Hello Everyone,
I have 20 samples that I measured the antioxidant activity for using DPPH. I calculated the IC50 using Excel by setting up a linear chart of my sample concentration vs % inhibition. Some previous papers mentioned that this relationship should n't be linear, while others referred to its linearity. I am confused and which way is the right and accurate to calculate IC50? Some of the samples I used showed very high antioxidant activity which means that the relationship I mentioned is not linear and semi-plateau appears with the 2nd or 3rd concentration (I used 8 concentrations for each sample to draw this chart).
Any suggestions? and advice with a good reference to rely on to solve this issue.
Thanks!
dear Fatma,
in the case of this specific sample, the calculated IC50 will not be accurate. For this sample with high activity I recommend you to use successive dilutions u,ntil you get at least 3 linear points. Ideally they have to be arround the IC50. I recommend you to check visually (by eye) if the calculated IC50 is close to the actual (experimentally determined values). For instance, in some cases I have calculated the IC50 using the excel and found a value tha was superstimating the real values. By eye you can see easily the approximate IC50 -and you must have at least 2 points arround theIC50- if your values do not reach close to the IC50 you have to increase the concentrations tsted. But as in the case of your sample, where you had a plateaunfrom the the 2nd point of the ccurve, you have to use lower concentrations until you get points close to the IC50.
Thanks Joao. I will do that. I am just wondering if DPPH IC50 is different from IC50 or EC50 for drug-response curve? Papers mentioned that drug-response curve shouldn't be linear (sigmoid or logarithmic) but I didn't see that in DPPH IC50. That's why I am confused.
Thanks a lot.
Hi Fatma,
I think non linear regression is the best approach for calculating IC50 or EC50 in dose response curve. You can do this using drc package installed in R. Here some reference for analyzing dose response curve using R:
https://www.jstatsoft.org/article/view/v012i05/v12i05.pdf
Article Dose-Response Analysis Using R
Good luck.
Dear Fatma,
The nature of graph is depend on the potency of the tested extract. if the extract is potent you find out the graph is non-linear and you cannot use the equation of the graph for IC50 determination because is going to be negative. However, if the tested extract is not potent then you will find out that the nature of the graph is linear and in such situation you can use the equation of the graph. Moreover, in respect of the accuracy of determination of IC50 the best way is to use software such GraphPad software.
Regards.
Dear Dr. Fatma Elzahraa Mohamed Elessawy ,
Theoretically, for calculating the IC50 values we need to have linear relation between the concentration (X) and % inhibition (Y), and the equation obtained is y=mx+c.
where 'X' is concentration and Y is response (% Inhibition), and based on the linearity of the DRC/CRC, you will get varied values of m and c. To this equation we sustitute 50 in place of Y. Example. say if you have got equation as Y=2.33x + 0.34, in this we have to substitute 50 in place of y, which gives the concentration corresponds to 50% inhibition, and it is called as ''Inhibitory concentration 50''.
Ideally, if you have perfect CRC, irrespective of which software you use, you must get similar IC50 values.
Regards
Vishwanatha
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