title: Preparative isolation and purification of indigo and indirubin from Folium isatidis by high-speed counter-current chromatography.
abstract: BACKGROUND
  Indigo and indirubin are the main active ingredients found in traditional Chinese herbal medicine Folium isatidis. An effective method for the isolation and purification of indigo and indirubin from Folium isatidis is needed. Compared with the conventional column chromatographic techniques, high-speed counter-current chromatography (HSCCC) is a suitable alternative for the enrichment and purification of these target compounds, and eliminates the complications resulting from a solid support matrix.
  
  
  OBJECTIVE
  To develop a reliable HSCCC method for isolation and identification of indigo and indirubin in a one-step separation from Folium isatidis.
  
  
  METHODS
  The optimum extracting conditions of indigo and indirubin from Folium isatidis were investigated by orthogonal test L(16) (4(5)). The target compounds were isolated and purified with a solvent system of n-hexane:ethyl acetate:ethanol:water (1:1:1:1, v/v) and the lower phase was used as the mobile phase in the head-to-tail elution mode. The purities of target compounds were tested by HPLC and their structures were identified by UV, IR, electrospray ion source (ESI)-MS, (1) H-NMR and (13) C-NMR analyses.
  
  
  RESULTS
  From 165 mg of the crude extract, 5.65 mg of indigo and 1.00 mg of indirubin were obtained by HPLC analysis with purities of 98.4% and 99.0% respectively, and their mean recoveries were 91.0% and 90.7%, respectively.
  
  
  CONCLUSIONS
  The HSCCC method is effective for the preparative separation and purification of indigo and indirubin in a one-step separation from Folium isatidis.
authors.affiliation: College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, 266109, People's Republic of China. htlu6@sina.com
authors.forename: Hai-tao
authors.initials: HT
authors.lastname: Lü
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Rui
authors.initials: R
authors.lastname: Deng
authors.forename: Ji-ying
authors.initials: JY
authors.lastname: Song
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004365:Drugs, Chinese Herbal / Q000737:chemistry*; D006586:Hexanes / Q000737:chemistry; D007203:Indigo Carmine; D007211:Indoles / Q000737:chemistry / Q000302:isolation & purification*; D031219:Isatis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D004365:Drugs, Chinese Herbal; D006586:Hexanes; D007211:Indoles; D012997:Solvents; C026385:n-hexane; D007203:Indigo Carmine; C027185:indirubin