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

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 6.1 mg of hellebrigenin‐3‐O‐ β ‐D‐glucoside ( 1 ), 2.2 mg of tigencaoside A ( 2 ), 8.3 mg of deglucohellebrin ( 3 ), 3.5 mg of 14 β‐hydroxy‐3β‐[β‐D‐glucopyranosyl‐(1→6)‐(β‐D‐glucopyranosyl)oxy]‐5α‐bufa‐20,22‐dienolide ( 4 ), and 3.0 mg of 14β‐hydroxy‐3β‐[(β‐D‐glucopyranosyl)oxy]‐5α‐bufa‐20,22‐dienolide ( 5 ), were effectively separated from 300 mg 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.