Chemical Composition of the Essential Oils of Three Popular Sideritis Species Cultivated in Greece Using GC-MS Analysis

(1) Background: The essential oils (EOs) of Sideritis L. have attracted great interest due to their pharmacological activities and potential applications in the cosmetic and perfume industries. The aim of this work was to study the EO chemical composition of three of the most popular, in Greece, mountain tea species: namely, these include Sideritis scardica, Sideritis raeseri, and Sideritis syriaca. (2) Methods: The EOs were obtained from the aerial parts of three Sideritis species that were cultivated in various regions of Greece by hydrodistillation, and the chemical composition was studied by gas chromatography–mass spectrometry (GC-MS) analysis. (3) Results: The EOs of the Sideritis species—S. scardica (SSC1, SSC2, SSC3), S. raeseri (SR1, SR2, SR3), and S. syriaca (SS1, SS2, SS3)—were analyzed by GC-MS, and they showed both qualitatively and quantitatively high variation in their chemical composition. (4) Conclusions: The EOs of S. scardica and S. raeseri from three different regions of Greece, and the S. syriaca from three different localities of Crete Island in Southern Greece, showed high chemical variability. Although 165 different components were found to be present in the nine samples through GC-MS analysis, only 7 (1-octen-3-ol, linalool, trans-pinocarveol, p-mentha-1,5-dien-8-ol, α-terpineol, myrtenol, and verbenone) were common components in the nine EOs, which were identified to be highly variable in different percentages among the samples. Even the EOs of SS1 and SS2, which were cultivated nearby, showed different GC profiles. The composition variation observed might be attributed to differentiations in the soil and climatic conditions.


Introduction
Mountain tea, which belongs to the genus Sideritis spp. (Lamiaceae family), and is also known as Dioscorides siderite, is very popular in the Mediterranean area for its use as an herbal tea. The genus name, which comprises more than 150 species and several subspecies, derives from the Greek word for "iron", thanks to the plant's healing action against wounds caused by iron weapons [1]. Siderites, Olympus tea, malotira, good sleeper, Malevos tea, and Taygetus tea, are some of the local names used in Greece to describe mountain tea. In the Mediterranean region and the Balkan Peninsula, many locally endemic species of the genus Sideritis exist. In Greece, apart from the species S. scardica and S. raeseri, which are the most widespread, several species of mountain tea can be found, most of which are collected wild and consumed on a local scale, and they are restricted to narrow mountainous areas or (uniquely to) islands, such as S. syriaca in Crete Island and S. euboea in Evia Island [2].
The therapeutic use of Sideritis species was first mentioned by Dioscorides in his book "De Materia Medica" [3]. Over the years, Sideritis species have found applications in Mediterranean traditional medicine [4] for their anti-inflammatory, antirheumatic, and antimicrobial activities [1,5]. Several investigations into plants belonging to the genus For this study, the aerial parts of three Sideritis species were harvested from different regions of Greece. The first sample of S. scardica (SSC1) was cultivated in the area of Mount Olympus in Central Greece (Olympus tea), while the second sample of S. scardica (SSC2) was cultivated in the area of Mount Mainalo in Peloponnesos; finally, the third sample of S. scardica (SSC3) was cultivated in the area of Kastoria in Northern Greece. The first sample of S. raeseri (SR1) was cultivated in the foothills of Mount Othrys in Central Greece, the second sample of S. raeseri (SR2) was cultivated in the area of Kastoria in Northern Greece, and the third sample of S. raeseri (SR3) was cultivated in the area of Elassona, Larissa, in Central Greece. The three samples of S. syriaca were cultivated in Crete island: the first sample of S. syriaca (SS1) was cultivated in the southern part of the White Mountains (Lefka Ori), the second sample of S. syriaca (SS2) was cultivated in the area of Anopoli Sfakion near to White Mountains (Lefka Ori), and the third sample of S. syriaca (SS3) was cultivated in the area of Omalos Chanion (Figure 1).  (Table 1).  (Table 1).

