title: Application of high-speed counter-current chromatography coupled with a reverse micelle solvent system to separate three proteins from Momordica charantia.
abstract: High-speed counter-current chromatography (HSCCC) coupled with a reverse micelle solvent system was successfully developed to separate three proteins from Momordica charantia. Suitable HSCCC conditions were carefully optimized as follows: the stationary phase was a reverse micellar phase composed of isooctane and 50mM bis-(2-ethylhexyl)-1-sulfosuccinate sodium (AOT). The mobile phase contained mobile phase A (50mM Tris-HCl buffer containing 50mM KCl at pH 7.0) for forward-extraction and mobile phase B (50mM Tris-HCl buffer containing 0.5M KCl at pH 10.0) for back-extraction. The flow rate, detection wavelength and column temperature were set at 1.5 ml/min, 280 nm and 4 °C, respectively. Under these conditions, three fractions (I, II and III) were separated, which showed high purity when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The structures of these proteins were then identified by MALDI-TOF/TOF-MS/MS and compared with the NCBInr database. Fractions I and III were identified as resistance-like protein P-B and pentatricopeptide repeat-containing protein, respectively, which were found in M. charantia for the first time. However, fraction II, which is thought to be a new protein, was not identified, and further investigations on this fraction are required. The anticancer activities of these three proteins on the human gastric cancer cell line SGC-7901 were evaluated in vitro. The results indicated that fraction II has excellent anticancer activity (IC(50)=0.116 mg/ml for 48 h treatment). This is the first report on the use of HSCCC to isolate proteins from M. charantia.
authors.affiliation: College of Pharmacy, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China.
authors.forename: Yingnan
authors.initials: Y
authors.lastname: Li
authors.forename: Lianhong
authors.initials: L
authors.lastname: Yin
authors.forename: Lingli
authors.initials: L
authors.lastname: Zheng
authors.forename: Lina
authors.initials: L
authors.lastname: Xu
authors.forename: Youwei
authors.initials: Y
authors.lastname: Xu
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yan
authors.initials: Y
authors.lastname: Qi
authors.forename: Jihong
authors.initials: J
authors.lastname: Yao
authors.forename: Xu
authors.initials: X
authors.lastname: Han
authors.forename: Kexin
authors.initials: K
authors.lastname: Liu
authors.forename: Jinyong
authors.initials: J
authors.lastname: Peng
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D003377:Countercurrent Distribution / Q000379:methods*; D006801:Humans; D008823:Micelles; D029777:Momordica charantia / Q000737:chemistry*; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology; D010940:Plant Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D012997:Solvents / Q000737:chemistry
chemical_list: D008823:Micelles; D010936:Plant Extracts; D010940:Plant Proteins; D012997:Solvents