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

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.

Lipase-Catalyzed Synthesis, Antioxidant Activity, Antimicrobial Properties and Molecular Docking Studies of Butyl Dihydrocaffeate

“…Wołosiak et al [ 16 ] evaluated the applicability of ABTS·+ (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) and DPPH· methods to assess the activity of 14 various antioxidants (i.a. phenolic acids, flavonoids, L-ascorbic acid, amino acids, α-tocopherol, their analogs, and derivatives) and mixtures of these compounds.…”

“…Interestingly, the ABTS method proved to be more suitable for amine compounds and ascorbic acid. On the other hand, the DPPH assay is more applicable for phenolic compounds and other compounds of limited polarity, and the use of an appropriate solvent should be matched to the polarity of the antioxidant [ 16 ].…”

Optimization of Ultrasonic-Assisted Extraction of Active Components and Antioxidant Activity from Polygala tenuifolia: A Comparative Study of the Response Surface Methodology and Least Squares Support Vector Machine

“…The antioxidant activity of Yuanzhi extracts was evaluated using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity (DPPH) and 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) antioxidant power (ABTS) [ 46 ]. The DPPH assay was introduced by Brand Williams [ 47 ].…”

Two Natural Flavonoid Substituted Polysaccharides from Tamarix chinensis: Structural Characterization and Anticomplement Activities

“…The antioxidant activities of MBAP-1 and MBAP-2 were evaluated in vitro by the ABTS and FARP methods [ 35 ]. As displayed in Figure 8 A, FRAP values of MBAP-1 were from 0.0145 ± 0.002 mmol/L to 4.483 ± 0.348 mmol/L, while that of MBAP-2 were from 0.124 ± 0.012 mmol/L to 3.455 ± 0.230 mmol/L.…”