Essential Oil Composition of Clinopodium vulgare L . subsp . arundanum ( Boiss . ) Nyman from Bingöl ( Turkey )

Abstract: Many of the medicinal and aromatic plants from Lamiaceae taxa are uses presumed to be connected to the terpenic constituents of the essential oils. In this study aerial parts essential oil of Clinopodium vulgare L. subsp. arundanum was analyzed by HS-SPME. As a result thirty seven components were identified. caryopyllene (16.1%), -terpinene (15.4%), germacrene D (10.3%), p-cymene (8.6%) and thymol (6.4%) were detected the major constituents. With this study, chemotypes of studied sample were detected -caryopyllene, terpinene, germacrene D, p-cymene and thymol. In addition studied plant sample was found to be rich in respect to essential oils and the results discussed natural product, renewable resources and chemotaxonomy


INTRODUCTION
Clinopodium vulgare L. is one of the two Clinopodium spp.(Lamiaceae) growing wild in Anatolia.In Flora of Turkey, two subspecies are defined: vulgare and arundanum.The latter is widespread in Anatolia [1].Until recently, essential oils have been studied most from the point of view their flavour and fragrance chemistry for flavouring foods, drinks and other goods.Actually, however, essential oils and their components are gaining increasing interest because of their relatively safe status, wide acceptance by consumers, and exploitation for potential multi-purpose functional uses [2].Many authors, in fact, have reported antimicrobial, antifungal, antioxidant and radical-scavenging properties by spices and essential oils and, in some cases, a direct food-related application has been tested [3].Clinopodium vulgare is one of the curative plants used in Turkish folk medicine, mainly during wars for the purposes of healing wounds; this plant also showed a very strong action on bacteria [4].Aqueous extract of C. vulgare showed strong antitumor activity [5].A literature survey has shown that there is one report on the volatile constituents of C. vulgare subsp.arundanum Boiss.growing in Turkey [6].
In our study, essential oil composition of C. vulgare subsp.arundanum which was collected from Bingöl (Turkey) was undertaken for the first time.

Plant Material
C. vulgare subsp.arundanum was collected from Bingöl-Solhan, vicinity of Hazarşah village, dry slopes, 1700-1750 m, July 2015, O.Kilic.The taxonomic description of the plant sample was made according to volume 4 of Flora of Turkey [1].Voucher specimen was deposited in the Bingöl University, Department of Park and Garden Plants.

HS-SPME Procedure
Dried aerial part powder of five grams plant samples were carried out by a head space solid phase microextraction method using a divinyl benzene/carboxen/polydimethylsiloxane fiber, with 50/30 um film thickness; before the analysis the fiber was conditioned in the injection port of the gas chromatography (GC) as indicated by the manufacturer.For each sample, 5 g of plant samples, previously homogenized, were weighed into a 40 ml vial; the vial was equipped with a ''mininert'' valve.The vial was kept at 35°C with continuous internal stirring and the sample was left to equilibratefor 30 min; then, the SPME fiber was exposed for 40 min to the headspace while maintaining the sample at 35°C.After sampling, the SPME fiber was introduced into the GC injector, and was left for 3 min to allow the analyzes thermal desorption.In order to optimize the technique, the effects of various parameters, such as sample volume, sample headspace volume, sample heating temperature and extraction time were studied on the extraction efficiency as previously reported by Verzera et al.,[7].

GC-MS Analysis
A Varian 3800 gas chromatograph directly inter faced with a Varian 2000 ion trap mass spectrometer was used with injector temperature, 260°C; injection mode, splitless; column, 60 m, CP-Wax 52 CB 0.25 mm i.d., 0.25 mm film thickness.The oven temperature was programmed as follows: 45°C held for 5 min, then increased to 80°C at a rate of 10°C/min, and to 240°C.at 2°C/min.The carrier gas was helium, used at a constant pressure of 10 psi; the transfer line temperature, 250°C; the ionisation mode, electron impact (EI); acquisit ion range, 40 to 200 m/z; scan rate, 1 us -1 .The compounds were identified using the NIST library, mass spectral library and verified by the retention indices which were calculated as described by Van den Dool and Kratz [8].The relative amounts were calculated on the basis of peak-area ratios.The identified constituents of C. vulgare subsp.arundanum is listed in Table 1.