Dear Anna

Yes, there are a huge variety between procedures; I can suggest you these main key points

1. About concentration : It depends directly on the source and potent of your scavenging compound like phenolic or even peptides...The more your source is reach of these bioactive compounds, the higher concentration you can use.I recommend you to use 60 microM which is very common in papers

2. Ethanol is a better solvent than methanol, also check the solvent purity.

3. Incubating of DPPH solution must be done in ambient temperature and at darkness( this is very important point , because in higher temperature you will see more discolouring of DPPH)

4. ABTS is somehow more preferable than DPPH because of the selectiveness of the compounds which scavenges...

Have A good Job

I have one. Do you mind contacting me later so I can send it to you? I am at a meeting atm and cannot do it right now.

Dear Anna,

First, you must know that to determine antioxidant activity of any material you have to test various methods besides DPPH to accurate your results about antioxidant activity.

1- You should clarify enough information about the nature of material you used.

2- ethanol and methanol are both used in DPPH solution to obtain 0.004% (W/v).

3- I am totally agree with Ehsan Moghaddas kia in the third point, concerned with darkness.

these papers should help.

have an interested work

Another paper

Another paper

Another paper

Hello.

In my study, i was using protocol of preparing DPPH solution according to this document (see bellow).

Hello,

Take (0.3mM) DPPH in a conical flask (Brown r Black) u grind it with 100% ethanol completely ur dye should be dissolved. now ur DPPH is ready. Incubate the test sample for 30 min in a dark room reading at 517nm. sample conc may vary from 25-400ug/ml(u decide the conc). DPPH should be in equal conc to all the tubes.

calculate IC50. decolouration must occur .DPPH is purple good antioxidant test sample may decolourise that v can visuvalise.

Respected Sir,

We use to follow the following method:

The free radical scavenging activity of all the extracts was evaluated by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) according to the previously reported method by Shen et al., 2010. Briefly, an 0.1mM solution of DPPH in methanol was prepared and 1mL of this solution was added to 3 ml of the solution of all extracts in methanol at different concentration (50,100,200,400 & 800μg/mL).The mixtures were shaken vigorously and allowed to stand at room temperature for 30 minutes. Then the absorbance was measured at 517 nm using a UV-VIS spectrophotometer. Ascorbic acid was used as the reference. Lower absorbance values of reaction mixture indicate higher free radical scavenging activity. The capability of scavenging the DPPH radical was calculated by using the following formula.

DPPH scavenging effect (% inhibition) = {(A0 –A1)/A0)*100}

Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged

Reference

Shen Q, Zhang B, Xu R, Wang Y, Ding X, Li P. Antioxidant activity in vitro of selenium-contained protein from the se-enriched. Bifodobacterium animalis 01. Anaerobe, 2010; 16: 380-386.

Good Luck

With regards,

Dr. Tresina

Respected Wilkinson,

You can follow this procedure

DPPH radical scavenging activity

The free radical scavenging activity of methanol extract was measured by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) using the method of Blois (1958). 0.1 mM solution of DPPH in methanol was prepared and I ml of this solution was added to 3 ml of various concentrations of methanol extract and reference compound (125, 250, 500 and 1000 µg/ml). After 30min, absorbance was measured at 517 nm. BHT was used as a reference material. All the tests were performed in triplicate and the graph was plotted with mean value. The percentage of inhibition was calculated by comparing the absorbance values of reference compound (control) and samples.

% of inhibition = [(A blank/A sample)/A blank] x 100

Where ‘A blank’ is the absorbance of the control reaction (containing all reagents except the test sample) and ‘A sample’ is the absorbance of the extracts/standard.

Blois, M.S. 1958. Antioxidant determination by the use of a stable free radical. Nat. 181: 1199-1200.

Dear All

I just used DPPH method but the result was not good.  My sample was hot water plant extracts with brown color. Lower concentration resulted lower absorbance. When I checked the absorbance of my sample (wl 517) in a series of concentration without DPPH, their responds was in line with when I added DPPH solution.  So, it is possible that the absorbance level was not indicating the Radical scavenging activity of the extracts but depend on concentration of the extracts.

I need suggestion to solve this problem. Should I solubilize my extracts in ethanol before test?

