The polyphenols were extracted from the oils according to the
method of Vazquez Roncero, Janer del Valle, and Janer del Valle
(1973). Ten grams of oil was dissolved in 50 ml n-hexane and the
solution was extracted successively with three 20 ml portions of
60% aqueous methanol. The mixture was shaken each time for
2 min. The solvent was removed from the combined using a vacuum
rotary evaporator (LABOROTA 4001- Efficient, Heidolph Co.)
at 40 C. The residue was dissolved in 1 ml methanol and was
stored frozen until the moment of the analyses. The concentration
of total polyphenols in the methanolic extract was estimated with
Folin Ciocalteau reagent. The procedure consisted of dilution of
0.1–0.4 ml methanolic extract with water to 5 ml in a 10 ml volumetric
flask, and addition of 0.5 ml Folin Ciocalteau reagent. After
3 min, 1 ml of saturated (ca. 35%) Na2CO3 Solution was added.
The content was mixed and diluted to volume (10 ml) with water.
The absorbance was measured after 1 h at 725 nm against a reagent
blank. Caffeic acid served as a standard for preparing the calibration
curve ranging 0–100 lg/10 ml assay solution (Gutfinger,
1981).