Targeted purification of glabridin and congeneric metabolites from Glycyrrhiza glabra L.

Abstract

Reported purification processes of glabridin (1) from the crude extract of European licorice (Glycyrrhiza glabra L.) generally involves a minimum of three liquid chromatography (LC) steps. Isolation methods using high speed counter current chromatography (HSCCC) have not been reported. The present study explores HSCCC as a powerful, targeted method for the two step isolation and purification of 1 as well as congeneric metabolites 4'-O-methoxyglabridin (2), 3'-hydroxyl-4'-O-methoxyglabridin (3). For targeting, the partition coefficient, K, of compounds 1 to 3 was calculated in different solvent systems (SSs) using UHPLC-UV through the shake-flask methodology. On the basis of the K value distribution of the screened metabolites (K(1):1.13, K(2):1.80, K(3):2.10), the SS composed of Hexanes/Ethyl Acetate/Methanol/Water 7:5:6:4, v/v, was used for the fractionation of G. glabra crude extract. A semi-preparative HPLC method was developed to provide an orthogonal approach for the purification of 1 from co-eluting metabolites, isoglabrone (4) and glabrocoumarin (5) from which the purity was determined by qHNMR. The results obtained highlight that countercurrent separation enables the targeted isolation of structurally related metabolites.