Evaluation of antioxydant activity?

It's intriguing that you did not observe any color change when using high concentrations of the ethanolic extract with DPPH (2,2-diphenyl-1-picrylhydrazyl), especially considering the high content of polyphenols and flavonoids in your extract. The DPPH assay is a widely used method for evaluating the antioxidant activity of compounds because it is based on the ability of antioxidants to scavenge the stable free radical DPPH, causing a color change from purple to yellow.

Several factors could potentially explain the lack of color change in your experiment:

Saturation Point: At very high concentrations of the ethanolic extract, it is possible that the DPPH radicals are already fully scavenged by the available antioxidants in the extract. Once all the DPPH radicals are neutralized, there would be no further color change even with higher concentrations of the extract.

Prooxidant Effects: In some cases, high concentrations of antioxidants can paradoxically exhibit prooxidant effects. Instead of neutralizing free radicals, they may actually promote the generation of reactive oxygen species (ROS) under certain conditions, leading to a lack of color change in the DPPH assay.

Interference: Some compounds in the extract might interfere with the DPPH assay, preventing the color change from occurring. Certain chemical constituents in the extract might react with DPPH or affect the stability of the radical, leading to ambiguous results.

Solubility: It's crucial to ensure that the extract is well dissolved in the ethanol solvent before performing the assay. Insoluble particles or aggregates in the extract might not interact effectively with DPPH, affecting the assay's outcome.

To troubleshoot and get a better understanding of the situation, you could try the following:

Lower Concentrations: Test the ethanolic extract at lower concentrations (e.g., 500 µg/ml, 100 µg/ml) to see if there is a concentration-dependent response in the DPPH assay. This may help identify whether the lack of color change is specific to higher concentrations.

Positive Control: Use a known antioxidant compound (e.g., ascorbic acid or quercetin) as a positive control in the DPPH assay. This will ensure that your DPPH reagent is active and capable of showing a color change under appropriate conditions.

Different Solvents: If possible, try using other solvents (e.g., water or methanol) for the extraction process. It's possible that the ethanolic extraction might be affecting the interaction between the extract and DPPH.

Other Antioxidant Assays: Consider performing additional antioxidant assays (e.g., ABTS assay, FRAP assay, or total phenolic content determination) to corroborate the results and gain a broader understanding of the antioxidant activity of your extract.

Remember that interpreting antioxidant activity data can be complex, and it's crucial to understand the limitations of each assay and consider the overall context of your research.

Ankit Negi

Can you please explain how you performed this experiment? What was the concentration of DPPH you used, and what color is your extract?

try with lower concentration 50, 100, 150......1000ug/ml

Khadija Boualam

The DPPH solution you used, did you prepare it or it was already in the refregirator? Maybe it's a stock solution then it's too concentrated. Did you try a standard such as ascorbic acid just to make sure that the problem isn't your DPPH solution? And look in litterature about DPPH IC50 values of your plant, when you say high content in polyphenols it's too relative.

Also, try lower concentrations, your extract at 1000 or 2000 ug/ml has already its own dark color that may that won't let the yellow color show through

Joan Imah-Harry

iI HAVE SEEN FROM EXPERIENCE THAT IT IS BETTER TO MOVE FROM A LOW TO A HIGH CONCENTRATIONS OF EXTRACT STOCK, TO BE SURE OF THE POTENCYOF THE POLYPHENOLIC CONTENTS OF THE EXTRACT ON DPPH. LIKE FROM 200, 1000, 800, 600, 400 AND 200 ETC.

Abhinay Thakur

Test a wider range of concentrations to determine if there's a threshold concentration for the color change to occur and also check the solubility of your extract in ethanol and consider adjusting the solvent or using a different method to dissolve your extract if needed.

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