Effect of Extraction Technique on Composition of Volatile Constituents of Oleoresin from Pinus Brutia

In this study, volatile constituents of oleoresin from Pinus brutia Ten. were extracted by solid phase microextraction (SPME) and hydrodistillation. Gas chromatography-mass spectroscopy (GC/MS) was performed to determine volatile constituents of turpentine oil (obtained by hydrodistillation) and extract from oleoresin (obtained by SPME). Eleven volatile compounds constituted the total of turpentine oil obtained by hydrodistillation, whereas SPME extract contained 32 volatile compounds. Three distinctive volatile constituents in the turpentine oil were α-pinene (49.12 %), β-pinene (19.12 %) and Δ3-carene (15.33 %). They were represented in the amount of 44.35 %, 18.59 % and 14.58 % in the SPME extract. The volatile constituents of turpentine oil were monoterpenes and sesquiterpenes. Monoterpenes, sesquiterpenes, terpene oxides, alcohols, aldehydes, esters and ethers were identifi ed as main groups in the SPME extract by GC/MS. The results of chromatographic analysis showed that solid phase microextraction was a more effective extraction technique than hydrodistillation for isolating volatile constituents from oleoresin.


Effect of Extraction
. Besides hydrodistillation technique, the volatile constituents of oleoresin were isolated from brutian pine using SPME in this study.This was followed by determination of volatile constituents of turpentine oil (obtained by hydrodistillation) and extract (obtained by SPME) by gas chromatographymass spectroscopy (GC/MS).Finally, both applications were compared to reveal which extraction technique is more effective for isolating the volatile constituents of oleoresin.

Materijali
Oleoresin of brutian pine (Pinus brutia Ten.) was obtained from Karaisali-Adana (37°17'40.0"N35°09'15.0"E) in Turkey.Tapping was applied using an acid paste method after bark chipping.The bark of each tree was chipped 10 cm in width and 5 cm in height using a wounding apparatus at 1m height above ground.Afterwards, a collector was placed under the wound for gathering fl owed oleoresin and the chemical stimulant.The acid paste was applied as chemical stimulant using plastic injectors.Acid paste contained 65 % solution of sulphuric acid, coal, dust, barley-rice bran and dyotamide soil.Sample trees were aged 35, 35 and 37 years, and they were 16, 17 and 18 meters in height and 30, 35 and 38 cm in diameter.

SPME and GC/MS analysis 2.2. SPME i GC/MS analiza
The solid phase microextraction (SPME) apparatus, equipped with a fi ber coated and a 75 μm-thick layer of Carboxen/Polydimethylsiloxane (CAR/PDMS), was used for the isolation of volatile constituents from oleoresin. 2 g of oleoresin were used in the experiment.Firstly, oleoresin was placed in a 10 mL vial, which was sealed with a silicone septum and a crimp cap and heated at 60 °C for 15 min.Secondly, SPME fi ber was pushed through the headspace of a sample vial.The volatile constituents were adsorbed at 60 °C for 30 min.Then fi ber was inserted directly into the injection port of the Shimadzu 2010 Plus GC/MS.GC/MS device was equipped with a Restek Rx-5Sil MS capillary column (30 m x 0.25 mm i.d., 0.25 μm fi lm thickness) coupled to a mass spectrometer with an ion trap detector in full scan under electron impact ionization (70 eV).Helium was used as carrier gas with a fl ow rate of 1.61 mL/min.The injection and detection were performed at the temperature of 250 °C.Quadrupole temperature was also 250 °C.Mass range was between 35 and 450 m/z.The temperature of column was kept at 40 °C for 2 min, subsequently raised to 250 °C at a rate of 4 °C/min and then held at 250 °C for 5 min.The software used was LabSolutions GCMSsolution Version 2.7.

