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The Chemistry behind antioxidant capacity assays

The Chemistry behind antioxidant capacity assays . Presented by: Alana Bryant . Introduction . Antioxidant- A substance that opposes oxidation or inhibits reactions promoted by oxygen or peroxides.

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The Chemistry behind antioxidant capacity assays

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  1. The Chemistry behind antioxidant capacity assays Presented by: Alana Bryant

  2. Introduction • Antioxidant- A substance that opposes oxidation or inhibits reactions promoted by oxygen or peroxides. • Recent studies show that there may be a link between oxidative stress and the development of several types of chronic diseases. • Research has also shown that diets rich in fruits and vegetables may decrease the incidence of these diseases

  3. Introduction • Antioxidant such as polyphenolic compounds, Vit. E & C and carotenoids are believed to be effective nutrients in preventing oxidative stress related diseases • It is of importance to know how much capacity of antioxidant in the foods we consume.

  4. Methods of Measuring AOX assays Antioxidant can deactivate radicals by two major mechanisms, • Hydrogen Atom Transfer (HAT) • Electron Transfer (ET) Important: The end results is the same, regardless of the mechansim, but kinetics and potential for side reactions differ.

  5. Hydrogen Atom Transfer (HAT) • Measure the classical ability of an antioxidant to quench free radicals by hydrogen donation (AN = any H donor) • HAT reactions are solvent and pH independent and are usually quite rapid (sec to minutes).

  6. HAT Methods • Oxygen radical absorbance capacity (ORAC) assay • Total oxyradical scavenging capacity (TOSC) assay

  7. ORAC • It is a widely accepted tool for measuring the antioxidant activity of various vitamins and other organic and inorganic compounds. • It measures the oxidative degradation of a fluorescent molecule (Usually fluorescein sodium salt or beta-phycoerythrin) after being mixed with an oxygen radical initiator

  8. ORAC METHOD • The antioxidant (Trolex and samples) are mixed with the fluorescence solution and incubated at a constant temperature before the initiator solution is added.

  9. Total Oxyradical Scavenging Capacity Assay (TOSC) • In contrast to ORAC assay but it measures the decrease of ethylene production caused by inhibition of thermal hydrolysis of ABAP (2,2’ – Azobis(2 –methyl-(propionamidine) dihydrochloride) by KMBA (alpha-keto-y-(methylthio) butyric acid sodium salt)(substrate) in the presence of antioxidant compounds • It has the ability to distinguish between faster acting and slower acting antioxidant as it is kinetically based assay

  10. ORAC & TOSC • Calculating the Trolox • AUC = ( 0.5 + F1 /F2 +F2/F1 +…FN/F1) x CT • In order to calculate Trolox a Trolox standard curve must first be obtained and calculating individual the individual AUC for each concentration.

  11. Assays for Hydrophilic and Lipophilic Antioxidant Capacity (oxygen radical absorbance capacity (ORACFL)) of Plasma and Other Biological and Food Samples • The FL decay curve in the presence of AAPH and antioxidant

  12. ELECTRON TRANSFER (ET) • Measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. • Probe (oxidant) + e (from antioxidant) reduced probe + oxidized antioxidant

  13. ELECTRON TRANSFER (ET) • ASSAYS • Ferric Reducing Ability of Plasma (FRAP) • Copper (II) reduction capacity assays

  14. Ferric Reducing Ability of Plasma (FRAP) • At a low pH when a ferric-tripyridyltriazine (Fe-TPTZ) complex is reduced to ferrous (Fe) form an intense blue color develops. • The color development represents that an antioxidant is present

  15. Ferric Reducing Ability of Plasma (FRAP) • To measure the FRAP • Samples are diluted in the provided Assay Buffer and added to the wells. The FRAP Color Solution is made by mixing Reagent A and B with Assay Buffer. The FRAP Color Solution is added to all wells and the plate incubated at room temperature. Antioxidant power in the samples reacts with the FRAP Color Solution to generate a blue colored product which is read at 560 nm. • 4 min

  16. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of ‘‘Antioxidant Power’’: The FRAP Assay • Figure 1 shows the rate of increase in absorbance at 593 nm for 100 mmol/liter solutions of bilirubin (crossed squares), ascorbic acid (filled squares), uric acid (trian- gles), a-tocopherol (open circles), and albumin (plus sign) compared to the monitored absorbance of reagent only (diamonds), all measured in parallel

  17. FRAP FRAP assay results • From the results from the FRAP assay, we conclude that the dandelion has a great potential. The results indicated that compound number two, out of the four compounds, (the flavonoid) exceeded the standard maximum level in our FRAP assay 1.6 times. Therefore, dandelions have a very powerful antioxidant property, and can be used to make effective medicine.

  18. Cu (II) reduction capacity assays • Method is based on reduction of Cu (II) to Cu (I) by antioxidants present in the samples. • It was found that 1 mol of alpha-tocopherol can reduce 2 mol of Cu (II) to Cu(I)

  19. Conclusion • ET based assays measure an antioxidant reducing capacity and the HAT- based assays quantify hydrogen atom donating capacity. • Based on current research HAT method is more relevant to antioxidant capacity

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