I have a question about the DPPH assay. Recently, I have been busy with literature and several preliminary experiments to be able to choose the best conditions (extraction method, solvents) to analyse my plant extracts, however, I have some questions,
1) To perform the analyse I use 96 -well plates, to perform Trolox standard curve I use 100µL of the Trolox solution ( in different concentrations in range 20-150µM) and 100µL of DPPH (100µM), in total I have volume 200µL what means that I dilute my Trolox concentrations ( 10-75µM) and DPPH (50µM), is it right? So when I calculate IC 50 should I refer to start concentrations 20-150µM of Trolox or to 10-75µM? And if I analyse different concentration for my sample, should I refer to the primer concentration or the final one?
2)I would also ask hat is the best way to express the results for the plant extracts? IC 50 or the equivalent of Trolox?
I will be thankful for your advice and any tips.
Kind regards,
Anna
Hi Anna,
For your first question, the final concentration of both extract and Trolox found in the reaction medium must be considered in calculations.
For your second question, the Trolox equivalent is more common and better for comparing your results with those of literature. The IC50 doesn't show the amount of antioxidant found in extract (as the Trolox equivalent is) but it shows the power or the ability of this extract to scavenge 50% of the free radical. These two concepts are different.
Hello Duried,
Thank you a lot for your answer, it helped me to clear some confusions that I had.
Have a nice day.
Greetings,
Anna
Hello Duried,
Could you still give me some tips about correct calculations?
If I want to express my results as Trolox equivalent, then I should use Trolox concentration vs abs curve and use abs of my sample to calculate x from the equation y=ax+b of the Trolox curve? Then I will get my results in Trolox µg/mL. Or it is better to express my results as a µM Trolox/g of the sample?
Thank you once again for your help.
Greetings,
Anna
Hello Anna,
µM Trolox equivalent/g of extract (sample) or mg Trolox equivalent/g extract (sample) for expressing the antioxidant activity is better.
Best wishes
Duried
Anna, please use mg (or µg) Trolox equivalent/g extract (sample) instead of µM Trolox equivalent/g of extract (sample).
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