Isolation and purification of clemastanin B and indigoticoside A from Radix Isatidis by high-speed counter-current chromatography

Abstract

A preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:7:9, v/v/v) was successfully performed to isolate and separate clemastanin B and indigoticoside A from the plant of Radix Isatidis, a traditional Chinese medicine. A total of 59.2 mg clemastanin B and 66.1 mg indigoticoside A with purities of 94.6% and 99.0% determined by high performance liquid chromatography (HPLC) were obtained in one-step elution from 250 mg crude extract, which contained clemastanin B 24.8% and indigoticoside A 28.4%, and the recoveries of clemastanin B and indigoticoside A were 90.3% and 92.2%, respectively. The chemical structure was identified by IR, MS, 1H NMR and 13C NMR.

Fig. 1

The chemical structures of clemastanin B and indigoticoside A.

Fig. 2

HPLC chromatograms of the crude extract and the fractions obtained by HSCCC. Column: reversed-phase Lichrospher C₁₈ (6.0 mm × 150 mm i.d. 5 μm); mobile phase: CH₃CN–H₂O–HAC (25:75:1, v/v/v); flow rate: 1.0 ml/min; UV wavelength: 254 nm; (A) crude extract; (B) fraction “I” obtained by HSCCC; (C) fraction “II” obtained by HSCCC; peak 1: clemastanin B; peak 2: indigoticoside A.

Fig. 3

Chromatogram of the crude extract by preparative HSCCC. Solvent system: ethyl acetate–n-butanol–water (2:7:9, v/v/v); stationary phase: upper phase; mobile phase: lower phase; flow rate: 2.0 ml/min; revolution speed: 800 rpm; separation temperature: 25 °C; sample size: 250 mg; sample loop: 20 ml; detection wavelength: 254 nm; retention of the stationary phase: 38.6%.