No abstract

pH-Zone-refining counter-current chromatography (pH-zone-refining CCC) was successfully applied for the large-scale separation of alkaloids from Corydalis bungeana. The crude extract was separated by a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (5:5:2:8, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as a displacer. As a result, 285 mg of protopine, 86 mg of corynoloxine, 430 mg of coryno1ine, and 115 mg of acetylcorynoline were obtained from 3.0 g of crude extract in a one-step separation. The purities of these compounds were 99.1%, 98.3%, 99.0% and 98.5%, respectively, as determined by HPLC. The chemical structures of these isolated compounds were confirmed by ESI-MS, 1H-NMR and 13C-NMR.

In this study, supercritical fluid extraction (SFE) was used to extract essential oil from Acorus tatarinowii Schott under the pressure of 25 MPa and temperature of 35°C. Two (E)- and (Z)-diastereomers of α-asarone and β-asarone were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.2:1:0.3, v/v). The separation yielded a total of 39 mg of α-asarone and 463 mg of β-asarone from 1.0 g of essential oil in one-step separation with the purity of 98.9 and 99.1%, respectively, as determined by HPLC. The chemical structures of these compounds were identified by EI-MS, (1)H-, and (13)C-NMR.

Two high-speed countercurrent chromatography (HSCCC) modes were compared by separation of major alkaloids from crude extract of Picrasma quassioides. The conventional HSCCC separation was performed with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (5:5:4.5:5.5, v/v/v/v) with 200 mg loading. pH-Zone-refining CCC was performed with two-phase solvent system composed of petroleum ether-ethyl acetate-n-butanol-water (3:2:7:9, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase and hydrochloric acid (5 mM) was added to the lower aqueous phase with 2 g loading. From 2 g of crude extract, 87 mg of 5-methoxycanthin-6-one (a), 38 mg of 1-methoxy-β-carboline (b), 134 mg of 1-ethyl-4,8-dimethoxy-β-carboline (c), 74 mg of 1-ethoxycarbonyl-β-carboline (d), 56 mg of 1-vinyl-4,8-dimethoxy-β-carboline (e) and 26 mg of 1-vinyl-4-dimethoxy-β-carboline (f) were obtained with purities of over 97.0%. The results indicated that pH-zone-refining CCC is an excellent separations tool at the multigram level.

Abstract:The aim of this paper was to seek an efficient method to preparative separation of alkaloid compounds from Aconitum coreanum (Guanbaifu), a well-known traditional Chinese medicinal plant for heart disease. Seven alkaloid compounds were successfully purified by pH-zone-refining counter-current chromatography with two-phase solvent system of petroleum ether-ethyl acetate-methanol-water (5:5:1:9, v/v/v/v), 10 mM triethylamine in upper phase and 10 mM hydrochloric acid in lower phase. From 3.5 g of crude extract, 356 mg of Guanfu base I, 578 mg of Guanfu base A, 74 mg of atisine, 94 mg of Guanfu base F, 423 mg of Guanfu base G, 67 mg of Guanfu base R and 154 mg of Guanfu base P were obtained with the purity of 96.40%, 97.2%, 97.5%, 98.1%, 98.9%, 98.3% and 98.4%. Their chemical structures were identified by TOF-MS and 1 H-NMR. This study indicated that pH-zone-refining counter-current chromatography was an efficient method for separating the kind of alkaloids with low absorbance values.