Development of an effective method based upon second-order overlapping repeated sample injections for isolation of carotenoids from Lycium barbarum L. fruits with elution-extrusion counter-current chromatography.

Carotenoids are one of the main active components in Lycium barbarum L. fruit, which has a wide range of excellent biological activities. In this study, a novel second-order overlapping repeated injection method with elution-extrusion counter-current chromatography was developed for isolation and preparation of carotenoids from L. barbarum fruits. And three carotenoids were successfully separated using the solvent system composed of n-hexane/dichloromethane/acetonitrile (10:3.5:6.5, v/v) with the injection before equilibrium method. The entire separation process consisted of three complete elution-extrusion cycles with a total of 9 injections (80 mg crude extract per injection). Finally, three target compounds including zeaxanthin (28.5 mg), zeaxanthin monopalmitate (45.8 mg), and zeaxanthin dipalmitate (161.5 mg) with average purities of 87.9%, 88.9%, and 91.2% were successfully obtained in one complete second-order overlapping repeated elution-extrusion CCC process within 651 min. The result indicated that this second-order overlapping repeated method is efficient for large-scale preparation of carotenoids based on its advantages of large amount of sample injection and low solvent consumption. So this novel second-order overlapping repeated elution-extrusion counter-current chromatography separation method has enormous potential for largely preparative separation of natural bioactive compounds, such as carotenoids, which have good biological activity but possess unstable or other special chemical structure. It is worth noting that this overlapping repeated injections method requires target compounds to meet the requirements of elution-extrusion counter-current chromatography, and the normal implementation of this method is closely related to the sufficient interval of elution time between the target compounds.