Preparative Separation of Phenylpropanoid Glycoside from Scrophularia ningpoensis Hemsley by High Speed Countercurrent Chromatography and ESI-MSⁿ Analysis

Abstract

The characteristic constituent angoroside C of the famous traditional Chinese medicine Scrophularia ningpoensis hemsley was separated from the crude extract by high speed countercurrent chromatography using reverse phase elution combined with silica gel column chromatography. Meanwhile, harpagoside was also obtained using the normal phase elution method. A two phase solvent system containing n-butanol:acetic acid:ethyl acetate:water (8:1:1:10) was selected according to the partition coefficient of angoroside C. A 311 mg quantity of the crude extract was loaded onto a 250 mL HSCCC column and yielded 49 mg angoroside C at over 89.1% purity, and 14 mg harpagoside at over 96.5% purity. Angoroside C was further purified by silica gel column chromatography and the purity reached 98.5% with high recovery. The chemical structures were determined by ESI/MS ⁿ and ¹H-NMR. The characteristic ESI/MS ⁿ fragmentation pattern of angoroside C and the proposed fragmentation mechanism are proposed.

Keywords:

• Angoroside C
• Countercurrent chromatography
• ESI-MS ⁿ analysis
• Preparative chromatography
• Scrophularia ningpoensis Hemsley

ACKNOWLEDGMENT

This research was funded by “the priority among priorities” pharmaceutical program of Zhejiang province established in Zhejiang University of Technology (56311601015).

Notes

K: upper phase as the stationary phase.

1/K: lower phase as the stationary phase.