Thanks you All

@ Samsu Nurdin : As said by you, Your sample itself is having strong coloration which may be hindering the normal absorbance readings. Try a lower concentration checks and also ethanol can also be tries.

what is the concentration of ascorbic acid used in the DPPH assay 

I think this link is useful, 

http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/

Hello Anna.Unfortunately, owing to the diverse chemical nature of many of the substances investigated for antioxidant activity, no single assay is enough to make a substantive conclusion.You need to perform a battery of assays (Total phenolic content, total flavonoid content, hydroxyl radical scavenging assay, hydroxyl radical assays, superoxide radical assays...etc) then go on to do a regression analysis of the IC50 values that you obtain from the tests.And another thing is that you need to make your own decision on the concentrations you will test as copying someone else's protocol of concentration is tantamount to plagiarism.Kindly find attached one of my articles on total phenolic,flavonoid and ascorbic acid content determination.

Good luck!

Article Phytochemical profile and antioxidant capacity of leaves of ...

 Sir,

Try the following method slightly modified from the author i used the solvent ethanol that of methanol  and based on diution of Dpph in the ethanol also interferce the scavenging action.

The free radical scavenging activity of all the extracts was evaluated by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) according to the previously reported method by Shen et al., 2010. Briefly, an 0.1mM of DPPH is dissolved in  ethanol (ethanol should be slightly warm) was prepared and 1mL of this solution was added to 3 ml of the solution of all extracts in ethanol at different concentration (50,100,200,400 & 800μg/mL).The mixtures were shaken vigorously and allowed to stand at room temperature for 30 to 45 minutes. Then the absorbance was measured at 517 nm using a UV-VIS spectrophotometer. Ascorbic acid was used as the reference. Lower absorbance values of reaction mixture indicate higher free radical scavenging activity. The capability of scavenging the DPPH radical was calculated by using the following formula.

DPPH scavenging effect (% inhibition) = {(A0 –A1)/A0)*100}

Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged

Reference

Shen Q, Zhang B, Xu R, Wang Y, Ding X, Li P. Antioxidant activity in vitro of selenium-contained protein from the se-enriched. Bifodobacterium animalis 01. Anaerobe, 2010; 16: 380-386.

Thank you,

What can I do if my freeze-dried extracts solubilize only in water

HI,

Antioxidant activity is based on the decrease in the absorbance when the diphenyl-picrylhydrazyl

radical (DPPH • ) is reduced at 517 nm. This assay depends on the fact that the purple color of the

DPPH • radicals is bleached to a yellow color in presence of any molecule that can donate an electron or

proton. Equal volumes of propolis extract were mixed with 0.2 mM methanol solution of DPPH • and

incubation in the dark for at 37

◦ C for 30 min [ 13 ]. After incubation, the absorbances of the mixtures

were measurement against a blank at 517 nm using a Tecan ® Safire II Reader. DPPH • with methanol

was employ as control, while ascorbic acid was employ as reference in comparing to the propolis

extract. The efficacy of antioxidant in the samples were expressed in percentage of DPPH • reduction

and was calculated using the formula below:

Inhibition (%) = 100 × [A517 (control) − A517 (sample)/A517 (control)]

More details

Medicines 2018, 5(1), 2; doi:10.3390/medicines5010002

And what about incubation time? In papers most popular is 30 min, somethimes people make 24h incubation. Of course in dark.

But isnt it too long? Is someone made shorter incubation time?

Article DPPH antioxidant assay revisited

Hello All..

I wanna ask some question.

I have crude extract that I got from endophytic fungi extracted with ethyl acetate. After done with rotary evaporator, I still maintain the extract in liquid form rather than solid or powder form.

Now, I will do DPPH assay, can I use my extract in ethyl acetate to that assay? I've read that ethyl acetate can be used in DPPH assay, but how with positive control? Can I use ascorbic acid in positive control, or the other?

Or there are other method I can use to do DPPH assay?

Thanks.

