Galls of European Fraxinus trees as new and abundant sources of valuable phenylethanoid and coumarin glycosides

Abstract

Irregular tissue outgrowths (called galls) in the inflorescences of three European Fraxinus species (F. angustifolia, F. excelsior and F. ornus), were analyzed for their phytochemical composition for the first time. The main goal of this study was to demonstrate the significance of Fraxinus galls as the new and abundant sources of drug candidate metabolites. The comparable quantitative results of nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC)-mass spectrometry (MS) analyses confirmed extraordinarily high amounts of the valuable phenylethanoid glycoside (PhEG) acteoside (in the galls of F. angustifolia 86.7 mg/g – 139.9 mg/g and F. excelsior 71.7 mg/g – 161.2 mg/g) and that of the coumarin glucoside cichoriin (in the galls of F. ornus 143.0 mg/g – 232.0 mg/g). Optimized, consecutive treatments of acteoside, in distilled water (100 °C, 300 min) and acidic media (2 M trifluoroacetic acid, 100 °C, 15 min), resulted in an equilibrium of acteoside and isoacteoside as well as that of desrhamnosyl acteoside and desrhamnosyl isoacteoside, following isomerization and derhamnosylation, respectively. Three related PhEGs could thus also be isolated after optimized treatments, in addition to acteoside, in the highest yield reported so far. Their structures were identified by NMR spectroscopy and high-resolution Orbitrap-MS/MS techniques, also confirming the distinction of the regioisomers desrhamnosyl acteoside and desrhamnosyl isoacteoside by MS/MS. Molecular modeling was used to study the mechanism of isomerization. Isolated PhEGs and cichoriin showed no cytostatic activity in non-human primate Vero E6 cells and no hemolyis of human erythrocytes. Our results highlight the significance of Fraxinus galls in the production of PhEGs and cichoriin as easily available, high-yield harvestable, new raw materials for these pharmacologically important metabolites.