Hi.
I have a methanolic extract that I have fractioned it following proper protocol. The problem is that the extract gave total polyphenols contents and flavonoids lower than its fractions but a higher antioxidant activity that evaluated by four methods. How I can explain these results? (the same result was found with other samples).
Thank you for help.
Hello, Im not sure if I understood your question, but an assay to measure the total amount of polyphenols, not strictly represents the antioxidant activity of a sample. Many of the phenolic or hydroxyl groups do not necessarily have antioxidant properties. It could also happen, that your extract has antioxidant activity that is not related to its hydroxyl groups. The mechanism is very complex, there may be several mechanisms at once and your assay detects one, the solvent also influences, the acidity constants etc. What protocol are you using?
Thank you for your answer.
To understand, I evaluate the content of polyphenols and flavonoids and antioxidant activity (DPPH, reduced iron, beta-carotene and CAT) of a plant extract, I fractioned this latter following Merkham protocol, that based on the degree of solubility of flavonoids, which is exhausted to extract successively with three solvents of different polarity: chloroform, ethyl acetate and n-butanol.
the fractions obtained have contents of polyphenols and flavonoids superior (quite logical), but lower antioxidant activity using the DPPH test and higher using other tests.
The following table is a sample of results found (attached file)
You will have different results with different methods. DPPH is a stable radical, is very big and reacts only through the electron transfer mechanism. whereas for example hydroperoxyl radical can react by several mechanisms, hydrogen atom transfer, electron transfer, and radical adduct formation. Polyphenols in some cases "prefer" to lose a hydrogen atom, to donate an electron, and this depends on the pKa of the flavonoid. At different pH values will act one way or the other and depending also of the nature of the radical
The dielectric constant of the solvents employed, the acidity constants of different analytes and the method used are variables that can give contradictory results. The order in the antioxidant activity of a group of flavonoids can be inverted depending on the method you used.
Morin for example is a flavonoid with the first pKa in 4.98, in aqueous medium and at pH value of 7.4 is one of the most powerful antioxidants, but in methanol against DPPH it is not a big thing.
Concluding, the estimation of antioxidant activity is a very controversial issue.
antioxidant assay is true as i see because the extracts usually contain combination of all phytochemicals the deactivating dpph but the other results must be repeated
you need to identify antioxidant components in your extract, may synergism in the component cause high activity
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