# How do I calculate DPPH assay IC50?

After I plot % inhibition versus concentration (ug/ml) in excel. What do I do next to find out IC50? Please help!

Question

After I plot % inhibition versus concentration (ug/ml) in excel. What do I do next to find out IC50? Please help!

- Hi Dep, you have to find the mathematical relationship between the two parameters. Regression lines can be used as a way of visually depicting the relationship between the independent (x) and dependent (y) variables in the graph. You can simply build a Scatter Plot of % inhibition versus concentration (ug / ml). Then you add the best regression line which fit for your data by following these steps: 1) select one of the points on the graph, 2) click on the right mouse button and from the menu select "Add Trend line", 3) select the type of regression (Have you any idea about the type of relationship between the parameters?) tick both "Show graph's equation " and "Display R-squared value" , that is very important parameter because closer R2 is to 1.00, the better the fit; 4) use the Regression Equation to Calculate Concentrations.

I hope that these tips will help you...further information on http://www.ncsu.edu/labwrite/res/gt/gt-reg-home.html

Good luck

B. - Hi Ms Barbara,

I plotted a scattered plot using %inhibition versus concentration (ug/ml). After i have the linear regression equation y= mx +b, what is the value of my y?

Concentration (ug/ml) --> x

25

50

75

100

and

% Inhibition --> y

4.06

23.74

29.00

31.78

How to i get the 50% number of the % inhibition values to put into the equation as y? I couldnt use mean or median.. - after you plot graph use cursor to find 50% inhibition (on y axis)

when u see it draw a vertical line . The point where vertical line touches x-axis gives u IC50.

There is one more mathematical method will update u with ref.

- jus checked ur data u canit get IC50 from results u have pasted becoz u have not achieved 50 percent inhibtion even at 100 ug/ml. so better find IC25.

- What should i do now?
- your comppound or extract is not potent antioxidant as revealed by data so find IC25 if u need a quantitative parameter to define potency ...
- But can i find IC50 by taking middle value of the data set of y and input into the linear trendline equation?

or

cant i use the "excel add-in" to calculate? http://www.sciencegateway.org/protocols/cellbio/drug/hcic50.htm - My another set of values are

Concentration (µg/ml)

25

50

75

100

and

% Inhibition

2.28

6.39

6.07

6.89

I dont know what should i do with calculating IC50

Please help - i guess i foung EC50 for ur data

Logit Regression

Exp((1.759 * Log(X) -8.555)) / (1 + Exp((1.759 * Log(X) -8.555)))

R = 0.886481

EC50 = 129.6411 (0÷35.0719)

Angular Regression

Y = Sin(0.29 * Log(X) -0.695)^2

R = 0.919899

EC50 = 164.18 (60.0194÷246290.4)

Probit Regression

Y = CumNorm(0.931 * Log(X) -4.607)

R = 0.899136

EC50 = 141.1165 (44.2249÷1.410962E+21)

enjoyy!!!! - oh my.. I dont understand =(
- Barbara

If you want a reasonably accurate estimate of the IC50 you need to arrange your analyses so that the domain of your results includes 50% inhibition and the range of % inhibitions are approximately symmetrical around the 50% level. You then fit a logit or probit regression to calculate the IC50. - Dear Peter I agree with you.. I have just seen the results obtained by Dep. Obviously there is not a linear relationship, it seems that the % inhibition reaches up to a plateau value despite the concentration increase.. I’d not trust an IC50 extrapolation from these data.

I could suggest Dep to add other experimental points (concentrations exceeding 100 ug / ml) .. so he can experimentally verify whether your data fit with those calculated by Oyasis..what do you think about?

Also, there is another problem .. he gets two completely different data sets with the same concentrations of substance: is impossible! .. However, the % inhibition values indicate that the substance is not a good antioxidant considering the selected concentrations.

But I can also think that perhaps Dep’s making a mistake during the pre-analytical phase-

Dep should check your protocol: vials, solvent , substance stability , blank sample - As dear Barbara said, you should add more concentrations and look the results if it reaches to 50% that is ok if you can not have 50% scavenging then you can conclude your compound is not a good antioxidant in term of radical scavenging activity. Also may, your concentrations is too high and it appears pro-oxidation activity therefore you should check for lower concentration (lower than 25 ug/ml) (this assumption is less likely). You should also check the solubility of your antioxidant in the solvent and check other solvents also. Also some antioxidants scavenge radicals slowly and other quickly, you should check the radical scavenging after longer time for example after 4 hours of reaction. (or even more, you can check when the your absorbance become stable during time)

I observed this condition when I check an antioxidant, which it did not reach to the 50% inhibition in any concentration.

The regression for radical scavenging activity is exponential for most of antioxidants.

It should be added that Radical Scavenging activity do not tell the antioxidant activity and it should be use beside other tests.

Some times an antioxidant does not show a good radical scavenging activity but in other test such as oven test , shows a strong antioxidant activity since its main antioxidative activity is done by other mechanisms rather than radical scavenging .

good luck - surely you know that the absorbance of spectrophotometer must be between 0.3 - 0.7 and if your absorbance be more than 1.5 you will get wrong results with no sense.

