Hederacoside c

Quantification of saponin and protoanemonin contents was carried out by high pressure liquid chromatography with diode array detection (HPLC-DAD) using a SunFire™ C18 (150 × 2.1 mm i.d., 5 μm particle size; Waters GmbH, Eschborn, Germany) column for saponin and a Reprosil-AQ reversed phase column (250 × 4 mm i.d., 5 μm particle size, Maisch, Ammerbruch, Germany) for protoanemonin analysis, respectively (Fig. 2). α-angelica lactone (PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany; purity 79%) was used as analytical reference for quantification of protoanemonin and the total saponin content was quantified at 210 nm using hederacoside C (PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany; purity 88%) as a reference. Gradient elution was carried out for both analyte groups, and detection wavelengths were 210 nm (saponins) and 261 nm (protoanemonin). Further details on both HPLC methods as well as method validation data have been reported earlier [21 (link)].Fig. 2

HPLC-DAD of an aqueous fermented Helleborus niger extract. a: HPLC-DAD chromatogram (λ = 261 nm) of an aqueous fermented Helleborus niger extract (1:20 dilution in water) and UV/Vis spectrum of protoanemonin. b: HPLC-DAD chromatogram (λ = 210 nm) of an aqueous fermented Helleborus niger extract and exemplary UV/Vis spectrum of a saponin. * Saponins

Nano-Glo^® Luciferase Assay was obtained from Promega (Promega GmbH, Mannheim). Ivy leaves dry extract EA 575^® (CID-100048) was obtained from Engelhard (Engelhard Arzneimittel GmbH & Co. KG Niederdorfelden).
The reference substances rutin, hederacoside C, hederacoside D, and α-hederin were obtained from Phytolab (Vestenbergsgreuth, Germany).
All other reagents were obtained from Merck (Darmstadt, Germany), if not stated otherwise.