I am working on the antioxidant activity of samples. I did the ORAC assay and DPPH assay. My samples work well in ORAC assay, and they have shown higher antioxidant activity than Trolox and Ascorbic acid. However, in the DPPH assay, they have not shown any activity and in some cases, the absorbance of DPPH increases when I add my samples.
Can we conclude that my samples do not have the ability to scavenge RNS?
Hello,
if your OD increases when you add your sample, I suspect that your control (the 0) is not adapted with your reaction mixture and what you are measuring, takes into consideration the OD of the DPPH and the OD of your sample.
What have you put in your control?
No, it is not possible for an antioxidant to scavenge only reactive oxygen species (ROS). Antioxidants are molecules that can neutralize or inhibit the harmful effects of free radicals, which include ROS as well as other reactive species such as reactive nitrogen species (RNS). Antioxidants work by donating an electron or hydrogen atom to stabilize and neutralize these reactive species. Therefore, antioxidants can scavenge both ROS and RNS to protect cells and tissues from oxidative damage.
The active species in these assays are very different. Therefore, the measured activity must be different.
First of all, thank you for all your responses.
The negative control is just water with radical DPPH (without antioxidants). I have the same control of Ascorbic acid and Trolox and they work really well.
Hello Zahra Kariminezhad,
I was talking about the "0" or the blank, what does it contain, to see if the problem that you have in measuring the DO, and its increase, when you add the antioxidant is not due to to that.
Also for the negative control you can add the antioxidant and measure at 0 min, i.e. without incubation
Dear Zahra Kariminezhad
yours "just water with radical DPPH (without antioxidants)." DPPH is not soluble in water. Antioxidant activity strongly depends on pH, you must use a buffer. EtOH or MeOH are the solvents for DPPH assay.
Whatever you do, the results from both assays will be different.
Yes, I dissolved radical DPPH in methanol, instead of antioxidant I added water. This is my negative control.
Blank contains just solvent which did not show absorbance. (Like an empty well).
"Can we conclude that my samples do not have the ability to scavenge RNS?" NO!
RNS stands for Reactive Nitrogen Species. None of your assay is based on RNS. RNS are NO2, ONOOH, O2NOOH, NO, etc
Dear Yurii V Geletii,
Thank you very much. You are totally right.
I have 3 antioxidant samples. I analyzed their antioxidant capacity through DPPH, ORAC, and FRAP assay. However, just ORAC assay worked well. I can not interpret the exact reason why my samples do not show their activity in the DPPH and FRAP assay.
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