I'm performing the DPPH Assay for my plant extract. So with an increase in the conc of extract (20,40,60,80,100), either the % is decreasing or showing an irregular pattern. Also, it is showing the least value for the highest concentration. But the standards are showing correct values. (inc with inc in conc)
I'm confused what and how to proceed further.
MM Rahman (2015) reported that all the plant extracts showed a good total antioxidant activity that increased with increasing concentration https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-015-1618-6
Check https://www.sciencedirect.com/topics/medicine-and-dentistry/dpph-radical-scavenging-assay
https://www.hindawi.com/journals/tswj/2014/279451/
The radical scavenging activity is measured as a decrease in the absorbance of DPPH in the presence of test sample/compound. You can follow my paper:Article Phytochemical Studies and In Vitro Evaluation of Antioxidant...
I think it is normal result. Here some discussions from Researchgate about your question may help you to solve the problem:
https://www.researchgate.net/post/DPPH_increasing_absorbance2
https://www.researchgate.net/post/Can-anyone-help-me-solve-an-issue-Ive-been-having-with-DPPH-assay-and-spectroscopy-analysis
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3551182/
Article Total Phenolic, Anthocyanin, Catechins, DPPH Radical Scaveng...
Article A new colorimetric DPPH• scavenging activity method with no ...
Yes. It is suppose that if you increase the concentration of your extracts (with antioxidants) it will have greater antioxidant capacity (less absorbance). However, if you find an erratic pattern, your sample may be interfering with the method. Try measuring absorbance during an hour every 5 minutes and not only final absorbance or try another method if you can too
I recommand to test the dpph for your extract then initial concenyration /2 for see the results
Yes.when you increase the concentration of your extracts, you Will be find an increased antioxidant capacity (decreased absorbance) until arrived to a saturation activity which corresponded to 0 concentration of dpph in your reaction.
Hello Mandeep Gulati ;
As María Belén Gutiérrez Barrutia pointed out, if you have antioxidant compounds in your plant extract, you are supposed to obtain higher antioxidant activities. So it appears that something is interfering with the reaction. In order to be able to assist you, please elaborate on the following:
1) What are the units of the extract concentrations you quote in your question (20, 40 ..)? how did you prepare such extracts? what dilutions were prepared?
2) Describe the DPPH antioxidant method you are using (concentrations and conditions)
3) Quote the equation for the DPPH inhibition value (% of inhibition).
Regards.
The increase in the Conc is directly proportional to scavenging activity. So you will find the lowest value of absorbance for the highest concentration.
Dear Sabrine Ben Lataief maam,
Thanks for clarifying my doubt.
As you said it'll increase until saturation comes but when I compare with the standards (BHT & Ascorbic acid), the pattern is different and it is increasing only.
Dear Rogelio Valadez-Blanco sir,
thank you for your response!
1. extracts conc are 20uL extract in 920uL of solvent , 40extract in 920uL of solvent, 60, 80, 100
2. 1ml test sample + 3ml DPPH alcholic solution = abs in dark after 30 minutes
3. dpph sca activity% = Abs C - Abs S / Abs C X 100
4. negative control is DPPH alcholic soln
5. positive control= BHT and Ascorbic acid (same conc as extract samples)
Dear Mandeep Gulati sir,
when you do the antioxidant activity of the standards, you will find that the saturation is well accelerated because the molecules of the standars are of nature a strong antioxidant molecules. In your case that the model only increases, here you have not yet arrived at the saturated concentration of your sample and in this case it is necessary to arrive at higher concentrations to have the saturation of your sample. and therefore you will conclude here that your sample has a lower antioxidant activity compared to standars.
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