Quantitative Analysis of Shikonin Derivatives

Shikonin derivatives were extracted from both the cells and culture media. For obtaining dye fractions, a powdered sample of lyophilized cells was sonicated with n-hexane. The extraction was done for 15 min at 40°C until the red coloration had faced. The media samples were similarly extracted with n-hexane. The extracts were evaporated from the extract solution under reduced pressure. Dry residue was dissolved in methanol and analyzed in a DIONEX HPLC system (Sunnyvale, CA), equipped with an automated sample injector (ASI-100), and UVD 340S detector using the following conditions: gradient elution—acetonitrile (40–0 ml) + 0.04 M orthophosphoric acid (60–100 ml); flow rate, 1.5 ml min⁻¹; column, EC 250/4.6 Nucleosil 120–127 mm C18 (Macherey-Nagel, Düren, Germany), and monitoring eluent at 215, 278, 514, and 320 nm. Shikonin (Wako, Tokyo, Japan) and its two derivatives ACS and isobutyrylshikonin (IBS), isolated previously from natural roots of Lithospermum canescens (Pietrosiuk and Wiedenfeld 2005 (link)), were used as standards and analyzed under the same conditions. Peaks were assigned by spiking the samples with the standards and comparison of the retention times and UV spectra.