2 thiobarbituric acid tba

Hydroxyl radical scavenging activity (HRSA) was determined according to the method of Halliwell and Gutteridge [17 (link)]. The reaction mixture consisted of 100 μl of 50 mM 2-deoxyribose (Sigma-Aldrich, USA), 100 μl of 50 mM hydrogen peroxide (Merck, Germany), 100 μl of 3.2 mM iron chloride (Sigma-Aldrich, USA), and 100 μl of 1 mM disodium EDTA (Sigma-Aldrich, USA), with or without 100 μl of plant extract at various concentrations. Butylated hydroxytoluene (BHT) (Sigma-Aldrich, USA) was used as a positive control. The reaction was triggered by adding 100 μl of 1.8 mM L-ascorbic acid (Ajax Finechem, USA) and incubated at 37 °C for 60 min. The reagent mixture, containing 500 μl of 10% trichloroacetic acid (TCA) (Merck, Germany) and 500 μl of 5% 2-thiobarbituric acid (TBA) (Merck, Germany), was added and boiled in a water bath at 95 °C for 30 min. After cooling at room temperature for 10 min, the absorbance was measured at 532 nm. The assays were performed in triplicate. The percentage of HRSA was calculated by using the following formula, and the results are shown as IC₅₀ values:
$$\%\text{HRSA}=\frac{\mathrm{Abs}\text{control}-\mathrm{Abs}\text{sample}}{\mathrm{Abs}\text{control}}\times 100$$
where Abs control is the absorbance without sample and Abs sample is the absorbance with sample.

Iron nitrate enneahydrate, nitrilotriacetic acid, oxidized and reduced glutathione, glutathione reductase, and hydrogen peroxide were purchased from Wako (Osaka, Japan). Nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt, glucose-6-phosphate, 1-chloro-2,4-dinitrobenzene (CDNB), 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), bovine serum albumin, trichloroacetic acid, and Tween 20 were purchased from Sigma (St. Louis, MO). 2-Thiobarbituric acid (TBA) was purchased from Merck (Darmstadt, Germany). The basal MF diet (powder) and Vitamin E (VE)-deficient diet were purchased from Oriental Yeast (Tokyo, Japan). The VE-stripped corn oil was purchased from TAMA Biochemicals (Tokyo, Japan). The BCA assay kit was purchased from Pierce (Rockford, IL). The Ax-C-8 was a kind gift from the Institute for Health Care Science, Suntory (Osaka, Japan). Normal goat serum was purchased from Vector Laboratories (Burlingame, CA), and Histofine Simple Stain rat Max-PO (multi) was purchased from Nichirei Biosciences (Tokyo, Japan). Liquid DAB was purchased from DAKO Japan (Tokyo, Japan). The two monoclonal antibodies against 8-OHdG (N45.1) and HNE-modified proteins (HNE-J2) were from the Japan Institute for the Control of Aging (Shizuoka, Japan). All of the other chemicals were of the highest quality available from Wako (Osaka, Japan).

The reagents sodium carbonate, di-sodium hydrogen phosphate, sodium carbonate, potassium ferricyanide, potassium persulfate, ethylenediamine tetra acetic acid (EDTA) and gallic acid were purchased from Scharlau (Barcelona, Spain). Folin–Ciocalteu’s phenol reagent, absolute ethanol and trichloroacetic acid (TCA) were purchased from Panreac (Castellar del Vallès, Barcelona, Spain). 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical, Iron (II) Chloride 4-hydrate, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylacid (Trolox), (L)-Dehydroascorbic acid and malondialdehyde (MDA) were purchased from Sigma Chemical Co. (Steinheim, Germany, and St. Louis, MO, USA). Propylgallate (PG) was purchased from Acrōs Organics (Fair Lawn, NJ, USA). 2-Thiobarbituric acid (TBA) was purchased from Merck KGaA (Darmstadt, Germany). The following standards were used: phenolic acids—hydroxybenzoic acid, vanillic acid, protocatechuic acid; cinnamic acids—chlorogenic acid, 4-coumaric acid; flavanols—catechin; flavonols—kaempferol, quercetin, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-rutinoside, rutin or quercetin-3-O-rutinoside; flavanones—naringenin, eriodictyol, eriodictyol-7-O-glucoside (Merck KGaA, Darmstadt, Germany).