You can see the protocol of DPPH assay in attachment

DPPH is a very good test to determine antioxidant capacity of plant extracts and its isolates. You can have a look to the link provided below-

Article A comparative study on the antioxidant activity of methanoli...

look through the article below

1 mL of extract was mixed with 2 mL of 0.5 mM DPPH ethanolic solution and incubated in dark for 30 min, the mixture was measured at 517 nm. The result was calculated by the following formula:

DPPH radical scavenging activity (%) = [1 - (A1 / A0)] * 100

In the formula, A1 indicates the absorbance of sample, A0 indicates the absorbance of control (ethanol solution of DPPH)

I have a question

The 5-7 dilutions of the samples to calculate the IC50: should be done in mg / ml of dry weight?

Thank you

Antioxidant Activity of DPPH in Vitro

Experimental method

(1) Solution preparation DPPH solution: DPPH standard reserve mother liquor was prepared with a mass concentration of 0.0625 mg/ml before each use.

(2) The steps of DPPH antioxidant experiment

The sample solution of 10 mu L polysaccharide and the DPPH solution of 0.0625 mg/ml were removed at 190 mu L at 96.

In the pore enzyme label plate, 5 holes were repeated for each concentration. Ap value was measured at 37 C for 30 minutes. Steam in the same volume

The absorbance value of blank group, Amax, was obtained by distillation instead of polysaccharide sample solution, and DPPH was replaced by anhydrous ethanol of the same volume.

The absorbance of Ac in the sample control group was obtained by the reaction of the solution with the sample solution, and the same volume of absolute ethanol was used instead of DPPH.

The absorbance of Ao in the zero-removal group was obtained by the reaction of the solution with distilled water .

The calculation of DPPH clearance rate is processed according to formula 4-1.

DPPH clearance (%)= 1-[(Ap-Ac)/((Amax-A0)]*100%

In formula (4-1), A0 represents the absorbance value of zero removal group; Amax represents the absorbance value of blank group; Ap represents the absorbance value of sample group; Ac represents the absorbance value of control group.

and i recommended these paper.

0.5 ml of the fraction solutions (10, 20, 40, 60, 80, 100, 200, and 400 μg/ml in ethanol) will be added to 0.5 ml of a DPPH solution (0.1mM in ethanol). Incubate for 30 min at room temperature. Measure the absorbance at 517 nm. The free radical scavenging activity of each fraction will be determine by comparing its absorbance with that of a blank solution (no sample). Ascorbic acid is used as standard. The ability to scavenge the DPPH radical was calculated using the following equation:

DPPH scavenging activity (%) = [(Ac – At) /Ac] ×100

Where Ac is the absorbance of the control and At is the absorbance of test sample. (Priya et al, 2010) (Shisode et al, 2011)

Shisode K. S. and B.M. Kareppa, (2011). In vitro antioxidant activity and phytochemical studies of Boerhaavia diffusa linn roots, ISPSR Vol.2 (12):3171-3176.

Kindly go through this reference, I hope it will help you better.

All the best

yes many references are available

https://www.banglajol.info/index.php/JBAS/article/view/48561

You may read

Hi, I have the question like how should we prepare the different concentrations of powder samples in mg/mL? Do we need to prepare them in the same concentration of ethanol as we use for DPPH solution preparation? Usually, 95% of ethanol is used for the DPPH solution. What if the sample is not soluble in the ethanol?

Dear @ Anna,

DPPH, the basic step for evaluating the antioxidant properties. Please find the answer.

1. DPPH radical scavenging activity of your desired sample was estimated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and done according to the procedure of Sharma and coworkers (Sharma et al., 2011).

2. Methanolic DPPH solution (1.3 mg/ml (3.3 mM), 150μl) was added to 3ml

of methanol and this was recorded as control.

3. Concentrations ranging from (5 to 500 μg/ml) of your desired sample and standards were tested.

4. The mixtures were vortex-mixed and kept for incubation in dark for 15 min after which the decrease in the DPPH absorption was measured at 517 nm.

5. A non-linear regression analysis method was employed to calculate the IC50 values for the estimation of the scavenging activity of your sample on DPPH

reduction.

Attached file

Sonu Sharma

Respected sir,

You can dissolving your sample in dmso, and followed same other steps in the origin procedure. because of dmso colorless and dont absorbed at 517 nm

Methanol

You can use this protocol: https://www.mdpi.com/1420-3049/26/10/3046/htm

You can actually request any experiment demonstration through http://labdemo.unaux.com for better understanding of the protocol.