PREPARATIVE ISOLATION AND PURIFICATION OF THREE GLYCINE-CONJUGATED CHOLIC ACIDS FROM PULVIS FELLIS SUIS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY COUPLED WITH ELSD DETECTION

Abstract

Coupled with evaporative light scattering detection, a high-speed counter-current chromatography (HSCCC) method was developed for preparative isolation and purification of three glycine-conjugated cholic acids, glycochenodeoxycholic acid (GCDCA), glycohyodeoxycholic acid (GHDCA), and glycohyocholic acid (GHCA) from Pulvis Fellis Suis (Pig gallbladder bile) for the first time. The separation was performed with a two-phase solvent system consisted of chloroform-methanol-water-acetic acid (65:30:10:1.5, v/v/v/v) by eluting the lower phase in the head-to-tail elution mode. The revolution speed of the separation column, flow rate of the mobile phase, and separation temperature were 800 rpm, 2 mL/min and 25°C, respectively. In a single operation, 33 mg of GCDCA, 38 mg of GHDCA, and 23 mg of GHCA were obtained from 200 mg of crude extract with the purity of 95.65%, 96.72%, and 96.63%, respectively, in one-step separation. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three glycine-conjugated cholic acids were identified by ESI-MS, ¹H NMR, and ¹³C NMR.

Keywords:

• glycine-conjugated cholic acids
• high-speed countercurrent chromatography
• Pulvis Fellis Suis
• purification
• separation
• two-phase solvent system

ACKNOWLEDGMENT

This work was financially supported in part by the Project of Graduate Innovation and Creativity Funds of Northwest University of the People's Republic of China (09YZZ61) and the Project of Scientific Research Plan of Education Department of Shaanxi Provincial Government of the People's Republic of China (2010JS096).