Characterization, Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves

<i>Moringa oleifera</i> leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from <i>Moringa oleifera</i> leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in <i>Moringa oleifera</i> leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in <i>Moringa oleifera</i> leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from <i>Moringa oleifera</i> leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from <i>Moringa oleifera</i> leaves (MLPE) were verified to have strong neuroprotective activities against H₂O₂-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of <i>Moringa oleifera</i> leaves.