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

Sheng, Feng, Wang, Yuning, Zhao, Xingchen, Tian, Na, Hu, Huali, Li, Pengxia
Journal of agricultural and food chemistry 2014 v.62 no.28 pp. 6813-6819
acetonitrile, binding properties, circular dichroism spectroscopy, countercurrent chromatography, cyanidin, fluorescence, high performance liquid chromatography, human serum albumin, keracyanin, mulberries, petunidin, solvents, van der Waals forces
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-α-rhamnopyranosyl-β-galactopyranoside) (also known as keracyanin) is the major component present in mulberry (41.3%). Other isolated pigments are cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) and petunidin 3-O-β-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 α-helical structure occur in the presence of keracyanin.