Counter‐Current Chromatography Separation of Arenobufagin Glycosylation Products With Improved Safety and Anti‐Inflammatory Activity

ABSTRACT Arenobufagin, a typical bufadienolide from toad venom, exhibits excellent anti‐inflammatory activity; however, its inherent toxicity severely limits its further application. To improve its safety profile, this study employed glycosylation for the structural modification of arenobufagin. Given the complexity of the reaction matrix, counter‐current chromatography (CCC) was selected for the efficient separation of the glycosylated products. First, response surface methodology was employed to optimize the glycosylation reaction conditions, which significantly enhanced the conversion rate. The glycosylation products were successfully separated using a solvent system composed of dichloromethane/methanol/water (8:4:8, v/v/v). From a 550 mg crude sample, 14.6 mg of arenobufagin‐3‐O‐β‐D‐glucosyl (1→2)‐β‐D‐glucoside (Arenobufagin‐2G), 80.3 mg of arenobufagin‐3‐O‐β‐D‐glucoside, and 17.5 mg of the unreacted arenobufagin substrate were obtained. Biological evaluation revealed that arenobufagin conjugated with two sugar moieties achieved a favorable balance between safety and anti‐inflammatory efficacy. Mechanistically, Arenobufagin‐2G suppressed inflammatory responses in Tohoku Hospital pediatrics‐1‐derived macrophages and attenuated lipopolysaccharide‐induced nucleotide‐binding domain, leucine‐rich repeat, and pyrin domain‐containing protein 3 inflammasome activation. This study not only demonstrates that CCC is a powerful strategy for separating complex glycosylation products but also lays the foundation for the future research and development of safe arenobufagin‐based therapeutics.