Isolation, identification and pharmacokinetic analysis of fructosyl puerarins from enzymatic glycosylation.

A method of using high-speed counter-current chromatography (HSCCC) was established for preparative isolation and purification of puerarin glycosides from the crude sample after enzymatic glycosylation of puerarin. Four fructosyl puerarins were successfully purified for the first time by HSCCC with a two-phase-solvent system composed of n-butanol-acetic acid-water (4:1.5:6, v/v/v). A total of 5mg of puerarin (1), 20mg of β-d-fructofuranosyl-(2→6)-puerarin (2), 41mg of β-d-difructofuranosyl-(2→6)-puerarin (3), 18mg of β-d-trifructofuranosyl-(2→6)-puerarin (4) and 15mg β-d-tetrafructofuranosyl-(2→6)-puerarin (5) were obtained in one-step separation from 100mg of the crude sample with purities of 98.5%, 98.3%, 98.9%, 97.8%, 97.5% and 97.2%, respectively. Among them, compounds 2-5 are novel compounds, and their chemical structures were identified by HRMS, (1)H NMR, (13)C NMR and 2D NMR. Pharmacokinetic analysis showed that β-d-fructofuranosyl-(2→6)-puerarin (2) was able to maintain higher plasma concentrations and have a longer mean residence time in the blood than puerarin.