Phytochemical Study of Eight Medicinal Plants of the Lamiaceae Family Traditionally Used as Tea in the Sharri Mountains Region of the Balkans

In the present study, eight plant species belonging to Lamiaceae family were identified as ingredients for herbal teas in the region of Sharri Mountains: Thymus serpyllum, Rosmarinus officinalis, Melissa officinalis, Origanum vulgare, Mentha longifolia, Ocimum basilicum, Teucrium chamaedrys, and Sideritis scardica, respectively. Chemical composition of essential oils obtained from these species was analyzed using GC-MS and GC-FID with the aim of examining their volatile compound profiles, responsible for their respective flavors and fragrance. Principal Component Analysis (PCA) was performed with the aim of grouping plant species under study on the basis of their chemical composition. Experimental data revealed the typical volatile constituent pattern for the Lamiaceae family. Monoterpenes and sesquiterpenes, responsible for flavor and medicinal use of these plants, were the most abundant groups of the volatile constituents. PCA data analysis resulted in the grouping of these analyzed species in four principal clusters.


Introduction
e Sharri Mountains region (in Albanian known as Malet e Sharrit; in Macedonian and Serbian asŠar-Planina) is one of the biggest mountain massifs in Balkans (1,600 km 2 ) and is situated in the Republic of North Macedonia (in a total area of 826.8 km 2 or 51.44%) and Republic of Kosovo (in a total area of 780.4 km 2 or 48.56%) [1]. is region serves as a tremendous reservoir of Traditional Environmental Knowledge (TEK) related to wild plants because of its complex biocultural diversity (hotspot of biodiversity and home to a variety of different ethnic and religious groups), the socioeconomic environment, and the long held traditions in the collection of wild plants by local inhabitants.
To date, there are roughly 1,500 vascular plant species known to grow in the Sharri Mountains [2,3]. In addition to local species richness, the region is also rich in cultural, linguistic, and religious diversity. Various ethnic groups, including Albanians, Bosniaks, and Gorani (Muslims) and Serbs (Christian Orthodox) live on the Kosovo side of the Sharri, while the Macedonian side is inhabited by Albanians and Gorani (Muslims) and Macedonians (Christian Orthodox). ese ethnic groups have lived in close contact over a span of many centuries in villages that are isolated from urban areas.

Plant Material.
Plants species were chosen based on the review of numerous ethnobotanical studies and published ethnographies, as well as unpublished fieldwork results originating from Sharri (Kosovo and North Macedonia). Species used in water infusions as aromatic and refreshing hot beverages (recreational tea) consumed in food and not for specific medicinal purposes were selected.
Plant material (specific plant tissues used to prepare tea) was either collected in the Sharri region from wild populations or purchased from the local markets, provided by local residents in Prizren (Kosovo) and Tetovo (Macedonia) that wildcrafted or cultivated the materials in Sharri for personal or family use. Plant material was collected from May to September and stored in a dry place protected from light until analyzed.
Taxonomic identification followed relevant standard botanical literature of the area [20]. Botanical nomenclature and family assignments followed the Plant List database [21], and voucher specimens were deposited at the University of Prishtina Herbarium.

Plant Material Extraction.
Plant material was air-dried in the shade at room temperature, and volatile compounds were obtained by hydrodistillation, standard extraction method of volatile compounds, using a Clevenger apparatus for 2 hours. e extracts were stored at − 18°C in a freezer until further analysis.

GC-MS Analysis.
GC-MS analysis using an Agilent 7890A GC system coupled to a 5975C MSD (Agilent Technologies) was used for the separation and identification of essential oils constituents. e ionization energy was 70 eV. e separation was conducted in HP-5MS column (30 m × 0.25 mm i.d., film thickness 0.25 mm). e carrier gas used was He, with an initial flow rate of 0.6 mL/min and subsequently at a constant pressure of 50 psi. e carrier gas used was He with a constant flow rate 1.0 mL/min, injection temperature 280°C, injection volume 1.0 μL, and a split ratio of 25:1. e initial GC oven temperature was 60°C (5 min) and was increased from 60°C to 200°C at a rate of 5°C/min.

