A Strategy for Preparative Separation of 10 Lignans from Justicia procumbens L. by High-Speed Counter-Current Chromatography

Abstract

Ten compounds, including three lignan glycosides and seven lignans, were purified from Justicia procumbens L. in 8 h using an efficient strategy based on high-speed counter-current chromatography (HSCCC). The two-phase solvent system composed of petroleum-ethyl acetate-methanol-H₂O (1:0.7:1:0.7, v/v) was firstly employed to separate the crude extract (320 mg), from which 19.3 mg of justicidin B (f), 10.8 mg of justicidin A (g), 13.9 mg of 6'-hydroxyjusticidin C (h), 7.7 mg of justicidin E (i), 6.3 mg of lignan J₁ (j) were obtained with 91.3 mg of enriched mixture of compounds a-e. The enriched mixture (91.3 mg) was further separated using the solvent system consisting of petroleum-ethyl acetate-methanol-H₂O (3:3.8:3:3.8, v/v), yielding 12.1 mg of procumbenoside E (a); 7.6 mg of diphyllin-1-O-β-d-apiofuranoside (b); 7.4 mg of diphyllin (c); 8.3 mg of 6'-hydroxy justicidin B (d); and 7.9 mg of diphyllin acetyl apioside (e). The purities of the 10 components were all above 94%, and their structures were identified by NMR and ESI-MS spectra. The results demonstrated that the strategy based on HSCCC for the separation of lignans and their glycosides was efficient and rapid.

Keywords: Justicia procumbens L.; high-speed counter-current chromatography; lignans and their glycosides; preparative separation.

Figure 1

Chemical structures of 10 lignans from Justicia procumbens.

Figure 2

HPLC chromatograms of crude sample, enriched mixture (compounds a–e) and purified lignans (compounds a–j) from J. procumbens. Experimental conditions: RP-C₁₈ column (5 µm, 4.6 mm × 250 mm; Waters Technologies, USA); mobile phase: acetonitrile (A) 0.1% TFA water solution (B) (0–10 min, 30–45% A; 10–25 min, 45–60% A; 25–40 min, 60–100% A); RP-C₁₈ column temperature: 25 °C, UV monitor wavelength: 254 nm; flow-rate: 1 mL·min⁻¹; injection volume: 20 µL. a. procumbenoside E; b. diphyllin-1-O-β-d-apiofuranoside; c. diphyllin; d. 6′-hydroxy justicidin B; e. diphyllin acetyl apioside; f. justicidin B; g. justicidin A; h. 6′-hydroxyjusticidin C; i. justicidin E; j. lignan J₁.

Figure 3

HSCCC chromatograms of crude sample and enriched mixture (compounds a–e). Experimental conditions of crude sample (A): solvent system: Pet–EtOAc–MeOH–H₂O (1:0.7:1:0.7, v/v); sample size: 320 mg; retention of the organic phase: 62.5%. Conditions of the enriched mixture (B): solvent system: Pet–EtOAc–MeOH–H₂O (3:3.8:3:3.8, v/v); sample size: 91.3 mg. retention of the organic phase: 53.1%. In both separations: flow-rate: 3.0 mL∙min⁻¹; rotational speed: 810 rpm; UV monitor: 254 nm.