Volatile aroma compounds of flowering stems and rosette leaves of Sideritis raeseri , Headspace GC/FID/MS profile

Sideritis raeseri Boiss. & Heldr. is endemic to the Balkan Peninsula and is reported to grow in Greece, R.N. Macedonia and Albania (Obon de Castro and Rivera Núńez, 1994). Mountain tea is a traditional beverage in the Balkan countries, prepared as a refreshing herbal tea. Aerial parts of this plant are widely utilized in folk medicine in the form of a decoction or infusion (Karapandzova et al., 2013; Romanucci et al., 2017). The specific and particular aroma is maybe the most important reason for the wide use of the flowering stems of S. raeseri by the Balkan peoples. Rosette leaves traditionally is not used (Qazimi et al., 2014). There are not data about the volatile aroma compounds in the dried rosette leaves of S. raeseri. To analyze these compounds a refined method of headspace sampling hyphenated with GC/FID/MS analysis can be utilized (Watson, 2005). The aim of this work was the determination of the volatile aroma compounds in the dried flowering stems and rosette leaves of spontaneous S. raeseri using a headspace (HS) method with GC/FID/MS. Materials and methods


Introduction
Sideritis raeseri Boiss. & Heldr. is endemic to the Balkan Peninsula and is reported to grow in Greece, R.N. Macedonia and Albania (Obon de Castro and Rivera -Núńez, 1994). Mountain tea is a traditional beverage in the Balkan countries, prepared as a refreshing herbal tea. Aerial parts of this plant are widely utilized in folk medicine in the form of a decoction or infusion (Karapandzova et al., 2013;Romanucci et al., 2017). The specific and particular aroma is maybe the most important reason for the wide use of the flowering stems of S. raeseri by the Balkan peoples. Rosette leaves traditionally is not used (Qazimi et al., 2014). There are not data about the volatile aroma compounds in the dried rosette leaves of S. raeseri. To analyze these compounds a refined method of headspace sampling hyphenated with GC/FID/MS analysis can be utilized (Watson, 2005).
The aim of this work was the determination of the volatile aroma compounds in the dried flowering stems and rosette leaves of spontaneous S. raeseri using a headspace (HS) method with GC/FID/MS.

Materials and methods
Plant material: The flowering stems (S-f) and rosette leaves (S-r) of Sideritis raeseri were collected in 3 different localities from National Park Galichica in R. N. Macedonia (Kazani, Krstec and Vojtino). The plant material was air dried, packed in paper bags and kept in a dark and cold place until analysis.

Analyses of aroma compounds:
GC and GC-MS analyses: 0.3 g of dried flowering stems ((homogenized samples from flower, leaf and stem) (S-f) and rosette leaves (S-r) was put in sealed vials, warmed for 5 minutes and the gas phase (highly volatile compounds) was investigated on Agilent 7890А Gas Chromatography system equipped with flame ionization detector (FID) and Agilent 5975C Mass Quadrupole detector as well as capillary flow technology which enable simultaneous analysis of the sample on both detectors. HP-5ms (30 m x 0.25 mm, film thickness 0.25 m) capillary column was used. Operating conditions were as follows: oven temperature 60C, 20C/min to 280C; helium as carrier gas at a flow rate of 1 mL/min; injector temperature 260C and FID temperature 270C. 1000 L of gas phase was injected at split ratio 1:1. 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. Headspace method: Incubation temperature 80˚C; incubation time 5 min; syringe temperature 85˚C; agitator speed 500 rpm.
Identification of components: Identification of the components was made by comparing the mass spectra of components with those from NIST, Wiley and Adams mass libraries, by AMDIS (Automated Mass Spectral Deconvolution and Identification System) and by comparing literature and estimated Kovat's (retention) indices that were determined using a mixture of a homologous series of normal alkanes from С9 to С25 in n-hexane, under the same conditions. The percentage ratio of the components was computed by the normalization method of the GС/FID peak areas and average values were taken.

Conclusion
Rosette leaves of S. raeseri exhibit very similar volatile aroma compounds profile with the flowering stems, comprising β-pinene, α-pinene, limonene and α-copaene as predominate components and accordingly can be consider as an additional plant material source of this aromatic plant. For complete assessment additional phytochemical analysis are require.