Re-evaluation of ABTS•+ Assay for Total Antioxidant Capacity of Natural Products

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS*+) is a stable free radical frequently used for estimating the total antioxidant capacity (TAC) of natural products. The existing methods for ABTS*+ radical-scavenging activity assays are diverse in pre-diluting solvents and reaction time, which lead to errors in the TAC estimations. To develop an effective and universal method for estimating the ABTS*+ capacity accurately and reasonably, five pre-dilution solvents [methanol, ethanol, phosphate buffer (Na2HPO4-NaH2PO4, 200 mM, pH = 7.4), PBS buffer (Na2HPO4-NaH2PO4- NaCl, 200 mM, pH = 7.4), and distilled water] and different reaction times were investigated in ABTS*+ assays of five typical antioxidants. The results showed that the solvent effects were very significant. When using different pre-diluting solvents, different detection wavelengths should be selected. ABTS*+ assay could be measured within 2-10 min to obtain a rough result, which was mostly 6 min in the literature. However, full and accurate evaluation of antioxidant reactivity rather than capacity requires recording ABTS*+ loss continuously during the whole reaction period. The present study makes a recommendation for estimating the ABTS*+ capacity accurately and reasonably.