Applied Microbiology and Biotechnology

, Volume 97, Issue 24, pp 10391–10398 | Cite as

Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

  • Kateřina Purchartová
  • Leonie Engels
  • Petr Marhol
  • Miroslav Šulc
  • Marek Kuzma
  • Kristýna Slámová
  • Lothar Elling
  • Vladimír KřenEmail author
Biotechnologically relevant enzymes and proteins


Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation—the Phase II conjugation reaction—of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3′-phosphoadenosine-5′-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.


Silybin Biotransformation Diastereoisomers Sulfation Aryl sulfotransferase Rat liver 



This work was supported by the grant from the Czech Science Foundation P301/11/0767; and projects M200201204 of the Academy of Sciences of the Czech Republic, RV06138897 of the Institute of Microbiology, and by EU ESF COST project MultiGlycoNano CM1102.

Supplementary material

253_2013_4794_MOESM1_ESM.pdf (313 kb)
ESM 1 (PDF 313 kb)


  1. Abourashed EA, Mikell JR, Khan IA (2012) Bioconversion of silybin to phase I and II microbial metabolites with retained antioxidant activity. Bioorg Med Chem 20:2784–2788PubMedCrossRefGoogle Scholar
  2. Agarwal C, Wadhwa R, Deep G, Biedermann D, Gažák R, Křen V, Agarwal R (2013) Anti-cancer efficacy of silybin derivatives—a structure-activity relationship. PlosOne submittedGoogle Scholar
  3. Baer-Dubowska W, Szaefer H, Krajka-Kuzniak V (1998) Inhibition of murine hepatic cytochrome P450 activities by natural and synthetic phenolic compounds. Xenobiotica 28:735–743PubMedCrossRefGoogle Scholar
  4. Bradford MM (1976) Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Burkart MD, Wong CH (1999) A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV. Anal Biochem 274:131–137PubMedCrossRefGoogle Scholar
  6. Coughtrie MW, Sharp S, Maxwell K, Innes NP (1998) Biology and function of the reversible sulfation pathway catalyzed by human sulfotransferases and sulfates. Chem Biol Interact 109:3–27PubMedCrossRefGoogle Scholar
  7. De Groot H, Rauen U (1998) Tissue injury by reactive oxygen species and the protective effects of flavonoids. Fundam Clin Pharmacol 12:249–255PubMedCrossRefGoogle Scholar
  8. Dorai T, Aggarwal BB (2004) Role of chemopreventive agents in cancer therapy. Cancer Lett 215:129–140PubMedCrossRefGoogle Scholar
  9. Fiebrich F, Koch H (1979) Silymarin, an inhibitor of lipoxygenase. Experientia 35:1548–1560PubMedCrossRefGoogle Scholar
  10. Flora K, Hahn M, Rosen H, Benner K (1998) Milk thistle (Silybum marianum) for the therapy of liver diseases. Am J Gastroenterol 93:139–143PubMedCrossRefGoogle Scholar
  11. Fraschini F, Demartini G, Esposti D (2002) Pharmacology of silymarin. Clin Drug Invest 22:51–65CrossRefGoogle Scholar
  12. Gažák R, Walterová D, Křen V (2007) Silybin and silymarin— new and emerging applications in medicine. Curr Med Chem 14:315–338PubMedCrossRefGoogle Scholar
  13. Goretti M, Branda E, Turchetti B, Cramarossa MR, Onofri A, Forti L, Buzzini P (2012) Response surface methodology as optimization strategy for asymmetric bioreduction of (4S)-(+)-carvone by Cryptococcus gastricus. Biores Technol 121:290–297CrossRefGoogle Scholar
  14. Gunaratna C, Zhang T (2003) Application of liquid chromatography-electrospray ionization-ion trap mass spectrometry to investigate the metabolism of silibinin in human liver microsomes. J Chromatogr B Anal Technol Biomed Life Sci 794:303–310CrossRefGoogle Scholar
  15. Hahn G, Lehmann HD, Kürten M, Uebel H, Vogel G (1968) Zur Pharmakologie und Toxikologie von Silymarin, des antihepatotoxischen Wirkprinzips aus Silybum marianum (L.) Gaertn. Arzneimittel-Forsch 18:698–704Google Scholar
  16. Jančová P, Anzenbacherová E, Papoušková B, Lemr K, Luzná P, Veinlichová A, Anzenbacher P, Šimánek V (2007) Silybin is metabolized by cytochrome P450 2C8 in vitro. Drug Metab Dispos 35:2035–2039PubMedCrossRefGoogle Scholar
  17. Kauffmann FC, Whittaker M, Anundi I, Thurman RG (1991) Futile cycling of a sulfate conjugate by isolated hepatocytes. Mol Pharmacol 39:414–420Google Scholar
  18. Kim N-C, Graf TN, Sparacino CM, Wani MC, Wall ME (2003) Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum). Org Biomol Chem 1:1684–1689PubMedCrossRefGoogle Scholar
  19. Křen V, Kubisch J, Sedmera P, Halada P, Přikrylová V, Jegorov A, Cvak L, Gebhardt R, Ulrichová J, Šimánek V (1997) Glycosylation of silybin. J Chem Soc Perkin Trans 1:2467–2474Google Scholar
  20. Křen V, Ulrichová J, Kosina P, Stevenson D, Sedmera P, Přikrylová V, Halada P, Šimánek V (2000) Chemoenzymatic preparation of silybin β-glucuronide and their biological evaluation. Drug Metab Dispos 28:1513–1517PubMedGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  22. Lee DYW, Liu Y (2003) Molecular structure and stereochemistry of silybin A, silybin B, isosilybin A, and isosilybin B, isolated from Silybum marianum (milk thistle). J Nat Prod 66:1171–1174PubMedCrossRefGoogle Scholar
