Separation and structural analysis of lignan glycoside isolated from Vaccinium myrtillus L. rhizomes

Lignans are a group of important plant polyphenols, occurring widely in vascular plants. The procedure of extraction and further analysis of these compounds is sometimes complicated due to the risk of contamination, losses in yield and unexpected chemical reactions. Therefore, we applied the method of the droplet counter-current chromatography, DCCC, to separate the lignan from ethanol extract of V. myrtillus rhizomes in only one chromatographic step. As a liquid-liquid partition technique without solid stationary phase, it operates under gentle conditions allowing non-destructive isolation of labile compounds. 30% aqueous EtOH extract was obtained from air-dried and crushed V. myrtillus rhizomes after preliminary extraction with ethyl ether, and separated by DCCC equipped with 95 tubes (400×2mm), operating with the solvent system CHCl₃:MeOH:H₂O (43:37:20, v/v/v) in the descending mode. After injection of 1g of the extract dissolved in 20ml of the solvent system, 370 fractions of 7ml each were collected during 40 hrs. Collected fractions were analysed by TLC (silica gel, CHCl₃:MeOH:H₂O 61:32:7, v/v/v). Fractions 15–31 contained a pure compound crystallizing after concentration. The analysis of HR-ESI-MS, 1D and 2D NMR spectra and the measurement of the optical rotation allowed to identify the purified compound as lyoniside (9-O-β-D-xylopyranosyl (+) lyoniresinol). V. myrtillus is very rich in phenolic compounds, however, to our knowledge the occurrence of described compound is presented in this plant for the first time. Its exclusive occurrence in woody organs (stems and rhizomes) points to its role in chemical protection of the winter-persistent part of the plant.