Silymarin was obtained from the following suppliers: Sigma-Aldrich (SM 1; St. Louis, MO, USA, batch No. BCBJ0393V), Liaoning Senrong Pharmaceuticals (SM 2; Panjin, China, batch No. 120501), INDENA (SM 3; Settala, Italy, batch No. 32621/M5), Panjin Huacheng Pharmaceutical Company (SM 4; with an additive for better solubility in water, Panjin, China, batch No. E5S66), Takeda (SM 5; Konstanz, Germany, Flavobion
® coated tablets, batch No. 383036), Panjin Huacheng Pharmaceutical Company (SM 6; supplied in August 2019 without batch No., Panjin, China). Flavobion
® coated tablets (25 tablets, 11.360 g) were powdered and subjected to Soxhlet extraction with acetone (450 mL) for 2 h, the extract was evaporated in vacuo to yield the sample SM 5 (126 mg of dry extract). The extract was stored at −80 °C. Standards of silybin A, silybin B, 2,3-
cis-silybin A, 2,3-
cis-silybin B, 10,11-
cis-silybin A, 10,11-
cis-silybin A, silychristin A, silychristin B, isosilychristin, silydianin, isosilybin A, isosilybin B, silyhermin, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B, 2,3-dehydrosilychristin A, 2,3-dehydrosilychristin B, 2,3-dehydroisosilybin, and 2,3-dehydrosilydianin were prepared and fully characterized in the Laboratory of Biotransformation, Institute of Microbiology, Prague, CZ [11 (
link),21 (
link),22 (
link),23 (
link),24 (
link),25 (
link)]. Taxifolin was purchased from Amagro (Prague, Czech Republic) and coniferyl alcohol from Sigma-Aldrich (Merck, Kenilworth, NJ, USA). All standard solutions for calibration curves were prepared in dimethyl sulfoxide in volumetric flasks. The silymarin preparations SM 1–SM 6 were also dissolved in dimethyl sulfoxide and their concentrations were 10.3, 6.5, 13.3, 8.3, 15.5, and 23.1 mg/mL, respectively. All substances dissolved in dimethyl sulfoxide were stable during the measurement; no new peaks appeared in repeated measurements even after several weeks. The concentrations of the flavanonol, flavonolignans, and 2,3-dehydroflavonolignans were calculated using seven-point calibration curves.
Silymarin fraction containing concentrated 2,3-dehydroderivatives of flavonolignans was obtained from silymarin (SM 2 preparation) as described previously [23 (
link)] using Sephadex LH-20 glass column XK 50 (100 × 5 cm, bead size 25–100 μm, GE Healthcare Life Sciences, Pittsburgh, PA, USA) equipped with a thermostatic jacket (23 °C). Isocratic elution with methanol, flow rate 3 mL/min, volume of each fraction was 30 mL, UV detection at 254 nm, run-time 28 h [23 (
link)]. Briefly, 6 g of “silybin free” silymarin (see
Appendix B) was loaded onto the column and eluted with methanol to obtain a fraction enriched in 2,3-dehydroderivatives of flavonolignans (typically 0.8 g).
Acetonitrile, methanol, formic acid, dimethyl sulfoxide (Avantor, Radnor, PA, USA), and deionized water were of LC-MS grade.
Petrásková L., Káňová K., Biedermann D., Křen V, & Valentová K. (2020). Simple and Rapid HPLC Separation and Quantification of Flavonoid, Flavonolignans, and 2,3-Dehydroflavonolignans in Silymarin. Foods, 9(2), 116.