Myrtus communis L . : Characterisation of Essential Oil of Leaves and Fatty Acids of Seeds Using Gas Chromatography-Mass Spectrometry ( GC / MSD )

In this study, the chemical contents of essential oil acquired from Myrtus communis leaves using hydrodistillation and seed oil obtained using cold press method analyzed with gas chromatography-mass spectrometry (GC/MSD). According to the analyse result, forty-five component were identified and 1,8-cineole (21.68%), alpha-pinene (18.02%), linalool (14.12%) and alpha-terpinyl acetate (10.40%) were detected as major compounds in the M. communis leaf essential oil. On the other hand, seven different fatty acids were determined in seed oil. Linoleic acid (77.59%) and palmitic acid (10.36%) were detected to be major fatty acids in the M. communis cold-pressed seed oil. The present study has shown that the nutritional and other industrial use of M. communis leaves and seeds are possible due to their rich phytochemical contents of essential oil and seed oil.


Introduction
Essential oils are secondary plant metabolites that found in various portions of plants such as leaves, flowers, roots, seeds, fruit and wood [1].Essential oils, also known as etheric oils by people, can contain terpenic hydrocarbons and their oxygenated derivatives as well as alcohols, organic acids, phenols and ketones [2].The basic components of essential oils are commonly mono and sesquiterpenes [3,4] and the main plant families which essential oils derived Asteraceae, Myrtaceae, Lauraceae, Lamiaceae, Rutaceae and Zingiberaceae [5].
Myrtus communis, one of the main sources essential oils, is a member of the Myrtaceae family and naturally distributed in the Mediterranean region as an evergreen shrub with bright green leaves and edible fruits in black and white [6].M. communis is an evergreen shrub, common in Mediterranean woodlands, maquis and garrigue [7,8].Myrtus communis (myrtle) is a common endozoochorous shrub species of forest patches in lowland agricultural Mediterranean areas [9].It is also called "mersin" or "murt" by the people in Turkey.M. communis has been used for medicinal and nutritional purposes since ancient times.The leaves and fruits of M. communis are traditionally used as antiseptic, disinfectant, and hypoglycemic agents [10,11].In the previous literatures, Elfellah et al. were studied on anti-hyperglycaemic effect of an extract of Myrtus communis in Libya [12], and Al-Hindawi et al. were described an anti-inflammatory activity of some Iraq plants using intact rats [13].
The leaves of M. communis are widely known for the presence of essential oils, and their composition determines the specific aroma and flavor of the plant.The various applications of this oil are generally valued for kitchen purposes.Fresh and/or dried leaf oils are used in cosmetics, and beverage industries [11].Some of the known biological activities of M. communis leaf essential oil include antioxidant activity and anti-mutagenic activity [11], antimicrobial activity, antibacterial activity and antifungal activity [14].
M. communis is an important plant not only because of the essential oils of leaves, but also because of the fatty acids contained in the fruits.Together with the increasing interest of consumers in natural products, seed oils of plants have found application field in food, medicine and cosmetic industries.Linoleic acidrich oils are used as raw materials in the production of conjugated linoleic acid, a therapeutic nutrient with antioxidant and anti-tumor characters [15,16].Certain polyunsaturated fatty, vitamin F, are essential for skin growth and guard [17].
Fatty acid composition of plants is widely affected by the geographical origin of the plant [18][19][20].Most of the studies on M. communis fruit has focused on it's volatile components and phenolics.However, few work has been done on the fatty acid contents of the M. communis fruits and seeds.M. communis fruits have a significant oil content about 15.40% and are rich in polyunsaturated linoleic acid [21,22].Messaoud et al. were identified approximately 79.10% of the oil studied that the major constituents were α-pinene and 1,8-cineole in Tunisia [Messaoud].Plants rich in linoleic acid are being cultivated commonly and their oils are extensively used [23].However, except for a few studies, it is difficult to find data in the literature on M. communis seed oil composition, especially from Turkey [22,24].
In this study, chemical contents of M. communis leaf essential oil and seeds oil were investigated from Gökova (Muğla), Turkey to reveal potential use of M. communis essential oil and seed oil in food, industrial, and pharmacological applications.

Plant material
M. communis leaves and fruits were gathered from plants growing wild in November 2016 in Gökova (Muğla), Turkey.Leaves of M. communis have been air-dried in room temperature (25 °C) for seven days.
Seeds were cleaned manually to remove mesocarp and other materials, then allowed to air-dry for ten days in ambient temperature (25 °C).

