Evaluation of radical scavenging properties of shikonin

With the aim of developing effective anti-inflammatory drugs, we have been investigating the biochemical effects of shikonin of "Shikon" roots, which is a naphthoquinone with anti-inflammatory and antioxidative properties. Shikonin scavenged reactive oxygen species like hydroxyl radical, superoxide anion (O-2(center dot-)) and singlet oxygen in previous studies, but its reactivity with reactive oxygen species is not completely understood, and comparison with standard antioxidants is lacking. This study aimed elucidation of the reactivity of shikonin with nitric oxide radical and reactive oxygen species such as alkyl-oxy radical and O-2(center dot-). By using electron paramagnetic resonance spectrometry, shikonin was found unable of reacting with nitric oxide radical in a competition assay with oxyhemoglobin. However, shikonin scavenged alkyl-oxy radical from 2,2'-azobis(2-aminopropane) dihydrochloride with oxygen radical absorbance capacity, ORAC of 0.25 relative to Trolox, and showed a strong O-2(center dot-)-scavenging ability (42-fold of Trolox; estimated reaction rate constant: 1.7 x 10(5) M(-1)s(-1)) in electron paramagnetic resonance assays with CYPMPO as spin trap. Concerning another source of O-2(center dot-), the phagocyte NADPH oxidase (Nox2), shikonin inhibited the Nox2 activity by impairing catalysis when added before enzyme activation (IC50: 1.1 mu M; NADPH oxidation assay). However, shikonin did not affect the preactivated Nox2 activity, although having potential to scavenge produced O-2(center dot-). In conclusion, shikonin scavenged O-2(center dot-) and alkyl-oxy radical, but not nitric oxide radical.