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

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Abstract

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.

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References

  • Abourashed EA, Mikell JR, Khan IA (2012) Bioconversion of silybin to phase I and II microbial metabolites with retained antioxidant activity. Bioorg Med Chem 20:2784–2788

    Article  PubMed  CAS  Google Scholar 

  • 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 submitted

  • Baer-Dubowska W, Szaefer H, Krajka-Kuzniak V (1998) Inhibition of murine hepatic cytochrome P450 activities by natural and synthetic phenolic compounds. Xenobiotica 28:735–743

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Burkart MD, Wong CH (1999) A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV. Anal Biochem 274:131–137

    Article  PubMed  CAS  Google Scholar 

  • 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 109:3–27

    Article  PubMed  CAS  Google Scholar 

  • De Groot H, Rauen U (1998) Tissue injury by reactive oxygen species and the protective effects of flavonoids. Fundam Clin Pharmacol 12:249–255

    Article  PubMed  Google Scholar 

  • Dorai T, Aggarwal BB (2004) Role of chemopreventive agents in cancer therapy. Cancer Lett 215:129–140

    Article  PubMed  CAS  Google Scholar 

  • Fiebrich F, Koch H (1979) Silymarin, an inhibitor of lipoxygenase. Experientia 35:1548–1560

    Article  PubMed  CAS  Google Scholar 

  • Flora K, Hahn M, Rosen H, Benner K (1998) Milk thistle (Silybum marianum) for the therapy of liver diseases. Am J Gastroenterol 93:139–143

    Article  PubMed  CAS  Google Scholar 

  • Fraschini F, Demartini G, Esposti D (2002) Pharmacology of silymarin. Clin Drug Invest 22:51–65

    Article  CAS  Google Scholar 

  • Gažák R, Walterová D, Křen V (2007) Silybin and silymarin— new and emerging applications in medicine. Curr Med Chem 14:315–338

    Article  PubMed  Google Scholar 

  • 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 121:290–297

    Article  CAS  Google Scholar 

  • 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 794:303–310

    Article  CAS  Google Scholar 

  • 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 18:698–704

    CAS  Google Scholar 

  • 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 35:2035–2039

    Article  PubMed  Google Scholar 

  • Kauffmann FC, Whittaker M, Anundi I, Thurman RG (1991) Futile cycling of a sulfate conjugate by isolated hepatocytes. Mol Pharmacol 39:414–420

    Google Scholar 

  • 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 1:1684–1689

    Article  PubMed  CAS  Google Scholar 

  • 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 1:2467–2474

    Google Scholar 

  • 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 28:1513–1517

    PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • 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 66:1171–1174

    Article  PubMed  CAS  Google Scholar 

  • Lee N, Kim EJ, Kim BG (2012) Regioselective hydroxylation of trans-resveratrol via inhibition of tyrosinase from Streptomyces avermitilis MA4680. ACS Chem Biol 7:1687–1692

    Article  PubMed  CAS  Google Scholar 

  • 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 34:2113–2117

    Article  PubMed  CAS  Google Scholar 

  • 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 89:24–27

    Article  CAS  Google Scholar 

  • 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 36:2219–2226

    Article  PubMed  CAS  Google Scholar 

  • Morazzoni P, Bombardelli E (1995) Silybum marianum (Carduus marianus). Fitoterapia 66:3–42

    CAS  Google Scholar 

  • 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 168:785–796

    Article  PubMed  CAS  Google Scholar 

  • Pietrangelo A, Borella F, Casalgrandi G (1995) Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 109:1941–1949

    Article  PubMed  CAS  Google Scholar 

  • Schriewer H, Badde R, Roth G, Raven HM (1973) Antihepatotoxic effect of silymarin in thioacetamide-damaged liver. Arzneimittel-Forsch 23:160–161

    Google Scholar 

  • Sekura RD, Jakoby WB (1981) Aryl sulfotransferase-IV from rat liver. Arch Biochem Biophys 211:352–359

    Article  PubMed  CAS  Google Scholar 

  • 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: 442–444

    Google Scholar 

  • Strott CA (2002) Sulfonation and molecular action. Endocrin Rev 23:703–732

    Article  CAS  Google Scholar 

  • 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 29:335–346

    PubMed  CAS  Google Scholar 

  • Vogel G, Tuchweber B, Trost W, Mengs U (1984) Protection by silibinin against Amanita phalloides intoxication in beagles. Toxicol Appl Pharmacol 73:355–362

    Article  PubMed  CAS  Google Scholar 

  • 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 18:688–695

    CAS  Google Scholar 

  • Wang LQ, James MO (2006) Inhibition of sulfotransferases by xenobiotics. Curr Drug Metab 7:83–104

    Article  PubMed  Google Scholar 

  • 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 30:134–138

    CAS  Google Scholar 

  • Wieland T, Faulstich H (1978) Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit Rev Biochem 78:185–260

    Article  Google Scholar 

  • Wieland T (1972) Struktur und Wirkung der Amatoxine. Naturwissenschaften 59:225–231

    Article  PubMed  CAS  Google Scholar 

  • 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 45:635–641

    Article  PubMed  CAS  Google Scholar 

  • 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 8:423–429

    Article  PubMed  CAS  Google Scholar 

  • 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 21:4389–4392

    Article  Google Scholar 

Download references

Acknowledgments

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.

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Authors and Affiliations

  1. Institute of Microbiology, Laboratory of Biotransformation, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ 14220, Prague, Czech Republic

    Kateřina Purchartová, Petr Marhol, Miroslav Šulc, Marek Kuzma, Kristýna Slámová & Vladimír Křen

  2. Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Worringer Weg 1, D 52074, Aachen, Germany

    Leonie Engels & Lothar Elling

  3. Department of Biochemistry, Faculty of Sciences, Charles University in Prague, Prague, Czech Republic

    Kateřina Purchartová & Miroslav Šulc

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  1. Kateřina Purchartová
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  2. Leonie Engels
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  3. Petr Marhol
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  4. Miroslav Šulc
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Correspondence to Vladimír Křen.

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Purchartová, K., Engels, L., Marhol, P. et al. Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver. Appl Microbiol Biotechnol 97, 10391–10398 (2013). https://doi.org/10.1007/s00253-013-4794-0

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  • DOI: https://doi.org/10.1007/s00253-013-4794-0

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