Preparative separation of alkaloids from Picrasma quassioides (D. Don) Benn. by conventional and pH-zone-refining countercurrent chromatography

Abstract

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.

Figure 1

Conventional HSCCC chromatogram of separation crude extracts from Picrasma quassioides; Conditions: Two-phase solvent system: Pet–EtOAc–MeOH–H₂O (5:5:4.5:5.5, v/v/v/v); the retention of the stationary phase: 46%; flow rate: 2 mL/min; detection wavelength: 254 nm; revolution speed: 850 rpm; sample size: 200 mg.

Figure 2

Chemical structures of alkaloids from Picrasma quassioides.

Figure 3

pH-zone-refining CCC chromatogram of preparative separation of alkaloids compounds from Picrasma quassioides; Conditions: Two-phase solvent system: petroleum ether–ethyl acetate–n-butanol–water at volume ratios (3:2:7:9, v/v/v/v); the retention of the stationary phase: 34%; 10 mM TEA in stationary phase and 5 mM HCl in lower phase; flow rate: 2 mL/min; detection wavelength: 254 nm; revolution speed: 850 rpm; sample size: 2.0 g.

Figure 4

HPLC chromatograms: (1) Crude extracts from Picrasma quassioides; (2) compound a: 5-methoxycanthin-6-one; (3) compound b: 1-methoxy-β-carboline, 1-ethyl-4; (4) compound c: 8-dimethoxy-β-carboline; (5) compound d: 1-ethoxycarbonyl-β-carboline; (6) compound e: 1-vinyl-4,8-dimethoxy-β-carboline; (7) compound f: 1-vinyl-4-dimethoxy-β-carboline.