Production of Cytoprotective, Antioxidant, and Anti-Inflammatory Shikonin Derivatives in Root Cultures of Plagiobothrys arizonicus: A Pilot Study

The naphthazarins shikonin and alkannan are strongly chromogenic, dark red enantiomers, each of which has biological activity, that are found primarily in the plant family Boraginaceae. These compounds and their many chemical metabolites, derivatives, oligomers, and analogs (“shikonoids”) are an important group of phytochemicals, utilized since antiquity as components of dyes, traditional medicines, and food and cosmetics. They are now recognized for their potent anti-inflammatory and regulatory activity on a variety of molecular signaling pathways in humans. Since many Boraginaceae species are overly exploited or endangered, we developed a pilot-scale in vitro shikonoid production system using Plagiobothrys arizonicus (Gray) Greene ex A.Gray, the Arizona popcorn flower, native to the southwestern USA and the Sonoran floristic province in the Madrean region of Mexico. Aseptic root cultures were initiated from fresh leaf tissue and stimulated to continuously produce shikonoids in liquid shake cultures layered under paraffin oil from which the shikonoids were extracted and concentrated. The crude, red extracellular product from these rapidly expanding root masses was also fractionated by Centrifugal Counter-Current Chromatography (CCC) into its component shikonin derivatives. A number of these shikonoids profoundly up-regulated detoxification and antioxidant proteins (phase 2 enzymes) and inhibited inflammation in mammalian cell bioassay systems. This prototype shikonoid production methodology can be readily scaled to either batch or chemostat culture.