Chemical Composition of the Essential Oil of Satureja myrtifolia (Boiss. & Hohen.) from Lebanon

Abstract Satureja myrtifolia (Boiss. & Hohen.) Greuter & Burdeta medicinal plant belonging to the Lamiaceae family was collected from south of Lebanon and hydro-distilled by Clevenger method. Essential oil composition from aerial parts was analyzed by GC-MS technique. The odor of essential oil is characteristic, and clear yellow liquid oil was obtained after hydro-distillation. The yield of the essential oil was 1.25±0.02 % of dry matter (w/w). Thirty nine volatile components were identified in the Satureja myrtifolia oil, which shows a high amount of hydrocarbons class (57.82±0.1 %). Other classes were also identified such assesquiterpene hydrocarbons (12.96±0.1 %), oxygenated sesquiterpenes (10.65±0.2 %), phenolic compounds (10.32±0.1 %), acids (5.53±0.1 %), and monoterpenes hydrocarbons (2.21±0.1 %).In addition, a comparison with the unique study performed on Satureja myrtifolia was also carried out.


Introduction
Lebanon is known for its rich biodiversity especially with plants having medicinal properties. In fact, 2607 wild species from which 92 are endemics can be found in only 10452 km 2 1 . Among this biodiversity, S .myrtifolia (Syn: Micromeria myrtifolia Boiss. & Hohen) is a perennial plant that grows in rocky slopes (often limestone) exposed to sunlight, and mainly found in the Eastern Mediterranean regions (Turkey, Crete, Karpathos, Cyprus, Greece, Palestine, Jordan, Egypt, Lebanon and Syria). The aerial parts of S. myrtifolia are used as medicinal teas and infusions, in folk medicine for its carminative, appetizer, stimulant and pain relieving such as stomach ache [2][3][4][5][6] . The ethno medicinal use of S. myrtifolia for digestive perturbation, nervous system regulation, headache and hepatic diseases with Ruta chalapensis 5 . In Lebanon this plant is known as "Zoufa, Zoufi or Achnan Daoud" and is used in the traditional medicine for pneumonia, respiratory infections, cough, stomach ache, mouth ulcer, gastritis, cardiotonic, febrifuge, diuretic, stomachic, expectorant internally, while the cooled infusion is used as a gargle for laryngitis and as antiseptic externally 1 .
Chemical composition of aromatic and medicinal plants depends on cultivation area, climatic conditions, vegetation phase, genetic modifications and other factors 7,8 ; therefore characterization of flora present in different growing sites, countries and geographical zones is an important task 9 . Factors that determine the composition and the yield of essential oil are numerous and in some instances it is difficult to segregate these factors from each other, since many are inter dependent and influence one another 10 . These variables may include seasonal and maturity variation, geographical origin, genetic variation, growth stages, part of plant utilized and post harvest drying and storage 10,11 . There are many reports in the literature showing the variation in the yield and chemical composition of the essential oil with respect to geographical regions. Ozek et al. 12 have studied the essential oil composition of S. myrtifolia from Turkey chemotype. They found that β-caryophyllene (42.56 %), patchoulane (8.69 %), germacrene D (7.00 %), and δ-cadinene (6.99 %) were the main components of the oil. To the best of our knowledge, since 1992 (Ozek study) to date, no other study was carried on essential oil composition of S. myrtifolia from other Mediterranean countries.
The main goal of this study is to examine the chemical composition of essential oil of S. myrtifolia from Lebanon origin and to compare it to the unique Turkish study performed on this species.

Plant material
S. myrtifolia plant biomass was collected in south region of Lebanon from kfarhatta village (350 m altitude, longitude 35° 26'47.04", and latitude 33° 30'28.08") during July 2011. S. myrtifolia has been authenticated and cataloged in the herbarium of the Faculty of Agronomy (Holy Spirit University of Kaslik, Lebanon) under voucher specimen number MNV175a 1 .

Essential oils extraction
Air dried plant (40 g) was mixed with 500 mL of distilled water and the essential oil was isolated by hydrodistillation in a Clevenger type apparatus during 3 h. The obtained essential oil was separated from the water and dried over anhydrous sodium sulphate. Isolation of essential oils was performed in duplicate and the samples of essential oil were stored in a freezer prior to further analysis. The yield of the oil was 1.25±0.02 % of dry matter (w/w).

Essential oil analysis GC analysis
Analytical gas chromatography was carried out on a Thermo Electron Corporation gas chromatograph fitted with a DB-5 MS capillary column (30 m × 0.25 mm), 0.1 μm film thickness. Helium was the carrier gas (0.7 ml/min). Column temperature was initially 35 o C, then gradually increased to 85 o C at 5 o C /min rate, held for 20 min and finally raised to 300 o C at 10 o C /min held for 5 min. Diluted samples (1/100, v/v) of 1 μl were injected at 250 o C, manually and in the splitless mode. Flame ionization detection (FID) was performed at 310 o C.

