2.5 Sterols determination
In order to determine the sterols content, 0.5 ml of dihydro-cholesterol (2 mg/mL) was added to 250 mg of oil and saponification was conducted at room temperature (Sander, Addis, Park & Smith, 1989). After about 20 h, the organic fraction was washed with 10 ml of diethyl ether and 10 ml of water. The unsaponifiable fraction was further extracted twice with 10 ml of diethyl ether, 10 ml of 0.5 N aqueous KOH and 10 ml of distilled water, respectively. The organic solvent was removed under vacuum and the unsaponifiable fraction was used for the sterols analysis. Before injection, samples were silylated according to Sweeley, Bentley, Makita & Wells (1963) and the sterol separation was performed by GC/MS (GCMS-QP2010 Plus, Shimadzu, Tokyo, Japan) in the same chromatographic conditions reported by Cardenia, Rodriguez-Estrada, Baldacci, Savioli & Lercker et al. (2012). Sterols identification was achieved by comparing peak mass spectra with peaks of standard mixture and by comparing them to the GC–MS data reported by Pelillo, Iafelice, Marconi & Caboni (2003). An internal standard was used to quantify all the sterols identified in seed samples. Sterols composition was measured in 2 replicates for each lipid extract (n = 4) and expressed in mg/100g of oil.