Isolation and purification of acetylshikonin andb-acetoxyisovalerylshikonin from cell suspensioncultures of Arnebia euchroma (Royle) Johnstonusing rapid preparative HPLC

Shikonin and its derivatives are important red colored naphthoquinone pigments found in a large number of Arnebia species, including A. euchroma, that are responsible for the various pharmacological activities exhibited by the plant. The precise separation of each naphthoquinone is essential for total quality evaluation and bioactivity analysis of herbal formulations of A. euchroma. Furthermore, the overexploitation of this useful plant has resulted in species becoming endangered. With this in mind, a simple and rapid preparative scale HPLC method with single compound recovery for the isolation and purification of two shikonin derivatives (i. e. acetylshikonin, b-acetoxyisovalerylshikonin) from cell suspension cultures of A. euchroma is presented. The compounds were separated on a C18 column within 10 min using acetonitrile/ methanol (95:5) as mobile phase in isocratic mode. The isolated compounds were found to be more than 98% pure. The LOD for acetylshikonin and bacetoxyisovalerylshikonin was estimated at 0.063 and 0.146 lg/mL, respectively, while the LOQ was found to be 0.209 and 0.487 lg/mL, respectively. The recoveries accomplished for both the shikonin derivatives were in the range of 94.7–96.8%. The repeatability, expressed as %RSD, of acetylshikonin and b-acetoxyisovalerylshikonin was found to be 1.74 and 1.27, respectively.