At maturity, grains of plants in 1 m2
of the middle part of four central rows of each plot were harvested in the
second year and total grain yield per unit area was determined. Grain oil content was determined by AOAC method (AOAC 1990)
with a soxhlet apparatus. The borage grains were
ground using a mortar. Then 2 g powdered grains were packed in a thimble and the
oil was extracted by using 300 ml diethyl ether as solvent for
6 hours. The solvent was evaporated by rotary evaporator to obtain
oil. Oil percentage was determined by weight difference and then oil yield per
unit area was calculated.
2.5. Fatty acids
Borage oil was extracted according
to the method described by Savage et al. (1997) from borage grains. In brief, 10
g powdered grain was extracted with 30 ml hexane/isopropanol (3:2, v/v) at room
temperature with vigorous shaking for 1 h in steel tubes containing four steel
balls to facilitate homogenization. After filtration of homogenates through
defatted filter papers on a Buchner funnel under vacuum, the residues were
washed twice with 20 ml of the same solvent; then 35 ml of 6.7% sodium sulfate
was added and the upper layer was separated and rotary-evaporated under reduced
pressure at 40°C. The extracted oils were stored at -20°C for further analysis.
2.6. Analysis of fatty
acid methyl esters
The modified version of the method
by Savage et al. (1999) was used for fatty acids analysis. Approximately, 10 mg
of extracted oil was dissolved in 0.5 ml of hexane and then 2 mL 0.01 M NaOH-methanol
solution was added. Test tube containing the solution was maintained at 55°C
for 10 minutes in water bath. Subsequently, 3 ml BF3 reagent was
added and again was kept in water bath for 10 minutes. The test tube was cooled
under a stream of cold water and after adding 2 ml of 20% sodium chloride
solution and 1 ml hexane, it was mixed properly. The mixture was centrifuged
and hexane layer containing the fatty acid methyl ester was isolated.
Fatty acids of the grain
oil were analyzed using gas chromatography (South Korea, YL-6100GC) with a
flame ionization detector (FID). Analysis of FAME was achieved with the fused
silica capillary column BP × 70 with length 30 m × i.d 0.25 mm and film
thickness 0.2 ?m. Helium was used as a carrier gas at a flow rate of 1.0 ml min–1.
The oven temperature was held initially at 80°C for 1 min, then increased to 230°C
at 5°C min-1 and maintained at 230°C for 10 min. The injector and
detector temperatures were 250 and 240°C, respectively. The area of each fatty
acid peak was expressed as a percentage of the total area. The peaks were identified by retention times and comparing
them with authentic standards analyzed under the same conditions.