For doing DPPH free radical scavenging assay, I have used ascorbic acid as the standard. and took different concentrations (5,10,15,20,25 and 30 microgram/ml) for both standard as well as for plant extract. Above 30 microgram/ ml, the OD value for ascorbic acid remains to be same for all concentrations. For standard (Ascorbic acid) the % inhibition values are 24.04, 54.06, 86.68, 96.63, 96.78 and 96.93.And that for sample, values are 9.63, 17.34, 27.50, 33.51, 41.68 and 46.47. I plotted the scattered plot using % inhibition versus concentation (microgram/ml). Do I need to modify anything? Please help me!!
Either You can use the TREND option in MS Excel to know the IC50 value(50% activity) or You can use the Probit analysis in Excel (https://probitanalysis.wordpress.com/).
My dear you have to prepare higher concentration of the plant extract because the 30 microgram/ ml sample is not able to scavenge the DPPH radical and to make such concentrations until is reached higher than 90 % inhibition. Obviously the plant extract has lower radical scavenging activity than ascorbic acid.
I agree with Soleya. Your plant extract is not concentrated enough.
Moreover, DPPH assay can give you 2 interesting parameters : EC50, but also the reaction speed which is interesting to take into consideration.
Ascorbic acid oxidized rapidly; use a freshly prepared stock solution for your dilutions, overexposing your solutions may affect your results. Run OD vs [Ascb. A] to make sure you get the corresponding slope and continue with the % inhibition by DPPH essay. This kind of steady state from 96.63 % seems like something may have happened, have a look on your last dilutions unless 100% activity. As Soleya said, you will need to increase your concentrations to optimize your results as some plant extracts may give you very low scavenging activities. From your plot % inhibition Vs [Con, ug/ml], your IC50 value will be the corresponding [Con] for 50% activity of your 100 % inhibition. Moreover, 3 replicates with a calculated SEM will be useful .
Regards
Thank you all for your valuable comments. But the main problem that I am facing is that, while taking higher concentrations of ascorbic acid the OD value is seems to be stable as concentration increases. I have tried higher concentrations but the the OD values are not fare, that is, beyond 30 ug/ml, the OD value is becoming stable for further higher concentrations. So in this situation what could I do.. Please share your valuable suggestions.
Please refer our publication IJPPR, Volume 7, Issue 5, October 2015- November 2015
Dear Geethika your problem is not with ascorbic acid, with 30 ug/ml it was scavenged all the DPPH that is why the OD value is stable. The problem is with the plant extract, you need to prepare it at higher concentrations.
Please read some publications.
This happens because the whole quantity of DPPH reacted, the color turned yellow, right? For higher quantities of ascorbic acid, where are the absorbance values on standard curve? You should prepare some dilutions for vit C and than multiply with the dil. factor.
Dear Geethika,
Ti get better IC50 you have to get inhibition rate % series ranged from 0-100 , use the better conc. to have such inhibitions, so in case of ascorbic acid use less than 5 microgram, and for the extract use conc. more than 30 microgram, finally you have inhibition rate more and less than 50%
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