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.