Antioxidant assays

Each sample was dissolved in 95% methanol to make a concentration of 1 mg/ml and then diluted to prepare the series concentrations for antioxidant assays. Reference chemicals were used for comparison in all assays.

DPPH radical scavenging activity assay

The free radical scavenging activity of the fractions was measured in vitro by 2,2′- diphenyl-1-picrylhydrazyl (DPPH) assay according to the method described earlier [18,19]. The stock solution was prepared by dissolving 24 mg DPPH with 100 ml methanol and stored at 20°C until required. The working solution was obtained by diluting DPPH solution with methanol to attain an absorbance of about 0.98±0.02 at 517 nm using the spectrophotometer. A 3 ml aliquot of this solution was mixed with 100 μl of the sample at various concentrations (10 - 500 μg/ml). The reaction mixture was shaken well and incubated in the dark for 15 min at room temperature. Then the absorbance was taken at 517 nm. The control was prepared as above without any sample. The scavenging activity was estimated based on the percentage of DPPH radical scavenged as the following equation:Scavengingeffect(%)=[(controlabsorbance−sampleabsorbance)/(controlasbsorbance)]×100

(1)

Superoxide anion scavenging assay

The assay for superoxide anion radical scavenging activity was supported by riboflavin-light-NBT system [20]. Briefly, 1 ml of sample was taken at different concentrations (25 to 500 μg/ml) and mixed with 0.5 ml of phosphate buffer (50 mM, pH 7.6), 0.3 ml riboflavin (50 mM), 0.25 ml PMS (20 mM), and 0.1 ml NBT (0.5 mM). Reaction was started by illuminating the reaction mixture using a fluorescent lamp. After 20 min of incubation, the absorbance was measured at 560 nm. Ascorbic acid was used as standard. The scavenging ability of the plant extract was determined by the following equation:Scavengingeffect(%)=(1−absorbanceofsample/absorbanceofcontrol)×100

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Phosphomolybdate assay (total antioxidant capacity)

The total antioxidant capacity of the fractions was determined by phosphomolybdate method using ascorbic acid as a standard [21]. An aliquot of 0.1 ml of sample solution was mixed with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 765 nm against a blank. A typical blank contained 1 ml of the reagent solution and the appropriate volume of the solvent and incubated under the same conditions. Ascorbic acid was used as standard. The antioxidant capacity was estimated using following formula:Antioxidanteffect(%)=[(controlabsorbance−sampleabsorbance)/(controlabsorbance)]×100

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Hydroxyl radical scavenging assay

Hydroxyl radical scavenging activity was measured by the ability of the different fractions of T. leptophylla extract to scavenge the hydroxyl radicals generated by the Fe3+-ascorbate-EDTA-H2O2 system (Fenton reaction) [22]. The reaction mixture contained; 500 μl of 2-deoxyribose (2.8 mM) in phosphate buffer (50 mM, pH 7.4), 200 μl of premixed ferric chloride (100 mM) and EDTA (100 mM) solution (1:1; v/v), 100 μl of H2O2 (200 mM) with or without the extract solution (100 μl). The reaction was triggered by adding 100 μl of 300 mM ascorbate and incubated for 1 h at 37°C. 0.5 ml of the reaction mixture was added to 1 ml of TCA (2.8%; w/v; aqueous solution), then 1 ml of 1% aqueous TBA were added to the reaction mixture. The mixture was heated for 15 min on a boiling water bath. After the mixture being cooled the absorbance at 532 nm was noted against a blank (the same solution but without reagent). The scavenging activity on hydroxyl radical was calculated as follows:Scavengingactivity(%)=(1−absorbanceofsample/absorbanceofcontrol)×100

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Hydrogen peroxide scavenging activity

Hydrogen peroxide solution (2 mM) was prepared in 50 mM phosphate buffer (pH 7.4). Aliquots (0.1 ml) of different fractions was transferred into the test tubes and their volumes were made up to 0.4 ml with 50 mM phosphate buffer (pH 7.4) After addition of 0.6 ml hydrogen peroxide solution, tubes were vortexed and absorbance of the hydrogen peroxide at 230 nm was determined after 10 min, against a blank [23]. The abilities to scavenge the hydrogen peroxide were calculated using the following equation:Hydrogenperoxidescavengingactivity=(1−absorbanceofsample/absorbanceofsample)×100

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ABTS radical scavenging activity

The 2,2′-azinobis (3-ethylbenzthiazoline-6-sulphonic acid), commonly called ABTS cation scavenging activity was performed [24]. Briefly, ABTS solution (7 mM) was reacted with potassium persulfate (2.45 mM) solution and kept for overnight in the dark to yield a dark coloured solution containing ABTS radical cations. Prior to use in the assay, the ABTS radical cation was diluted with 50% methanol for an initial absorbance of about 0.70±0.02 at 745 nm, with temperature control set at 30°C. Free radical scavenging activity was assessed by mixing 300 μl of test sample with 3.0 ml of ABTS working standard in a microcuvette. The decrease in absorbance was measured exactly one minute after mixing the solution, then up to 6 min. The percentage inhibition was calculated according to the formula:Scavengingeffect(%)=[(controlabsorbance−sampleabsorbance)/(controlasbsorbance)]×100

(6)

The antioxidant capacity of test samples was expressed as EC50 (anti-radical activity), the concentration necessary for 50% reduction of ABTS [25].

Article Antioxidant activity, total phenolic and total flavonoid con...

Dear

The approve mentioned methods are the most common, I would like to add the iron chelating methodology which tell the possibility to chelate metals. The reducing power procedure is also known. The determination of total phenolic and flavonoids content is useful to compare.

I think DPPH, FRAP and ABST are the most common. you need to adjust the concentrations of your samples to get the readings into an appropriate range of absorbance values.