Essential oils composition of Pinus peuce Griseb . ( Pinaceae ) growing on Pelister Mtn . , Republic of Macedonia

The composition of essential oils obtained by hydrodistillation from needles, from branches without needles and from branches with needles of Pinus peuce Griseb. (Pinaceae) from Pelister Mtn. (R. Macedonia) was analyzed by GC/FID/MS. One hundred and seven components (40 monoterpenes, 37 sesquiterpenes, 9 diterpenes and 21 other components aliphatic and cyclic hydrocarbons; aliphatic alcohols, aldehydes, and acids; phenols and other oxygenated benzene derivates) were identified. The most abundant constituents were terpene hydrocarbons, encompassing the monoterpenes: α-pinene, β-pinene, limonene + β-phellandrene and bornyl acetate and the sesquiterpenes: trans (E)-caryophyllene and germacrene D.


Introduction
Pinus peuce Griseb.(Pinaceae), commonly called Macedonian pine or Balkan pine is an endemic conifer tree inhabiting the southern and western parts of the territory of R. Macedonia.This conifer is autothonus to the Baba Mountain (Pelister), Nidze and Shara Mountain.It can be also found in Bulgaria, Albania, Serbia and Greece, growing typically at 1000 -2200 m altitude.It is a member of the white or soft pine group (subgenus Strobus, section Strobus, subsection Strobi), and thus differes from the other pine species (P.mugo, P. nigra and P. sylvestris) growing in R. Macedonia, that belong to the yellow or hard pine group (subgenus Pinus, section Pinus, subsection Pinus).
For the first time, Pinus peuce was described on Baba Mountain (Pelister) in 1839 by Grisebach, thus this location is locus classicus for this species.
Nowdays, there is uprising trend of consumption of value added natural products complying different standards as Sustainable, Organic/Biological, Fare Wild etc.The populations of P.peuce in the National Park (NP) Pelister have a great resource potential and sustainable use of these natu-ral resources can have a significant socio-economic impact on local community.In this direction, assessment of the essential oil of P.peuce would greatly contribute in its value added portfolio.
Up to present, there are no evident data about essential oil composition of Pinus peuce population growing on NP Pelister (locus classicus) neither from any locality of the territory of R. Macedonia.On a subject of all previously mentioned, the aim to this study was to obtain data for the yield and composition of the essential oils isolated from needles, from branches without needles and from branches with needles of Pinus peuce from NP Pelister.

Plant material
Only terminal, up to three years old branches were collected in July, 2008and 2009, in the NP Pelister on 4 different altitudes (1208m.a.s.l., 1723m.a.s.l., 1943m.a.s.l. and 2042 m.a.s.l.).Plant identity was verified as Pinus peuce Grisebach and voucher specimens (N o 2008/Pp, N o 2009/ Pp) were deposited at the Herbarium at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, Skopje.Plant material was dried at room temperature and on draft for two weeks.The needles were separated from the branches just before the hydrodestillation and were properly minced.

Essential oil isolation
Essential oil isolation from plant material was made by hydrodestillation in all-glass Clevenger apparatus (Ph.Eur 7).For that purpose, 20 g of plant material (needles, branches without needles and branches with needles) was distilled for 4 hours.After isolation, anhydrous sodium sulfate was added to remove residual water and to dry essential oil.
For GC/FID/MS analysis, the essential oil was dissolved in xylene (1: 1000 v/v).

Gas chromatography
Essential oil samples were analyzed on Agilent 7890А Gas Chromatography system equipped with FID detector and Agilent 5975C mass spectrometer as well as capillary flow technology which enables simultaneous analysis of the sample on both detectors.HP-5ms 5% phenyl 95 % dimethylpolysiloxane bonded phase capillary column (30 m x 0.25 mm, film thickness 0.25 µm) was used.Operating conditions were as follows: oven temperature at 60 °C for 5 min, then increased to 80 °C at rate of 1 °C/min and held 2 min and at the end increased to 280 °C at rate of 5 °C/min and held 5 min; helium as carrier gas at a flow rate of 1ml/ min; temperature of the injector 260 °C and that of the FID detector 270 °C; the GC split ratio 1:1.1µl of each sample was injected per GC run.
The mass spectrometry conditions were: ionization voltage 70 eV, ion source temperature 230 °C, transfer line temperature 280 °C and mass range from 50 -500 Da.The MS was operated in scan mode.
Identification of the components present in essential oils was made by comparing mass spectra of components in essential oils with those from Nist, Wiley and Adams mass spectra libraries, by AMDIS (Automated Mass Spectral Deconvolution and Identification System) and by comparing literature and estimated Kovat′s (retention) indices that were determined using mixture of homologous series of normal alkanes from C 9 to C 25 in hexane, under the same above mentioned conditions.
The percentage ratio of essential oils components was computed by the normalization method of the GC/FID peak areas and average values were taken into further consideration (n=4).

