Antibacterial , Antifungal Properties and Chemical Composition of Essential Oils of Satureja hortensis L . and Satureja khuzestanica Jamzad

Background & Aim: The aim of this study was to investigate the antibacterial, antifungal properties and chemical composition screening of essential oils of Satureja khuzestanica Jamzad and Satureja hortensis L. Experimental: For determination of antibacterial and antifungal activity of these essential oils, Staphylococcus aureus and Candida albicans were targeted, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each essential oil were determined individually. Also, chemical composition of essential oils was identified and characterized by gas chromatography armed by mass spectrometry (GC–MS). Results: The total of 22 and 21 compounds were identified in the essential oils of aerial parts of S. hortensis and S. khuzestanica, respectively. The major components of S. hortensis essence were carvacrol (56.36%), γterpinene (24.75%), p-cymene (5.81%) and the major constituents of S. khuzestanica essence was reported carvacrol (69.62%), γ-terpinene (9.25%) and p-cymene (8.36%). The obtained results showed the antibacterial and antifungal activity of both extracted essential oils against the tested pathogens. The MIC and MBC of S. hortensis and S. khuzistanica essential oils against S. aureus were determined 0.1 and 0.5 μl/ml, and 0.1 and 0.2 μl/ml against C. albicans, respectively. Recommended applications/industries: The results showed that S.hortensis oil had higher antimicrobial activity compare to S. khuzistanica. Type: Original Research Topic: Medicinal Plants Received December 18 2017 Accepted February 2


Introduction
The genus Satureja belonging to the (Lamiaceae) family, subfamily Nepetoideae, tribe Mentheae, consists of more than 200 species of herbaceous perennials worldwide.Distribution of the genus Satureja overlaps the region of southern and southeastern Europe, Asia Minor, and northern Africa, with the center of the genus area predominantly in the Mediterranean (Senatore et al., 1998).The genus of Satureja with name of "Marze" in Persian, consists of fourteen species has been reported in Flora Iranica (Rechinger, 1982).The species Satureja khuzestanica Jamzad is an endemic plant that widely distributed in the northern Khuzestan and southern Lorestan provinces of Iran (Mozaffarian, 2008).The essential oils extracted from some species of Satureja have showed wide range of biological activity including the antibacterial and antifungal activities (Sefidkon and Jamzad, 2000;Behravan et al., 2004), analgesic and anti-inflammatory (Ghazanfari et al., 2006), antispasmodic and antidiarrhoea (Hajhashemi et al., 2002), antioxidant, antidiabetic, antihyperlipidemic and reproduction-stimulatory activities (Abdollahi et al., 2003).The antimicrobial effects of essential oils derived from medicinal and aromatic plants are the basis of copious applications, in various revenue generating sectors such as pharmaceutical, nutraceutical, cosmetic and agronomy (Raut and Karuppayil, 2014).Carvacrol is a monoterpenoid phenol biosynthesized via aromatization of γ-terpinene to p-cymene and subsequent hydroxylation of pcymene.This phenol along with its two precursors γterpinene and p-cymene appeared as the major components in numerous phenolic essential oils of the Lamiaceae family (e.g., in thymus, oregano, and savory oil) (De Vincenzi et al., 2004).Hence carvacrol has a wide range of activities including antimicrobial, antioxidant, anticandidal, and anti-inflammatory properties which already investigated (Di Pasqua et al., 2007).Many of the previous studies demonstrated that the members of the genus Satureja showed a high antimicrobial activity due to the presence of thymol, carvacrol, and their precursors (Gulluceet al., 2003;Sahin et al., 2003).Also, several studies have been performed concerning the antimicrobial activity of essential oils of other Satureja species while antibacterial and antifungal effects of S. khuzistanica as an endemic plants in Iran has been never investigated.
The aim of the present investigation is evaluation of antibacterial and antifungal properties and also essential oil composition in Satureja khuzestanica Jamzad and Satureja hortensis L.

Plant material
The aerial parts (leaves and flowers) of two Satureja species including Satureja khuzestanica Jamzad and Satureja hortensis L.were harvested in July 2015 from Isfahan province in center Iran.Collection site information and soil physical and chemical characteristics field including pH and EC were determined (Table 1).

Essential oil extraction
Air-dried plant material was subjected to hydrodistillation for 2 h using a Clevenger-type apparatus according to the method recommended in BP (British Pharmacopoeia, 1988).Samples were dried with anhydrous sodium sulfateand kept in amber glass vials at 4 • C ± 1 • C until use.