Then your absorance must be between 0.3 - 0.7 (optimum) for your sample. if it not you should bring in this region by diluting or concentrating. - well guys i in my earlier comment have already highlighted that the compound is poor antioxidant ..... and i had also emphasised on calculation of IC25 values which is equally valid as 50% inhibition was achieved nevea. Dep u cen get tentitatve idea of the potency of ur compound by values provided by different mathematical procedures from R values given in there u can assess that fitting is not good (nature of ur data) but yes it is valid.....
- DPPH RADICAL SCAVENGING ACTIVITY

The solution of DPPH was prepared by adding 4.3 mg of DPPH (1, 1-Diphenyl –2-picrylhydrazyl) was dissolved in 3.3 µl methanol; it was protected from light by covering the test tubes with aluminum foil. 150 µl DPPH solution was added to 3ml methanol and absorbance was taken immediately at 517nm for control reading. 50 µl of various concentrations such as 40μg/mL, 80μg/mL, 120μg/mL, 160μg/mL, 200μg/mL, 240μg/mL, 280μg/mL, 320μg/mL and 360μg/mL. of the plant sample extracts as well as standard compound (Ascorbic acid) were taken and the volume was made uniformly to 150 µl using methanol. Each of the samples was then further diluted with methanol up to 3µl and to each 150 µl DPPH was added. Absorbance was taken after 15 min. at 517nm using methanol as blank on UV-visible spectrometer Shimadzu, UV-1601, Japan. The IC50 values for each drug compounds as well as standard preparation were calculated. The DPPH free radical scavenging activity was calculated using the following formula:

% scavenging = [Absorbance of control - Absorbance of test sample/Absorbance of control] X 100

The effective concentration of sample required to scavenge DPPH radical by 50% (IC50 value) was obtained by linear regression analysis of dose-response curve plotting between %inhibition and concentrations (Iranshahi et al., 2009). - In excel, plot the scatter curve. Draw the linear trend line and then find the equation. Next, in place of y, put 50 and then find the value of x. That gives the IC50. This is in fact finding the median value. for different percentiles, just replace y in the equation with the intended percentile and calculate x. This is very simple, but I hope it will be helpful...BOL
- Try these concentrations: 25, 50, 100, 250, 500 and 1000. This is to ensure that % inhibition would fall within the 50% range. i.e. 50 plus minus some value.
- I couldn't repeat my experiments for higher concentrations due to lack of time.

Currently now, I only could calculate IC50 by input 50 in y for the linear equation, although it is not accurate. =( - no need to take O.D. of higher concentration.calculateIC50 value by help of equation in excel

enjoy - Hi!!! you can use Ghap Pad Prism program, is very easy and you can dowload one month free on the web.......
- Hi Dep lin

If you are hopeless from all the analysis, Pls follow the old method. A bit time consuming but you will get the exact result. Use a graph paper to plot the curve between % inhibition and concentration. Then manually find the 50% inhibitroy concentration for each sample. It is simple and easy. Other wise you can use the Graph Pad Prism or Instats statistical softwares for this purposes. They also work well.

The manual method is also very well accepted by high impact international journals. So don't worry about acceptability of this method. Here I have shared my experience. haha

GUD LUCK

regards - Hi all.,For my results, i think my % inhibition did not even met 25%.. how should i display my results in my report?

Can i use IC10? - for DPPH assay even 2 ug to 10ug concentration of ascorbic acid is sufficient ,it will give good result and IC50, for test compound take same concentration as ascorbic acid and compare the result....

it also depends which protocol you are following and what instrument you are using to take O.D. - , to calculate IC 50 value, just do low range concentration to high range concentration assay and plot the graph i.e. Concentration vs OD or percent inhibition
- Hi Dep, I think it is not advisable to calculate IC10. If the results do not show significant antioxidant activity, and if you are still intended to use the same plant extract, you can try some other experiments to find its other bioactivities. There are several compounds/extracts with no antioxidant properties but having other major bioactivites. I hope you will find some good activity with those extracts. Best of Luck!
- Hey start from lower concentration 2 ppm ,4 ppm ,6 ppm ,8 ppm , 10 ppm 20 ppm , 40 ppm , 60 ppm , 80 ppm 100 ppm , 200 ppm ,400 ppm , 600 ppm , 800 ppm and see the % scavanging activity .
- we have a formula that absorbance of Control- Absorbance of test / absorbance of control * 100
- That shows percentage of inhibition......how to calculate ic 50of it?
- In excel manually i know to calculate in graph

.. - I agree with atul. If the values are low, then that plant extract might have no antioxident property and the phenolic compounds might be present in low conc in that plant.
- Dear,Satyajit Kanungo but the problem with my experiment is my control value is coming less then the test sample so it will be -ve if i will put this in formula for calculation of scavenging activity what should i do???
- You don't need it. Assumed that you can have only one trend line passing through your 2 points (each one for each experiment), you can calculate IC50 by an extrapolation of your values.

However, as somebody suggested before, it is better to make an intrapolation, so you should have at least one point at a concentration where the IC is higher then 50%.

Usually, I have an excel sheet with an atomation for this calculation... if you need it, just ask me. - Yes mr. Antonio i need excel automated...help me out sir..plz.
- Get your line graph then from the line graph you can do a line equation and obtain your line equation of y=mx+c . from this you can easily obtain your value.

Note: this val;ue is only reliable where the R2 value of the curve is close to 1

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