Influence of Harvest Term on the Content of Carvacrol, p-Cymene, γ-Terpinene and β-Caryophyllene in the Essential Oil of Satureja montana

The aim of the studies was to determine the optimal term of harvest for Satureja montana L. (winter savory) in order to obtain the essential oil rich in antioxidative compounds such as carvacrol, p-cymene, γ-terpinene and β-caryophyllene. Essential oils of S. montana aerial parts were obtained by hydrodistillation in Deryng-type apparatus and analyzed by gas chromatography/mass spectrometry (GC/MS). In total, 30 compounds were identified in the savory volatile oil under different harvesting terms (before flowering, during flowering and after the flowering), that represented 94.61 to 97.55% of the oils. The major components were carvacrol (65.43 to 69.99%), its precursors: p-cymene (3.69 to 9.69%) and γ-terpinene (1.51 to 5.92%) as well as β-caryophyllene (2.74 to 4.71%). Moreover, the term of harvest had a significant effect on the content of main essential oil constituents. The highest concentrations of carvacrol, γ-terpinene and β-caryophyllene were observed in the herb collected before flowering, while the highest amounts of p-cymene were noted after the flowering.


Introduction
The genus Satureja belongs to the family Lamiaceae that comprises numerous species growing wild in Mediterranean area. Among them, many are used worldwide as medicinal and spice plants (Vidic et al., 2010).
Satureja montana L., commonly called winter or mountain savory, is a bushy perennial subshrub with woody stems at the base, linear leaves and pale pink flowers (Lawless, 2002). This aromatic plant can be found in nature, but it is also cultivated as a culinary herb having strong and spicy taste . The leaves and flowering tops are used as flavoring agents in salads, soups, sauces, stews, and lentil dishes (Small and Deutsch, 2001). Savory is one of the best honey plants and its honey is wellknown as folk remedy for bronchitis (Mastelic and Jerkovic, 2003). The whole plant is mildly antiseptic, carminative, digestive, expectorant and stomachic .
Winter savory oil is used in the food industry to flavor condiments, canned meats, sausages, soups, in the making of liqueurs and in the perfumery (Ciani et al., 2000;Small and Deutsch, 2001). It is also applied as natural conservation agent in cosmetic and food industries and as active ingredient in medicinal preparations (Chorianopoulos et al., 2004). According to Lawrence (1979), the value of S. montana oil is due to its high carvacrol content and its fresh, spicy notes reminiscent of oregano and thyme oils.
The application of savory oil in different industries as well as its quality and biological activity depends on the oil composition which is affected by vegetative stage of the plant (Kustrak et al., 1996;Milos et al., 2001;Slavkovska et al., 2001).
The aim of this work was to determine the optimal term of harvest for winter savory herb to gain the oil with high content of biologically active compounds, such as carvacrol, p-cymene, γ-terpinene and β-caryophyllene. time started after the falling of the first drop of distillate. The essential oil obtained this way was separated from water (due to its immiscibility with water) and then dried over anhydrous sodium sulphate, filtered, and stored in sealed vial at 4 °C until GC-MS analysis.
Tree replicates were carried out. Essential oil percentage was calculated based on dry weight of plant material and expressed as (% v/w) in Tab. 2.

Gas Chromatography/Mass Spectrometry (GC/MS) analyses of essential oils
The qualitative GC-MS analysis of the volatile oils was performed using an HP 6890 gas chromatograph coupled with HP 5973 Mass Selective Detector operating at 70 eV mode. Compounds were separated on 30 m long capillary column (HP-5MS), 0.25 mm in diameter and with 0.25 µm thick stationary phase film ((5% phenyl)-methylpolysiloxane).
The initial temperature of the column was 40 °C for 5 minutes, then increased to 60 °C at a rate of 20 °C min -1 , next to 230 °C at a rate of 5 °C min -1 (kept constant for 20 min), and then increased to a final temperature of 280 °C at a rate of 10 °C min -1 .
The flow rate of helium through the column was kept at 1.2 mL min -1 . Samples of 2 μL (30 mg of oil dissolved in 1.5 mL of dichloromethane) were injected with a split ratio of 5:1. The temperatures of the injector, transfer line and ion source were maintained at 250, 280 and 230 °C, respectively. The solvent delay was 3 min. The scan range of the MSD was set from 40 to 550 m/z. The total running time for a sample was 65 minutes.
The identification of the components in the sample was based on the computer matching with the Wiley NBS75K.L and NIST/EPA/NIH (2002 version) mass spectral libraries, as well as by comparison of their mass spectra with authentic compounds available in our laboratory (thymol, carvacrol and p-cymene), purchased from Fluka and Sigma-Aldrich. The identity of compounds was also confirmed by retention indices from literature data (Adams, 2007;Cavar et al., 2008).
Retention indices (RI) values were measured on HP-5MS column. For RI calculation, a mixture of n-alkanes (C8-C20) was used, under the same chromatographic conditions which were used for the analysis of the essential oils.
The relative percentage amounts of the essential oil constituents were evaluated from the total peak area (TIC) by apparatus software.

