## 2 Materials and Methods
### Methods  
#### 2.2.1 Determination of Lipid Composition  
* _Composition of fatty acids_ Gas chromatography (GC) was used for determination of the fatty acid composition of the oil. Briefly, the vegetable oil (100 mg) was pre-esterified with 5 mL methanol in the presence of sulfuric acid in order to obtain fatty acid methyl esters (FAMEs) [30; 31]. The deter mination of FAMEs was carried out using an Agilent 8860 gas chromatograph (Santa Clara, CA, USA) equipped with a capillary column DB-FastFAME (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m (film thickness)) and a flame ionization detector (FID). Nitrogen was the carrier gas. The temperature of the inlet was 270 \({}^{\circ}\)C and that of the detector was 300 \({}^{\circ}\)C. The temperature program of the column was as follows: 70 \({}^{\circ}\)C (1 min), followed by increasing of the temperature up to 180 \({}^{\circ}\)C at a rate of 6 \({}^{\circ}\)C/min, and then, at a rate of 5 \({}^{\circ}\)C/min, increasing of the temperature up to 250 \({}^{\circ}\)C. The volume of the sample injected in the system was 1 \(\upmu\)L. For the identification of the FAMEs, we used a standard mixture from Supelco, USA (FAME mix 37 components, Supelco, Bellefonte, PA, USA).  
* _Content of sterols_ Unsaponifiables were determined according to the ISO standard [32]. A total of 5 g of the oil was subjected to saponification with 50 mL of 2N KOH, and then, the unsaponifiable matter was separated from the solution through extraction with 50 mL of \(n\)-hexane. The extraction was repeated five times, and then, the solvent was removed using a rotary-vacuum evaporator. The rest of the unsaponifiables were dried at 105 \({}^{\circ}\)C to a constant weight and their content was determined gravimetrically. The sterols were isolated from the unsaponifiable matter via thin-layer chromatography (TLC) [33] and their total content was determined spectrophotometrically at a wavelength of 597 nm. Individual sterol composition was determined using an HP 5890 gas chromatograph (Santa Clara, CA, USA) equipped with a DB-5 capillary column (25 m \(\times\) 0.25 mm) (Santa Clara, CA, USA) and FID. The carrier gas was hydrogen and the temperatures of the inlet and the detector were as follows: 300 \({}^{\circ}\)C and 320 \({}^{\circ}\)C. The temperature program of the column was 90 \({}^{\circ}\)C (3 min), increasing up to 290 \({}^{\circ}\)C at a rate of 15 \({}^{\circ}\)C/min, and then, increasing at a rate of 4 \({}^{\circ}\)C/min up to 310 \({}^{\circ}\)C (5 min). The volume of the sample injected was 1 \(\upmu\)L. Identification was established by comparing the retention times with those of a standard sterol mixture (Acros Organics, New Jersey, USA) [34].
* _Content of tocopherols_ Individual tocopherols were determined using a high-performance liquid chromatograph (HPLC) from Merck-Hitachi (Merck, Darmstadt, Germany). The column was a Nucleosil Si 50-5 (250 mm \(\times\) 4 mm). Fluorescence detection was used (excitement at 295 nm and emission at 330 nm). The mobile phase was \(n\)-hexane:dioxane, 96:4 (\(v/v\)) and the flow rate was set at 1 mL/min. The content of the tocopherols was determined after diluting 30 mg of the oil with \(n\)-hexane (2% solution) and injecting 20 \(\upmu\)g of the solution into the system under the described conditions. The identification was carried out by comparing the retention times of the peaks with those of a standard solution of tocopherols [35].