The Effects of Cultivation Area and Altitude Variation on the Composition of Essential Oil of Laurus nobilis l . Grown in Eastern , Western and Central Karadeniz Region

Laurus nobilis L. is one of the most valuable non-wood forest products on world export market and Turkey. Turkey is the biggest provider country for Laurus nobilis in the world. Therefore, laurel is an important commercial product for our country. In this study, the effects of cultivation area and altitude variation on essential oil content and quantity of laurel leaves were examined which grown in Trabzon, Bartın and Samsun. It was aimed to determine chemical composition of laurel’s leaves grown in Karadeniz region. Laurus nobilis L. leaves were collected in three different height ranges. These were 0-100 m, 100-300 m, 300-600 m. Leaves were shade-dried and crushed. A device called ‘’Clevenger’’ was used for getting volatile oil and their yields were calculated according to dry weight. The yields of essential oils ranged between 0.91% to 1.66 %. These essential oils were obtained from Bartin (B2) (100-300 m) and Artvin (A1) (0-100 m) respectively. The major components of these essential oils were 1,8cineole (19.71%-35.63%), α-terpinyl acetate (12.86%-21.24%), sabinene (5.98%-9.40%), αpinene (3.67%-8.45%) and βpinene (2.91%-5.87%) were the most abundant volatile compounds in the leaves of bay.


INTRODUCTION
Laurel has an important product in the trade of non-wood forest products in terms of Turkey and this respect laurel plant is one of the high value-added products for our country.According to foreign trade statistics of Turkish Statistical Institute (TSI) the year of 2015, 2,207,550 kg of bay leaf were exported from Turkey and the financial provision corresponds to 6,365,257 $.
The bay, known as Laurus nobilis L., is a peculiar plant of Mediterranean Region.It belongs to Magnoliophyta (closed seeds) branch and it is in the Magnoliopsida class.Laurus nobilis which is a member of the Lauraceae family of Laurales, is a genus of Laurus [1].The leaves are short and thick.The fresh leaves are thin and they have light green vein and they have red tinged yellow color, then their color turns into light green, with little aromatic odor.The fresh shoots are green, the next is red, black and hairless.Their maximum length is 2 cm [2].The place of use of Laurus nobilis L. is, thanks to its phenolic compounds which are taking place in substances of laurel leaves, in food industry as natural antioxidants with antioxidant properties [3].In particular, thanks to its volatile compounds in dried leaves, they are used in meat, soup, candy and sauce making as a flavoring [4][5][6][7].Bay leaves and volatile oils of leaves have an effects of antiepileptic, anticonvulsive [8][9][10], antimicrobial [10], antibacterial [11,12].
The antibacterial effect which are found in bay leaf essential oil, thanks to the high ratio of methyl eugenol and 1,8-cineole in the bay volatile oil.Thus, they have antibacterial properties against bacteria that cause foodborne diseases such as, Staphylococcus aureus, Staphylococcus intermedius, Klebsiella neumonia, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus [12,13].
The antimicrobial effect is due to the presence of compounds such as 1,8-cineole, camphor, myrcene, β-caryophyllene, eugenol, α-pinene, β-pinene and p-cymene in the content of volatile oil in bay leaf [2,14].Apart from beneficial properties, there have lots of unique properties of bay leaf volatile oil, such as having thermal stability, does not showing phototoxic effect, positive effects against migraine, headache, high blood sugar, bacterial and fungal infections [9,15].Leaf volatile oil is advised to use as a mucolytic agent (antipyretic) in advanced asthmatic disorders, upper and lower respiratory tract disorders since it has a 1,8cineole (volatile compound) in it.So it has an anti-inflammatory effect (pain, fever cutter) which is from oxygenated monoterpenes and also it is a high concentration in bay volatile oil [16].Recent studies have shown that bay leaf and its' essential oil have a positive effect against colitis which causes flatulence in stomach [17].
In recent years, it is indicated that the high rate of volatile oil of the leaves in the southern regions of our country makes bitter taste in the bay leaves.Thus, tendency towards the bay leaves in the EU countries is shifted progress to the laurel leaves grown in Russia.These leaves have less essential oils than Mediterranean Regions.Russia is on the shore to the black sea.Therefore, the Karadeniz Region may also be a suitable region for bay exports.Based on this, Laurel leaves were collected from Artvin, Trabzon, Samsun and Bartın from Karadeniz Region and then essential oils were obtained and they were compared in oil yields and essential oil compositions.

