Separation and Purification of Ombuoside from Gynostemma Pentaphyllum by Microwave-Assisted Extraction Coupled with High-Speed Counter-current Chromatography

Abstract

A rapid and efficient method for the separation and purification of ombuoside from Gynostemma pentaphyllum by microwave-assisted extraction coupled with high-speed counter-current chromatography (HSCCC) was successfully developed. Using an orthogonal array design L₉ (3⁴), the extraction conditions, including microwave power, irradiation time, solid-to-liquid ratio and extraction times, were optimized. Ombuoside was isolated and purified from the crude extraction by HSCCC with two-phase solvent system composed of n-hexane:ethyl acetate:ethanol:water (5:6:5:5, v/v) in a single run. A 210 mg quantity of the crude extract containing 2.16% ombuoside was loaded, yielding 3.9 mg of ombuoside at 96.7% purity. The chemical structure of ombuoside was determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as UV, FT-IR, ESI-MS, ¹H NMR and ¹³C NMR spectra. The purified ombuoside had strong 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radical scavenging activities.