Identification of the oil components
Volatile compounds from the aerial parts of plants were analyzed by GC/MS.The GC/MS analysis was carried out with an Agilent 5975 GC-MSD system equipped with.HP-5MS column (30 m x 0.25 mm, 0.25 μm filmthickness).Helium was used as carrier gas with flow rate of 1.0 mL/min.The oven temperature was kept at 50 °C for 4 min and programmed to 280 °C at a rate of 5 °C /min, and kept constant at 280 °C for 5 min, at split mode.The injector temperature was set at 280 °C.MS were taken at 70 eV.Mass range was from m/z 35 to 450.Identification of the essential oil components was accomplished based on comparison of retention times with those of authentic standards and by comparison of their mass spectral fragmentation patterns (WILLEY/ChemStation data system) (Adams, 2007).

Antimicrobial assay
Essential oils were individually tested against a panel of microorganisms including one Gram-positive bacteria, Staphylococcus aureus and one fungal strain Candida albicans collected from Isfahan province.Stock cultures of the bacteria were kept in 10% glycerol PBS (phosphate buffered saline) at 37°C.The yeast was cultured overnight at 30°C in Sabouraud dextrose agar (SDB) (Merck, Germany).

Antimicrobial test
These experiments were performed by the disc diffusion method with some modification.For the experiments, antibacterial activity of the crude extract was investigated against Staphylococcus aureus bacterial strains by the paper disk diffusion technique.The extract was redissolved in methanol to make a 100 mg/ml solution and then filtered.From this solution, 40-μl aliquots were transferred onto blank paper disks with a diameter of 6 mm.Dried disks were placed onto Mueller Hinton agar medium (Merck) previously inoculated with a bacterial suspension (ca.108 CFU/ml) and incubated at 35±1 °C for 24 h.antimicrobial activity of the crude extract was investigated against C.albicansfungal strains the disc diffusion method with some modification.The yeast was cultured overnight at 30°C in Sabouraud dextrose agar (SDB) (Merck, Germany).The extracts were dissolved in dimethyl sulfoxide (DMSO, 20 μl) before testing for antimicrobial activity.Normal saline was used for the preparation of inoculants having turbidity equal to 0.5 McFarland standards.Disc assay was applied on nutrient agar media with adjusting pH at 7.0.The fungus was maintained on Potato Dextrose Agar (PDA) at 25 ± 1ᵒC.Disks were placed onto Mueller Hinton Broth and LB broth (MILLER) medium.

Composition of the essential oils
Qualitative and quantitative analysis of the essential oils volatile profile are listed in Table (2).A total of 22 and 21 compounds were identified in the essential oil from the aerial parts of S. hortensis and S. khuzestanica respectively.The yield of S. hortensis oil is 2.01% (v/w), and components corresponding to 98.07% and consisted mainly of oxygenated monoterpenes (60.05%) and monoterpene hydrocarbons (36.11%), with a small amount of sesquiterpene hydrocarbons (1.5%) and oxygenated sesquiterpenes (0.41%).The major constituents of S. hortensis oil were carvacrol (56.36%), γ-terpinene (24.75%) and p-cymene (5.81%) (Table 2).

