Integrated Approach for Separation of 2-O-β-D-glucopyranosyl-L-ascorbic Acid From Lycium barbarum L. Fruit Using High-Speed Countercurrent Chromatography Coupled With Aqueous Two-Phase System.

2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), a natural ascorbic acid derivative discovered in Lycium barbarum L., has been demonstrated to exhibit multiple biological activities. This study developed an integrated approach combining high-speed counter-current chromatography (HSCCC) and an aqueous two-phase system (ATPS) for the preparative isolation of AA-2βG from L. barbarum fruit. The solution system of ammonium sulfate/ethanol/water/acetonitrile (19.5:28.9:53.3:2.0, w/w) was established through systematic screening processes. To address the multi-parameter optimization challenges in HSCCC, this study employed the entropy weight method to calculate the information entropy and weight coefficients of five critical parameters, thereby establishing a comprehensive evaluation methodology for HSCCC separation efficiency. Subsequently, the response surface methodology was utilized to optimize HSCCC operational conditions. Under the optimized conditions, 2.66 mg of AA-2βG with a purity of 91.24% was successfully isolated from a 140 mg extract. Building on the optimized HSCCC conditions, an overlapping injection mode was implemented based on the conventional single-injection mode. Through cumulative purification of 700 mg extract, 10.65 mg of AA-2βG with an 84.53% purity was successfully obtained. Compared with the single-injection mode, this mode reduced separation time by 55.68% while decreasing solvent consumption by 36.22%. Finally, the antioxidant activity of AA-2βG was assessed through in vitro antioxidant activity experiments. These results demonstrate the synergistic potential of the HSCCC-ATPS integration as an efficient strategy for bioactive compound purification. The established protocol provides a robust method for AA-2βG preparation from Lycium barbarum L. fruit. Furthermore, it demonstrates the broader applicability of this hybrid technique in isolating bioactive constituents from complex biological matrices.