title: An efficient and target-oriented sample enrichment method for preparative separation of minor alkaloids by pH-zone-refining counter-current chromatography.
abstract: An efficient and target-oriented sample enrichment method was established to increase the content of the minor alkaloids in crude extract by using the corresponding two-phase solvent system applied in pH-zone-refining counter-current chromatography. The enrichment and separation of seven minor indole alkaloids from Uncaria rhynchophylla (Miq.) Miq. ex Havil(UR) were selected as an example to show the advantage of this method. An optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v) was used in this study, where triethylamine (TEA) as the retainer and hydrochloric acid (HCl) as the eluter were added at the equimolar of 10mM. Crude alkaloids of UR dissolved in the corresponding upper phase (containing 10mM TEA) were extracted twice with lower phase (containing 10mM TEA) and lower phase (containing 10mM HCl), respectively, the second lower phase extract was subjected to pH-zone-refining CCC separation after alkalization and desalination. Finally, from 10g of crude alkaloids, 4g of refined alkaloids was obtained and the total content of seven target indole alkaloids was increased from 4.64% to 15.78%. Seven indole alkaloids, including 54mg isocorynoxeine, 21mg corynoxeine, 46mg isorhynchophylline, 35mg rhynchophylline, 65mg hirsutine, 51mg hirsuteine and 27mg geissoschizine methylether were all simultaneously separated from 2.5g of refined alkaloids, with the purity of 86.4%, 97.5%, 90.3%, 92.1%, 98.5%, 92.3%, and 92.8%, respectively. The total content and purities of the seven minor indole alkaloids were tested by HPLC and their chemical structures were elucidated by ESI-HRMS and (1)H NMR.
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Rui-Hong
authors.initials: RH
authors.lastname: Feng
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Jin-Jun
authors.initials: JJ
authors.lastname: Hou
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Yi-Bei
authors.initials: YB
authors.lastname: Zhang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Hui-Qin
authors.initials: HQ
authors.lastname: Pan
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Wenzhi
authors.initials: W
authors.lastname: Yang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Qi
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Yao
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Lu-Ying
authors.initials: LY
authors.lastname: Cai
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Bao-Hong
authors.initials: BH
authors.lastname: Jiang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China. Electronic address: wanyingwu@simm.ac.cn.
authors.forename: Wan-Ying
authors.initials: WY
authors.lastname: Wu
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China. Electronic address: daguo@simm.ac.cn.
authors.forename: De-An
authors.initials: DA
authors.lastname: Guo
mesh_terms: D000085:Acetates; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003377:Countercurrent Distribution / Q000379:methods; D006586:Hexanes; D006863:Hydrogen-Ion Concentration; D026121:Indole Alkaloids / Q000302:isolation & purification*; D000432:Methanol; D010936:Plant Extracts / Q000737:chemistry; D012997:Solvents; D032070:Uncaria / Q000737:chemistry
chemical_list: D000085:Acetates; D006586:Hexanes; D026121:Indole Alkaloids; D010936:Plant Extracts; D012997:Solvents; C026385:n-hexane; C007650:ethyl acetate; D000432:Methanol
keywords: Indole alkaloids; Target-oriented sample enrichment method; Uncaria rhynchophylla; pH-zone-refining counter-current chromatography