Main content area

Application of high-speed counter-current chromatography coupled with a reverse micelle solvent system to separate three proteins from Momordica charantia

Li, Yingnan, Yin, Lianhong, Zheng, Lingli, Xu, Lina, Xu, Youwei, Zhao, Yanyan, Qi, Yan, Yao, Jihong, Han, Xu, Liu, Kexin, Peng, Jinyong
Journal of chromatography 2012 v.895-896 pp. 77-82
Momordica charantia, antineoplastic agents, countercurrent chromatography, databases, humans, in vitro studies, iso-octanes, micelles, pH, polyacrylamide gel electrophoresis, potassium chloride, proteins, sodium dodecyl sulfate, solvents, stomach neoplasms, temperature, wavelengths
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 1.5ml/min, 280nm 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 (IC₅₀=0.116mg/ml for 48h treatment). This is the first report on the use of HSCCC to isolate proteins from M. charantia.