Evaluation of radical scavenging properties of shikonin

Abstract

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 (O2•−) 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 O2•−. 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 O2•−-scavenging ability (42-fold of Trolox; estimated reaction rate constant: 1.7 × 105 M−1s−1) in electron paramagnetic resonance assays with CYPMPO as spin trap. Concerning another source of O2•−, the phagocyte NADPH oxidase (Nox2), shikonin inhibited the Nox2 activity by impairing catalysis when added before enzyme activation (IC50: 1.1 µM; NADPH oxidation assay). However, shikonin did not affect the preactivated Nox2 activity, although having potential to scavenge produced O2•−. In conclusion, shikonin scavenged O2•− and alkyl-oxy radical, but not nitric oxide radical.

Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca²⁺ fluxes

“…As we ( 16 ) and others ( 14 , 15 ) have previously reported, shikonin scavenges O 2 •− , with IC 50 values of 2.9~17 µM depending on the source, assay conditions and O 2 •− detection system used. To know the contribution of the chemical scavenging of O 2 •− by shikonin in RH buffer with 1 µM CLA used in assays with cells, we first investigated the quenching of O 2 •− -produced chemiluminescence of the hypoxanthine-xanthine oxidase reaction ( 3 ) in the CFL-C2000 (Fig.…”

“…Previous studies have indicated that shikonin chemically scavenges ROS such as O 2 •− ( 14 – 16 ) and hydroxyl radical, ( 15 , 17 ) and biologically behaves as an anti-inflammatory compound by directly targeting these oxidative molecules. The effects of shikonin on gene expression of key molecules involved in the inflammatory response have also been described; ( 11 , 13 ) however, the mechanisms of how shikonin suppresses O 2 •− generation of cells remained unclear.…”

“…We have also been investigating the anti-inflammatory effects of shikonin, the major active substance of the herbal medicine “Shikon” ( Lithospermum erythrorhizon ), ( 11 – 13 ) for its therapeutic potential in inflammatory diseases. Shikonin scavenges ROS such as O 2 •− , ( 14 – 16 ) hydroxyl radical, ( 15 , 17 ) singlet oxygen, ( 15 ) and alkyl-oxy radical ( 16 ) . Moreover, shikonin also inhibits the activities of enzymes that produce ROS such as Nox2 ( 16 , 18 , 19 ) and nitric oxide synthase ( 20 ) by mechanisms likely independent of ROS-scavenging effects.…”

“…Shikonin scavenges ROS such as O 2 •− , ( 14 – 16 ) hydroxyl radical, ( 15 , 17 ) singlet oxygen, ( 15 ) and alkyl-oxy radical ( 16 ) . Moreover, shikonin also inhibits the activities of enzymes that produce ROS such as Nox2 ( 16 , 18 , 19 ) and nitric oxide synthase ( 20 ) by mechanisms likely independent of ROS-scavenging effects. The inhibition of Nox2 by shikonin (IC 50 range, 0.4~2 µM) was reported to occur before activation of the enzyme, ( 16 , 18 , 19 ) a process that requires assembly of cytosolic and membrane components to form a complex with O 2 •− -generating activity.…”

“…Moreover, shikonin also inhibits the activities of enzymes that produce ROS such as Nox2 ( 16 , 18 , 19 ) and nitric oxide synthase ( 20 ) by mechanisms likely independent of ROS-scavenging effects. The inhibition of Nox2 by shikonin (IC 50 range, 0.4~2 µM) was reported to occur before activation of the enzyme, ( 16 , 18 , 19 ) a process that requires assembly of cytosolic and membrane components to form a complex with O 2 •− -generating activity. ( 21 ) Although the inhibition targets are believed to be processes and/or molecules relevant to enzyme activation, such targets remain unknown and might differ depending on the stimulant used for activation of Nox2.…”