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

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• 作者：Yingnan Li^a ; Lianhong Yin^a ; Lingli Zheng^b ; Lina Xu^a ; Youwei Xu^a ; Yanyan Zhao^a ; Yan Qi^a ; Jihong Yao^a ; Xu Han^a ; Kexin Liu^a ; Jinyong Peng^a ; ^{jinyongpeng2005@163.com} ; ^{jinyongpeng2008@126.com}
• 关键词：Anticancer activity ; HSCCC ; Momordica charantia ; Protein separation and purification ; Reverse micelles
• 刊名：Journal of Chromatography B ; Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2012
• 期刊代码：124_15700232
• 类别：ch
• 出版时间：1 May, 2012
• 卷：895-896
• 期：Complete
• 页码：77-82
• 文件大小：887 K

摘要

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 50 mM bis-(2-ethylhexyl)-1-sulfosuccinate sodium (AOT). The mobile phase contained mobile phase A (50 mM Tris-HCl buffer containing 50 mM KCl at pH 7.0) for forward-extraction and mobile phase B (50 mM Tris-HCl buffer containing 0.5 M 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 掳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 = 0.116 mg/ml for 48 h treatment). This is the first report on the use of HSCCC to isolate proteins from M. charantia.