Separation and Identification of Anthocyanin Extracted from Mulberry Fruit and the Pigment Binding Properties toward Human Serum Albumin

Purple pigments were isolated from mulberry extracts using preparative high-speed countercurrent chromatography (HSCCC) and identified by ESI-MS/MS and high performance liquid chromatography (HPLC) techniques. The solvent system containing methyl tert-butyl ether, 1-butanol, acetonitrile, water, and trifluoroacetic acid (10:30:10:50:0.05; %, v/v) was developed in order to separate anthocyanins with different polarities. Cyanidin 3-O-(6 ''-O-alpha-rhamnopyranosyl-beta-galactopyranoside) (also known as keracyanin) is the major component present in mulberry (41.3%). Other isolated pigments are cyanidin 3-O-(6 ''-O-alpha-rhamnopyranosyl-beta-glucopyranoside) and petunidin 3-O-beta-glucopyranoside. The binding characteristics of keracyanin with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopy. Spectroscopic analysis reveals that HSA fluorescence quenched by keracyanin follows a static mode. Binding of keracyanin to HSA mainly depends on van der Waals force or H-bonds with average binding distance of 2.82 nm. The results from synchronous fluorescence, three-dimensional fluorescence, and CD spectra show that adaptive structure rearrangement and decrease of alpha-helical structure occur in the presence of keracyanin.