Large-scale isolation of high-purity anthocyanin monomers from mulberry fruits by combined chromatographic techniques.

Anthocyanins have attracted attention over the past several decades because of their beneficial health effects. In this research, a strategy combining column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity anthocyanin monomers from mulberry fruits. After purification using Amberlite XAD-7HP column with 80% ethanol (0.1% HCl), a fraction of anthocyanins mixtures with a purity of 68.6% was obtained. High-speed countercurrent chromatography with a biphasic solvent system of n-butanol/methyl tert-butyl ether/acetonitrile/water/trifluoroacetic acid (30:10:10:50:0.05, v/v) was used to separate the anthocyanin monomers. Three monomers of delphinidin-3-O-rutinoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-glucoside were obtained, and identified by 1 H and 13 C NMR spectroscopy and high-performance liquid chromatography with electrospray ionization-mass spectrometry. The method developed in this work can be used to conduct large-scale separations of anthocyanin monomers from mulberry fruits and other plants.