Essential oil composition of wild growing Sage from R . Macedonia

The main objective of this study was to analyze and identify the essential oil composition of S. officinalis populations growing in Republic of Macedonia and to evaluate these data according to different standards’ requirements for, commercially most utilized, Dalmatian sage. The essential oil yield, obtained after hydrodestilation from leaves, of three different populations of Salvia officinalis L. from Republic of Macedonia was determined, varying from 1.40 to 3.46%. The GC/FID/MS analysis of the composition of the essential oils revealed 63, 57 and 51 components in Galicica Mtn., Jablanica Mtn. and Karaorman Mtn. sage populations, respectively. The main components of the oil, in all three samples, were the terpene hydrocarbons, encompassing the monoterpenes: camphor (13.15 25.91%), α-thujone (19.25 26.33%), β-thujone (2.03 5.28%), 1,8-cineole (6.51 – 13.60%), α-pinene (0.93 – 1.47%), borneol (1.07 – 4.67%), then sesquiterpenes: trans (E)-caryophyllene (1.72 – 5.33%), α-humulene (2.89 – 7.99%), viridiflorol (4.27 – 7.99%), and the diterpene manool (2.13 3.79%). Thus, our results for the essential oil composition of sage complied with the reference values specified in the DAC 86 monograph for Salvia essential oil.


Introduction
Most of the species from genus Salvia have medicinal and horticultural importance as they produce many useful natural constituents including terpenes and flavonoids (Kelen and Tepe, 2008).They are counted as one of the largest members of the Lamiaceae family that includes around 900 species and has an almost cosmopolitan distribution (Mediterranean, Asia Minor, Central Europe and America etc).
"Sage", the dialect name of the genus Salvia is attributed to different species that are widely used in the food, drug and fragrance industry.The high diversity in secondary metabolites (essential oils and the phenolic derivatives) isolated from sage plants, possess excellent antimicrobial activity as well as antioxidant capacity and some are used as anticancer agents or have hypoglycemic effect (Kintzios, 2000;Miladinovic and Miladinovic, 2000;Khalil and Li, 2011).Of the many existing Salvia species, Salvia officinalis also known as "Dalmatian sage" or "Garden sage", has economic importance and can be used for preparation of various extracts and herbal remedies with antiseptic and antibacterial properties which are attributed to the rich chemical content of the essential oil and proven by the modern medical science (Velickovic et.al., 2003;Avato et.al., 2005;Delamare et.al., 2005;Bernotiene et.al., 2007).Although sage is an ancient spice and remedy, its importance today is quite limited to the Medi terranean countries (starting from Italy till Greece) (Mockute et.al., 2003;Hager, 2006).Some of the Salvia species, including S. officinalis (S. officinalis folium) and S. triloba (S. triloba folium) can be found in many pharmacopeias (Flamini et.al, 2005;Eur.Ph.7.0, 2010) Salviae officinalis aetheroleum -DAC 86) are available, as well.The essential oil can be obtained by steam distillation of the leaves of S. officinalis (Helv VII), or from aboveground parts of the sage thus obtaining essential oil rich in thujone (DAC 86).
The Macedonian flora includes 37 species of genus Salvia.Up to present there are no evident data about essential oil composition of S. officinalis populations growing in Republic of Macedonia.The importance of S. officinalis drew our attention to examine and identify the active ingredients of the essential oils obtained by hydrodistillation of sage leaves collected from R. Macedonia.

Plant material
The plant material from three different populations was harvest from Galicica Mtn., Jablanica Mtn., and Karaorman Mtn., located in the western part of Macedonia, nearby the Albanian border, during June year 2009 and 2010.The leaves were air dried and stored in a cool and dark place until distillation.The herb was authenticated as Salvia officinalis L. (Lamiacecae) by Dr. Gjoshe Stefkov, and voucher specimens (No.So-MKD 13/10; So-MKD 14/10; So-MKD 15/10) were deposited at the Herbarium at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, Skopje, Macedonia.

Essential oil isolation
Essential oil isolation from sage leaves was performed by hydrodistillation in all-glass Clevenger apparatus following the procedure from European Pharmacopeia (Ph.Eur.7.0, 2010).

