I performed a DPPH assay for plant extract prepared in methanol and aqueous solvent. For the experiment, I dissolve DPPH in DMSO. I prepared a stock solution of 5mg/ml conc. for each extract and then take the 20, 40, 60, 80 and 100 ug/ml conc. for experiment. To make the volume 1ml, I added respective solvents (i.e. methanol & aqueous ) in the test tubes. 1ml of DPPH was added to each tube. 1ml DPPH+ 1ml methanol and 1ml DPPH+ 1ml aqueous used as control.
But the order of absorbance is not decreasing. Almost equal abs. was shown by 3-4 tubes. Even all the tubes show yellowish color of the solution. Almost similar results were shown by aqueous as well as methanol extract.
I just want to know that which factors contribute to these type of results.
Is DMSO is interfering with the antioxidant potential of plant extract?
Looking forward for your valuable suggestions.
Thanks..
Hello Preety Rohilla,
As per my suggestion, DPPH should not be dissolved in DMSO, as DMSO itself has its antioxidant properties. So you can be misguided with the false lower absorbance in a spectrophotometer.
As per literature, 0.1%-0.5% DMSO is still safe. That means if you dissolve your plant extract in DMSO, that will be okay. Because during dilution of extract into DPPH-methanol/ethanol, the concentration of DMSO comes around 0.5% only. We usually take 200µl extract +1.5ml DPPH-methanol.
But if you use 1ml DPPH-DMSO and mix it with 1ml methanol/water, the con of DMSO would be 50% which can cause free radicle scavenging of DPPH by DMSO itself. Better to dissolve DPPH in methanol or ethanol.
when you were observing equal absorbance in 2-3 cuvets, that means DPPH has already come to the saturation point, in that case, you have to increase the molarity of DPPH in the stock and control.
Hope this will help you,
Thanks
Esha Sarkar Thank You so much Mam for such an elaborative answer.
But the absorbance of the control (1ml methanol/water + 1ml DPPH) is ok (i.e. higher as compared to sample and control solution is of purple color). I think in that case DMSO is not interfering with the solvent. Problems come only in test samples.
Thanks
Check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3551182/
Also check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648874/
Hi Dr Preety Rohilla . I hope the following article could help you:
https://www.orientjchem.org/download/62805
Dear all, according to many studies (see attached), DMSO is considered as the best solvent for DPPH assay. My Regards
https://www.researchgate.net/post/Did_anyone_know_does_DPPH_assay_can_be_used_to_determine_antioxidant_activity_of_the_cream_formulation
You can try this paper
http://www.orientjchem.org/vol36no3/comparison-of-different-organic-solvents-on-antioxidant-activity-of-astaxanthin-extracted-from-hematococcus-pluvialis-using-colorimetric-and-non-colorimetric-methods/
The standard DPPH assay uses methanol or ethanol as solvents, however if compd. is insolublr one can use high polar solvent like DMSO.
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