Plant Material
The areas where the bay leaves were collected are given in Table 1.Laurel leaves were collected at three altitudes.These were 0-100 m, 100-300 m, 300-600 m but in Artvin (A3) (300-600m) plant material were not collected because this plant were not encountered.
Dried by leaves (11 samples of 75 gr each) were subjected to the hydro distillation for 3 hours on a Clevenger-type apparatus.The essential oils were removed from the water and stored at 4ºC until gas chromatography-mass spectrometry (GC-MS) analysis.

GC-MS Analysis
GC-MS analysis was carried out with a 5975 Agilent apparatus equipped with İnowax FSC column (60 m long x 0.25 mm i.d.x 0.25 μm film thickness).The column temperature program was 60 ºC during 10 min, with 4 º C/min increases to 220 º C, then wait 10 min in 220 º C and 1 º C/min increases to 240 º C. The carrier gas was helium at a flow-rate of 0.8 ml/min.Split mode injection (ratio 1:50) was employed.Injector temperature was 250 º C. Mass spectra were taken over the m/z 35-450 range with an ionizing voltage of 70 eV.The relative delay times for defining the essential oil components were determined by comparing the mass spectrum profiles of the materials using Wiley GC/MS Library, Adams Library, and Mass Finder 2.1 Library mass spectrum libraries on the computer to which the device was connected [18].

Extraction Yield
In our study, volatile oil yield which was obtained by Hydro distillation method are shown in Table 2.The highest yield of volatile oil in our work was 1.66 % Artvin (0-100 m) and the lowest amount was 0,91% Bartın (100-300 m) was obtained.Our volatile oil yield results vary between 0.91% and 1.66%.
Fiorini et al., investigated laurel leaf which were collected from France.They reported that the yield of laurel leaf essential was 0.57% [19].Our results are much higher than this research.

GC-MS Analysis
Fifty-six compounds accounting for 78.07%-98.78% of the essential oils were identified by capillary GC-MS.Components are listed in Table 3 according to their retention times.There are lots of factors affecting the production of secondary metabolites in plants.It can be explained as an environmental, geographical, physiological, genetic, political and social factors [20].The percentage of volatile compounds also depends on climate conditions and edaphic factors.Our results show high similarity in Karadeniz region.The highest volatile compounds were 1,8-cineole (19.71%-35.63%),α-terpinyl acetate (12.86%-21.24%),sabinene (5.98%-9.40%).Yilmaz et al., reported that 1.8-cineole (51.8 %), α-terpinyl acetate (11.2 %) and sabinene (10.1 %) as the major compounds in their research which is smilar with the results of our study but results is much higher than our results [21].
Therefore, we detected some sesquiterpenes in lower amounts of our research.These are β-elemene (0.36%-1.89%), caryopyllene oxide (%0.46-2.63%),βeudesmol (0.58%-1.91%).These compounds have been detected in several essential oils and the other oils have not been contained this compounds.These compounds have inhibitory effect on ethanol absorption [31].Methyl eugenol (0.49% -6.00%) from the phenylpraponoids was a compound that we have detected in our analysis as a high amounts.Methyl eugenol has an anesthetic and relaxing effect on muscles [23].

CONCLUSION
In this study, volatile compounds which were in the content of volatile oil in the leaves of the Laurus nobilis L. that grow in Karadeniz Region (Eastern, Western and Central), were revealed and we also aimed to determine if there is any similarity in the essential oils of laurel leaves which are grown in Karadeniz Region.We determined similarities in substances of essential oils.1,8 cineole is the major compound but the amount of 1.8 cineole is considerably less than most of the previous studies.Since 1.8-cineole is a flavoring compound, if the compound is found in a small amount in volatile oil, it promotes that the taste of the oil can be much softer.The results of our research support, exporting laurel leaf to the EU countries from Karadeniz Region.

Table 1 .
Locations and altitudes of collected laurel leaves.

Table 2 .
Avarage yield of Laurus nobilis L. essential oil.

Table 3 .
The chemical composition of essential oils of leaves from laurel (Laurus nobilis L.).