Si 60

Fat extracted from muffins (200 mg) was dissolved in n-hexane, and diluted up to 10 mL. Tocopherols were qualitatively and quantitatively identified using a Waters HPLC system consisting of a pump (Waters 600), a fluorimetric detector (Waters 474), an autosampler (Waters 2707), a column oven (Waters Jetstream 2 Plus), and a LiChrosorb Si 60 column (250 × 4.6 mm, 5 μm) by Merck (Darmstadt, Germany). The mobile phase was a mixture of n-hexane with 1,4-dioxane (96:4, v/v). The excitation and emission wavelengths were 295 and 330 nm, respectively for the fluorescence detection of tocopherols [18 (link)].

Substrates (10 mg) dissolved in 200 μl DMSO were fed to 2-day old Streptomyces sp. MBT76 culture broth. This biotransformation period lasted for another 3 days. The control experiment included both negative control of feeding 200 μl DMSO solvent to MBT76, and positive control of 10 mg substrates dissolved in 200 μl DMSO added to sterile culture media. Each experiment was done in triplicate. The method used to harvest the biotransformation products was as described for the metabolomics study.
To isolate the biotransformation products of genistein (Sigma), the combined mixture in 50 ml methanol was first defatted twice with 20 ml of n-hexane, which was subsequently fractionated by Sephadex LH-20 chromatography (GE Healthcare Life Sciences, Eindhoven, The Netherlands) eluting with methanol, to give seven fractions fr1–7. These fractions were subjected to NMR profiling to discard fr1–3, and fr6 that gave no isoflavone signals. Fr7 was purified by silica gel (pore size 60 Å, 70–230 mesh, St. Louis, MO, USA) to yield compound 15 (0.7 mg). Fr4 was separated by preparative TLC on a silica gel plate (Si60, Merck, Darmstadt, Germany), migrated with solvent system of CHCl₃-MeOH (20:3) to give pure compound 16 (0.5 mg) and semi-pure compound 18 (0.2 mg). Fr5 was purified by preparative TLC to give pure compound 17 (0.3 mg) using the same TLC conditions.

Based on UHPLC-MS results, VPPR_B_2017 underground parts were selected for semi-preparative purposes. The total amount of 12.875 g of underground parts was macerated three times with 70% methanol in water in the ratio 1:10. Combined extracts were diluted with water and applied to SPE. The fraction eluted with 70% to 85% methanol was concentrated to dryness in vacuo in 40 °C (Rotavapor V-100; Büchi, Flavil, Swiss), giving 346 mg (2.7% of initial dry mass). The first separation was performed by FC on 85 g of Si₆₀ (Merck, Darmstadt, Germany), with chloroform→methanol gradient at flow rate 5 mL/min. Further, selected fractions were purified by semi-preparative HPLC on RP-18 in 75% methanol (isocratically) with a flow of 3 mL/min. As a result, 36.1 mg of 13 and 40.8 mg of 12 were obtained, corresponding to 0.28% and 0.32% of starting dry mass. To assure, that no artifacts were collected, 12 and 13 were confronted with primary extract by UHPLC-MS (conditions as described in Section 4.5).