Linoleic acid

The inhibition of linoleic acid peroxidation of samples was measured according to the ferric thiocyanate method [29 ,30 (link)], with some modifications. Briefly, 50 µL of sample (1000, 500, 250, 125, 62.5 and 31.25 µg/mL) was added to 100 µL of linoleic acid emulsion (0.02 M, pH 7.0) and 100 µL of phosphate buffer (0.2 M, pH 7.0). The mixture was then incubated at 37 °C for 24 h. Previously, 0.02 M linoleic acid emulsion was prepared by mixing 0.2804 g of linoleic acid (Sigma Aldrich, USA) with the same weight of Tween 20 and a 50 mL phosphate buffer (0.2 M, pH 7.0), after which the mixture was homogenized. Following incubation, 1 mL of ammonium thiocyanate (3%, w/v) and 100 µL of FeCl₂ (0.02 M in 1 M HCl) were added to 100 µL of the mixture. The inhibition activity was then determined based on the absorbance at 500 nm in a spectrophotometer. ASA was used as a positive control and the solution without the samples was used as a negative control.
The inhibition of linoleic acid peroxidation was calculated as follows: inhibition percentage (%) = [1 − (A_sample − A_blank)/A_control] × 100%, where A_sample was the absorbance of reaction mixture or standards and A_control was the absorbance of the negative control. The IC₅₀ was calculated using the SPSS 17.0 software (regression analyses).

Ethanol, mEthanol, chloroform, and ethyl acetate were purchased from Merck KGaA (Darmstadt, Germany). Catechin, gallic acid, linoleic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, vanillin, linoleic acid, β-carotene, streptozotocin, and tyloxapol (Triton WR-1339) were acquired from Sigma Chemical Co. All other reagents were of analytical grade. Kits for the determination of total cholesterol, cholesterol HDL, and triglycerides were from RANDOX.