Antimicrobial activity
The results of antimicrobial activity of the essential oils obtained from S. hortensis and S. khuzistanica are shown in Table 3-6.The results showed that the essential oil of S. hortensis were most active against S. aureus (Table 3).According to these results, MIC and MBC of S. hortensis and S. khuzistanica oil on S. aureus were estimated 0.1 and 0.5 μl/ml, respectively.MIC and MBC of S. hortensis and S. khuzistanica oil on C. albicans were estimated 0.1 and 0.2 μl/ml, respectively.The results of antimicrobial activity of the essential oils obtained from S. hortensis and S. khuzistanica determined by Mueller Hinton Broth medium were shown in Table 5 and 6.Correlation coefficient between essential oil concentration of S. hortensis and diameter of inhibition zones in S. aureus and C. albicans were 85% and 90%, respectively.Correlation coefficient between essential oil concentration of S. khuzistanica and diameter of inhibition zones in S. aureus and C. albicans were 96% and 92%, respectively.The dominant monoterpene produced in glandular trichomes on the surface of the leaves, geraniol, α-terpineol, thuyanol-4, linalool, carvacrol, and thymol, is named after its dominant monoterpene.The six monoterpenes are all produced from geranyl pyrophosphate via a series of changes in configuration and hydroxylation and have fairly similar molecular structures.A major distinction is the phenolic nature of carvacrol and thymol, and the nonphenolic nature of the four other monoterpenes (Thompson et al., 2003).Carvacrol and thymol are structural isomers and have a phenolic hydroxyl at a different location on the phenolic ring.The hydroxyl group increased their hydrophilic ability, which could help them dissolve in microbial membrane and impair them (Sikkema et al.,1995).
According to Ghasemi Pirbalouti and Moalem (2013), analyzed the essential oil of the aerial parts of S. khuzistanicafrom different natural habitats in Southwest Iran, results showed that the carvacrol contents in different ecotypes ranged (42.5 -94.8 mg/ml) oil.Carvacrol and γ-terpinene were found in two oils but the amount of carvacrol in S. khuzestanica oil is higher than that of S. hortensis oil (Table 2).Hassanzadeh- Khayyat et al. (2012), analyzed the essential oil of the aerial parts of S. hortensis from the Khorasan province, Northeast of Iran and twenty-one compounds were identified at which the main oil constituents were carvacrol (55.69 %), γ-terpinene (24.93 %) and p-cymene (4.07 %).Biological activity of essential oils depends on their chemical composition which is determined by the genotype and influenced by environmental and agronomic conditions.It is well known that yield and yield components of plants are determined by a series of factors, including plant genetic, climate, edaphic, elevation, and topography and also an interaction of various factors (Golparvar and Hadipanah, 2016;Ardalani et al., 2017).
Previously, antimicrobial properties of essential oils from plants of the genus Satureja have been reported, although in variable degrees and spectrum of activity according to plant species and their composition.The previous study showed that essential oil and extract of S. khuzistanicaexhibited antimicrobial activities against Staphylococcus aureus subsp.aureus, Bacillus cereus, Escherichia coli, Salmonella enterica subsp.enterica, Shigella flexneri, Candida albicans, and Aspergillus brasiliensis (Hadian et al., 2011;Saei-Dehkordi et al., 2012;Ghodrati et al., 2015;Mahboubiand Kazempour, 2017) and Aspergillus flavus, A. niger, Penicillium sp., Fusarium sp.,Alternaria sp., Rhizopus sp., and Mucor sp (Sadeghi-Nejad et al., 2010).The results of the antimicrobial activity of S. hortensis L. extract were published.The effect of the essential oil was investigated by the more or less precise disk diffusion method.It was limited to food borne pathogens (Oussalah et al., 2007;Adiguzel et al., 2007).
Most of the studies on the mechanism of this phenolic compound focused on its effects on cellular membranes which alters its function and, in some instances, structure, causing swelling as a result of its increased permeability.Increases in cytoplasmic membrane permeability appear to be a consequence of the loss of the cellular pH gradient, proton motive force and decreased ATP levels, resulting in the death of the cell (Ultee et al., 2000).Also, synergism between carvacrol and its precursor p-cymene has been noted.P-cymene is a very weak antibacterial, and swells bacterial cell membranes to a greater extent than carvacrol does.By this mechanism p-cymene probably enables carvacrol to be more easily transported into the cell so that a synergistic effect is achieved when the two are used together.Carvacrol, which is the main component of Satureja species essential oils, has been considered as a biocidal, resulting in bacterial membrane perturbations that lead to leakage of intracellular ATP and potassium ions and ultimately cell death (Ultee et al., 2002).Carvacrol and thymol have strong antibacterial activities against Pseudomonas aeruginosa, in vitro (Kotan et al., 2013).Moreover, carvacrol exhibits antioxidant, antibacterial and antifungal activities (Ramak, 2013).

Conclusion
In the present work, the carvacrol, γ-terpinene and pcymene were the major components of S. hortensis and S. khuzistanica oil.The essential oils of these Satureja species can be placed into the phenolic class with regard to their high contents of monoterpene phenols and their precursors (p-cymene and γ-terpinene).The antimicrobial effect of carvacrol is due to damage in membrane integrity with changing in pH hemostasis and also equilibrium of inorganic ions.P-cymene does not have antimicrobial activity but it increases the antimicrobial activity of carvacrol.The obtained data showed that the tested oil was active against all the tested microorganisms.

Acknowledgement
This research project has been supported by Islamic Azad University, Jahrom branch, Iran.

Figure 1 .
Figure 1.Inhibition diameter zones obtained by paper disk diffusion method.

Table 1 .
Collection site information, some physical and chemical properties of soil collection site in the

Table 2 .
Chemical composition of essential oils of two Satureja species cultivated from Iran.

Table 3 .
Antimicrobial activity of the essential oil of S. hortensis determined by the disc diffusion method.

Table 4 .
Antimicrobial activity of the essential oil of S. khuzistanica determined by the disc diffusion method.

Table 5 .
Antimicrobial activity of S.hortensis essential oil determined by Mueller Hinton Broth medium.

Table 6 .
Antimicrobial activity of S. khuzistanica essential oil determined by Mueller Hinton Broth medium.