Chemical composition of volatile extract from Inula aschersoniana Janka var. aschersoniana growing in Bulgaria

The chemical composition of the volatile oil of Inula aschersoniana was studied by GC and GC-MS. Forty-five constituents representing 92.9% of the total oil were detected. The oil contained fatty acids (55.2%) and alkanes (14.1%), followed by oxygenated monoterpenes (9.6%), sesquiterpenoids (7.1%) and aromatic compounds (4.5%). It is characterized by relatively low content of terpenoids in total 16.7% only of which linalool (2.1%) and τ-cadinol (2.2%) were the dominant components in this class of compounds. Cluster analysis (CA) was used for determination of the relationship between the species in Inula verbascifolia aggregate.


INTRODUCTION
The genus Inula (Asteraceae) consists of 100 species, some of which are known as plants used in traditional medicine.A literature survey revealed that Inula plants are rich source of biologically active compounds, mainly sesquiterpene lactones, flavonoids, and triterpenoids (Seca et al., 2014;2015).Surprisingly, the information on essential oil composition of Inula species is scarce and only few biological activities have been reported -antibacterial (Deriu et al., 2008;Priydarshi et al., 2016), antifungal (Chauhan and Saxena, 1985;Haoui et al., 2016), and influence of I. helenium essential oil on electroencephalographic activity of the human brain (Sowndhararajan et al., 2016).Inula aschersoniana Janka is a species with areas of distribution in Bulgaria, Greece, Macedonia, and Turkey (The Global Species, 2009).Three varieties of I. aschersoniana -var.madarense, var.macedonica and var.aschersoniana are identified in Bulgaria (Delipavlov et al., 2003;Stojanov et al., 1967).Our previous phytochemical investigation of I. aschersoniana Janka var.aschersoniana afforded parthenolide, diepoxycostunolide, inusoniolide, chrysosplenol C and four pseudoguaiane derivatives (Trendafilova et al., 2014).The essential oil composition of I. aschersoniana has not been studied yet.In continuation of our phytochemical work on Inula essential oils, herein we report the chemical composition of the volatile fraction of I. aschersoniana var.aschersoniana growing in Bulgaria.

Plant material
Flowers of I. aschersoniana were collected in July 2015 near Marziganiza hut (41°53'32.8"N 24°52'47.8"E) in Rhodope Mts, Bulgaria.A voucher specimen (SOM169980) has been deposited in the Herbarium of the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria.

Preparation of the essential oil
Air-dried flowers were subjected to a micro hydrodistillationextraction in a Likens-Nickerson apparatus for 3 hours using diethyl ether as a solvent.The essential oil dissolved in diethyl ether was dried over anhydrous Na 2 SO 4 .After filtration, the solvent was removed under N 2 flow, and the essential oil was stored at 4°C before analysis.The oil yield was 0.6 mg/g.

Gas chromatography (GC) and Gas chromatographymass spectrometry (GC-MS)
The GC analysis was performed with a Shimadzu 17A gas chromatograph equipped with FID and an HP5-MS fusedsilica capillary column ((5%-phenyl)-methylpolysiloxane, 30 m x 25 mm i.d., film thickness 0.25 µm).The column temperature was programmed from 50°C (4 min isotherm), to 240°C at a rate of 4°C/min, to 300°C at a rate of 10°C/min, and held at this temperature for 10 min, the injector and detector temperatures were 260°C, carrier gas -N 2 (linear velocity, 33 cm/s), split ratio, 1:100.Relative percentage amounts were calculated from peak area without the use of correction factors.
GC-MS analysis was performed on a HP 6890 Plus instrument with MS detector 5973 of the same company.Mass detector was operated in electron ionization mode (70 eV) at a mass range of 30-450 Da.All chromatographic conditions and the column were as described above (for GC Analysis) except for the carrier gas, which was He.Retention indices (RI) of the oil components were calculated by using retention times of C 8 -C 30 n-alkanes under the same chromatographic conditions according to Van den Dool's method (van Den Dool and Dec. Kratz, 1963).The individual components were identified by their RI, referring to known compounds from the literature (Adams, 2009;Tkachev, 2008) and by comparison of their mass spectra with those of NIST 98, WILEY and homemade MS databases.

Statistical analysis
The single linkage clustering method based on linkage distances was used to determine similarities between the examined samples (Statistica 10.0 package).

RESULTS AND DISCUSSION
Hydrodistillation of I. aschersoniana flowers gave pale yellow oil, which was further analyzed by GC and GC-MS.Forty-five volatile constituents in concentration more than 0.1%, representing 92.9% of the total oil were listed in Table 1.Forty-three compounds were identified, and the chemical type of two components was determined.The oil was primarily composed of fatty acids (55.2%) and alkanes (14.1%), smaller amounts of oxygenated monoterpenes (9.6%), sesquiterpenoids (7.1%) and aromatic compounds (4.5%).It should be noted the abundance of fatty acids, of which octanoic, dodecanoic, and decanoic acids were principal constituents of the oil (16.8, 13.7 and 9.3%, respectively).The aliphatic hydrocarbons pentacosane (5.4%), heptacosane (3.5%) and nonacosane (2.5%) also presented in the oil in significant concentrations.The oil is characterized by relatively low content of terpenoids in total 16.7% only.Linalool (2.1%) and τ-cadinol (2.2%) were the dominant components in this class of compounds.Megastigmatrienone-2 and megastigmatrienone-4 were detected now for the first time in Inula species.Megastigmatrienones are dinor-isoprenoids derived from carotenoids and have been found mainly in members of the Solanaceae (D'Abrosca et al., 2004;Osorio, 2003) but also have been identified in Lamiaceae (Stachys palustris) (Senatore et al., 2007) and Asteraceae (Bidens pilosa) (Deba et al., 2008) essential oils.
The observed differentiation of the samples was further supported by cluster analysis (Fig. 2), which showed three distinct groups based on the Euclidian distance.The first group (Iv and Ia) was characterized by high content of alkanes and fatty acids.The second cluster (Ivp) contained almost equal amounts of oxygenated monoterpenes, sesquiterpene hydrocarbons and aromatic compounds.The third cluster (Ivm) contained primarly terpenoids (91.6%).