The 2-deoxyribose degradation assay (2-DR assay) is an in vitro method broadly used for evaluating the scavenging activity against the hydroxyl radical (HO). One of the major drawbacks of the assay, however, is that only water soluble compounds can be tested for their radical-scavenging activity. Lipoic acid (LA) is an excellent scavenger of HO but it exhibits a low solubility in the aqueous milieu of the 2-DR assay and a high tendency to polymerize under a variety of conditions. We used LA as a paradigmatic substrate to evaluate the effect of several organic co-solvents in increasing its solubility. Most of these solvents, however, demonstrated to be potent scavengers of HO making their use in the 2-DR assay improper. On the other hand, acetonitrile showed a remarkably low reactivity toward HO (rate constant ~8.7×106M-1s-1) which allowed us to use it as a co-solvent in the preparation of stock solutions of LA ~5mM. We therefore evaluated the radical-scavenging activity of LA by the 2-DR assay in a relatively large range of concentrations, 1-200μM. We found that the rate constant for LA+HO is diffusion-controlled (~1×1010M-1s-1 in water at 25°C) and uninfluenced by the presence of small quantities of acetonitrile. Therefore, the use of acetonitrile in the 2-DR assay does not interfere with the test and may increase the solubility of the radical scavengers.

Four natural N(6)-substituted adenine derivatives (cytokinins) were evaluated for the first time in vitro for they antioxidant capacity by using fluorimetric and spectrophotometric assays, i.e., the oxygen radical absorbance capacity (ORAC), trolox equivalence antioxidant capacity (TEAC) and the 2-deoxyribose degradation (2-DRA) assays. The results from the TEAC assay show that only N(6)-(4-hydroxybenzyl)adenine (p-topolin) shows an electron transfer capacity due to the presence of a phenolic moiety in the N(6)-position. The results from the ORAC test show that the antioxidant activity of N(6)-furfuryladenine (kinetin, K) is the highest up to a concentration of 1 μM, whereas at concentrations higher than 1 μM p-topolin is the most efficient antioxidant. Analysis of the kinetic data suggests that, compared to the other cytokinins, more sites of the molecular structure of p-topolin are available for the quenching of peroxyl radicals. The hydroxyl radical scavenger ability, as measured by the 2-DRA assay, showed that all tested cytokinins react in this test and that N(6)-(Δ(2)-isopentenyl)adenine is slightly more potent, probably because of the allylic methylene group present in the N(6)-isopentenyl moiety. Our data suggest that a part of the biological activity of the evaluated cytokinins is likely to be related to an intrinsic antioxidant capacity.