TITLE: Antioxidant activity by DPPH assay: in vitro protocol
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Ana Herrmann, Angelo Piato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Considering the role of oxidative stress in the pathology of several diseases and the use of antioxidants as treatment and/or adjuvants in these conditions. Here we propose a protocol to evaluate the antioxidant capacity of compounds by the DPPH method through the scavenging capacity of free radicals by reducing the DPPH radical. This protocol was standardized at LAPCOM (Psychopharmacology and Behavior Laboratory at UFRGS) to assess biochemical parameters </span><span style = "font-style:italic;">in vitro.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh3.googleusercontent.com/ygcUCEMjEDjiVqIHP2EbToWbeJfuAJnpLY6hbKiJLD8i4gnL6lqtWAUUMcKXU8QZwDHzcMcdognhs_gwISFI6hIBHaFWThVQcZBDI3KVtLZgPxpwVpv1rl6lqxagWhL5jgT_-6EW0hI" /></div></div>

[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in this protocol;
?. [Preparing the reagents]
2,2-Diphenyl-1-picrylhydrazyl (DPPH)  :    1.1.1 Weigh  of DPPH in a piece of aluminum foil;    1.1.2 Add  of absolute ethanol to the beaker to dissolve the salt;    1.1.3 Transfer your solution to a  volumetric flask;    1.1.4 Using absolute ethanol, complete the solution's volume to reach ;    1.1.5 Read the absorbance of the solution at  in a microplate reader;                If the absorbance of the solution is above 1.12, dilute the solution by adding absolute ethanol;                If the absorbance of the solution is lower than 1.08, add DPPH;
0.024 g
50 mL
100 mL
100 mL
517
?. [Incubation of the samples]
To optimize the reaction, an incubation step is needed.
?. [Incubation of the samples]
Prepare  microtubes, to be used to store the samples, with the correct information. Wrap the microtubes in aluminum foil. The number of microtubes depends on the number of samples. You should provide at least five replicates (n = 5) of each sample with at least one control tube per sample. Perform the test at least two times to ensure the results are as correct as possible.
1.5 mL
?. [Incubation of the samples]
For each sample, fill the plastic microtubes as described below. Using a micropipette fill the tubes in this order: Absolute ethanol + DPPH + Sample. Mix the solution with the pipette tip to homogenize the content; MicrotubesEtOH (µL)DPPH (µL)Sample (µL)Control20280-Sample1028010
MicrotubesEtOH (µL)DPPH (µL)Sample (µL)Control20280-Sample1028010
?. [Incubation of the samples]
Incubate all your samples in the dark at  for ;
25 °C
?. [Reading your samples]
Prepare to read the absorbance of your samples in a microplate reader;
?. [Reading your samples]
Use a conventional 96-well microplate to run your samples. Before start pipetting, each well of the microplate should be marked for sample identification.
?. [Reading your samples]
Add  of the content of each microtube to the wells needed. Fill two additional wells with  of absolute ethanol to work as the negative control for the absorbance reading of all of your samples;
250
250
?. [Reading your samples]
Read the absorbance of the samples at  in a microplate reader;
517
?. [Calculating data and determinig results]
Prepare to analyze the results obtained after reading the absorbance of the samples;
?. [Calculating data and determinig results]
Determine the percentage of inhibition of the DPPH radical:
?. [Calculating data and determinig results]
Results should be expressed as % of inhibition of the DPPH radical.
?. [Calculating data and determinig results]
Calculate the mean absorbance of your replicates;
?. [Calculating data and determinig results]
Determine the median effective concentration (EC50) for each sample, which expresses the minimum amount of the extract capable of reducing the initial concentration of DPPH radical by 50%. Calculated in a nonlinear regression model using GraphPad Prism.
?. [Calculating data and determinig results]
On an XY graph, plot the concentrations of the samples in column X and the results of the DPPH assay (% inhibition) in column Y.
?. [Calculating data and determinig results]
Transform the values to logarithmic scale: Analyze -> Transform -> Ok -> transform X values using = X = Log (x) -> Ok.
?. [Calculating data and determinig results]
Perform a nonlinear regression analysis: Analyze -> XY analyzes -> Nonlinear regression -> Ok -> Dose responds stimulation– Log (agonist) VS. Normalized response -> variable slope -> Ok.
?. [Calculating data and determinig results]
Results should be expressed as EC50.