GC-MS analysis
GC-MS was performed using an Agilent gas chromatograph 6890 coupled with Mass Detector 5975. The 7683 B auto sampler was injecting each time 1 μL of oil sample. GC-MS analysis was carried out using a fused silica capillary column Factor DB-5 MS, measuring 30 m x 0.25 mm internal diameter, film thickener of 0.1 μm; the oven temperature program adopted was 35 o C with an increase of 5 o C /min until 85 o C (20 min) and then to 300 o C (10 min) with an increase of 10 o C / min. Mass spectra were recorded at 70 eV, Ion source temperature 310 o C, Transfer line 320 o C, Acquisition: Full Scan 50-400 amu.
GC and GC-MS analysis was also run by using a fused silica HP Innowax polyethylen glycol capillary column (50 m × 0.20 mm), 0.20 μm film thickness. In both cases helium was used as carrier gas.
Most constituents were identified by gas chromatography by comparison of their retention indices (RI) with those of the literature 13 or with those of authentic compounds obtained from Sigma-Aldridch (Lebanon). The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) under the same operating conditions. Further identification was made by comparison of their mass spectra on both columns with those stored in NIST and Wiley 275 Libraries and our home-made library or with mass spectra from literature 13 . Standards of some EOs (Essential Oil) of known composition (such as EO of Rosmarinus officinalis L. -Phytosun' Aroms, Plelo, France) have been injected in similar conditions to check the retention times and the mass spectra. Component relative concentrations were calculated based on GC peak areas without using correction factors.

Statistical analysis
For GC-MS raw data were imported into the statistical software package MS Stat (ANALYT MTC, Muehlheim, Germany, MS Statistical Software Version 3.02u); the software was looking for differences in the mass fragments (m/z), the retention time and the abundance.

Results and discussion
The yield of the essential oil from air-dried aerial parts of S. myrtifolia was 1.25±0.02 % (dry wt. basis). Thirty nine components were identified in oil and their composition is presented in Table 1. Five major volatile compounds, namely Tricosane (13.65±0.02 %), tetracosane (12.31±0.05 %), caryophyllene oxide (10.65±0.02 %), thymol (10.32±0.03 %), and β-caryophyllene (8.99±0.04 %), were found in oil. Volatile constituents identified in the S. myrtifolia belong to the following classes of organic compounds: Hydrocarbons, sesquiterpenes hydrocarbons, oxygenated sequiterpenes, phenolic compounds, acids and monoterpenes hydrocarbons. Hydrocarbons were the most abundant class of organic compounds in S. myrtifolia oil which consist 57.82±0.01 % of total oil, followed by sesquiterpene hydrocarbons (12.96±0.01 %) and oxygenated sequiterpenes (10.65±0.01 %). About 10.32±0.03 % of total oil composition consists of thymol, a typical phenolic compound. Also minor classes of organic compounds were identified such acids (5.53±0.01 %), and monoterpenes hydro- Comparison of the percentages of the main components in S. myrtifolia oil analyzed in our study and previously reported in literature by Ozek et al. 12 are summarized in Table 2. Air-dried parts of S. myrtifolia Turkish plant where found to produce an oil yield of 0.03 % while the same parts of S. myrtifolia Lebanese plant produce 1.25±0.02 % of oil. In present study tricosane was the dominat constituent (13.65±0.02 %) in Lebanese oil, while β-caryophyllene (42.56±0.04 %) was the major constituent in Turkish oil. βcaryophyllene was also present in Lebanese oil but about five fold less (8.99±0.04 %) with regard with the one obtained from Turkish oil (42.56 %). Besides, five other major volatile compounds such It should be noted that the Lebanese plant contained interesting amount of thymol, which was not the case for the Turkish plant. Thymol is characterized by a pleasant aromaticsmell and is known for its antimicrobial, antiseptic and antioxidant properties. This variation in component level is significant, but not impossible because chemical polymorphism is significantly characteristic of many Labiatae family species 14 .
The major components in both Turkish and Lebanese S. myrtifolia oils were different. The odor of these compounds was not similar. Indeed, the absence of thymol compound in Turkish oil which has astronger, spicy and woody smell, suggests that the odor perception of S. myrtifolia oil reported from Turkey is different than that of Lebanese oil. However, five major volatile compounds (germacrene-D; δ-cadinene; patchoulane; α-humulene and nerodiol) were not present in the Lebanese oil. These compounds have woody, spicy, smoky, flowery, herbal, green, waxy, floral and citrusodor (Table 1). A remarkable quantitative and qualitative difference exists between both oils ( Table 2). These differences may be dependent on climatic conditions prevailing during plant development and their genetic potential 7,8,9 . Indeed, the composition of essential oils as well as other chemical molecules of plants may vary considerably depending on annual climatic changes and genotype 15,16 . These parameters influence the total content of essential oils and their compositions. Unfortunately, weather conditions were not reported in studies performed in Turkey.

Conclusion
The present study contains some new information and gives a significant amount of information about the essential oil chemical composition of S. myrtifolia species from Lebanon geographical origin. Our study highlighted the differences in essential oils composition by comparison with the unique study performed up till now. Nevertheless, this comparison may gain in precision if the same analytical methods are used. Further investigations are necessary to ascertain our results and therefore could help for S. myrtifolia genetic programs improvement and may be to diversify the industrial uses.