Analysis of alkannin derivatives from Alkanna species by high-performance liquid chromatography/photodiode array/mass spectrometry.

Alkannin, shikonin (A/S) and their derivatives are enantiomeric hydroxynaphthoquinone red pigments found in the roots of almost 150 species of the Boraginaceae family. A/S have been shown to exhibit strong wound healing, antimicrobial, anti-inflammatory and antioxidant activities and recent extensive research has well established their antitumor properties. A/S and their derivatives comprise the active ingredients of several pharmaceutical and cosmetic preparations. Although A/S have been efficiently synthesized and have been produced by cell tissue cultures in high yield, most of the pharmaceutical preparations worldwide contain A/S extracted from the roots of Boraginaceous species, found in nature. In the present study, a high-performance liquid chromatography/photodiode array/mass spectrometry (HPLC/PDA/MS) method was established to identify monomeric hydroxynaphthoquinones of the alkannin series and other metabolites from Boraginaceous root extracts. This method can be applied for the identification of alkannin derivatives and other metabolites from Boraginaceous cell cultures, and also to determine active ingredients in pharmaceutical preparations containing A/S derivatives. A phytochemical investigation of the Alkanna genus grown in Greece was also performed. Fifty-three root samples belonging to 10 species of the genus Alkanna (A. calliensis, A. corcyrensis, A. graeca, A. methanaea, A. orientalis, A. pindicola, A. primuliflora, A. sieberi, A. stribrnyi and A. tinctoria) were collected from several regions of the Greek flora and analyzed for their constituent hydroxynaphthoquinones and other metabolites. In most of the above Alkanna samples tested, the main hydroxynaphthoquinones were determined to be beta,beta-dimethylacrylalkannin, isovalerylalkannin + alpha-methyl-n-butylalkannin and acetylalkannin. The hydroxynaphthoquinone constituents and their proportions were found to vary among Alkanna species. Unknown metabolites (not monomeric hydroxynaphthoquinones) were detected by HPLC-PDA-MS, while in several Alkanna species hydroxynaphthoquinones were detected for the first time.