Essential fatty acid components and antioxidant activities of eight Cephalaria species from southwestern Anatolia

Pure and Applied Chemistry, Dec, 2007 by Suheyla Kirmizigul, Nazli Boke, Huseyin Sumbul, R. Suleyman Gokturk, Nazli Arda

All the methyl esters, especially 9,12,15-octadecatrienoic and 9-octadecenoic (Z) methyl esters, were also verified by GC-6890 Agilent, with DB-23 polar column (30 m x 0.25 mm x 0.25 [micro]m). The detector and injector temperatures were set at 280 [degrees]C, and the flow of hydrogene was 40 mL/min. The other characteristics of the program were adjusted as air flow 450 mL/min, total flow 66 mL/min, oven temperature 50 [degrees]C (1 min waiting), rising from 25 [degrees]C/min to 175 [degrees]C. The program was continued heating the system to 230 [degrees]C with 4 [degrees]C/min rising (20 min waiting). Inlet temperature, pressure, and split were 250 [degrees]C, 15 psi, and 1/50, respectively.

Antioxidant activity tests

The antioxidant activity of hexane extracts of Cephalaria species was assessed by three different methods. Radical scavenging activity was measured by DPPH test [21]. Inhibitory effect on lipid peroxidation was examined by FTC [22] and TBA methods [23]. The main chemicals; ammonium thiocyanate, ferrous chloride, hydrochloric acid, and potassium phosphate salts (Merck), DPPH, linoleic acid (LA), Trolox, butylated hydroxytoluene (BHT), L-ascorbic acid (Sigma), and ethanol (Carlo Erba) were used during the following antioxidant activity tests.

Free radical scavenging activity

The DPPH assay was carried out according to the modified method of Cheung et al. (2003). Briefly, 0.5 mL of DPPH in ethanol (0.1 mM) was added to 1 mL of hexane extract in different concentrations (0.1-1.6 mg/mL) and kept in the dark for 10 min. The absorbance of the resulting solution was recorded on a spectrometer at 520 nm against a blank of hexane. Vitamin C was used as reference antioxidant. DPPH scavenging activity was expressed as I[C.sub.50] values ([micro]g extract/mL) for comparison. I[C.sub.50] value of each sample defined as the concentration of sample required for the 50 % decrease in absorbance of the blank, was calculated.

Inhibitory effect on lipid peroxidation

Four milligrams of each extract were dissolved in 4 mL of 99.5 % ethanol and added to lipid peroxidation system, containing 2.51 % LA in 99.5 % ethanol (4.1 mL), 0.02 M phosphate buffer (8.0 mL) (pH 7.0), and distilled water (3.9 mL), in a screw-cap vial (38 mm x 75 mm). Final concentration of the extract was 0.02 % (w/v) in the assay mixture. Hexane was used as negative control. This mixture was incubated in an oven at 40 [degrees]C in the dark until the end of the experiment (8th day) and used for FTC and TBA methods. Each experiment was repeated at least in duplicate, and mean values were used for the evaluation of the results.

FTC method

Hydroperoxides produced by LA oxidation system in the assay mixture were detected as described by Abas et al. (2006). To measure the extent of antioxidant activity, an aliquot (0.1 mL) was taken from the above-mentioned assay mixture and added to 75 % (v/v) ethanol (9.7 mL), followed by 30 % aqueous ammonium thiocyanate (0.1 mL) and 0.02 M Fe[Cl.sub.2] (0.1 mL) in 3.5 % HCl were added. After 3 min, absorbance was recorded at 500 nm, as the first measurement. Recordings were repeated at 24 h intervals, until the absorbance of the blank reached its maximum value (8th day). BHT and Trolox were used as reference antioxidants.