Isolation of High-Purity Silybin from Silymarin

We are grateful for the provision of silybin A/B by Dr. David Biedermann, Institute of Microbiology of the Czech Academy of Sciences, in the Laboratory of Biotransformations, Prague. The preparation of silybin from silymarin has been described previously [13 (link)]. Briefly, 200 mg of silymarin (Liaoning Senrong Pharmaceutical Co., Panjin, China) was suspended in 10 mL of methanol. The insoluble silybin was filtered, and the filtrate was collected and applied to a SEPHADEX HL-20 colony (Sigma Aldrich, Schnelldorf, Germany). The isocratic method with a mobile phase of water/methanol/cyanomethane/formic acid in the ratio 2/37/61/0.1 was used. The flow rate of the mobile phase was 1.2 mL/min, and the detection was performed spectrophotometrically at 285 nm SYNERGY HT (Biotek, Winooski, VT, USA). The purity of the obtained silybin (mixture of two diastereoisomers A and B) was 95 %, as determined by HPLC PDA. For further analyses, silybin was dissolved in dimethyl sulfoxide (Sigma Aldrich, Germany) to a 100 mM stock solution, which was subsequently diluted in the appropriate culture media.