Volatile constituents of three Thymus sipyleus Boiss. subspecies from different sites in Turkey

This study was designed to reveal the chemical diversity of some Thymus sipyleus subspecies growing wild in Turkey and to compare the volatile compound profiles by using micro(hydro)distillation technique. For this purpose, volatile compounds isolated by microdistillation from nine samples (three plant samples collected from different regions in Antalya) of Thymus sipyleus Boiss. subsp. sipyleus var. sipyleus, T. sipyleus Boiss. subsp. sipyleus var. davisianus Ronniger, and T. sipyleus Boiss. subsp. rosulans (Borbas) Jalas were analyzed by GC and GC-MS systems. 1,8-Cineole, p-cymene, α-terpineol and carvacrol were identified as major compounds in T. sipyleus subsp. sipyleus var. sipyleus samples. Geranial, neral, 1,8-cineole and β-caryophyllene, and α-terpineol and geranial were the main compounds in T. sipyleus subsp. sipyleus var. davisianus samples. β-Caryophyllene, intermedeol, 1,8-cineole and α-terpineol, α-pinene were the major compounds in T. sipyleus subsp. rosulans samples. As known, thymol is the main compound in most Thymus species in Turkey, but, according to our study, chemical polymorphism has been found among the T. sipyleus subspecies.

coughs in Sivas and Yozgat [14].In Ulukışla, Niğde, an infusion of the aerial parts of T. sipyleus subsp.sipyleus var.sipyleus is consumed three times a day for colds and stomach aches [15].T. sipyleus subsp.rosulans known as "catri" in the Eastern part of Turkey is used for diabetes, colds, abdominal ailments as an infusion and decoction [16].

Isolation of the volatiles
Each sample was obtained by microdistillation of the dried, ground plant material (50 mg) using an Eppendorf MicroDistiller with 10 mL distilled water per sample vial.The sample vial was heated to 108 °C at a rate of 20 °C/min and kept at this temperature for 90 min, then heated to 112 °C at a rate of 20 °C/min and kept at this temperature for 30 min.The sample was subjected to a final postrun for 2 min under the same conditions.The collecting vial, containing a solution of NaCl (2.5 g, Sigma-Aldrich) and water (0.5 mL, ultrapure) plus 350 µL of n-hexane [Sigma-Aldrich, ≥99% (GC)] to trap volatile components, was cooled to -5 °C during distillation.After the distillation was completed, the organic layer in the collection vial was separated and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) systems, simultaneously.

GC analysis
The GC analysis was carried out using an Agilent 6890N GC system.FID detector temperature was 300 °C.To obtain the same elution order with GC-MS, simultaneous autoinjection was done on a duplicate of the same column applying the same operational conditions.Relative percentage amounts of the separated compounds were calculated from FID chromatograms.The results of the analysis are shown in Table 2.

GC-MS analysis
The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system.Innowax FSC column (60 m × 0.25 mm, 0.25 mm film thickness) was used with helium as carrier gas (0.8 mL/min).GC oven temperature was kept at 60 °C for 10 min and programmed to 220 °C at a rate of 4 °C/min, and kept constant at 220 °C for 10 min and then programmed to 240 °C at a rate of 1 °C/min.Split ratio was adjusted at 40:1.The injector temperature was set at 250 °C.Mass spectra were recorded at 70 eV.Mass range was from m/z 35 to 450.

Identification of components
Identification of volatile compounds was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention indices (RRI) to series of n-alkanes (C 8 to C 25 ).Computer matching against commercial (Wiley GC-MS Library, Adams Library, MassFinder 3 Library) and in-house "Başer Library of Essential    [29], e [30], f [31], g [32], h [33], k [34], m [35], n [36], p [37]) for polar column values.tr: trace (<0.1 %).IM: identification method.t R : identification based on the retention times (t R ) of genuine compounds on the HP Innowax column.MS: identified on the basis of computer matching of the mass spectra with those of the in-house Baser Library of Essential Oil Constituents, Adams, MassFinder and Wiley libraries and comparison with literature data.

Table 2. (Continued).
Oil Constituents" built up by genuine compounds and components of known oils, as well as MS literature data were used for the identification [20].

Results and discussion
GC and GC-MS analysis of the samples obtained by microdistillation resulted in a total of one hundred fifteen volatile compounds were identified in Thymus sipyleus subspecies by using in house and commercial libraries.The elution of the compounds in the microdistilled oils was done by using an HP-Innowax FSC column.Table 2 shows the list of detected and identified volatile constituents with their RRI and relative percentages in the samples.Seventy seven total components of three T. sipyleus subsp.rosulans samples were identified by GC-MS.Forty-eight components of the KT199 sample were detected representing 90% of the oil.β-Caryophyllene (14.2%) and intermedeol (13.3%) were the major compounds of this sample.Twenty one volatiles are higher than 1% and other major compounds are 1,8-cineole (8.7%), caryophyllene oxide (6.2%), spathulenol (7.0%), α-humulene (3.7%), limonene (2.9%).

Conclusion
Thymol is the major compound of most Thymus species.According to published data and our present study, chemical polymorphism has been found among the Thymus sipyleus subspecies even though the samples were collected from the same region.Thymus populations collected from Turkey have a greater variation of the major components in volatile oils.The variation of volatile oil composition has great importance due to its uses as food and in food processes.The results obtained here suggest that the growing conditions of thyme may alter the volatile oil content and composition.

Table 1 .
Data on GPS and locations of the plant materials