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ESSENTIAL OILS CHEMICAL VARIABILITY OF SEVEN POPULATIONS OF SALVIA OFFICINALIS L. IN NORTH OF ALBANIA

Department of Agronomy Sciences, Faculty of Agriculture and Environment, Agricultural University of Tirana, Kodër Kamëz Tirana, Albania Department of Ecology and Agro-Environment, Faculty of Agriculture and Environment, Agricultural University of Tirana, Kodër Kamëz Tirana, Albania Department of Biology, Faculty of Mathematical and Natural Science, University of Prishtina “Hasan Prishtina”, Mother Theresa St., 10000 Prishtinë, Kosovo Department of Horticultural Sciences, University of Bonn, Bonn, Germany University of Zagreb, Faculty of Agriculture, Department of Seed Science and Technology, Svetošimunska cesta 25, 10000 Zagreb, Croatia Centre of Excellence for Biodiversity and Molecular Plant Breeding (CroP-BioDiv), Svetošimunska cesta 25, 10000 Zagreb, Croatia Centre for Natural Products, Faculty of Pharmacy, Ss. Cyril and Methodius University, Majka Tereza 47, 1000 Skopje, North Macedonia Institute of Pharmacognosy, Faculty of Pharmacy, Ss. Cyril and Methodius University, Majka Tereza 47, 1000 Skopje, North Macedonia


INTRODUCTION
Nowadays, Salvia officinalis L. (Lamiaceae), also known as Dalmatian sage, is a flavorful and much used medicinal plant throughout the world. It is a perennial evergreen subshrub, native to the Mediterranean region, up to 0.5-1.0 m high with woody stems and greyish leaves [1,2]. Common sage has been used for a long time as traditional herbal medicine against a variety of diseases, for culinary purposes, and as a condiment [1][2][3][4][5][6][7][8].
Based on its great economic and industrial value, S. officinalis L. is the most important medicinal and aromatic plant in Albania. Additionally, Albania is an important exporter of medicinal and aromatic plants in Europe and the most important exporter of sage to the USA [9][10][11]. In Albania, S. officinalis L., is one of the most frequently used herbs in folk medicine. It is used for different medicinal purposes like sore throats, tonsillitis, a cough, the flu, "good for the health", "to strengthen the stomach", digestion, etc. [12]. However, over the past 20 years, the wild populations of sage have declined so drastically that now it is classified in the Red Book of Albania as "vulnerable"-VU A1b [13].
Sage extracts and essential oils are phytotherapeutically useful. S. officinalis L. essential oil has a wide range of biological activities, and it is used in the treatment of many diseases because of its viricidal, cytotoxic, antimutagenic, antioxidant, antimicrobial, hypoglycaemic, anti-inflammatory, fungistatic, astringent, eupeptic and anti-hydrotic effects. Additionally, this plant is used as digestive stimulator, appetizer and performance enhancer [14][15][16].
Essential oils of more than 32 populations of S. officinalis from 11 Balkan countries (24 indigenous populations and eight non-indigenous, cultivated or naturalized, populations) including six wild growing populations from Albania were analyzed by Cvetkovikj et al. [19]. Schmiderer et al. [20] investigated the differences between the sage populations in the South and North of Albania. All of these studied populations are not the subject of the present work.
Previous reports published on the essential oil composition of sage in Albania and South-Eastern-European countries showed a variation in the content and chemical composition. Therefore, a number of classifications have been proposed [1,4,5,[18][19][20]. The most commonly reported major constituents of Dalmatian sage essential oil have been αthujone, β-thujone, camphor, and 1,8-cineole.
The aim of this study was to assess the yield and chemical composition of the essential oil of seven wild sage populations from the North of Albania and its compliance to ISO standards.  Table 1).
The leaves were air dried, packed in paper bags and kept in a dark and cold place until analysis. Voucher specimens were deposited at the Crop Production Department, Agricultural University of Tirana (Table 1). Plant identity was verified by A. Ibraliu according to Albanian Flora [24].

Essential oil isolation
Essential oil isolation from dried sage leaves, as an average sample of the 30 different individual samples of every population, was performed by hydrodistillation in an all-glass Clevenger apparatus for two hours, following the procedure of the European Pharmacopeia (2.8.12) [21]. These samples were stored at -18 ºC in the freezer until further analysis.

Analysis of essential oils' chemical composition
Each of the seven different essential oils was dissolved in xylene (10:990, v/v) for GC analyses. Oil samples were analyzed on a gas chromatography system (Agilent 7890А) equipped with FID detector and mass spectrometer detector (Agilent 5975C), using HP-5ms capillary column (30 m × 0.25 mm, film thickness 0.25 μm).
Analytical conditions were as follows: oven temperature was set at 60 °C for 0 min, then increased by 3 °C/min to 240 °C and held there for 1 min and then it was increased to 280 °C at a rate of 10 °C/min where was held for 1 min; helium was used as the carrier gas at a flow rate of 1 ml/min; temperature of the injector was 220 °C and that of the FID detector was 270 °C; the GC split was ratio 1:1. The injection volume was 1.0 μl.
The mass spectrometry conditions were as follows: ionization voltage 70 eV, ion source temperature 230 °C, transfer line temperature 280 °C, and mass range from 50-550 Da. The MS was operated in scan mode.