Results and discussion
The yield of essential oils calculated on a dry mass and obtained by hydrodistillation from needles, from branches without needles and from branches with needles of Pinus peuce, is given in Table 1.The obtained essential oils were transparent, light yellowish liquids with specific and strong odor.Table 2 shows components that were identified in the Pinus peuce essential oils isolated from needles, from branches without needles and from branches with needles with their percentage amount and Kovat′s retention indices.The estimated percentage values are averages of components that are present in the essential oils obtained from plant material (needles, branches without needles and branches with needles) collected from four various altitudes.
One hundred and seven components were identified in the Pinus peuce essential oils.Among them, 40 monoterpenes, 37 sesquiterpenes, 9 diterpenes and 21 other com- ponents (aliphatic and cyclic hydrocarbons; aliphatic alcohols, aldehydes, and acids; phenols and other oxygenated benzene derivates) were identified.Analysis of the essential oils composition shows variations in the content of components that are present, probably due to the impact of weather conditions throughout the year.Seasonal variability is mostly seen in the presence of monoterpenes cam-phene and α-terpenil acetate and sesquiterpenes δ-cadinene and α-cadinol (Table 2).
Dominant components in the essential oil obtained from branches with needles were the same 6 components that were found in the needle essential oils, representing 68.76 % of the total amount of the oil.Among them, α-pinene (23.77 %), β-pinene (11.85 %), limonene + β-phellandrene (13.94 %) were the most abundant, while germacrene D (9.47 %) and bornyl acetate (5.60 %) were present in smaller amounts.
The most abundant fraction among the total amount of monoterpenes and sesquiterpenes, diterpenes and other components that are present in needle essential oil were monoterpenes (43.28 %/52.39 %) (Fig. 2).
The major constituents in the essential oil obtained from branches with needles from Pinus peuce were also components that belong to terpene hydrocarbons fraction (78.39 %) and fraction of oxygenated terpenes (14.28 %) (Fig. 1).The same situation was with the essential oil isolated from branches without needles which contains 81.8 % of terpene hydrocarbons fraction and 9.1 % of oxygenated terpenes (Fig. 1).Diterpenes and other components were present in smaller amount (0.59 % diterpenes and 0.30 % other components in the essential oil from branches with needles and 1.23 % diterpenes and 0.08 % other components in the essential oil isolated from branches without needles).
The most abundant fraction among the total amount of monoterpenes and sesquiterpenes, diterpenes and other components that are present in essential oil obtained from branches with needles were monoterpenes (66.09 %) (Fig. 2), while essential oil isolated from branches without needles contains 53.33 % and 37.57 % of monoterpenes and sesquiterpenes, respectively (Fig. 2).
Compared with the chemical composition of needle essential oil isolated from Pinus sylvestris (Pini sylvestris aetheroleum) and from Pinus mugo (Pini pumillionis aetheroleum) for which the European Pharmacopoeia prescribes certain limits for same components (Table 3), it is evident that the composition of needle essential oil from P. peuce is closer to the chemical composition of Pini pumillionis aetheroleum.There is no big difference in presence of α-pinene and β-pinene between these two oils.Camphene and bornyl acetate are present in larger amount in needle essential oil from Pinus peuce while ∆ 3 -carene, β-myrcene, limonene + β-phellandrene and terpinolene are present in smaller amount.
Nowdays, there are a lack of data that are relate to chemical composition of Pinus peuce essential oil.Up to now, there are only five references that are concern to the composition of the essential oils isolated from needles and branches from Pinus peuce that grows on the territory of Greece, Serbia and Montenegro (Henning, 1994;Papadopoulou, 1996;Koukos, 2000;Petrakis, 2001;Nikolic, 2008).Seventy eight components were detected, but only fifty six components were identified in the needle essential oil.α-Pinene, camphene, β-pinene, β-phellandrene, bornyl acetate, β-caryophyllene, germacrene D and citronelol are declared as main components in the needle essential oil while α-pinene, β-pinene, β-phellandrene, β-caryophyllene and citronelol are concerned as main components in the essential oil isolated from branches from P. peuce.Compared with essential oils isolated from P. peuce from Macedonia, the needle essential oil isolated from P. peuce from Greece contains larger amount of α-pinene (23.07 %) and β-pinene (22.00 %) and large amount of citronellol (13.42 %), but it doesn't contain germacrene D, while essential oil isolated from branches contains larger amount of β-phellandrene (26.93 %) and large amount of citronellol (12.48 %), smaller amount of α-pinene (7.38 %), but it doesn't contain germacrene D and bornyl acetate.The needle essential oils isolated from P. peuce from Montenegro and Serbia contain larger amount of α-pinene (36.5 %) and camphene (8.5 %), but smaller amount of bornyl acetate (6.8 %).

Fig. 1 .
Fig. 1.Different classes of components that are present in Pinus peuce essential oils isolated from needles, from branches without needles and from branches with needles (THC -terpene hydrocarbons, OT -oxygenated terpenes, DT -diterpenes, OC -other components)

Table 1 .
The yield of Pinus peuce essential oils isolated from needles, from branches without needles and from branches with needles. *(n=3)

Table 2 .
Chemical composition of Pinus peuce essential oil from Pelister Mtn .