GC-FID Analysis.
GC-FID analyses were performed using an Agilent 7890A GC system equipped with an FID detector (Agilent Technologies). e separation was conducted on a HP-5MS column (30m × 0.1 mm, with 0.17 μm film thickness). e carrier gas used was He, with an initial flow rate of 0.6 mL/min and subsequently at a constant pressure of 50°psi. e GC oven temperature increased from 60°C to 160°C at a rate of 10°C/min and subsequently to 280°C at a rate of 20°C/min., and the FID operated at 250°C with an air flow of 350 mL/min and a hydrogen flow of 35 mL/min. e injection volume was 1.0 mL.

Identification of Essential Oil Components.
e identification of each of the components of the essential oil was performed by comparing their Kovats retention indices with those reported in the literature [22]. e Kovats index was calculated based on a linear interpolation of the retention times of a homologous series of n-alkanes (C9-C28) under the same operating conditions. e components were also identified by comparing the mass spectra of each constituent with those stored in the NIST 08 and WILEY MS 9 th databases and with mass spectra from the literature [22]. e percentage composition of the oils was computed using the normalization method from the GC peak areas, calculated as the mean of three samples, without correction factors.

Statistical Analysis.
Principal Component Analysis (PCA) was used for multivariate association between volatile constituents and plant species in the study. Chemical compounds present in percentages higher than 3% were selected for statistical analysis. e XLSTAT program (version 2014.2.03) was used for the PCA and HCA.

Results and Discussion
In total, eight plant species belonging to the Lamiaceae family were identified as being used for making recreational herbal teas (Tables 1 and 2). Members of the Lamiaceae (and Rosaceae) family are well-known as local herbal teas in eastern and central Europe [10]. Principal Component Analysis demonstrated that eight plant species examined in this study have been grouped in four principal groups. ymus serpyllum in the first group, Rosmarinus officinalis in the second group, and Melissa officinalis in the third group, whereas the fourth group included Origanum vulgare, Mentha longifolia, Ocimum basilicum, Teucrium chamaedrys, and Sideritis scardica, with greater similarities in relation to their chemical composition ( Figure 1).

Chemical Composition of Volatile Compounds of Lamiaceae Species.
Volatile compounds of eight species of the Lamiaceae family were analyzed in total, and results have been presented in Table 1.

Origanum vulgare L., Lamiaceae.
irty-nine compounds were identified using GC-MS analysis of essential oils obtained from the distillation of aerial parts of the oregano, Origanum vulgare L. ese compounds predominantly belonged to the class of sesquiterpenes and their oxygenated derivatives (36.92% and 10.57%, respectively) and monoterpenes and their oxygenated derivatives (23.32% e Scientific World Journal

Rosmarinus officinalis L., Lamiaceae.
irty compounds were identified in the essential oil obtained by hydrodistillation of the aerial parts of rosemary, Rosmarinus officinalis.

3.3.
Frequency of the Species Used as Tea. Some of the plant species still have an important use as herbs in the cultures of the Sharri region.
us, the Origanum vulgare and ymus serpyllum still are frequently used in regions, while Sideritis scardica is frequently used in Macedonia, as it is present in a larger population, while in Kosovo side of Sharri it is rarely used, as it grows in only a few small populations and this is an endangered species. Melissa officinalis , Ocimum basilicum , and Mentha longifolia are occasionally used to prepare tea. On the other hand, Teucrium chamaedrys and Rosmarinus officinalis are rarely used nowadays.

Conclusions
Over centuries, a considerable number of plant species in Sharri region have been used as traditional medicines, foods, and teas. In this study, eight plant species belonging to the Lamiaceae family were identified to be used as recreational tea based on review of the ethnobotanical literature. Chemical screening of the volatile constituents carried out with GC-MS and GC-FID revealed that most of these species have quite different chemical compositions (qualitatively and quantitatively) which contribute to the specific flavor and fragrance of these teas.
Further research is needed to address the preferences of the consumers on the tastes, fragrances, and colors of these recreational teas. Furthermore, research on the pharmacological, nutritional, and phytochemical properties of plants used for making tea is needed to ensure the safety and appropriateness of their use, especially for those consumed on a daily basis. e wild plant species used for the preparation of tea represent an important national resource and therefore they need to be preserved and used in a sustainable way. e promotion of the traditional values of this group of plant species, besides their scientific importance, also represents their socioeconomic importance, since their promotion ecotourism, rural tourism, and other economic activities can be stimulated. is baseline data can be used for projects intended to foster rural development programs focusing on sustainable valorization of local herbal and wild food resources.