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

•We firstly used HSCCC to get urolithins from intestinal metabolites of pomegranate ellagitannins.•We studied the metabolites of the herb extract with high resolution-MS, NMR and Single crystal x-ray.•Urolithins reduced the oxidative stress status in colon cancers.•The combination of urolithins might be beneficial to human health.

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 100 mg extract yielded 21 mg of pure urolithin A and 10 mg 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.