A Rapid and Simple Chromatographic Separation of Diastereomers of Silibinin and Their Oxidation to Produce 2,3-Dehydrosilybin Enantiomers in an Optically Pure Form

Abstract

Silybin A and B were separated from commercial silibinin using the preparative HPLC method. The described method is rapid and effective in obtaining gram-scale amounts of two diastereoisomers with minimal effort. In our approach, silibinin was dissolved in THF (solubility greater than 100 mg/mL), an alternative solvent to H₂O or MeOH in which silibinin has a very low solubility (ca 0.05–1.5 mg/mL), and then separated into its two components using preparative RP-HPLC. By starting with purified diastereoisomers, it was possible to obtain the two enantiomers of 2,3-dehydrosilybin in good yields and optically pure using an efficient oxidation procedure. All of the purified products were fully characterised using NMR (¹H, ¹³C), CD, [α]_D, and ESI MS analyses. The purities of the products, which were evaluated using analytical HPLC, were greater than 98 % in all cases.

Key words

prep-RP-HPLC - flavonolignans - Silybum marianum - Asteraceae - silibinin - silybin A - silybin B - 2,3-dehydrosilybin

• References

• 1 Gažák R, Walterová D, Křen V. Silybin and silymarin: new and emerging applications in medicine. Curr Med Chem 2007; 14: 315-338
• 2 Kroll JD, Shaw HS, Oberlies NH. Milk thistle nomenclature: why it matters in cancer research and pharmacokinetic studies. Integr Cancer Ther 2007; 6: 110-119
• 3 Napolitano JG, Lankin DC, Graf TN, Friesen JB, Chen SN, McAlpine JB, Oberlies NH, Pauli GF. HiFSA fingerprinting applied to isomers with near-identical NMR spectra: the silybin/isosilybin case. J Org Chem 2013; 78: 2827-2839
• 4 Kim NC, Graf TN, Sparacino CM, Wani MC, Wall ME. Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum). Org Biomol Chem 2003; 1: 1684-1689
• 5 Davis-Searles PR, Nakanishi Y, Kim NC, Graf TN, Oberlies NH, Wani MC, Wall ME, Agarwal R, Kroll DJ. Milk thistle and prostate cancer: differential effects of pure flavonolignans from Silybum marianum on antiproliferative end points in human prostate carcinoma cells. Cancer Res 2005; 65: 4448-4457
• 6 Thelen P, Jarry H, Ringert RH, Wuttke W. Silibinin down-regulates prostate epithelium-derived Ets transcription factor in LNCaP prostate cancer cells. Planta Med 2004; 05: 397-400
• 7 Faezizadeh Z, Mesbah-Namin SA, Allameh A. The effect of silymarin on telomerase activity in the human leukemia cell line K562. Planta Med 2012; 9: 899-902
• 8 Varghese L, Agarwal C, Tyagi A, Singh RP, Agarwal R. Silibinin efficacy against human hepatocellular carcinoma. Clin Cancer Res 2005; 11: 8441-8448
• 9 Singh RP, Gu M, Agarwal R. Silibinin inhibits colorectal cancer growth by inhibiting tumor cell proliferation and angiogenesis. Cancer Res 2008; 68: 2043-2050
• 10 García-Maceira P, Mateo J. Silibinin inhibits hypoxia-inducible factor-1alpha and mTOR/p 70S6K/4E-BP1 signalling pathway in human cervical and hepatoma cancer cells: implications for anticancer therapy. Oncogene 2009; 28: 313-324
• 11 Payer BA, Reiberger T, Rutter K, Beinhardt S, Staettermayer AF, Peck-Radosavljevic M, Ferenci P. Successful HCV eradication and inhibition of HIV replication by intravenous silibinin in an HIV-HCV coinfected patient. J Clin Virol 2010; 49: 131-133
• 12 Gažák R, Svobodová A, Psotová J, Sedmera P, Přikrylová V, Walterová D, Křen V. Oxidised derivatives of silybin and their antiradical and antioxidant activity. Bioorg Med Chem 2004; 12: 5677-5687
• 13 Dvořák Z, Vrzal R, Ulrichová J. Silybin and dehydrosilybin inhibit cytochrome P450 1A1 catalytic activity: a study in human keratinocytes and human hepatoma cells. Cell Biol Toxicol 2006; 22: 81-90
• 14 Huber A, Thongphasuk P, Erben G, Lehmann WD, Tuma S, Stremmel W, Chamulitrat W. Significantly greater antioxidant anticancer activities of 2,3-dehydrosilybin than silybin. Biochim Biophys Acta 2008; 1780: 837-847
• 15 Zhan T, Digel M, Küch EM, Stremmel W, Füllekrug J. Silybin and dehydrosilybin decrease glucose uptake by inhibiting GLUT proteins. J Cell Biochem 2011; 112: 849-859
• 16 Halbach G, Trost W. Chemistry and pharmacology of silymarin. Studies on various metabolites of silybin. Arzneimittelforschung 1974; 24: 866-867
• 17 Pelter A, Hänsel R. Struktur des Silybins: I. Abbauversuche. Chem Berl 1975; 108: 790-802
• 18 Maitrejean M, Comte G, Barron D, El Kirat K, Conseil G, Di Pietro A. The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of P-glycoprotein. Bioorg Med Chem Lett 2000; 10: 157-160
• 19 Džubák P, Hajdúch M, Gažák R, Svobodová A, Psotová J, Walterová D, Sedmera P, Křen V. New derivatives of silybin and 2,3-dehydrosilybin and their cytotoxic and P-glycoprotein modulatory activity. Bioorg Med Chem 2006; 14: 3793-3810
• 20 Li W, Han JP, Li ZW, Li XX, Zhou SP, Liu CX. Preparative chromatographic purification of diastereomers of silybin and their quantification in human plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B 2008; 862: 51-57
• 21 Graf TN, Wani MC, Agarwal R, Kroll DJ, Oberlies NH. Gram-scale purification of flavonolignan diastereoisomers from Silybum marianum (milk thistle) extract in support of preclinical in vivo studies for prostate cancer chemoprevention. Planta Med 2007; 73: 1495-1501
• 22 Křen V, Sedmera P, Kubisch J, Halada P, Přikrylová V, Jegorov A, Cvak L, Gebhardt R, Ulrichová J, Šimánek V. Glycosylation of silybin. J Chem Soc [Perkin I] 1997; 2467-2474
• 23 Monti D, Gažák R, Marhol P, Biedermann D, Purchartová K, Fedrigo M, Riva S, Křen V. Enzymatic kinetic resolution of silybin diastereoisomers. J Nat Prod 2010; 73: 613-619
• 24 Gažák R, Marhol P, Purchartová K, Monti D, Biedermann D, Riva S, Cvak L, Křen V. Large-scale separation of silybin diastereoisomers using lipases. Process Biochem 2010; 45: 1657-1663
• 25 Zarrelli A, Sgambato A, Petito V, De Napoli L, Previtera L, Di Fabio G. New C-23 modified of silybin and 2,3-dehydrosilybin: synthesis and preliminary evaluation of antioxidant properties. Bioorg Med Chem Lett 2011; 21: 4389-4392