Hello
In the tests related to Hydroxyl Radical Scavenging, two different methods were used.
In some articles, EDTA, deoxyribose, etc. have been used, but in other articles, these two substances have not been used at all and salicylic acid has been used (Hydrogen peroxide and iron chloride are common in both methods).
What is the reason for this difference?
And what is the difference between these two methods? Are both valid?
Thanks a lot
Those two are different methods. One of the assays uses the Fenton reaction to estimate hydroxyl radical with salicylic acid as trapping reagent, for example:
Article Effect of pH on Fenton process using estimation of hydroxyl ...
EDTA/deoxyribose is another method to assess hydroxy radicals:
Article The deoxyribose method: A simple “test-tube” assay for deter...
Dear Sahar
As Prof Shin wrote mostly we have more than one protocol , you should notice about the materials and all other points.
Deoxyribose assay is used to determine the hydroxyl radical scavenging activity in an aqueous medium. The reaction mixture containing FeCl3 (100 µM), EDTA (104 µM), H2O2 (1 mM) and 2-deoxy- D-ribose (2.8 mM) are mixed with or without the test sample (plant extract/fraction/compound) at various concentrations (10-250 µg) in 1 ml final reaction volume made with potassium phosphate buffer (20 mM, pH 7.4) and incubated for 1 hr at 37 0C. The mixture is then heated at 950C on water bath for 15 min followed by the addition of 1 ml each of Trichloroacetic acid (TCA) (2.8%) and Thiobarbituric acid (TBA) [0.5% TBA in 0.025 M NaOH containing 0.02% 2-Deoxy-D-ribose, butylated hydroxyanisole (BHA)]. Finally the reaction mixture is cooled on ice and centrifuged at 5000 rpm for 15 min. Absorbance of supernatant is measured at 532 nm. All readings are corrected for any interference from brown colour of the extract or antioxidant by including appropriate controls. The negative control without any antioxidant or sample is considered 100% deoxyribose oxidation. The % hydroxyl radical scavenging activity of test sample is determined accordingly in comparison with negative control. Ascorbic acid is taken as the positive control.
Hope it helps you.
Good Luck
Parisa Ziarati Thank you very much
And when is the salicylic acid method used?
Highly recommend to read some references such as
https://doi.org/10.1155/2011/809696
......is very likely that the effect of salicylates is due to the introduction of a negative charge to the protein, thus diminishing hydrophobic interactions. An example of the effect of negative charge of sulfate groups is sulfitolysis of proteins that completely unfolds their polypeptide chains and yet maintains their complete solubility. It should be noted that fibrin-like deposits, which are remarkably resistant to proteolytic degradation, have been observed in numerous degenerative......
to achieve the goal a correct protocol must be realized. the specifications of the materials within this protocol are important
Dear Sahar Kelvani
You have posted several questions on details of antioxidant assays. All your questions show that you have no ideas about the chemistry behind these assays. When you are doing something, you must understand what you are dealing with. You need to learn basics of antioxidant chemistry. Otherwise, you waste time.
https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-015-1618-6
Methods for trapping hydroxyl radicals use conventional redox reactions, where there are a reducing agent and an oxidizing agent. In particular, metal atoms and complexones are used to form colored oxo complexes of the metal, which are easily detected with a spectrophotometer. Amine compounds also act as radical traps, where the lone pair of electrons of the nitrogen atom at the aromatic ring can trap radicals. Various polysaccharides and carbohydrates can be used as reducing compounds or indicators where the iodine oxidation reaction is used. In addition, aromatic carboxylic acids can form standard peracids
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