Isolation of the Essential Oil
The fresh aerial parts of these plants were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The yields of EOs (Table 2) for these 9 samples ranged from 0.01% (v/w) (SS3) to 0.10% (v/w) (SR3). Due to their extremely low oil yield, EOs were obtained by liquid-liquid extraction using diethyl ether. The diethyl ether phase was then concentrated under a gentle flow of nitrogen stream, and the resulting solutions of oils were dried over anhydrous magnesium sulfate and stored in the freezer.

Gas Chromatography-Mass Spectrometry (GC-MS)
The EOs were analyzed using a gas chromatography instrument (SCION) coupled with a mass spectrometer detector and an autosampler (CP-8400) (Bruker, Carteret (NJ), USA). The capillary column used was OPTIMA-5 MS, 30 m × 0.25 mm, 0.25 µm. Helium (He) was used as the carrier gas, with a flow rate of 1.0 mL/min. The temperature at the injector was 220 • C and at the ionization source was 230 • C. The source was operated with an electric voltage of 70 eV. The analysis program, which had a total duration of 73.33 min, involved a rise in the temperature of the column, which was initially at 60 • C and increased gradually up to 250 • C at a rate of 3 • C/min and at a rate of 5 • C/min up to 300 • C. The volume of the sample to be analyzed was 1 µL.

Identification of the Components of the Essential Oils
The identification of volatile components of EOs was performed by comparing the mass spectra of the components with those from NIST and Adams mass spectral libraries and by comparing literature and estimated arithmetic (retention) indices that were determined using mixture of homologous series of normal alkanes from C 7 -C 24 in n-hexane under the same conditions.

