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To improve its safety profile, this study employed glycosylation for the structural modification of arenobufagin. Given the complexity of the reaction matrix, counter\u2010current 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\u00a0mg crude sample, 14.6\u00a0mg of arenobufagin\u20103\u2010O\u2010\u03b2\u2010D\u2010glucosyl (1\u21922)\u2010\u03b2\u2010D\u2010glucoside (Arenobufagin\u20102G), 80.3\u00a0mg of arenobufagin\u20103\u2010O\u2010\u03b2\u2010D\u2010glucoside, and 17.5\u00a0mg 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\u2010inflammatory efficacy. Mechanistically, Arenobufagin\u20102G suppressed inflammatory responses in Tohoku Hospital pediatrics\u20101\u2010derived macrophages and attenuated lipopolysaccharide\u2010induced nucleotide\u2010binding domain, leucine\u2010rich repeat, and pyrin domain\u2010containing 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\u2010based therapeutics.<\/jats:p>","DOI":"10.1002\/jssc.70544","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T17:45:08Z","timestamp":1790703908000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Counter\u2010Current Chromatography Separation of Arenobufagin Glycosylation Products With Improved Safety and Anti\u2010Inflammatory Activity"],"prefix":"10.1002","volume":"49","author":[{"given":"Feilong","family":"Zhao","sequence":"first","affiliation":[{"name":"Hubei Shizhen Laboratory College of Pharmacy Hubei University of Chinese Medicine  Wuhan P. R. 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China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiqi","family":"Wang","sequence":"additional","affiliation":[{"name":"Hubei Shizhen Laboratory College of Pharmacy Hubei University of Chinese Medicine  Wuhan P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Denglang","family":"Zou","sequence":"additional","affiliation":[{"name":"Hubei Shizhen Laboratory College of Pharmacy Hubei University of Chinese Medicine  Wuhan P. R. 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