Analysis of shikonins by chiral-HPLC and development of easy method for preparing (R)-shikonin and (S)-shikonin using characteristic of the RS mixture in the shikonins from "Koshikon" (the roots of Lithospermum erythrorhizon) and "Nanshikon" (the roots of Arnebia euchroma)

Abstract

"Koshikon" (the roots of Lithospermum erythrorhizon) and "Nanshikon" (the roots of Arnebia euchroma) have been used as natural dye and medicine. Interestingly, shikonins, the components of those species, exist as mixture of enantiomers. In this report, we isolated six shikonins from "Koshikon", and five shikonins from "Nanshikon", and determined the enantiomer ratios of them by chiral-HPLC analysis. From the "Koshikon", acetylshikonin and β-hydroxyisovalerylshikonin were obtained with the low R/S ratio (74/26 and 90/10), while isobutyrylshikonin, β,β-dimethylacrylshikonin, α-methyl-n-butyrylshikonin and isovalerylshikonin were obtained as almost pure R-form (>95%). On the other hand, the "Nanshikon" gave acetylshikonin with the low R/S ratio (34/64), and isobutyrylshikonin, β,β-dimethylacrylshikonin, α-methyl-n-butyrylshikonin and isovalerylshikonin as almost pure S-form (just about 100%). Generally, enantiopure shikonins have been obtained by hydrolysis of shikonin derivatives followed by several recrystallizations. We established an easy and efficient method for preparing enantiomerically pure (R)-shikonin and (S)-shikonin by removing low-enanntiopure acetylshikonin and β-hydroxyisovalerylshikonin from shikonins before the hydrolysis.