title: Efficient purification of active bufadienolides by a class separation method based on hydrophilic solid-phase extraction and reversed-phase high performance liquid chromatography.
abstract: Traditional Chinese medicines (TCMs) have played a significant role in the process of discovering natural bioactive compounds, especially in anticancer therapeutics. However, the components of TCMs are complex mixtures with wide variation in polarity and content, which leads to inefficiency in the process of active compound discovery from TCMs. In this paper, the popular strategy of utilizing "pre-fractionated natural product libraries" has been improved by a new class separation approach to accelerate the process. As an example, the skin of Bufo bufo gargarizans Cantor, a well-known TCM, mainly contains two distinct bufadienolide classes: amino acid-conjugated bufadienolides (AACBs) and free form bufadienolides (AAUBs). We utilized hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE) to resolve the two types of bufadienolides, which co-eluted on C18 columns. By this strategy, twelve bufadienolides of the two types were purified via prep-HPLC from one active fraction, and eight of them were identified by (1)H NMR and (13)C NMR. These results indicated that the class separation method not only overcame the limited orthogonality in a 2D-RPLC×RPLC system but also accelerated the process of active compound discovery.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; School of Pharmaceutical Science and Technology, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: liuyanfang@dicp.ac.cn.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Aijin
authors.initials: A
authors.lastname: Shen
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Chaoran
authors.initials: C
authors.lastname: Wang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Jingyu
authors.initials: J
authors.lastname: Yan
authors.affiliation: School of Pharmaceutical Science and Technology, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
authors.forename: Weijie
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: liangxm@dicp.ac.cn.
authors.forename: Xinmiao
authors.initials: X
authors.lastname: Liang
mesh_terms: D000818:Animals; D002018:Bufanolides / Q000737:chemistry / Q000302:isolation & purification*; D002023:Bufo bufo; D056148:Chromatography, Reverse-Phase; D009682:Magnetic Resonance Spectroscopy; D008516:Medicine, Chinese Traditional; D012867:Skin / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D002018:Bufanolides
keywords: Active bufadienolides; Class separation; Hydrophilic solid-phase extraction; Purification; Toad skin