Dear Vandana, you need a laboratory protocol, which is quite different from the descriptions you can read in the Material & Methods sections of the published papers.
I have a detailed protocol for 96wells microplate but needs of a microplate reader. I am attaching it for you.
If you only have a classic UV spectrophotometer working with cuvettes then you need another one.
IMPORTANT NOTE: not all the concentrations of DPPH follow a linear trend. YOU HAVE TO CHECK WHICH IS THE LINEAR RANGE OF DPPH FOR YOUR INSTRUMENT, and work only within this range.
Best wishes, Jose.
DPPH radical-scavenging activity was performed by the method described by Akter et al. (2010). For each determination, the stock solution (1mg/ml) was diluted to a dilution series (50μg-1000μg/ml) with 60% (v/v) ethanol. An aliquot of each dilution (0.5 mL) was mixed with methanolic solution of DPPH (5 mL, 0.06 mM). The mixtures were shaken vigorously and incubated at 37 °C in the dark for 30 min. At the same time, a control containing 60% (v/v) ethanol (0.5 mL) and methanolic solution of DPPH (5 mL, 0.06 mM) was run. The absorbance was measured at 517 nm against methanol as a blank. The percentage of DPPH scavenging was calculated as follows:
DPPH radical scavenging activity (%) = [(Abscontrol- Abssample) /Abscontrol] x 100
The percentage of DPPH scavenging versus concentration of samples was plotted. The concentration of the sample necessary to decrease the DPPH concentration by 50% was obtained by interpolation from linear regression analysis and denoted IC50 value (μg/mL). All determinations were carried out in triplicate. Ascorbic acid was used as reference compound.
References
Akter, S., Ahmed, M., Eun, J.B., 2010. Solvent effects on antioxidant properties of persimmon (Diospyros kaki L. cv. Daebong) seeds. Int. J. Food Sci. Tech. 45,2258–2264.
Dear Vandana, Hope you will be alright. method given above is alright. However you can find several papers on it on it complete detail.
Dear Vandana, you need a laboratory protocol, which is quite different from the descriptions you can read in the Material & Methods sections of the published papers.
I have a detailed protocol for 96wells microplate but needs of a microplate reader. I am attaching it for you.
If you only have a classic UV spectrophotometer working with cuvettes then you need another one.
IMPORTANT NOTE: not all the concentrations of DPPH follow a linear trend. YOU HAVE TO CHECK WHICH IS THE LINEAR RANGE OF DPPH FOR YOUR INSTRUMENT, and work only within this range.
Best wishes, Jose.
and a very good article about the experimental complexities of the DPPH assay and the best ways to perform it. Read it well before trying it.
Best, Jose
Protect the reagent from light. Use amber colored bottles or wrap your reagent bottle with aluminium foil. Fresh preparation of the reagent is also advised.
Whatever method u r using for DPPH, u mst care for concentraton of prepared sample and a fixed time interval for OD. U can check variations in OD by taking multiple OD depends upon diff. time interval. Such as in 5 then 15 and 30 mins. All jst u cn compare the data and fix the time interval for OD without any fluctuations in OD values. Thanks
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
Free radical scavenging potential of each test compound was tested using DPPH assay.1 The degree of decoloration of ethanolic solution of DPPH (Sigma) indicates the scavenging efficiency of the added substance. For each compound, appropriate volume of solution in methanol was added to 2 mL of DPPH solution (10 mg/L). Five minutes later, the absorbance was measured at 515 nm. A reference sample was prepared with same volume of methanol as above without adding compound. The antiradical activity was calculated as a percentage of DPPH decoloration using the following equation: antiradical activity = 100 x (1 - absorbance of sample/absorbance of reference).
1Burda S, Oleszek W. J. Agric. Food Chem. 2001, 49:2774.
@Jose Prieto: YOU HAVE TO CHECK WHICH IS THE LINEAR RANGE OF DPPH FOR YOUR INSTRUMENT, and work only within this range.
Sir, Kindly suggest me how to do the above linear range in our instrument? we are having Thermo Scientific-Evolution 201 UV-Visible spectrophotmeter.
Simply make up a solution of DPPH which gives you a value of 1 unit. Then from this double dilute serially and plot your different abs vs conc. until you get 0. Find the linear part in this plot. This is the valid range of conc you can realistically work with using your equipment.
Could anybody please help me with the detailed lab protocol to perform the ABTS antioxidant technique?
