LC–MSn characterization of steroidal saponins in Helleborus niger L. roots and their conversion products during fermentation

• 38 steroidal saponins were identified in H. niger roots by LC-MSn analyses.
• Besides diosgenyl-type saponins, acetylated polyhydroxy saponins were predominant.
• Enzymatic treatment of isolated saponins led to a first idea of conversion paths.
• During fermentation, furostanol saponins were converted to spirostanols.
• Polyhydroxy saponins were partly cleaved to result in a stable final structure.

Steroidal saponins comprise a substantial part of the secondary metabolite spectrum in the medicinal plant Helleborus niger L. (black hellebore). The saponin fraction from the roots was investigated by LC–MSn resulting in 38 saponins and β-ecdysone. Nine diosgenyl-type glycosides, mainly furostanols consisting of the aglycones diosgenin, macranthogenin, sceptrumgenin, and sarsasapogenin were accompanied by 5 diosgenyl-type saponins exhibiting an aglycone with an additional OH group. However, the most relevant compounds were 24 acetylated polyhydroxy saponins including hellebosaponins A and D.The enzymes glucuronidase, β-glucosidase, and pectinase were used to obtain an idea on potential fermentative transformation reactions by incubation of the isolated model saponins macranthosid I and hellebosaponin A. In a second step, aqueous H. niger extracts containing a much greater range of saponins were monitored during fermentation and 12 months of storage. The metabolites were examined and assigned by LC–MSn and targeted extracted ion current (EIC) scan analyses. Good agreement was found among the results from the model compounds and the whole aqueous fermented extracts.The native diosgenyl-type furostanol saponins were converted to spirostanols under scission of hexoses. Alteration of the acetylated polyhydroxy saponins, exclusively spirostanols, took place following cleavage of acetyl groups and terminal deoxyhexoses. Most interestingly, the pentoses of the sugar chain at C(1) were not affected. Conversion of acetylated polyhydroxy saponins resulted in a final structure type which was stable and detectable, even after 12 months of fermentation and storage.