al. [28] reported a higher IC 50 for S. montana L. EO with 13-folds that of the present study (5490 g/mL).More recently, Serrano et al. [29] found an IC 50 value of 508.45 ± 5.80 g/mL for the same species.
Table 1 shows that EO of S. montana L. was markedly rich in oxygenated terpenes which may act as radical scavenging agents.However, Tepe et al. [30] indicate that EOs containing oxygenated monoterpenes and/or sesquiterpenes have greater antioxidative properties.This EO antioxidant property (DPPH radical-scavenging activity) is well needed for food industry as possible alternative to synthetic preservatives.In this context, S. montana L. EO gave interesting results, indicating the presence of potent antioxidant compound(s) that can be exploited as alternatives for use in the food and cosmetic industries and/or as nutraceuticals.

Cytotoxic Activity.
Cell viability, determined by the ability of the cells to metabolically reduce MTT to a formazan dye, was performed after 24, 48, and 72 h exposure to EO at different concentrations ranging from 12.5 to 800 g/mL.A concentration-and time-dependent inhibitory effect on human respiratory epithelial cell line (A549) was observed.After 24 h of incubation, cytotoxicity was considered whenever cell survival percent was less than 50.The extract was not cytotoxic towards A549 cell line in all tested concentrations.But after 48 and 72 h EO exposure, the S. montana L. IC 50 is 400.00 ± 0.02 g/mL and 11.00 ± 0.01 g/mL, respectively (Figure 1).
Koparal and Zeytinoglu [31] demonstrate that carvacrol, the predominant monoterpene in S. montana L., was very potent cell growth inhibitor of A549 cell line.
The cytotoxicity is likely due to the relatively high phenolic compounds concentrations, particularly carvacrol as described previously [31].The cytotoxic effects of carvacrol have been previously described in different cellular models, especiallyin tumor cell lines.Comparative evaluation of its components' cytotoxicity (generally recognized as safe) showed that this type of oil and its major component carvacrol (which constitutes 53.35% of the oil) were highly cytotoxic against human metastatic breast cancer cells, MDA-MB 231, and that the compound could have a potential therapeutic significance in treating cancer [32].

Antimicrobial Activity.
In the present study, S. montana L. EO antimicrobial activities against microorganisms examined and its potency were qualified by inhibition zone diameter and quantified by MIC and MBC values.The results given in Table 2 showed that the EO of S. montana L. had substantial antimicrobial activity against 13 reference strains and 31 strains belonging to Salmonella genus; with 12 The maximum activity of this oil was observed against Gram-positive L. monocytogenes, but this oil had poor activity on the growth of Enterococcus faecium resistant to ciprofloxacin.The oil of S. montana L. was generally active against the majority of food intoxitcation isolated S. enteritidis.The diameters of growth inhibition were ranging from 20 mm to 50 mm.Concerning 19 food isolated strains of Salmonella spp., the EO (7.9 mg/disc) was very active showing a clear zone of inhibition ranging from 15 to 35 mm.
The MIC and MBC values of S. montana L. EO against all the studied strains were summarised in Table 2.The EO was efficient against all tested bacteria with MIC about 0.39 mg/mL to 1.56 mg/mL for Gram-positive bacteria and from 0.78 to 1.56 mg/mL for Gram-negative bacteria.For Salmonella spp.strains, MIC values were ranging from 0.39 to 0.78 mg/mL.The MBC values were also important, and low concentration of S. montana L. EO was sufficient to eliminate the growth of Gram-positive bacteria (MBC: 0.78 mg/mL) and E. coli, E. feacalis, V. alginolyticus, and S. typhimurium (MBC: 0.78 mg/mL).It has shown also that 0.39 mg/mL of EO was sufficient to stop the growth of several pathogenic Salmonella species including S. enteritidis responsible for collective food intoxication.
A number of reports indicate that EOs containing carvacrol, eugenol, or thymol have the highest antimicrobial properties [58,59].The chemical composition of this sample of S. montana L. oil demonstrates this relationship between high antimicrobial activity and the presence of phenolic components since savory oil was active against methicillinresistant S. aureus, vancomycin-resistant Enterococcus faecium, and multidrug-resistant Serratia marcescens [12].However, the antimicrobial activities of Satureja species do not arise only from the carvacrol and thymol content since the oil of S. cuneifolia, which is relatively rich in -cubebene, limonene, -pinene, spathulenol, and -caryophyllene, also displayed relatively good activity.It has also to be considered that minor components, as well as a possible interaction between the substances, could also affect the antimicrobial properties.It has also been reported that Gram-positive bacteria are more susceptible to EOs than Gram-negative bacteria [60].The relative tolerance of Gram-negative bacteria to EOs has been ascribed to the presence of a hydrophilic outer membrane which can block the penetration of hydrophobic components through the target cell membrane [61].We conclude that the oils investigated can be used as a complementary therapy where a single antimicrobial agent is often ineffective; however, it is most important that the composition of the oil is known for maximum efficacy.

Conclusion
In conclusion, S. montana L. EOs represent a source of natural antibacterial and antioxidant substances with potential applicability in food systems to prevent the growth of foodborne pathogenic and spoilage bacteria, as well as oxidation, and to extend food shelf life.However, further research is still required to confirm such applicability and to evaluate the safety of S. montana L. EO.

Table 1 :
Mean percentage of the Satureja montana L. (France) essential oil components and RI comparison according to the literature.

Table 2 :
Antibacterial activity of S. montana L. essential oil against human pathogenic bacteria using agar disc diffusion method and determination of MIC (mg/mL) and MBC (mg/mL) values.