Analysis of tocopherols and DPPH radical scavenging activity

Tocopherols of the oils were analyzed using an HPLC (Ultimate 3000; Thermo Scientific Dionex, Waltham, MA, USA) equipped with a silica-based column (ZORBAX Eclipse Plus C18, Agilent Technologies, Santa Clara, CA, USA) [11]. Each oil was dissolved in 1 mL 2-propanol, followed by filtering the solution using a 0.2 μm hydrophobic syringe filter (Advantec MFS Inc., Dublin, CA, USA). Mobile phase was a mixture of methanol and acetonitrile (1:1) at 1.5 mL/min. A fluorescence detector was set at an emission wavelength of 325 nm and an excitation wavelength of 295 nm. Tocopherols were identified by comparing their retention times with those of corresponding standards.

DPPH radical scavenging activity of the oils were determined according to a method by Brand-Williams et al. [12]. Fifty μL of each oil was added with 950 μL 0.2 mM DPPH in methanol. The mixture was vortexed and held at room temperature for 5 min in the dark. Absorbance was measured at 515 nm using a UV–vis spectrometer (Spectramax 190, Molecular Devices, Sunnyvale, CA, USA) against a blank sample. DPPH radical scavenging activity (%) was calculated as follows: DPPH radical scavenging activity (%) = (1 − absorbance of the sample/absorbance of the DPPH solution) × 100.