  23. Lee N, Kim EJ, Kim BG (2012) Regioselective hydroxylation of trans-resveratrol via inhibition of tyrosinase from Streptomyces avermitilis MA4680. ACS Chem Biol 7:1687–1692PubMedCrossRefGoogle Scholar
  24. Mao SH, Hu XJ, Hua BY, Wang N, Liu XG, Lu FP (2012) 5α-Hydroxylation of a steroid (13-ethyl-gon-4-en-3,17-dione) by Penicillium raistrickii in an ionic liquid/aqueous biphasic system. Biotechnol Lett 34:2113–2117PubMedCrossRefGoogle Scholar
  25. Marhol P, Hartog AF, van der Horst MA, Wever R, Purchartová K, Fuksová K, Kuzma M, Cvačka J, Křen V (2013) Preparation of silybin and isosilybin sulfates by sulfotransferase from Desulfitobacterium hafniense. J Mol Catal B Enzymat 89:24–27CrossRefGoogle Scholar
  26. Miranda SR, Lee JK, Brouwer KLR, Wen ZM, Smith PC, Hawke RL (2008) Hepatic metabolism and biliary excretion of silymarin flavonolignans in isolated perfused rat livers: role of multidrug resistance-associated protein 2 (Abcc2). Drug Metab Dispos 36:2219–2226PubMedCrossRefGoogle Scholar
  27. Morazzoni P, Bombardelli E (1995) Silybum marianum (Carduus marianus). Fitoterapia 66:3–42Google Scholar
  28. Neunzig I, Widjaja M, Dragan CA, Peters FT, Maurer HH, Bureik M (2012) Engineering of human CYP3A enzymes by combination of activating polymorphic variants. Appl Biochem Biotechnol 168:785–796PubMedCrossRefGoogle Scholar
  29. Pietrangelo A, Borella F, Casalgrandi G (1995) Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 109:1941–1949PubMedCrossRefGoogle Scholar
  30. Schriewer H, Badde R, Roth G, Raven HM (1973) Antihepatotoxic effect of silymarin in thioacetamide-damaged liver. Arzneimittel-Forsch 23:160–161Google Scholar
  31. Sekura RD, Jakoby WB (1981) Aryl sulfotransferase-IV from rat liver. Arch Biochem Biophys 211:352–359PubMedCrossRefGoogle Scholar
  32. Šimánek V, Křen V, Ulrichová J, Vičar J, Cvak L (2000) “What is in the name...?” An appeal for a change of editorial policy. Hepatology 32: 442–444Google Scholar
  33. Strott CA (2002) Sulfonation and molecular action. Endocrin Rev 23:703–732CrossRefGoogle Scholar
  34. Tan E, Pang KS (2001) Sulfation is rate limiting in the futile cycling between estrone and estrone sulfate in enriched periportal and perivenous rat hepatocytes. Drug Metab Dispos 29:335–346PubMedGoogle Scholar
  35. Vogel G, Tuchweber B, Trost W, Mengs U (1984) Protection by silibinin against Amanita phalloides intoxication in beagles. Toxicol Appl Pharmacol 73:355–362PubMedCrossRefGoogle Scholar
  36. Wagner H, Hörhammer L, Münster R (1968) Zur Chemie des Silymarins (Silybin), des Wirkprinzips der Früchte von Silybum marianum (L.) Gaertn. (Cardus marianus L.). Arzneimittel-Forsch 18:688–695Google Scholar
  37. Wang LQ, James MO (2006) Inhibition of sulfotransferases by xenobiotics. Curr Drug Metab 7:83–104PubMedCrossRefGoogle Scholar
  38. Weyhenmeyer R, Mascher H, Birkmayer J (1992) Study on dose-linearity of the pharmacokinetics of silibinin diastereomers using a new stereospecific assay. Int J Clin Pharmacol Ther Toxic 30:134–138Google Scholar
  39. Wieland T, Faulstich H (1978) Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit Rev Biochem 78:185–260CrossRefGoogle Scholar
  40. Wieland T (1972) Struktur und Wirkung der Amatoxine. Naturwissenschaften 59:225–231PubMedCrossRefGoogle Scholar
  41. Wu JW, Lin LC, Hung SC, Chi CW, Tsai TH (2007) Analysis of silibinin in rat plasma and bile for hepatobiliary excretion and oral bioavailability application. J Pharm Biomed Anal 45:635–641PubMedCrossRefGoogle Scholar
  42. Yang YS, Marshall DA, McPhie P, Guo WXA, Xie XF, Chen X, Jakoby WB (1996) Two phenol sulfotransferase species from one cDNA: nature of the differences. Protein Expr Purif 8:423–429PubMedCrossRefGoogle Scholar
  43. Zarelli A, Sgambato A, Petito V, De Napoli L, Previtera L, Di Fabio G (2011) New C-23 modified of silybin and 2,3-dehydrosilybin: synthesis and preliminary evaluation of antioxidant properties. Bioorg Med Chem Lett 21:4389–4392CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Kateřina Purchartová
    • 1
    • 3
  • Leonie Engels
    • 2
  • Petr Marhol
    • 1
  • Miroslav Šulc
    • 1
    • 3
  • Marek Kuzma
    • 1
  • Kristýna Slámová
    • 1
  • Lothar Elling
    • 2
  • Vladimír Křen
    • 1
    Email author
  1. 1.Institute of Microbiology, Laboratory of BiotransformationAcademy of Sciences of the Czech RepublicPragueCzech Republic
  2. 2.Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical EngineeringRWTH Aachen UniversityAachenGermany
  3. 3.Department of Biochemistry, Faculty of SciencesCharles University in PraguePragueCzech Republic

Personalised recommendations