I was performing DPPH assay taking ascorbic acid as a posititve control. I found that many of the articles have expressed their values in mainly three categories; Ascorbic acid equivalent, percentage inhibition and IC50. While I was carrying out the procedure I tried with few methods and according to the principle of DPPH assay my assumption was that whatever the concentration of DPPH is used, while performing it do not affect the final calculation of percentage inhibition as percentage inhibition depends upon absorbance of conntrol(Only DPPH and solvent) and absorbance of sample assayed. But I carried out two experiments taking two different DPPH concentrations one 0.004% and another 0.02% in methanol solvent. I found that the initial absorbance at 515nm for 0.004%DPPH is 0.642 and for 0.02%DPPH is 3.280. After addition of 50microlitre of 200microgram/ml ascorbic acid to 800microlitre of DPPH in both cases and incubated at dark for 30mins, I found that the absorbance decreased to 0.108 and 2.334 respectively for 0.004%DPPH and 0.02% DPPH. When percentage inhibition was calculated it was found that there was vast difference 83% and 28%.
So, following the same protocol but only changing DPPH concentration changes the percentage inhibition? If so evaluating the assay in terms of percentage inhibiton and also IC50 is good? Or it is best to evaluate in terms of Ascorbic acid equivalent as it is not affected by above mentioned problem.
Dear Ankit
As per my suggestion, you should use a concentration of DPPH and calculate the percentage inhibition of the test drug and standard ascorbic acid. Calculate IC50 value of test and standard from graphs. Suppose you get IC50 value of ascorbic acid and test drug as 50 microgram/ml and 500 micrograms/ml, respectively. You can interpreat the results by calculating the potency in respect to standard. In this case it is 1/10th of the standard. Suppose you are evaluating 5 formulations, you just calculate the potency in respect to standard and compare them and conclude that which is having more scavanging activity. I suppose this explanation will help you in interpretation of your results.
Good luck.
Dear Yadu
Thank you for your explanation, so I perceived that whether I follow any protocol it will give results in reference to the standard used following same protocol. So in order to compare antioxidant activity of two different samples done by two different protocols, it is best to calculate ascorbic acid equivalent for both. Since this will make both results in same term or reference to ascorbic acid. For example if a certain plant has antioxidant activity of 30microgram ascorbic acid equivalent per gram dry weight and if another plant has antioxidant activity of 50microgram ascorbic acid equivalent per gram dry weight then these two results can be compared saying that later one has more antioxidant potential although protocol for DPPH assay was different.
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