Isolation and purification of oridonin from the whole plant of Isodon rubescens by high-speed counter-current chromatography

Abstract

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of oridonin from Isodon rubescens by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (2.8:5:2.8:5, v/v/v/v). The targeted compound isolated, collected and purified by HSCCC was analyzed by high performance liquid chromatography (HPLC). A total of 40.6 mg of oridonin with the purity of 73.5% was obtained in less than 100 min from 100 mg of crude Isodon rubescens extract. The chemical structure of the compound was identified by IR, 1H-NMR and 13C-NMR.

Figure 1

The structure of oridonin.

Figure 2

HPLC chromatograms of the crude extract from the whole plant of Isodon rubescens and the two targeted compounds purified by HSCCC. Conditions: column: reversed phase Agilent ODS C18 column (250 mm × 4.6 mm I.D., 5 μm); mobile phase: methanol-water formic acid (0.5%) in gradient mode as follows: 0–10 min, 20–40% methanol; 10–20 min, 40–60% methanol; 20–35 min, 60–70% methanol.; flow-rate: 0.8 mL/min; detection wavelength: 238 nm. (A) The crude extract from the whole plant of Isodon rubescens; (B) the shaded area of fraction I separated by HSCCC (Figure 3); (C) the pure compound oridonin after PTLC purification.

Figure 3

HSCCC chromatogram of the crude extract from the whole plant of Isodon rubescens. Two-phase-solvent system: n-hexane-ethyl acetate-methanol-water (2.8:5:2.8:5, v/v/v/v); mobile phase: the lower phase; flow-rate: 2 mL/min; revolution speed: 850 rpm; detection wavelength: 254 nm; sample size: 100 mg of crude sample dissolved in 5 mL of the lower phase; separation temperature: 25 °C; retention of the stationary phase: 61%. I, II: collected fractions.