Statistical analysis
Several results of the study (Tab. 2, 4) were subjected to an analysis of variance which was performed with AWAR software, made by Department of Applied Informatics,

Plant material
The experiment was conducted at the Horticultural Experimental Station near Szczecin (north-western Poland), which belongs to the West Pomeranian University of Technology Szczecin. The plants of Satureja montana L. (winter savory) were grown in experimental plots of area of 1.44 m 2 , in four replications.
The savory seeds (purchased from Herb Factory 'Kawon-Hurt', Gostyń, Poland) were sown on seedbed at the first decade of April 2008-2009. In the same years, the obtained seedlings were planted into the open field in the first decade of June, at row spacing of 40×30 cm. For laboratory analyses a herb from two-year old plants was collected before flowering (harvest dates: 11 July 2010 and 13 July 2011), during flowering (harvest dates: 6 August 2010 and 5 August 2011), and after the flowering (harvest dates: 13 September 2010 and 15 September 2011). The field was prepared according to agrotechnique proper for perennial plants from Lamiaceae family. Mineral fertilization was quantified according to the results of the chemical analysis of the soil samples. In the first year nitrogen fertilizer (80 kg N ha -1 ) was applied in three equal doses: ⅓ before sowing, ⅓ three weeks after sowing and ⅓ after harvest. Phosphorous fertilizer (60 kg P2O5 ha -1 ) and potassium fertilizer (100 kg K2O ha -1 ) were applied once, during spring cultivation treatment. In the second year of cultivation the whole phosphorous fertilizer and potassium fertilizer were applied once (on early spring) before plants vegetation. Nitrogen fertilizer was applied in two doses: ½ before plants vegetation and ½ after harvest.
The experiment was performed on sandy clay soil which is characterized by low water-holding capacity. During the growing season manual weeding and irrigation were performed.
After the harvest, plant material was dried in a shady and well ventilated place at room temperature (drying room). Dry herb was cut into small pieces and stored (in paper bags in a dry and cool place) until chemical analyses were performed.

Essential Oil Extraction
The essential oil was isolated from dry winter savory herb (collected before flowering, during flowering and after the flowering, separately) by hydrodistillation for 4 hours (after which no more essential oil was obtained) using a Deryng-type apparatus (Polish Pharmacopoeia VI, 2002). Each sample (30 g) was placed in 1 liter round-bottomed flask containing 500 mL of distilled water. The measurements of the distillation Institute of Soil Science and Plant Cultivation in Puławy. The means of two years were separated by the Tukey's test at p=0.05.