Isolation of essential oil and seed oil
Air-dried M. communis leaves were subjected to hydrodistillation for 3h to obtain essential oil while the seed oil of was obtained by cold press of air-dried seeds.The resulting oils were dried with anhydrous sodium sulfate, filtered and stored in a brown glass pot at +4 °C until analysis by Gas Chromatography-Mass Spectrometry (GC/MSD).

Analytical procedure for essential oil
M. communis leaf essential oil was weighed (30 mg) into a volumetric flask, dissolved in hexane (2 mL) for injection to the gas chromatography mass spectrometry instrument.GC/MS analyses were carried out using GC equipped with a Multi Mode Inlet (MMI) (280 °C).The carrier gas was He (2.1 mL/min), and the oven temperature was held at 60 °C for 5 min, then increased up to 220 °C at a rate of 2 °C/min and held at this temperature for 10 min.The injected volume was 2 μl and the split ratio was 40:1.The library search was carried out using NIST and Wiley 2008 GC/MS libraries.

Analytical procedure for seed oil
100 µL of seed oil was weighed into a volumetric flask and 9.80 mL of hexane was added.This mixture was vortexed for 5 min and then 100 µL of 2N KOH (dissolved in methanol) solution was added.The lidded tube was vortexed for 1 min followed by this addition, centrifuged at 4000 rpm for 10 min, the supernatant was removed for injection.
A quadrupole mass spectrometer (MS) and a J&W 112-88A7, HP-88 (60 m x 250 µm x 0.25 µm) column were used for the gas chromatography.For GC/MS detection, an EI system with ionization energy of 70 eV was used. 1 μL of sample was injected automatically in the split mode (50:1).Mass range was from m/z 50 to 650 amu.The library search was carried out using NIST and Wiley 2008 GC/MS libraries.

Results
In this study, chemical compounds of essential oil and seed oil of M. communis were examined using GC/MS.Forty-five components include alpha-pinene, 1,8cineol, linalool, alpha-terpinyl acetate were determined in the essential oil of M. communis leaves.All results were given in Table 1.Gas chromatography-mass spectrometry examination of M. communis leaf essential oil showed 1,8-cineol (21.68%), alpha-pinene (18.02%), linalool (14.12%), alpha-terpinyl acetate (10.40%), myrtenol (8.59%) and alpha-limonene (4.92%) as major compounds among forty-five components.Linalyl acetate (4.71%), alpha-terpineol (3.09%) and p-cymene (2.64%) were determined as secondary major components.The other compounds such as spathulenol (1.91%) and geranyl acetate (1.31%) were present in minor percentages.All results were summarized in Table 1 and total ion chromatograms with retention times of the components above the concentration of 3% were given in Figure 1.Seven fatty acid methyl esters include palmitic acid methyl ester, oleic acid methyl ester and linoleic acid methyl ester were detected in the seed oil obtained by cold press method from the seeds (Table 2).

Discussion and Conclusion
Although the results vary depending on factors such as the oil extraction techniques, analysis conditions, geographical factors, in previous researches [25] it was determined that basic constituents of this essential oil were 1,8-cineole, alpha-pinene, linalool, bornyl acetate, alpha-terpineol, linalyl acetate and limonene.In another study [26], alpha-pinene and 1,8-cineol were reported to be present in high proportions and agreed on our results.
Linoleic acid, as a major fatty acid methyl ester component, has been shown in previous studies with its properties as skin moisturizing and skin healthiness activity in addition to high nutritional properties [3,27].
Results given in this work shown that leaf essential oil of naturaly grown M. communis in Gökova (Muğla), Turkey is rich in 1,8-cineole, alpha-pinene, linalool and poor in sabinene, camphene and ocimene.Also seed oil of M. communis is rich in linoleic acid, palmitic acid and oleic acid.
In conclusion, essential oil and seed oil of M. communis may be considered as a natural raw material source for food, pharmaceuticals and cosmetic products.GC/MSD analyse results verified nutritional and industrial usage of M. communis has great advantages due to its significant chemical composition.Essential oil and seed oil of M. communis worth studying further.

Table 1 .
Essential oil composition of Myrtus communis leaves.

Table 2 .
Fatty acid composition of M. communis seeds.