Isolation of turpentine oil and GC/MS analysis Izolacija terpentinskog ulja i GC/MS analiza
For the isolation of turpentine oil, 100 g of oleoresin were submitted to hydrodistillation for 5 h using Oleoresin is obtained as exudates from pine trees by tapping the bark (bark chipping).Turpentine, essential oil, is produced by steam/hydro distillation from oleoresin.Turpentine is also known commercially as the "spirits of turpentine", "pine tree terpenic", "pine oleoresin", "gum turpentine", "terpenes oil", "turpentine from Bordeaux" or "oil of turpentine".α-pinene and β-pinene are the major constituents of turpentine.Pinenes are mostly used as starting materials to produce more valuable components (Jantam and Ahmad, 1999 Brutian pine (Pinus brutia Ten.) is especially used for the production of oleoresin in Turkey ( Oz et al., 2012;Deniz, 2013;Oz et al., 2015).It grows widely in the Mediterranean region, Aegean region, Marmara region and also in some localities of the western Black Sea region in a total area of about 5.8 million ha in Turkey (OGM, 2015).
Solid phase microextraction (SPME) is a sampling technique, whereby a constituent is adsorbed onto the surface of the coated silica fi ber.Afterwards, constituents are desorbed into a suitable chromatography instrument coupled with an appropriate detector for identifi cation and quantifi cation.SPME is usually performed by gas chromatography (GC) in the applications.In SPME-GC analysis, the fi ber is introduced into the injection port of GC device and constituents are thermally desorbed from the coating for chromatographically determination (Malik et al., 2006).SPME was especially applied in environmental chemistry (Fattore et al., 1996;Abalos et al., 2002;Mousavi et al., 2007), for example for determining organic pollutants in environmental samples (Penalver et al., 1999) and phthalate esters in environmental waters (Polo et al., 2005).It was regularly used to isolate volatile and semivolatile components (Zhang and Pawliszyn, 1993;James and Stack, 1996), for example for determining volatile components of some medicinal and aromatic plants (Bicchi et al., 2007;Yasar et al., 2016) and semivolatile organics in environmental solids (Hageman et al., 1999).
The determination of volatile constituents of oleoresin, after isolation of turpentine oil by hydrodistillation, is a commonly used procedure (Rezzi et  GC/MS chromatograms of volatile constituents of turpentine oil obtained by hydrodistillation and extract obtained by SPME from oleoresin of Pinus brutia Ten. are demonstrated in Figure 1 and 2. Identifi ed volatile constituents of turpentine oil and SPME extract of oleoresin from Pinus brutia Ten. are given in Table 2. Through the application of SPME for the extraction of volatile compounds from oleoresin, it was possible to determine a total of 32 compounds by direct injection on GC/MS.α-pinene, β-pinene and Δ 3 -carene were the major constituents (44.35 %, 18.59 % and 14.58 %, respectively) of oleoresin.According to GC/MS analysis of the hydrodistilled turpentine oil, a total of 11 volatile constituents were characterized.At the highest percentage of monoterpenes, they were represented by α-pinene (49.12 %), β-pinene (19.2 %) and Δ 3 -carene (15.33 %).
a Clevenger-type apparatus.Hydrodistillation yielded 30.4 % (w/w) turpentine oil from oleoresin.30 μL turpentine oil were added to 970 μL hexane (GC grade) and 1 μL of this solution was submitted to injection port of GC/MS.GC/MS was performed under the same conditions as described above.

Identifi cation of constituents 2.4. Identifi kacija sastojaka
Retention indices of volatile constituents were determined using a series of saturated n-alkanes.n-alkanes were injected after each sample at the same chromatographic conditions as described above for GC/MS.The volatile constituents were identifi ed by comparison of their mass spectra with the Wiley, NIST, Tutor and FF-NSC libraries or with data already available in the literature (Adams, 2007).Percentage amounts were calculated based on peak areas from the GC/MS chromatogram by the computer software described above.

Statistical analysis 2.5. Statistička analiza
MiniTab 16 software was used for statistical analyses.Independent samples t test analysis was applied to determine the statistical signifi cance.For each variable, independent samples t test was separately done.Oz et al. (2015) determined, as major compounds, α-pinene, β-pinene and γ-terpinene with quantities of 19.7 %, 13.3 % and 10.2 % in the turpentine oil of oleoresin from Pinus brutia Ten.These results support the present fi ndings except for γ-terpinene and Δ 3carene.γ-terpinene was not obtained in the turpentine oil of this study.Δ 3 -carene was presented as 3.7 % in the work by Oz et al. (2015), whereas Δ 3 -carene was determined as a major compound in the turpentine oil of this study.
The main groups of determined volatile constituents of turpentine oil and SPME extract from oleoresin of Pinus brutia Ten. are listed in Table 3. Monoterpenes and sesquiterpenes were identifi ed in the turpentine oil, whereas monoterpenes, sesquiterpenes, terpene oxides, alcohols, aldehydes, esters and ethers were characterized in the extract by SPME.Based on independent samples t test, a signifi cant difference was found between hydrodistillation and SPME sampling for monoterpenes and sesquiterpenes (p<0.05).

ZAKLJUČAK
Using SPME for the extraction from oleoresin, 32 volatile constituents were determined by direct injection in GC/MS, whereas 11 volatile constituents were identifi ed in the turpentine oil by GC/MS after hydrodistillation of oleoresin from Pinus brutia Ten.Three major volatile constituents in the turpentine oil were α-pinene α-pinene, camphene, β-pinene, Δ 3 -carene, p-cymene, limonene, α-cubebene, α-gurjunene, β-caryophyllene, aromadendrene and α-humulene constituted the total of turpentine oil by hydrodistillation, whereas these compounds appeared as 90.25 % in the extract by SPME.The present fi ndings show that 9.75 % of volatile constituents cannot be extracted by hydrodistillation of oleoresin from Pinus brutia Ten.
DRVNA INDUSTRIJA 69 (3) 239-245 (2018) SPME was defi nitely a more effi cient sample preparation technique as compared to classical hydrodistillation, and it could be performed with a much smaller amount of oleoresin than hydrodistillation (Table1).For this reason, SPME technique offers more advantages than conventional hydrodistillation for extraction.

Table 3
Main groups of determined constituents of turpentine oil and SPME extract from oleoresin of Pinus brutia Ten.Tablica 3. Glavne skupine određenih sastojaka terpentinskog ulja i SPME ekstrakta iz oleoresina drva Pinus brutia Ten.They were represented by the amounts of 44.35%, 18.59% and 14.58% in oleoresin volatiles isolated by SPME.Monoterpenes and sesquiterpenes were determined in the turpentine oil, while monoterpenes, sesquiterpenes, terpene oxides, alcohols, aldehydes, esters and ethers were identifi ed as main groups in the extract by SPME.Regarding the quantities of identifi ed volatile compounds occurring in resin of Pinus brutia Ten., SPME has been shown as a more appropriate extraction technique than conventional hydrodistillation.