Relatively stable organic radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) has been widely used in the determination of antioxidant activity of single compounds, as well as of complex mixtures. The assay is based on the reduction of alcoholic DPPH solutions in the presence of a hydrogen donating antioxidant (13 (link)). The reduction of DPPH is followed by a decrease in its absorbance. According to the previously described procedure (14 (link)), an aliquot of each sample (0.2 mL) was mixed with 2.8 mL of DPPH ethanolic solution (Sigma-Aldrich Chemie, Merck, Steinheim, Germany). Ethanol (Vrenje Spiritana, Belgrade, Serbia) was used as a blank, while ethanol with DPPH solution was used as a control. Free radical scavenging activity was determined by measuring the absorbance of the solution using UV-Vis double beam spectrophotometer (HALO DB-20; Dynamica GmbH, Salzburg-Mayrwies, Austria) at 525 nm after 40 min of reaction at room temperature in the dark. Trolox (Sigma-Aldrich Chemie, Merck) was used as a standard and the results were expressed in mmol Trolox equivalents per litre of essential oil (mmol/L). The antioxidant potential of the non-encapsulated essential oils was measured before and after the storage. To evaluate the stability of the samples, the antioxidant activity of the essential oils released in water was determined after storage for 12 months in PET containers.
Dpph ethanolic solution
Manufactured by Merck Group
Sourced in United States
DPPH ethanolic solution is a laboratory reagent used to assess the antioxidant capacity of various substances. It is a stable free radical that absorbs light at a specific wavelength, and its absorbance decreases when it is reduced by an antioxidant. The core function of this solution is to provide a standardized method for measuring the free radical scavenging activity of samples.
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5 protocols using dpph ethanolic solution
1
Antioxidant Activity of Essential Oils
2
DPPH Radical Scavenging Assay for EPS
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The scavenging activity for DPPH was assayed as described in the study by Niknezhad et al. [36 (link)]. The reaction mixture contained 50 μL of EPSt at different concentrations (0.1, 0.25, 1.0, 2.5, 5.0, 7.5 and 10 mg/mL) and 100 μL of DPPH (100 μM DPPH–ethanolic solution) (Sigma Chemical, St Louis, MO, USA). The mixtures were shaken vigorously and incubated in the dark at 25 °C. After 30 min, the absorbance was recorded at OD525nm. Ascorbic acid (Vc) (Sigma Chemical, St Louis, MO, USA) was used as positive control.
The percentage of radical scavenging activity for DPPH was calculated according to Formula (1):
where
A1 = OD525 nm of reaction mixture.
A2 = OD525 nm of reaction mixture without DPPH.
A0 = OD525 nm of the reaction mixture with DPPH but without EPSt.
The percentage of radical scavenging activity for DPPH was calculated according to Formula (1):
where
A1 = OD525 nm of reaction mixture.
A2 = OD525 nm of reaction mixture without DPPH.
A0 = OD525 nm of the reaction mixture with DPPH but without EPSt.
3
DPPH Free Radical Scavenging Assay
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The free radical scavenging activity of extracts was evaluated by DPPH, according to the method previously described by Chen et al. (2005) (link) with some modifications. An aliquot of 100 µL of the sample was mixed with 900 µL of an DPPH ethanolic solution (100 μM) (Sigma Aldrich, St. Louis, USA) and was incubated for 30 min at 25 °C in darkness. Then, the absorbance of the reaction was measured in a spectrophotometer at 517 nm. A standard curve of ascorbic acid (10.5–176 µg/mL) was constructed as a reducing agent. The results were expressed as milligram ascorbic acid (Sigma Aldrich, St. Louis, USA) equivalents per 100 g dry weight (mg AAE/100 g DW).
4
DPPH Radical Scavenging Assay of AC
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The radical scavenging ability of AC was assessed by the method of Shimada et al. with slight modifications [52 ]. The radical scavenging ability of the extracts was determined at concentrations of 10 mg/mL in ethanolic DPPH solution (0.1 mM) (Sigma, Saint Louis, USA). In control, water was used in place of the sample in which the extract was prepared. Cuvettes were left in the dark at room temperature for 30 min and the resulting color was measured spectrophotometrically at 517 nm against blanks. A decreasing intensity of purple color was related to higher radical scavenging ability, which was calculated using the following equation:
Where AS:30 is absorbance of the sample and AB:30 is absorbance of the blank at 30 min reaction time.
Where AS:30 is absorbance of the sample and AB:30 is absorbance of the blank at 30 min reaction time.
5
Quantifying Antioxidant Capacity of Cheese Spreads
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The DPPH free radical scavenging was quantified following the method proposed by Suresh et al. [22 (link)] with some modifications. Briefly, an aliquot (0.1 mL) of each homogenized cheese spread (CSExt, CSFos, and CS) was added to 3.0 mL of 0.1 mM ethanolic DPPH solution (Sigma-Aldrich, St. Louis, MO, USA), and absorbance were measured at 515 nm after incubation for 30 min at 30 °C in the dark (A sample) in a UV-VIS Spectrophotometer (Shimadzu). Three replicates were performed. Using trolox (Sigma-Aldrich, St. Louis, MO, USA) as the standard (0–0.05 mg/mL), the results were expressed as the equivalent concentration of trolox (mg trolox equiv/mL), being the DPPH free radical scavenging percentage calculated using the following formula:
For each sample, the absorbance of 3 mL of DPPH plus 0.1 mL ethanol was measured as the control (A control), whereas the absorbance of 3 mL of ethanol plus 0.1 mL of homogenized cheese spread was measured as the blank (indicated as A blank).
For each sample, the absorbance of 3 mL of DPPH plus 0.1 mL ethanol was measured as the control (A control), whereas the absorbance of 3 mL of ethanol plus 0.1 mL of homogenized cheese spread was measured as the blank (indicated as A blank).
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