Isolation of High-Purity Betanin from Red Beet and Elucidation of Its Antioxidant Activity against Peroxynitrite: An in vitro Study

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO^-) is the most toxic molecular species among RNS. Betalains are known to possess ONOO^- scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO^- reaction, ONOO^--dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H₂O₂ and ONOO^- cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO^- toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.