I am working on DPPH assay with different antioxidants.I do not understand why some antioxidants like Ascorbic acid and Glutathione have high radical scavenging properties with IC50 less than 0.250 mM. However, D-Mannitol and vitamin B3 have no radical scavenging activities even at high concentration like 800 mM.
Is there any mechanism should know to understand this issue?
In order to understand, you need to read textbooks and learn what are the antioxidants. D-mannitol and vitamin B3 do NOT react with DPPH radical and are not antioxidants. This is another story, why DPPH reacts very slowly with the most of organic molecules.
DPPH (1,1-diphenyl-2-picrylhydrazyl) is a commonly used radical to assess compounds' antioxidant activity. The IC50 value, the antioxidant concentration required to scavenge 50% of the DPPH radical, is a measure of antioxidant potency. Lower IC50 values indicate higher antioxidant activity.
Ascorbic acid and glutathione are both known to have strong antioxidant properties and can scavenge free radicals effectively, hence their low IC50 values in the DPPH assay.
In the case of D-mannitol and vitamin B3, they may not exhibit antioxidant activity in the DPPH assay due for several reasons. Firstly, they may not be able to scavenge free radicals effectively and, therefore, cannot reduce the DPPH radical to its reduced form. Secondly, they may be unable to penetrate the cell membrane, where the DPPH radical is located, to scavenge it. Additionally, some compounds may interfere with the reaction between the DPPH radical and the antioxidant, leading to a decreased ability to scavenge the radical.
It's also important to note that the DPPH assay measures only one aspect of antioxidant activity. Some antioxidants may exhibit activity against other radicals or oxidative stress in different ways that the DPPH assay may not measure.
Short clarification of my first answer in simple words. DPPH is stable in solutions of EtOH/MeOH. Why it should react with such alcohol as mannitol?
DPPH is a stable compound in ethanol and methanol solutions because these alcohols are poor nucleophiles and do not readily react with the aromatic ring structure of the DPPH molecule. On the other hand, mannitol is a sugar alcohol and is much more reactive than ethanol or methanol. The hydroxyl groups in mannitol can potentially react with the DPPH molecule through hydrogen bonding, leading to a reaction between the two compounds. The reaction between DPPH and mannitol would likely lead to a change in the DPPH structure, reducing its stability and potentially leading to the degradation of the molecule.
Dear Geir Bjorklund , DPPH is reduced to colorless product in the reaction of hydrogen atom transfer from an antioxidant to N-centered radical of DPPH. The bond dissociation energy of formed N-H bond is only 75 kcal/mol, which is too week to cleave C-H or O-H bonds in alcohols . See for details https://encyclopedia.pub/entry/history/show/17803
Yes, that is correct. The reaction of hydrogen atom transfer from an antioxidant to the N-center radical of DPPH results in the reduction of DPPH to its colorless product. The bond dissociation energy of the formed N-H bond is indeed low, at 75 kcal/mol, which is not strong enough to cleave C-H or O-H bonds in alcohols. This makes it a useful tool for measuring the antioxidant activity of various compounds. The ability of a compound to reduce DPPH is used as a measure of its antioxidant activity, with more potent antioxidants leading to a greater reduction of DPPH to its colorless product.
Thank you for your response. The mentioned points are very useful🙏🙏
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