Optimisation of isolation of polyphenols from Malus pumila Mill. Leaves by high-speed countercurrent chromatography using response surface methodology

Considering comprehensive utilization of natural products, isolation and activity determination processes of bioactive compounds are essential. In this study, a combined high-speed countercurrent chromatography (HSCCC) with preparative HPLC method was developed to isolate the five antioxidant polyphenols from 75 % ethanol extract of Malus pumila Mill. leaves. The HSCCC conditions were optimized by response surface methodology (RSM) considering two response indexes including retention of stationary phase and analysis time. The optimal HSCCC conditions were flow rate of 2.11 mL/min, revolution speed of 717 rpm, and temperature of 25℃, with a solvent system of ethyl acetate/methanol/water (10:1:10, v/v/v). The unseparated fractions obtained from HSCCC were subjected to preparative HPLC for further isolation. As a result, phloridzin (15.3 mg), isoquercitrin (2.1 mg), quercetin-3-xyloside (1.9 mg), quercetin-3-arabinoside (4.0 mg), and quercetin-3-rhamnoside (2.0 mg) were isolated from 200.0 mg extracts. The purities of these compounds were all above 92 %. Their chemical structures were identified by mass spectrometer and nuclear magnetic resonance. The five isolated compounds were further investigated for their rat hippocampal neuroprotective effects against hydrogen peroxide-induced oxidative stress. No cytotoxicity was observed in all tested concentrations. While all five compounds except phloridzin showed significantly neurogenic activities and neuroprotective effects, especially at the concentration of 0.5 mg/L. These results demonstrate that RSM is a suitable technique for optimisation of HSCCC and the isolated polyphenols can be used as antioxidants in pharmaceutical and food products.