An efficient two-step approach for the preparative separation and purification of eight polyphenols from Hibiscus manihot L. flower with high-speed countercurrent chromatography

In the present study, a green and efficient ultrasonic-assisted extraction method of polyphenols from Hibiscus manihot L. flowers (HMLF) was performed, and the corresponding experimental parameters were optimized by Taguchi OA Design. Meanwhile, a two-step high-speed counter-current chromatography coupled with macroporous resin was successfully established with the optimal two-phase solvent systems comprising EtOAc-MeOH-H2O (100:3:100, v/v/v) and Hex-EtOAc-MeOH-H2O (1:5:1:5, v/v/v/v) to isolate and prepare eight polyphenols with purities exceeding 95%, including two phenolic acids and six flavonoids, respectively. The yield of the isolated polyphenols was determined to be 50.4 mg from the enriched extract in a single run. Moreover, the interactions of the eight isolated compounds with AChE and BuChE were explicitly evaluated and described by molecular docking. This study confirms that the proposed method is a cost-effective and powerful strategy for the scaled-up extraction and preparative separation of the high-purity polyphenols from HMLF, and provides an opportunity for automation and systematic preparation of natural products used in the food and pharmaceutical industries.