Essay ABTS
It is an analytical method that uses a spectrophotometric measurement to determine the antioxidant capacity of a sample. Using a UV-Vis spectrophotometer, the absorbance of a solution containing the radical ABTS • + is measured, generated by oxidation of the ABST (2, 2'-azinobis (3-ethylbenzothiazolin-6-sulfonate), a colorless substance in the form radicals are colored by absorbing at wavelengths characteristics in the visible range The addition to the solution of ABTS • + of antioxidant molecules, which can act by transferring both hydrogen and an electron, determines the reduction of the radical to the colorless form, with consequent discoloration of the reaction mixture .This bleaching, proportional to the amount of antioxidant present, can be measured as a decrease in absorbance over a certain time at a specific wavelength (734 nm) .The antioxidant power is expressed by comparison with the absorbance values measured for known quantities of an antioxidant molecule chosen as the reference standard, which is usually ascorbic acid or Trolox (in this case we speak of TEAC Trolox Equivalent Antioxidant Capacity antioxidant activity.
The antioxidant power measurement based on the use of ABTS has the advantage of being simple and fast. Moreover, it allows the measurement of antioxidant substances both hydrophilic and lipophilic in a wide pH range. However, it must be borne in mind that the radical used (ABTS • +) is not physiological and is not present in biological systems and that problems of repeatability of measurement due to the reaction kinetics of the various antioxidants involved are often highlighted.
FRAP (Ferric Reducing Antioxidant Power)
The FRAP test measures the reducing ability of antioxidants against iron ions. It is a method based on electron transfer, in which iron ions pass from Fe3 + to Fe2 +. Under certain conditions of pH (3.6) and in the presence of TPTZ (2, 4, 6-tris (2-pyridyl) -s-triazine), these ions form complexes with different characteristics, in particular the reduced derivative (Fe2 + -TPTZ) assumes a blue color that has a maximum absorption at 593 nm which can be measured spectrophotometrically. The reducing capacity of an antioxidant substance can therefore be measured as a change in the absorbance of the solution containing the oxidant at the wavelength established by comparison with the variation relative to a standard (eg ascorbic acid).
The FRAP test was designed to measure the reducing power of the plasma, but was then adapted to test the antioxidant capacity of pure compounds and complex matrices. In fact, since this method allows to evaluate only the reducing capacity by electron transfer, completely ignoring the action of antioxidants that act through hydrogen transfer, it does not allow to measure the contribution of molecules, such as thiols and proteins, which play an antioxidant role fundamental in biological fluids (eg blood). The advantage in using this method is that it is one of the simplest, fastest and least expensive methods for determining the antioxidant capacity in vitro.
DPPH TEST
2, 2-diphenyl-1-picrilhydrazyl (DPPH •) is a very stable and commercially available nitrogen radical, characterized by an intense purple-red color, which dulls when reduced in the presence of a molecule with antioxidant capacity. By spectrophotometric measurement at 517 nm of the change in absorbance of the DPPH solution after reaction with an antioxidant compound, it is possible to quantify the reducing capacity of the test substance whether it acts with hydrogen transfer or electron transfer. The result is generally expressed as IC50, ie the amount of antioxidant capable of reducing the initial DPPH concentration by 50%.
This is a quick, simple and inexpensive method. The limits of this analytical technique are given by the possibility that the results of the analysis are distorted in the case in which the molecules under examination absorb in the same wavelength range of the DPPH radical or in the presence of large molecules huddled sterically that do not they come to react with the reactive part of the radical. This causes the DPPH to react with antioxidants up to 1000 times more slowly than peroxyl radicals.
PCL TEST (Photochemiluminescence)
The PCL test is based on the reaction of a specific radical species, the superoxide anion (O2 • -), generated photochemically by UV radiation, with a compound capable of emitting chemiluminescence. The marker used is luminol, a molecule that when oxidized by free radicals emits a light that can be measured using a special instrument (Photochem®). The presence of antioxidant substances in the ration mixture deactivates the radical species inhibiting the emission of chemiluminescence. PCL analysis is very rapid and sensitive. Furthermore, with the application of two different analytical protocols, called ACW (Antioxidant Capacity Water soluble) and ACL (Antioxidant Capacity Lipid soluble), the contributions to the total antioxidant capacity of the water-soluble component (flavonoids, vitamin E) can be measured for the same compound. C, amino acids, etc.) than the liposoluble one (tocopherols, tocotrienols, carotenoids, etc.). The antioxidant capacity of the product under examination is obtained by comparing the values recorded with the measurements relating to standard reference molecules, ascorbic acid for the ACL protocol and Trolox for the ACW protocol.