Please see in attachment simple and lab applicable micro methods for these two parameters
Does anyone know how do you set the wave lenght. I have found multiple authors claiming 517 nm. Is this correct for all the cases?
Does anyone know how to do this method with DPPH in blood serum samples? What's the diference?
i would like to know more about free radical scavenging and antioxidant assays using DPPH,
help me please
Dear colleagues, good night!
I'm in trouble with how I will present my results from the antioxidant activity from an Hibiscus extract.
In fact, I used the Brand-Williams protocol for DPPH inhibition assay and I'm with some doubts.
First of all, I would wanna know if i have to consider the dilution of the extracts samples in the DPPH medium used during the assay. For example: if I have dissolved my extracts for concentrations ranging between 2000-125 micrograms/mL and I put 0,3 mL of these solutions together with 3 mL of DPPH, so I have to consider my extracts concentrations between 50-3,125 micrograms/mL (final concentrations after dilution)?
Best wishes.
I want to share some of my recent tries on determination of antioxidant activity assay. I tried the microplate protocol, by Dr. Perieto, mentioned earlier here, and also another one by W. Brand-Williams (1995). if you have so many samples the microplate is more convientent. But if not, I personally prefer the other one. I added 0.1 ml methanol extract of fruit juice to 3.9 ml DPPH of 62.5 uM, and expressed the result as Trolox equivalence. I used 0-0.6 mM Trolox solution as the standard curve. The standard curve was so accurate with a regression of 0.9999.
I hope it will be helpful,
Dear Jose Maria Prieto, please guide about the blank, when we are taking the absorbance of DPPH+ methanol (control), then the blank will be methanol. if we are taking the absorbance of DPPH+methanol+Extract (sample), then the composition of blank will be methanol+extract....... is it ?
if yes then we have to change the blank every time accordingly, when we are taking the absorbance of different concentration of same extract.
please enlighten me?
Dear Mueen,
Yes this is correct. Each concentration of extract will have a different absorption, so needs a matched blank consisting on MeOH+Extract at the right dilution.
Best wishes
Jose
Dear Jose Maria Prieto
kindly how to refer to your protocol above as a reference ? i mean paper ,volum, publisher, year, ....ect ?
Dear Jose Maria Prieto
My question is pertaining to the composition of blank in dpph assay. As you mentioned earlier that it should be composd of methanol+ extract(sample blank) at right dilution, in this way one can negate the effect of extract abs on overall scavenging potential of the extract. So can we make a sample blank seperately that is to be compose of methanol and extract and take OD of this then add dpph(sample test) then take OD. Finally minus the OD of sample blank from sample test to achieve the correct OD
I mean in order to calculate the OD of sample as given in the usual formula of % scavenging assay of Dpph.
SampleAbs = OD of sample test- OD of sample blank
DPPH free radical
scavenging activity of Leea macrophylla extract was
determined in comparison to standard antioxidant ascorbic
acid. The whole procedure was administrated according to
established procedure by Sanja et al., with slight modifications
[20]. Specific amount of ascorbic acid was dissolved
in methanol to prepare stock solution of 1 𝜇g/mL. Specific
amount of ascorbic acid was dissolved in methanol to prepare
stock solution of 1 mg/mL. The stock solution was diluted to
give the concentration of 30, 60, 120, 240, and 480 𝜇g/mL.
Similarly 10mg of dried different extracts was dissolved
in 10mL of specific solvent for the preparation of stock
solution of sample to give concentration of 1 mg/mL. The
stock solution was diluted to give the concentrations of 30,
60, 120, 240, and 480 𝜇g/mL. Required amount of DPPH was
dissolved in methanol and the solution was kept in conical
flax. It was protected from light by covering the flax with
aluminum foil.
The scavenging activity against DPPH was calculated
using the following equation:
%𝐼 = {
(𝐴0 − 𝐴1)
𝐴0
} × 100, (1)
where 𝐴0 is the absorbance of the control (freshly prepared
DPPH solution) and 𝐴1 is the absorbance of the
extract/standard.
Then the percentage of scavenging activity or inhibition
was plotted against log concentration and fromthe graph IC50
value was calculated by linear regression analysis.
You can have a look at the following article-
A comparative study on the antioxidant activity of methanolic extracts from different parts of Morus alba L.(Moraceae)
You should consult method by shad etal in 2011 published paper with modification.also it's light sensitive reaction so use dark room.check your chemical pH and composition if you prepare much before test
@ Muhammad Ali Khan . You can change your testing material like Vit C and DPPH. The deterioration of these chemicals can cause the fluctuated results.
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