[High performance liquid chromatographic method for determination of active components in lithospermum oil and its application to process optimization of lithospermum oil prepared by supercritical fluid extraction]

Lithospermum erythrorhizon has the functions of cooling blood, activating blood, as well as detoxifying and penetrating rash. Lithospermum oil extracted from Lithospermum erythrorhizon can prevent and treat diaper rash, skin ulceration, eczema, and other skin diseases. Supercritical fluid extraction is the optimal method for the extraction of active components from lithospermum. In this study, an analytical method was established for simultaneously determination of six active components in lithospermum oil with high performance liquid chromatography (HPLC), and the contents of the active components as the evaluation index were used to investigate several important factors in the preparation of lithospermum oil by supercritical fluid extraction. The optimized HPLC conditions were as follows: separation column, Diamonsil C18 (250 mm×4.6 mm, 5 μm); mobile phases, acetonitrile containing 0.1% (v/v) formic acid-0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium formate (75∶25, v/v); flow rate, 1 mL/min; injection volume, 15 μL; room temperature; photodiode array detector (PAD); detection wavelength, 275 nm. The supercritical fluid extraction was optimized for ensuring stability of the amounts of effective components and the reliability of the quality of lithospermum oil. This will serve as the basis for preparation and quality control processes. Three factors and three levels orthogonal tests were adopted to investigate the important factors, viz. the pressure, temperature and CO2 flow rate in the preparation of lithospermum oil. The results showed that the developed HPLC-PAD method can be used for the simultaneous determination of shikonin, acetylshikonin, β-acetoxyisovaleryl akanin, isobutyryl shikonin, β,β-dimethylacryl shikonin, and 2-methylbutyryl shikonin in 30 min. The method has good precision, accuracy and repeatability. The contents of the active components were the highest when the extraction pressure, extraction temperature, and CO2 flow rate were 23 MPa, 40 ℃, and 27 L/h, respectively. The optimized conditions are suitable for the preparation and actual production of lithospermum oil. The HPLC-PAD method is simple, feasible, accurate, and reliable. It can be used for the preparation and quality control of lithospermum oil by supercritical fluid extraction. Thus, with this method, the stability of the contents of active ingredients and the reliability of the quality of lithospermum oil can be ensured; moreover, safe and effective drug use can be realized. The established method has obvious advantages over the traditional process and is a good candidate for widespread use.