Analysis of essential oils' chemical composition
Each sample of essential oil was dissolved in xylene (1:1000 v/v) and further analyzed on Agilent 7890А Gas Chromatography system equipped with FID detector and Agilent 5975C mass spectrometer.HP-5ms 5% phenyl 95% dimethylpolysiloxane bonded phase capillary column (30 m x 0.25 mm, film thickness 0.25 µm) was used.Analytical conditions were: oven temperature at 60 °C for 5 min, then increased to 80 °C at rate of 1 °C/min and held 2 min and at the end increased to 280 °C at rate of 5 °C/min and held 5 min; helium as carrier gas at a flow rate of 1ml/ min; temperature of the injector 260 °C and that of the FID detector 270 °C; the GC split ratio 1:1.1µl of each sample was injected per GC run.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.
The compounds were identified on the basis of literature and estimated Kovat′s (retention) indices that were determined using mixture of homologous series of normal alkanes (C 9 -C 25 ) analyzed under Automated Mass Spectral Deconvolution and Identification System (AMDIS)' conditions.Confirmation was done by comparing the mass spectra obtained from AMDIS with the reference spectra from Nist, Wiley and Adams mass spectra libraries.
Quantification of the essential oils components was performed using the normalization method of the GC/FID peak areas.
By the means of GC-MS, total of sixty nine components, in all three samples of S. officinalis were identified and presented in Table 1.
The GC-MS analysis revealed sixty three components (37 monoterpenes, 17 sesquiterpenes, 5 diterpenes and 4 other components) in the essential oil from Galicica sage population, fifty seven (34 monoterpenes, 12 sesquiterpenes, 5 diterpenes and 6 other constituents) from Jablanica sage population and fifty one from Karaorman sage population (27 monoterpenes, 13 sesquiterpenes, 5 diterpenes and 6 other components -aromatic and aliphatic hydrocarbons, esters and etc).The quantities of the constitutive chemical groups of the essential oils of Salvia officinalis populations are shown in Fig. 1.
Our results for the essential oil composition of sage, thus, comply with the reference values specified in the German Drug Codex monograph for Salvia officinalis essential oil.The composition analysis of the volatile constituents of the three Macedonian sage populations showed that only the essential oil from Karaorman Mtn.population belongs to the thujone-rich oils, while essential oils isolated from Galicica Mtn. and Jablanica Mtn.populations had camphor as major component.Typically, according to some authors (Perry et.al., 1999;Walch et.al., 2011), three are three Dalmatian Sage chemotypes with low (9%), medium (22-28%), and high (39-44%) thujone contents.Concerning the latter, essential oils from S. officinalis samples from Galicica Mtn. and Jablanica Mtn.populations, belong to the Sage group containing medium thujone amounts and cannot meet with the requirements for the sage essential oil chemical composition reported in other available scientific literature where the demand of thujone content is stated as 35-60% (e.g.Radulescu et.al., 2004;Maksimovic et.al., 2007).Due to the camphor predominance in essential oil isolated from Galicica Mtn.population, this essential oil cannot meet the ISO 9909 standard (Table 2).
This inter-specific comparison of the essential oils isolated from three Salvia officinalis populations from Macedonia showed differences in the amount of the principle components especially in the oils obtained from Galicica and Jablanica populations what emphasize the role of the environmental factors (light, soil, water, time of harvest, drying, etc.) on the yield and chemical composition of the essential oil even when they are from same region.

Conclusion
The essential oils' yields obtained after hydrodestilation of leaves from three different populations of sage from Macedonia varied from 1.40 to 3.46%.The essential oil composition of all three sage populations complies with the reference values specified in the DAC 86 monograph for Salvia essential oil.Yet, the requirements of the ISO 9909 standard have not been met.Nevertheless, the set of components identified in the three samples of the Macedonian sage oil match up with the essential oil composition of the Dalmatian sage, though the set differs in relative content of the principle components.et.al., 2003; Bernotiene et.al., 2007; 3 Salvia officinalis essential oil from Serbia, Ref. : Miladinovic  and Miladinovic, 2000; 4 Salvia officinalis essential oil from Romania, Ref. : Radulescu et.al., 2004; 5 Salvia officinalis essential oil from Croatia, Ref. : Maksimovic et.al., 2007; So-MKD 13/10 -Salvia officinalis population from Galicica Mtn.; So-MKD 14/10 -Salvia officinalis population from Jablanica Mtn.; So-MKD 15/10 -Salvia officinalis population from Karaorman Mtn.

Table 1 .
Chemical composition of essential oils isolated from three different populations of S. officinalis from R.

Table 2 .
Comparison of the dominant constituents of the essential oils obtained after hydrodestilation of the three Salvia officinalis populations from R. Macedonia with referent literature Salvia officinalis essential oil in DAC 86 (German Drug Formulary 86); 2 ISO 9909 standard for medicinal uses regulates the amounts of nine constituents in the sage essential oil, Ref: Mockute 1 Monograph for