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Preparative isolation and purification of urolithins from the intestinal metabolites of pomegranate ellagitannins by high-speed counter-current chromatography

Zhao, Wenhua, Wang, Yuji, Hao, Weijia, Yang, Hua, Song, Xueying, Zhao, Ming, Peng, Shiqi
Journal of Chromatography B 2015 v.990 pp. 111-117
X-radiation, colorectal neoplasms, countercurrent chromatography, high performance liquid chromatography, human cell lines, hydrogen peroxide, malondialdehyde, metabolites, nuclear magnetic resonance spectroscopy, oxidative stress, pomegranates, solvents, superoxide dismutase
Urolithins were separated from the intestinal metabolites of pomegranate ellagitannins by high-speed counter current chromatography in two steps using two solvent systems composed of n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5, v/v/v/v/v) and n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:0. 8:0.25:5, v/v/v/v/v) for the first time. Each injection of 100mg extract yielded 21mg of pure urolithin A and 10mg of pure urolithin B. High-performance liquid chromatography analyses revealed that the purity of urolithin A and urolihtin B was over 98.5%. The structures of urolithin A and urolitihn B were identified by high resolution-MS, NMR and single crystal x-ray analysis. Urolithins reduced the oxidative stress status in colon cancer by decreasing the intracellular ROS and malondialdehyde levels, and increasing SOD activity in H2O2 treated Caco-2 cells.