Bioassay-Guide Preparative Separation of Hypoglycemic Components from Gynura divaricata (L.) DC by Conventional and pH-Zone Refining Countercurrent Chromatography

<i>Gynura divaricata</i> (L.) DC is a long-used medicinal and edible plant in China folk. Its hyperglycemic effects have garnered increasing public attention in recent years. This study revealed that the ethyl acetate (EtOAc) and butanol (BuOH) partition fractions of <i>G. divaricata</i> crude extract exhibited significantly higher α-glucosidase inhibition activity and enhanced glucose uptake ability compared to other fractions. Guided by the hypoglycemic bioassay, these two fractions were subjected to isolation of active compounds using high-speed countercurrent chromatography (HSCCC). A two-phase solvent system composed of hexane-methyl tert-butyl ether (MtBE)-methanol-0.1% TFA water was employed for the separation of the EtOAc fraction by conventional HSCCC through a gradient elution strategy. Five major compounds were obtained and identified as chlorogenic acid (1), 3,4-dicaffeoylquinic acid (2), 3,5-dicaffeoylquinic acid (3), 4,5-dicaffeoylquinic acid (4), and kaempferol-3-O-β-D-glucopyranoside (5) by ESI-MS, ¹HNMR, and ¹³CNMR. The chlorogenic acid and the three dicaffeoylquinic acids were found to display higher inhibitory activities against α-glucosidase compared to the flavonoid. Considering their acidic nature, pH-zone-refining CCC (PHZCCC) was then applied for further scale-up separation using a solvent system MtBE: n-butanol: acetonitrile: water with trifluoroacetic acid (TFA) as a retainer and ammonium hydroxide (NH₄OH) as an eluter. A significantly higher yield of chlorogenic acid was obtained from the BuOH fraction by PZRCCC. Molecular docking between the caffeoylquinic acids and α-glucosidase confirmed their hypoglycemic activities. This study demonstrates that CCC is a powerful tool for preparative separation of active constituents in natural products. This research presents a novel and effective method for the preparative isolation of hypoglycemic compounds from <i>Gynura divaricata.</i>