Separation of bufadienolides from Helleborus thibetanus Franch. by a combination approach involving macroporous resin column chromatography and gradient countercurrent chromatography.

Abstract

In this study, a combination approach involving macroporous resin (MR) column chromatography and gradient countercurrent chromatography (CCC) was employed to enrich and purify bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch. Initially, a D101 MR-packed column chromatography was utilized for fractionation and enrichment of the bufadienolides, which were effectively eluted from the column using a 60% ethanol solution. CCC was subsequently introduced to separate the enriched product using the ethyl acetate/n-butanol/water (EBuWat, 4:1:5, v/v) and EBuWat (5:0:5, v/v) solvent systems in a gradient elution mode. As results, five bufadienolides, including 6.1mg of hellebrigenin-3-O-β-D-glucoside (1), 2.2mg of tigencaoside A (2), 8.3mg of deglucohellebrin (3), 3.5mg of 14 β-hydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (4), and 3.0mg of 14β-hydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (5), were effectively separated from 300mg of the enriched product. The respective high-performance liquid chromatography purities were as follows: 95.2%, 75.8%, 85.7%, 82.3%, and 92.8%. This study provides valuable insights for the efficient enrichment and separation of bufadienolides from Helleborus thibetanus Franch.