To confirm the peaks in the HPLC analysis, comparison of its retention times and co-injection of standards were used. The spectroscopic data of carnosol and betulinic acid, used in this experiment, were previously reported by Benincá et al. (2011). Calibration curves were prepared with standard solutions of investigated compounds in the concentration range from 0.01 Trametinib to 1.5 mg ml−1. Five standard solutions

were injected in triplicate. The calibration curves were constructed by linear regression of the peak-area ratios (y) of each analyte, versus concentrations (x). The r2 values were in the range from 0.990 to 0.999, which confirmed the linearity of the method. Fresh aerial parts (200 g) were ground prior to the operation and then ground rosemary was submitted to water distilation for 4 h using a Clevenger apparatus. The distiled essential oil was dried over anhydrous sodium sulphate, filtered and stored at 4 °C. After removing the essential oil, the leaves were re-extracted with alcohol (96%) to obtain the essential oil-free fraction (13.2 g, 6.6%, yield). The GC-FID technique in this study was used to quantify the compounds present in the essential oil and it was performed on a gas chromatograph

(GC) -Shimadzu 14 B with a find more flame ionisation detector (GC-FID), column OV-5 (30 m × 0.25 mm i.d. × 0.25 μm film), N2 as flow gas with constant pressure of 80 kPa. The split ratio was 1/150 and injection volume was 0.3 μl of the oil. Injector and detector were held at

250 and 300 °C, respectively. The following programme was: 50 °C for 3 min, rate of 5 °C min−1 until 270 °C and held for 8 min. The GC–MS was used to identify the compounds and it was performed on a gas cromatograph coupled with mass spectrometer (GC–MS) (Varian®CP 3800-Saturn 2000). Scanning (1 scan s−1) was performed in the range of 39–400 m/z and using electron impact ionisation at 70 eV. The column used was CPSil 8CB (30 m × 0.25 mm triclocarban i.d., 0.25 μm film). Analyses were carried out using helium as carrier gas at a flow rate of 1.0 ml min−1 in a split ratio of 1:20 and the following programme rate was: 50 °C held for 1 min, rate of 3 °C min−1 until 240 °C; injector: 250 °C. The compounds were identified using the: (1) arithmetic indices (AI), (2) GC–MS retention indices (authentic chemicals), and mass spectra (authentic chemicals and NIST98 spectral library collection). The Sigma retention index standard (Sigma Aldrich, USA), used in this study, consisted of a mixture of aliphatic hydrocarbons, ranging from C10 through C30, dissolved in hexane. It is designed to be used to obtain the arithmetic indices type gas chromatographic retention indices, which are useful for preliminary identification of unknown compounds.