Identification of the components
The compounds were identified by comparing their Kovats (retention) indices with those in the literature [25]. The Kovats (retention) indices were determined using a mixture of a homologous series of normal alkanes (C9-C25) analyzed under the Automated Mass Spectral Deconvolution and Identification System (AMDIS) conditions [26]. Confirmation was done by comparing the mass spectra obtained from AMDIS with the reference spectra from NIST (National Institute of Standards and Technology), Wiley and Adams mass spectra libraries. Quantification of the essential oils components was performed using the normalization method of the GC/FID peak areas.

Statistical analysis
Correlations among seven main essential oil constituents were calculated by PROC Corr in SAS [27]. The Principal Component Analysis (PCA) based on the main constituents was performed using PROC PRINCOMP in SAS. Pairwise Euclidean distances among seven populations were calculated and used in unweighted pair-group average (UP-GMA) cluster analysis. Support for clades was evaluated by nonparametric bootstrapping [28] with 10.000 replicates in PAST ver. 2.01 [29].

RESULTS AND DISCUSSION
The yield of essential oil differed between sage populations and ranged from 1.95 to 2.75 % ( Table 2). The highest yield was determined in P2 (2.75 %), while the lowest yield of essential oils was observed in P5 (1.95 %). All populations met the requirements of the European Pharmacopeia 7.0 and ISO 6571/2008 standard for Dalmatian sage essential oil content [21,22]. In all of the essential oils analyzed, 42 constituents were identified in total. The number of identified constituents differed among populations. The lowest number of constituents were found in P1 (20 constituents); while the highest number was found in P7 (39).
Seven constituents were found at concentrations higher than 5.00 % in at least one population: camphene, 1,8-cineole, α-thujone, β-thujone, camphor, α-humulene, and viridiflorol. These constituents were chosen for further analysis and were hereafter referred to as the main constituents. They constituted 72.40 % (P6) to 78.39 % (P4) of the essential oils of individual populations. Camphor was the dominant component in populations P2, P5 and P6, while it had an equal share with α-thujone in population P3 (Table 2). On the other hand, in the populations P1, P4 and P7, α-thujone was the most abundant compound (Table 2). Table 3 shows the correlation between the seven main constituents of the essential oils of S. officinalis L. Strong negative correlations (r < -0.70) have been observed between 1,8-cineole and α-humulene.  The significance of the correlations is indicated as follows: *significance at the 5.00 % nominal level; nsnot significant.
The Principal Component Analysis (PCA) of the seven main constituents in seven Dalmatian sage populations revealed that three principal compo-nents had an eigenvalue greater than 1 and explained 86.01 % of the total variation (Table 4; Fig.  1A). The significance is indicated as follows: ***significance at the 0.10 % nominal level; **significance at the 1.00 % nominal level; *significance at the 5.00 % nominal level; nsnot significant. The first PC explained 47.73 % of the total variation and was strongly positively correlated with 1,8-cineole (r > 0.70; P < 0.05), while strongly negatively correlated with α-humulene (r < -0.70; P < 0.05). Strong negative correlation was observed between the second PC and β-thujone (r < -0.70; P < 0.05). The average Euclidean distance among populations based on seven main constituents was 6.12, ranging from 2.22 (between populations P3 and P4) to 11.83 (between P5 and P6). The highest net divergence defined as the sum of distances from a population to all the other populations was observed for population P6 (58.05) followed by P5 (45.27). As shown in the UPGMA dendrogram based on the Euclidean distance matrix, populations P5 and P6 were the most distant from the others ( Figure 1B). To characterize the natural variability between the S. officinalis L. populations, we compared the yield and chemical composition of the essential oils from seven locations in the North of Albania. The essential oil yield of the studied populations ranged from 1.95 to 2.75 %, and these data are very similar to the results reported for essential oil yield of sage populations from Croatia and Montenegro, ranging from 1.90 to 3.70 % and 1.98 to 2.84 %, respectively. This shows that the oil yield varies depending on the origin of the plant material [1,4,5]. However, these results were higher compared to the results published by Cvetkovikj et al. [19] where the oil yield ranged from 0.15 to 1.70 % for the six indigenous populations of S. officinalis L. from Albania.
According to Boszormenyi et al. [30], the chemical composition of the essential oils of S. officinalis L. that originated from different countries were divided into five groups according to the amount of the major constituents:  [32].
The relative amounts of camphor and αand ß-thujone determine the quality of essential oils of Dalmatian sage. The best quality of Dalmatian sage essential oil is in the cases where αand ß-thujone (together) are present at up to 50.00 % of the total percentage of essential oil, while the relative amount of camphor is not lower than 20.00 % [34,[41][42][43]. International standards require 18.00-43.00 % of α-thujone and 3.00-8.50 % of ß-thujone [23]. Thus, the essential oils of the populations P1, P3, P4, and P7 comply with the requirements of the ISO 9909/1997 standard for Dalmatian sage essential oil composition.

CONCLUSION
Variability between the chemical composition and content of the essential oil from the seven different Dalmatian sage populations of North Albania was recorded.
The yield of sage essential oils ranged from 1.95 to 2.75 %. All populations had possessed oil content within the requirements of the European Pharmacopeia 7.0. [21] and ISO 6571/2008 Standard [22]. P1, P2, P3 and P4 populations showed a similar chemical oil profile. On the other hand, it was confirmed by statistical analysis that populations P5 and P6 differed the most in the chemical composition of their oils. Finally, populations: P1, P3, P4 and P7 comply with the requirements of the ISO 9909/1997 Standard for the Dalmatian sage essential oil composition [23].