Results and discussions
The essential oil isolated by hydrodistillation from plant material collected at different growth stages (pre-flowering, during flowering and post-flowering) was found to be yellow liquid with strong, characteristic smell. As shown in Tab. 2, the 394 term of harvest had a significant effect on the content of essential oil in Satureja montana L. herb. Statistical analysis of the results obtained in the first year of the study as well as the synthesis of the two year study results showed that Satureja montana L. herb collected during the flowering phase and before flowering was characterized by significantly higher concentration of the essential oil in comparison with the herb collected after plant flowering. However, the differences between the content of essential oil in herb collected before and after flowering were not significant. In the second year of the 1.60 RI: retention indices relative to n-alkanes (C8-C20) on HP-5MS column study there were no significant differences found between the content of the essential oil according to the term of harvest.
Available literature data indicates that the essential oil content in S. montana herb varies depending on the place of origin. Skocibuasic and Bezic (2004) reported that the content of essential oil in some Croatian winter savories varied from 1.2 to 2.2%. In the study conducted by Damjanovic-Vratnica et al. (2011), the content of essential oil in the herb of S. montana from Montenegro ranged from 1.1 to 1.9%. Savory plants collected from north part of Albania  contained from 0.22 to 1.61% of essential oil, while plants harvested in the central part of Italy contains 0.59% of oil . The content of essential oil in our plants, especially in these collected during flowering stage, was higher compared to cited literature. Only winter savory cultivated in Egypt  had higher essential oil content (about 3%), in contrast to our results.
The chemical composition of isolated essential oil is shown in Tab. 3. The components are listed in order of their elution on the HP-5MS column.
The obtained results shows that the savory oils obtained from plants collected at different growth stages had similar 395 composition, however, thymoquinone was found only in the oils obtained from savory herb collected during (1.39 and 0.87% ) and after the flowering (2.86 and 2.46%) stage.
Because of high content of p-cymene, γ-terpinene, carvacrol and β-caryophyllene in the all oil samples, these major components were analyzed statistically (Tab. 4).
Statistical analysis of the results given in Tab. 4 showed that, in the first and the second year of the study as well as synthesis of both years of the study, that among four main constituents of Satureja montana L. essential oil, the highest concentration was noted for carvacrol, significantly lower for pcymene and the least for β-caryophyllene and γ-terpinene. Moreover, it was found that the content of these components differed significantly according to the stage of plant development. Significantly higher amounts were assessed for herb collected before and during the flowering while the leastfor the plant material collected after the flowering. However, the analysis of the interaction between the experimental factors gave us more detailed information. In the case of γ-terpinene, carvacrol and β-caryophyllene significantly higher concentrations were assessed for the herb collected before flowering and the least -after the flowering, while in the case of p-cymene the highest amounts were noted after the flowering and the least -before flowering.
According to literature data, the composition of Satureja montana L. oil shows large variations in the relative concentration of major components: carvacrol, linalool, γterpinene, p-cymene and β-caryophyllene, depending on the geographic origin and existence of different chemotypes (Cazin et al., 1985).
The effect of vegetation cycle on phytochemical composition of the essential oil obtained from wild-growing S. montana collected from Podgorica region (central part of Montenegro) was studied by Damjanovic-Vratnica et al. (2011). The higher content of thymol (37.36%), carvacrol (15.47%), γ-terpinene (11.75%) and β-caryophyllene (3.96%) they noted in the volatile oil obtained from plants collected before flowering, while content of p-cymene (31.37%) was higher in the essential oil extracted from plants collected during the flowering stage.
The content of carvacrol found in our oils was higher as compared to the results obtained by others Skocibuasic and Bezic, 2004;Damjanovic-Vratnica et al., 2011), although its highest concentration we also observed in volatile oil obtained from plants collected before flowering. Similarly, our plants contained more β-caryophyllene at preflowering stage. Moreover, β-caryophyllene was detected in all our oil samples in contrast to the results obtained by Milos et al. (2001). However, the content of p-cymene noted in our oils isolated from plants collected after the flowering, was lower compared to cited literature Skocibuasic and Bezic, 2004). Also, the content of this compound noted in our oils obtained from savory collected during flowering (8.65 and 8.01% in 2010 and 2011, respectively) was much lower compared to the results presented by Damjanovic-Vratnica and co-workers (2011).

Conclusion
The results presented in this study shows that the essential oils obtained from winter savory collected at different growth stages had similar compositions. The major compounds were carvacrol, p-cymene, m-cymene, γterpinene and β-caryophyllene. Significantly higher concentrations of carvacrol, γ-terpinene and βcaryophyllene were assessed for the herb collected before flowering, while in the case of p-cymene the highest amounts were noted after the flowering. The optimal time for harvesting of the plants with respect to high carvacrol, γterpinene and β-caryophyllene content is before flowering. If essential oil rich in p-cymene is necessary for any reason, then the plants should be harvested after the flowering It can be also concluded, that winter savory cultivated in north-western Poland may found wide industrial application due to the high content of phenolic compound -carvacrol, which is responsible mainly for high antimicrobial activity of savory's oil.