Hederagenin

Commercial seeds of fenugreek (Trigonella foenum-graecum L.) were purchased from Murciana de Herboristeria (Murcia, Spain) and seeds of red quinoa (Chenopodium quinoa Willd.) were purchased from Hijo de Macario Marcos (Salamanca, Spain). Trizma base, maleic acid, sodium chloride, calcium chloride, HCl, Amano lipase A from Aspergillus niger, pepsin, pancreatin from porcine pancreas, bile salts, phosphatidyl choline from egg yolk, β-sitosterol (≥70%), oleic acid, lysine, 1,3-diolein, 1-oleoyl-rac-glycerol, myo-inositol, d-glucose, quercetin, diosgenin, hederagenin, protocatechuic acid, squalane, and N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Sucrose was from Panreac (Barcelona, Spain). Dioscin, protodioscin, 5-pentadecylresorcinol and oleanolic acid were from Cymit Quimica S.L (Barcelona, Spain). Methanol, hexane, 1-butanol, chloroform were from Macron (Gliwice, Poland).

Saponins aescin (E1378), hederagenin (H3916), and soyasaponin I (S9951), and fungicides metconazole, fluconazole, boscalid, and fluopyram (all purchased from Sigma–Aldrich, St. Louis, MO, USA) were dissolved in 99.8% ethanol (EtOH) as 10 mg·ml⁻¹ stock solution. Tebuconazole, in the form of the commercially formulated product Horizon 250 EW (Bayer CropScience, Germany), was also prepared as 10 mg·ml⁻¹ stock solution in EtOH. Ergosterol (Sigma, St. Louis, MO, USA) was prepared as 5 mM stock solution in EtOH and used at the final concentration of 25 µg·ml⁻¹. Benzothiadiazole (BTH) was used in the form of the commercially formulated product Bion 50 WG (Syngenta, Switzerland) and prepared directly into the working solutions. The commercial peptide flg22 (EZBiolab) was diluted in Milli-Q water and used at the final concentration of 1 µM. All stock solutions were stored at −20°C.

Hedera helix leaves were harvested in the summer of 2021 at Hofigal S.A., Romania. The fresh leaves were dried in an air flow-heating oven at 60 °C to a constant weight (the final moisture content was 5.8%). The dried leaves were then ground using an electric grinder and sieved to a particle size under 315 μm. The ground leaves were dosed in samples of 25 g (in a sealed plastic vessel) and stored at 4–5 °C until they were used for extraction of the bioactive components.
The standard used for saponin determination was diosgenin, purchased from Sigma-Aldrich (Bucharest, Romania). The solvent ethanol, vanillin and sulfuric acid were of analytical grade and were purchased from Merck. For the HPLC quantification of saponins, the following standards were used: α-hederin, hederacoside C, and hederagenin (Sigma-Aldrich, Bucharest, Romania). Methanol, acetonitrile, water (all for chromatography, ultragradient grade), and formic acid (for analysis, 85%) were purchased from Carlo Erba (Cornaredo, Italy).