Antioxidant Activity

All assays were made using 96-well microplates (Nunclon, Nunc, Roskilde, Denmark) and were measured in an ELISA Reader Infinite Pro 200F (Tecan Group Ltd., Männedorf, Switzerland).
The DPPH^• (2,2-diphenyl-1-picryl-hydrazyl) radical scavenging activity was measured by a previously described method [57 (link)]. DPPH solution (180 μL of freshly prepared 0.07 mg/mL solution) was mixed with 20 μL of the examined extract in various concentrations in microplates. The absorbance at 517 nm was monitored after 30 min incubation at 28 °C and the results were expressed as an EC₅₀ value. Ascorbic acid was used as the control. The antiradical potential was also analyzed using the previously described ABTS^•+ (2,2′-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid) assay [57 (link)]. The absorbance was measured at 734 nm and the results were expressed as millimoles of Trolox equivalents per g of dry extract (TEAC).
The metal chelating activity was determined by the method described by Guo et al. [58 ] with some modifications. In this assay, 0.2 mM aqueous solution of ferric chloride and 0.5 mM aqueous solution of ferrozine were used. Twenty microliters of the 0.2 mM aqueous solution of ferric chloride (II) was mixed with 100 μL of extract at different concentrations. Next, 40 μL of 0.5 mM aqueous solution of ferrozine was added and microplates were shaken and incubated for 10 min in 24 °C. The absorbance was measured at 562 nm and the percentage of inhibition of ferrozine–Fe²⁺ complex formation was calculated using the following formula:
where A_c is the absorbance of the control (water instead of the extract), and A_s is the absorbance of the extract.
The results were presented as the concentration of the extract that causes metal chelating in 50% (EC₅₀) calculated on the basis of the linear correlation between the inhibition of ferrozine–Fe²⁺ complex formation and the concentrations of the extract. EDTA was used as a positive control.
Antioxidant activity was also assayed using the β-carotene bleaching method described and modified by Deba and co-authors [51 (link)]. Twenty microliters of extract at different concentrations were mixed with freshly prepared β-carotene-linoleic acid emulsion, and incubated for 20 min at 40 °C. The absorbance was measured at 470 nm. BHT was used as a positive control.

The antioxidant activity was determined using oxygen radical absorbance capacity (ORAC) assay [22] and modified in our laboratory [23] (link). Phenolic extract dilutions were prepared with 75 mM phosphate buffer (pH 7.4). The assay was performed in black-walled 96-well plates (Corning Scientific, Corning, NY). The outside wells of the plate were not used as there was much more variation from them than from the inner wells. Each well contained 20 µL extracts or 20 µLTrolox standard (range 6.25–50 µM), and 200 µL fluoroscein (final concentration 0.96 µM), which were incubated at 37°C for 20 min. After incubation, 20 µL of 119 mM ABAP was added to each well. Fluorescence intensity was measured using Fluoroskan Ascent FL plate-reader (Thermo Labsystems, Franklin, MA) at excitation of 485 nm and emission of 520 nm for 35 cycles every 5 min. ORAC values were expressed as milligrams of TE/100 g FW. Data were reported as mean ± SD with triplicates.

After 3-NPA (12.5 mg/kg) treatment for seven days, different tissues (ovary, brain, spleen, liver and kidney) were collected. Separately, tissue from each of the five sampled organs (ovary, brain, spleen, liver and kidney) was homogenized in cold saline to prepare for the assay for activity of antioxidant enzymes. The activities of three enzymes—T-SOD, GPx and CAT—were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). (1) T-SOD activity was assayed using the xanthine/xanthine oxidase method based on the production of O²⁻ anions. (2) GP_X activity was estimated based on its catalyzation by the oxidation of reduced glutathione in the presence of cumene hydroperoxide. The generation of nicotinamide adenine dinucleotide phosphate was measured spectrophotometrically at 340 nm. (3) CAT activity was measured by analyzing the rate at which it caused the decomposition of H₂O₂ at 240 nm, the substrate of the enzyme contained in various tissue samples. Activities of T-SOD and GP_X are expressed as units per milligrams of protein (U/mg protein). The activity of CAT is expressed as (U/g protein).

In plasma samples, the following oxidative stress markers were measured: nitrite (NO₂), superoxide anion radical (O₂⁻), hydrogen peroxide (H₂O₂), and the index of lipid peroxidation (measured as TBARS – thiobarbituric acid reactive substances).
Nitric oxide decomposes rapidly to form stable metabolite nitrite/nitrate products. The nitrite level was measured and used as an index of nitric oxide (NO) production using the Griess reagent. A total of 0.5 ml of plasma was precipitated with 200 μl of 30% sulphosalicylic acid, vortexed for 30 min, and centrifuged at 3000 × g. Equal volumes of supernatant and Griess reagent containing 1% sulphanilamide in 5% phosphoric acid/0.1% naphthalene ethylenediamine dihydrochloride were added and incubated for 10 min in the dark, and the sample was measured at 543 nm. The nitrite levels were calculated using sodium nitrite as the standard [13 (link)].
The O₂⁻ concentration was measured after the reaction of nitro blue tetrazolium in Tris buffer with the plasma at 530 nm. Distilled water served as the blank [14 ].
The measurement of H₂O₂ is based on the oxidation of phenol red by H₂O₂ in a reaction catalysed by horseradish peroxidase (HRPO). Two hundred μl of plasma was precipitated with 800 ml of freshly prepared phenol red solution, followed by the addition of 10 μl of (1:20) HRPO (made ex tempore). Distilled water was used as the blank instead of the plasma sample. H₂O₂ was measured at 610 nm [15 (link)].
The degree of lipid peroxidation in the plasma samples was estimated by measuring TBARS using 1% thiobarbituric acid in 0.05 NaOH, incubated with the plasma at 100 °C for 15 min, and measured at 530 nm. Distilled water served as the blank [16 (link)].
The activity of the following antioxidants in the lysate was determined: reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). The level of reduced glutathione was determined based on GSH oxidation with 5,5-dithiobis-6,2-nitrobenzoic acid using a method by Beutler [17 ]. The CAT activity was determined according to Aebi [18 (link)]. The lysates were diluted with distilled water (1:7 v/v) and treated with chloroform-ethanol (0.6:1 v/v) to remove haemoglobin, and then 50 μl of CAT buffer, 100 μl of sample and 1 ml of 10 mM H₂O₂ were added to the samples. The detection was performed at 360 nm. SOD activity was determined by the epinephrine method of Beutler [19 (link)]. Lysate (100 μl) and 1 ml carbonate buffer were mixed, and then 100 μl of epinephrine was added. The detection was performed at 470 nm.