Variation of the Chemical Composition of Thymus Vulgaris Essential Oils by Phenological Stages

CRISTIAN MOISA1,2, ANDREEA LUPITU1,2, GEORGETA POP2, DORINA RODICA CHAMBRE1, LUCIAN COPOLOVICI1, GABRIELA CIOCA3*, SIMONA BUNGAU4*, DANA MARIA COPOLOVICI1 1Aurel Vlaicu University, Faculty of Food Engineering, Tourism and Environmental Protection; Institute for Research, Development and Innovation in Technical and Natural Sciences, Romania, 2 Elena Dragoi Str., 310330, Arad, Romania 2Banat University of Agricultural Sciences and Veterinary Medicine King Michael 1st of Romania from Timisoara, 119 Calea Aradului Str., Timisoara, Romania 3Lucian Blaga University of Sibiu, Faculty of Medicine, 10 Victoriei Blvd., 550024, Sibiu, Romania 4University of Oradea, Faculty of Medicine and Pharmacy, 29 N. Jiga Str., 410028, Oradea, Romania

A lot of papers in the scientific literature focus on the content of essential oils and other components (especially antioxidant and therapeutics) existing in different parts of plants [1][2][3][4][5][6][7], these oils having numerous and diverse uses since ancient times [8]. Thymus vulgaris L. var. Donne Valley is a woody-based perennial evergreen subshrub rich in essential oil stored in glandular peltate trichomes located on both sides of the leaves, that blooms in clusters of pink and purple flowers in early summer (June and July) [9][10][11][12][13]. It belongs to the Lamiaceae family and is indigenous to the southern part of Europe and the Mediterranean area [6]. Both thymi herba (aerial plant parts) and thymi aetheroleum (thyme essential oil) are used in food industry as spices, pharmaceutics and medicine, cosmetics and perfumery [9,10,[14][15][16][17].
Chemical polymorphism is usual in Lamiaceae family when one or more chemotypes have been observed in Thymus species in accordance with the major component determined in the essential oil [23,24]. Thymol and carvacrol are the most frequent chemotypes of thyme species indicating them as major components of thyme essential oils, having the same molecular weight (M=150 g.mol -1 ) but with a different OH group position at the phenolic ring (meta and ortho). Thymol and carvacrol are attended by two precursors: p-cymene and γ-terpinene [10,25].
The aim of this study was to analyze the variations in chemical composition depending on phenological stages and different vegetation cycles (over a period of two years) for Thymus vulgaris L. var. Donne Valley. For this purpose, essential oils were obtained before and after flowering stages in two different years (2017 and 2018) under different meteorological conditions. The results obtained in this paper are meant to complete previous work reported by other authors [10,14,16,19,20,23] in relation to occurring differences in the chemical composition of thyme essential oils influenced by phenological or weather conditions.

Essential oil extraction
Leaves and flowers were removed from the aerial parts of thymi herba and were placed on the grid found in the column of a 5L copper alembic distillation equipment to be subjected to steam distillation. The essential oil and hydrolate mixture were placed in a separator funnel and left to separate in two layers based on their density. The collected pure essential oil was stored at 4 o C in brown glass vials until further analysis.

Annotations
Essential oil of Thymus vulgaris L. var. Donne Valley was denoted as TDL-x-y, where x stands for year of harvest and y stands for phenological stage, as is presented in table 1.

GC-MS analysis
The chemical composition for all TDL-x-y essential oil samples were determined by gas chromatography (Shimadzu 2010, Kyoto, Japan) coupled with mass spectrometer (TQ 8040, Shimadzu, Kyoto, Japan). The column was an Optima 1MS+WAX column (30 m x 0.25 mm i.d., 0.25 µm film thickness, Macherey-Nagel, Duren, Germany). As a carrier gas, He was used at 1 mL min -1 . The initial temperature of the oven was 70 o C (for 11 min); it was raised to 190 o C at a rate of 5 o C min -1 , then to 240 o C at a rate of 20 o C min -1 and left for 5 min. Injector temperature was 250 o C and MS source temperature was 200 o C. The volume of injection was 1 µL with a split ratio of 10:1. TDL-x-y compounds have been identified based on their mass spectra using NIST 14 library and Wiley 09 library.  ATR-FTIR analysis Essential oil samples were analyzed by FT-IR and their spectra were recorded using a Bruker Vertex 70 spectrophotometer equipped with an ATR cell on the 600-4000 cm -1 wavelength range, a 4 cm -1 resolution and 32 scans. Background measurements were performed before each analysis. Normalization (min-max) and baseline corrections were applied, and processing of the spectra was performed using OPUS software.

Results and discussions Essential oil composition
The yield for extraction of essential oils TDL-2017-BF/ AF was ~0.9% and for TDL-2018-BF/AF was ~0.3%. The chemical compositions of those oils are presented in table 2.
Based on literature, essential oils of Thymus species have been known to have six chemotypes, containing as major component a phenolic derivative (thymol or carvacrol) or an alcohol (linalool, geraniol, thujanol-4 or áterpineol). It is difficult to have a precise delimitation of chemotypes, therefore for essential oils, the distinction is done in accordance to the major compound detected in the essential oil, but one should also take into consideration the biogenetic pathways [26,27]. During phenological stages the chemical composition of the essential oil changes, influencing the percentage of major compounds. Differences were observed in the essential oil chemical composition before and after flowering stages from two consecutive years taken into this study. Differences are depicted in figure 1 and 2.
The chemical composition of TDL-2018-BF/AF also have shown modifications mainly in the major compounds. Furthermore, fewer compounds were detected compared with the essential oils from 2017 (table 1).
As it is presented in table 1 and figures 1 and 2, the chemical composition of Thymus vulgaris L. var. Donne Valley essential oil changes from one phenological stage to another giving options on choosing the ideal harvesting time. ATR-FTIR analysis ATR-FTIR spectroscopy is a simple, sensitive, fast and non-destructive method of analysis based on the vibrational spectra recording. As it is known, all major compounds present in essential oils dominate the obtained vibration spectra, while low concentration compounds, hardly influence the ATR-FTIR pathway [29]. In accordance with GC-MS analysis results, the major compounds found in TDL-x-y essential oils are: thymol, γ-terpinene and pcymene. A smaller impact could be attributed to: 3octanone, α-terpinolene, thymol methyl ether, carvacrol methyl ether, carvacrol and isocaryophyllene.
The ATR-FTIR spectra recorded for the TDL-x-y essential oil samples are presented in figure 3, with characteristic bands added. Because essential oils are complex mixtures of compounds, the absorption spectrum for some compounds overlap.

Conclusions
The present study revealed differences between the chemical compositions of essential oils obtained from plants harvested in different phenological stages and vegetation cycles. For this purpose, essential oils were obtained and analyzed before and after flowering stages in two different years (2017 and 2018). Through GC-MS and ATR-FTIR analysis the chemical composition of Thymus vulgaris L. var. Donne Valley essential oils was determined. Thymol was found as the major component with a high percentage 46.74% in essential oil obtained before flowering in 2018. This percentage changed through phenological stages and vegetation cycles. Due to the differences between chemical composition in phenological stages and vegetation cycles, and taking into consideration the desired compound of interest, the harvesting time could plan ahead.
Because there is an increasing demand on natural bioactive compounds derived from medicinal plants, Thymus vulgaris L. var. Donne Valley is a rich source of phenolic monoterpenes with many applications (food, pharmaceutical, cosmetic, perfumery, agriculture, etc).