Extraction and Identification of Volatile Components of Two Salvia Species Native to Iran (Salvia limbata and S. multicaulis) by Using Solid Phase Micro-Extraction Method

There are 58 species belonging to sage genus as annual and perennial plant in different regions of Iran that 18 species of them are endemic to Iran and they have different medicinal properties such as antibiotic, sedative, carminative, antispasmodic and commonly used in treatment of respiratory problems: infections, cough, cold and sore throat and cosmetics industries. The present study has aimed to evaluate the composition of essential oils achieved by Solid Phase Micro-Extraction method from aerial parts of two Salvia species native to Iran: Salvia limbata and Salvia multicaulis.
The experiments were carried out at the Research Station of Agriculture College, TarbiatModares University in Tehran, Iran during the years 2011-2013. The seeds of Salvialimbata and Salvia multicauliswere collected in Ardabil and Isfahan provinces in 2009. The seeds were sown in planting trays(filled with soil and cocopeat 1:1) under controlled greenhouse condition (temperature: 26±1°C, light: 3000 lux, relative humidity: 65%) in the last week of February 2011. The soil of experimental pots (soil and coco peat 2:1) was a clay silt loam with pH of 7.4. After two months,seedlings with uniform height and stem diameter with two true leaves were transferred to a growth chamber adjusted to 30/20 °C, 50% relative humidity, light intensity of approximately 3000 Lux and 16 h photoperiod.Aerial parts of two cultivated plantsincludingSalvia limbata and Salvia multicaulisat flowering stage were harvested in June 2012 and kept at 80°C until further experiments. Volatile compounds were extracted by solid phase micro-extraction (SPME) method for the first time in Iran for these species. Before the SPME, the leaves lyophilized and then were used. The optimization of SPME extraction and desorption conditions were performed by analyzing dried leaves of Salvia officinalis L., used as the matrix. The sample preparation procedure was as: 15 mg of dried sage leaves mixed into a 20 ml screw-on cap HS vial to 5 ml of 5% Ethanol and 0.5 mg of Na2SO4. The vials were sealed after stirringwith a Teflon (PTFE) septum and an aluminum cap (Chromacol, Hertfordshire, UK) for the production of headspace and the successive analysis. The sample vial was put in the instrument dry block-heater and held at 40°C for 20 min to come into equilibrium. The extraction and injection processes were automatically performed using an auto sampler MPS 2 (Gerstel, Mülheim, Germany). The fiber was, then, automatically inserted into the vial’s septum for 10 min, to allow the volatile compounds absorption onto the SPME fiber surface. Each SPME fiber was conditioned before its first use, as recommended by the manufacturer. In order to desorb the volatile metabolites, the SPME fiber was introduced into the injector port of the gas chromatograph device, model GC 7890A, Agilent (Agilent Technologies, Santa Clara, USA) coupled with a mass spectrometer 5975 C (Agilent) wherein the metabolites were thermally desorbed and transferred directly to a capillary column HP-Innowax (30m×0.25 mm×0.5µm Agilent J&W) and analyzed. The identification of VOMs was accomplished by comparing the retention times of the chromatographic peaks with those, when available, of authentic standards run under the same conditions. For volatiles for which reference substances were not available, the identification was performed by matching their retention indices (RI) determined relative to the retention time of a series of n-alkanes (C8–C20) with linear interpolation, with those of authentic compounds or literature data (Van Den Dool&Kratz, 1963). Confirmation of metabolites identification was also conducted by searching mass spectra in the available database (NIST, version 2005; Wiley, version 2007).
66 volatile components were identified in Salvia limbata, which Sabinene (19.16%), β-Pinene (19%), α-Pinene (16.3%), α-Terpinolene (14.41%), 1,8-Cineole (10.86%) and Limonene (3.73%) were highest amounts. 58 volatile compounds were identified inSalvia multicaulis thatCamphene (28.85%), α-Pinene (12.33%), Camphor (10.73%), Limonene (9.01%), 1,8-Cineole (5.47%), β-Pinene (4.58%) and Bornyl Acetate (3.75%) had the maximum amounts, respectively. The main part of volatile constituents of Salvia limbata and Salvia multicaulisplantsbelonged to monoterpenes (91.57 and 84.28% respectively) and sesquiterpenes (5.12 % and 7.58 %, respectively).
The results obtained in Salvia limbataand Salvia multicaulisin respect to main components are similar to previous papers (3, 16). However, there is some compound such as α-Terpineol in Salvia limbatawhich has not been reported. According to the obtained results by solid phase micro-extraction is closer to plant compounds.