Verification of the Conditions for Determination of Antioxidant Activity by ABTS and DPPH Assays-A Practical Approach.

Rafał Wołosiak,Paulina Pakosz,Marta Ciecierska,Beata Drużyńska,Dorota Derewiaka,Małgorzata Piecyk,Elwira Worobiej,Ewa Majewska

doi:10.3390/molecules27010050

Rafał Wołosiak, Paulina Pakosz + Show 6 more

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https://doi.org/10.3390/molecules27010050

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Abstract

The ABTS and DPPH methods are among the most popular assays of antioxidant activity determination. Attempts to adapt them to different analytes and the search for the highest values of antioxidant activity has resulted in a large variety of assay conditions to be presented in the literature, including the way the measurement is made. This makes it difficult to relate the results to real oxidation systems, and often makes it impossible to compare them. Such a comparison is limited in advance by the use of stable radicals that do not exist in nature and that react differently from those generated in food or in vivo. Therefore, it is important to introduce measures aimed at standardizing the conditions of the activity assay, including reaction time and several reaction environments suitable for testing different groups of compounds. In this study, we used natural antioxidants of various structures: phenolic acids, flavonoids, peptides and corresponding amino acids, ascorbic acid and α-tocopherol, and also synthetic analogues of selected compounds. The curves of dependence of the measured absorbance on the concentration of antioxidants were described, the ranges of linearity were determined, and the value of the error made when reading in various ranges of dependencies was estimated. We also determined and compared the activity values using two popular methods (IC50 and TEAC), taking into account different environments and reaction times. Based on the collected data, recommendations were formulated regarding the reaction conditions adapted to the studies of individual groups of antioxidants, and unified reaction times were proposed. Taking into account the state before reaching the equilibrium of antioxidants reacting in a complex manner, this approach may introduce a simplified reference to the competing reaction that occurs in reality.

Highlights

Aerobic cellular respiration, as adopted by highly organized organisms living on Earth, provides much energy for their functioning, and causes side effects from metabolism in the form of free radicals
The standard times for the deactivation of ABTS and DPPH radicals seem to be the most appropriate. They are widely used in the literature, and what is most needed in research on antioxidant activity is the unification of assay conditions
Apart from rapidly reacting substances, these times seem long enough to allow the more reactive phenolic compounds or peptides and amino acids to come closer to equilibrium, but short enough to take into account the different reaction rates of tested compounds, which takes place under real conditions

Summary

Introduction

As adopted by highly organized organisms living on Earth, provides much energy for their functioning, and causes side effects from metabolism in the form of free radicals. In defending themselves against the damaging effects of radical presence, living organisms use exogenous and endogenous antioxidants These compounds play a very important metabolic role, and studies on the antioxidant activity of substances extracted from food or biological material has been the subject of researchers’ interest for many years. Research with the use of stable free radicals ABTS and DPPH, which can be determined spectrophotometrically and discolored as a result of deactivation, is popular. Their popularity, ease of use and susceptibility to modification have resulted in the appearance of many publications in which, because of the different versions of the assays used, the results are not comparable

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