Results
The chemical composition (%) of the EOs of the nine Sideritis samples are summarized in Table 3. In the Sideritis scardica EOs (SSC1 and SSC2), others constituted the main group of components ( Figure 2) yielded in a total amount of 45.65% and 52.02%, respectively, while for the essential oil of SSC3, the group of components known as oxygenated monoterpenes was found as the predominant amount in a percentage of 35.46%. In the Sideritis raeseri EOs (SR1, SR2, and SR3), considerable variations were observed ( Figure 3). Oxygenated sesquiterpenes constituted the main group of components found in SR1 in a total amount of 35.1%, while in the SR2 and SR3, oxygenated monoterpenes and monoterpene hydrocarbons were estimated in a total amount of 31.91% and 28.12%, respectively. Considerable variations were also observed in the Sideritis syriaca EOs (Figure 4). In the essential oil of SS1, the main group of components was others (49.38%), because of the presence of 1-octen-3-ol (17.90%) and hexanol (16.23%) in a high percentage. In the SS2, oxygenated monoterpenes constituted the main group of components yielded in a total amount of 49.13%, while sesquiterpene hydrocarbons (32.87%) constituted the main group of components found in the SS3. Table 3. Chemical composition (%) of the essential oils of Sideritis samples.
The chemical variation and relationship between the Sideritis species are presented in the dendrogram ( Figure 5). As can be observed, based on their chemical composition the samples were initially divided into two groups. The 1st group included the sample SR1, and the 2nd group included all the other samples (SSC1, SSC2, SSC3, SR2, SR3, SS1, SS2, and SS3). Then, the samples of the 2nd group were divided into subgroups; the 1st subgroup had only one sample-SR3-and 2nd subgroup included the rest of the other samples (SSC1, SSC2, SSC3, SR2, SS1, SS2, and SS3). raeseri from Elassona; SS1: S. syriaca from Lefka Ori; SS2: S. syriaca from Anopoli Sfakion; SS3: S. syriaca from Omalos. c not identified. d correct isomer did not identify. e correct isomer did not identify.
Τhe chemical variation and relationship between the Sideritis species are presented in the dendrogram ( Figure 5). As can be observed, based on their chemical composition the samples were initially divided into two groups. The 1st group included the sample SR1, and the 2nd group included all the other samples (SSC1, SSC2, SSC3, SR2, SR3, SS1, SS2, and SS3). Then, the samples of the 2nd group were divided into subgroups; the 1st subgroup had only one sample-SR3-and 2nd subgroup included the rest of the other samples (SSC1, SSC2, SSC3, SR2, SS1, SS2, and SS3).
In this second subgroup, the samples were separated into smaller subgroups: one subgroup (3rd subgroup) consisted of the samples SS1, SS2, and SS3, and another subgroup included the samples SR2, SSC1, SSC2, and SSC3 (4th subgroup). The 3rd subgroup samples were also divided into smaller subgroups: the 5th subgroup included SS1 and SS2, and the 6th subgroup included SS3; the 4th subgroup samples were divided into the 7th subgroup-SR2-while the 8th subgroup included SSC1, SSC2, and SSC3. Finally, the 8th subgroup samples were divided into a 9th subgroup-including SSC1-and a10th subgroup-including SSC2, SSC3.
According to these results, the EO of the SR1 was differentiated due to its chemical composition from the rest of the eight EOs. Both of the other two EOs of the S. raeseri samples were quite different from the rest. The EOs of S. syriaca (SS1, SS2, and SS3) and S. scardica (SSC1, SSC2, and SSC3), showed more similar chemical composition, especially the EOs of SS1 and SS2, as well as SSC2 and SSC3. In the nine samples studied, 165 different compounds were found in total. However, only seven of them were identified to be present in all of the nine samples. Figure 6 depicts the contents of the seven common components, namely, 1-octen-3-ol, linalool, trans-pinocarveol, p-mentha-1,5-dien-8-ol, α-terpineol, myrtenol, and verbenone. In five out of the nine samples, 1-octen-3-ol was estimated as the most common component (>6%) ( Figure  6A). All of the rest of the common components (linalool, trans-pinocarveol, p-mentha-1,5dien-8-ol, α-terpineol, myrtenol, and verbenone) were found at percentages that were lower than 6% ( Figure 6A,B). In this second subgroup, the samples were separated into smaller subgroups: one subgroup (3rd subgroup) consisted of the samples SS1, SS2, and SS3, and another subgroup included the samples SR2, SSC1, SSC2, and SSC3 (4th subgroup). The 3rd subgroup samples were also divided into smaller subgroups: the 5th subgroup included SS1 and SS2, and the 6th subgroup included SS3; the 4th subgroup samples were divided into the 7th subgroup-SR2-while the 8th subgroup included SSC1, SSC2, and SSC3. Finally, the 8th subgroup samples were divided into a 9th subgroup-including SSC1-and a 10th subgroup-including SSC2, SSC3.
According to these results, the EO of the SR1 was differentiated due to its chemical composition from the rest of the eight EOs. Both of the other two EOs of the S. raeseri samples were quite different from the rest. The EOs of S. syriaca (SS1, SS2, and SS3) and S. scardica (SSC1, SSC2, and SSC3), showed more similar chemical composition, especially the EOs of SS1 and SS2, as well as SSC2 and SSC3.
Furthermore, it was found that the EOs of the S. scardica and S. raeseri from three different regions of Greece and the S. syriaca from three different localities of Crete Island, in Southern Greece, yielded different major components.
The essential oil's chemical variability in relation to the examined plant species was presented using a dendrogram dividing the samples into two main groups and of one group to six subgroups. Among the same species of different geographical origins, the observed qualitative and quantitative differences can be attributed to abiotic factors such as soil, temperature, humidity, rainfall, and altitude, which influence the biosynthetic pathways of certain essential oil constituents.
The present work presents new data for the EOs' chemical composition of the cultivated S. scardica, S. raeseri, and S. syriaca of different geographical origins in Greece, which therefore contribute to the existing literature.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.