Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

Abstract

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two-phase solvent systems composed of CHCl(3)-MeOH-(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl(3)-MeOH-0.2 M HCl (4:2:2, v/v) and CHCl(3)-MeOH-0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12-hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94-99% as determined by HPLC. Their chemical structures were characterized on the basis of (1)H-NMR, (13)C-NMR, and LC-ESI-Q-TOF-MS/MS analyses.

Figure 1

Chemical structures of alkaloids from C. bungeana Turcz.

Figure 2

Effects of the volume ratios of MeOH (A, B, C) and the concentrations of dilute HCl (D, E, F) on the K values of the target alkaloids.

Figure 3

Chromatograms of the separation of crude alkaloid extracts from C. bungeana Turcz. by preparative HSCCC using solvent systems of CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v/v) with stepwise elution. Experimental conditions: Stationary phase: upper phase of CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v/v); mobile phase: lower organic phase of the two-solvent systems; flow rate: 2 mL/min (A), 4.0 mL/min (B); revolution speed: 800 rpm; temperature of column oven: 26°C; sample size: 200 mg dissolved in equal volume of lower and upper phase (each 2 mL); retention of the stationary phase: 80% (flow rate: 4.0 mL/min).

Figure 4

The results of HPLC analyses of crude alkaloid extracts and HSCCC fractions from C. bungeana Turcz. (A) crude alkaloids; (B) peak 1; (C) peak 2; (D) peak 3; (E) peak 4; (F) peak 5; (G) peak 6; (H) peak 7 of the preparative HSCCC separation shown in Fig. 3; column: Ultimate RP C18 column (250 mm × 4.6 mm i.d., 5 µm, Welch Materials); mobile phase: solvent A: methanol containing 0.05% TEA; solvent B: water; gradient: 0.0 min (60% A)–25 min (100% A)–35 min (100% A); flow rate: 1.0 mL/min; UV wavelength: 289 nm.

Figure 5

Positive ESI-Q-TOF-MS/MS analyses of purified alkaloids from C. bungeana Turcz.