Biochemistry (Moscow)

, Volume 80, Issue 2, pp 233–241 | Cite as

Enzymatic polymerization of dihydroquercetin using bilirubin oxidase

  • M. E. Khlupova
  • I. S. Vasil’eva
  • G. P. Shumakovich
  • O. V. Morozova
  • V. A. Chertkov
  • A. K. Shestakov
  • A. V. Kisin
  • A. I. YaropolovEmail author


Dihydroquercetin (or taxifolin) is one of the most famous flavonoids and is abundant in Siberian larch (Larix sibirica). The oxidative polymerization of dihydroquercetin (DHQ) using bilirubin oxidase as a biocatalyst was investigated and some physicochemical properties of the products were studied. DHQ oligomers (oligoDHQ) with molecular mass of 2800 and polydispersity of 8.6 were obtained by enzymatic reaction under optimal conditions. The oligomers appeared to be soluble in dimethylsulfoxide, dimethylformamide, and methanol. UV-visible spectra of oligoDHQ in dimethylsulfoxide indicated the presence of highly conjugated bonds. The synthesized oligoDHQ was also characterized by FTIR and 1H and 13C NMR spectroscopy. Comparison of NMR spectra of oligoDHQ with DHQ monomer and the parent flavonoids revealed irregular structure of a polymer formed via the enzymatic oxidation of DHQ followed by nonselective radical polymerization. As compared with the monomer, oligoDHQ demonstrated higher thermal stability and high antioxidant activity.

Key words

dihydroquercetin enzymatic polymerization bilirubin oxidase oligomers 1H and 13C NMR spectra 



2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)


bilirubin oxidase








1,1-diphenyl-2-picrylhydrazyl radical


dihydroquercetin oligomers


thermogravimetric analysis


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Kudanga, T., Nyanhongo, G. S., Guebitz, G. M., and Burton, S. (2011) Potential applications of laccase-mediated coupling and grafting reactions: a review, Enzyme Microb. Technol., 48, 195–208.CrossRefPubMedGoogle Scholar
  2. 2.
    Hollmann, F., and Arends, I. W. C. E. (2012) Enzyme-initiated radical polymerizations: a review, Polymers, 4, 759–793.CrossRefGoogle Scholar
  3. 3.
    Rogovsky, V. S., Matyushkin, A. I., Shimanovsky, N. L., Semejkin, A. V., Kukhareva, T. S., Koroteev, A. M., Koroteev, M. P., and Nifant’ev, E. E. (2010) Antiproliferative and antioxidant activity of the new dihydroquercetin derivatives, Eksp. Klin. Farmakol., 73, 39–42.Google Scholar
  4. 4.
    Desentis-Mendoza, R. M., Hernandez-Sanchez, H., Moreno, A., del Rojas, C. E., Chel-Guerrero, L., Tamariz, J., and Jaramillo-Flores, M. E. (2006) Enzymatic polymerization of phenolic compounds using laccase and tyrosinase from Ustilago maydis, Biomacromolecules, 7, 1845–1854.CrossRefPubMedGoogle Scholar
  5. 5.
    Nicotra, S., Cramarossa, M. R., Mucci, A., Pagnoni, U. M., Riva, S., and Forti, L. (2004) Biotransformation of resveratrol: synthesis of trans-dehydrodimers catalyzed by laccases from Myceliophtora thermophyla and from Trametes pubescens, Tetrahedron, 60, 595–600.CrossRefGoogle Scholar
  6. 6.
    Jankun, J., Selman, S. H., Swiercz, R., and Skrzypczak-Jankun, E. (1997) Why drinking green tea could prevent cancer, Nature, 387, 561.CrossRefPubMedGoogle Scholar
  7. 7.
    Bordoni, A., Hrelia, S., Angeloni, C., Giordano, E., Guarnieri, C., Caldarera, C. M., and Bia, P. L. (2002) Green tea protection of hypoxia/reoxygenation injury in cultured cardiac cells, J. Nutr. Biochem., 13, 103–111.CrossRefPubMedGoogle Scholar
  8. 8.
    Ferry, D. R., Smith, A., Malkhandi, J., Fyfe, D. W., Takats, P. G., Anderson, D., Baker, J., and Kerr, D. J. (1996) Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition, Clin. Cancer Res., 2, 659–668.PubMedGoogle Scholar
  9. 9.
    Ono, K., Nakane, H., Fukushima, M., Chermann, J.-C., and Barre-Sinoussi, F. (1990) Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases, Eur. J. Biochem., 190, 469–476.CrossRefPubMedGoogle Scholar
  10. 10.
    Hagerman, A. E., Riedl, K. M., Jones, G. A., Sovik, K. N., Ritchard, N. T., Hartzfeld, P. W., and Riechel, T. L. (1998) High molecular weight plant polyphenolics (tannins) as biological antioxidants, J. Agric. Food Chem., 46, 1887–1892.CrossRefGoogle Scholar
  11. 11.
    Kurisawa, M., Chung, J. E., Uyama, H., and Kobayashi, S. (2003) Enzymatic synthesis and antioxidant properties of poly (rutin), Biomacromolecules, 4, 1394–1399.CrossRefPubMedGoogle Scholar
  12. 12.
    Kurisawa, M., Chung, J. E., Uyama, H., and Kobayashi, S. (2003) Laccase-catalyzed synthesis and antioxidant property of poly(catechin), Macromol. Biosci., 3, 758–764.CrossRefGoogle Scholar
  13. 13.
    Zhao, J., Wang, J., Chen, Y., and Agarwa, R. (1999) Antitumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5-3′-gallate as the most effective antioxidant constituent, Carcinogenesis, 20, 1737–1745.CrossRefPubMedGoogle Scholar
  14. 14.
    Hollman, P. C., and Katan, M. B. (1997) Absorption, metabolism and health effects of dietary flavonoids in man, Biomed. Pharmacother., 51, 305–310.CrossRefPubMedGoogle Scholar
  15. 15.
    Makena, P. S., Pierce, S. C., Chung, K.-T., and Sinclair, S. E. (2009) Comparative mutagenic effects of structurally similar flavonoids quercetin and taxifolin on tester strains Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA, Environ. Mol. Mutagen., 50, 451–459.CrossRefPubMedGoogle Scholar
  16. 16.
    Teselkin, Yu. O., Zhambalova, B. A., Babenkova, I. V., Klebanov, T. I., and Tyukavkina, N. A. (1996) Antioxidant properties of dihydroquercetin, Biofizika, 41, 620–623.PubMedGoogle Scholar
  17. 17.
    Vladimirov, Yu. A., Proskurina, E. V., Demin, E. M., Matveeva, N. S., Lyubitsky, O. B., Novikov, A. A., Izmajlov, D. Yu., Osipov, A. N., Tikhonov, V. P., and Kagan, V. E. (2009) Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at the key stages of apoptosis, Biochemistry (Moscow), 74, 301–307.CrossRefGoogle Scholar
  18. 18.
    Wang, Y.-H., Wang, W.-Y., Liao, J.-F., Chen, C.-F., Hou, Y.-C., Liou, K.-T., Chou, Y.-C., Tien, J.-H., and Shen, Y.-C. (2004) Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx, Biochem. Pharmacol., 67, 2251–2262.CrossRefPubMedGoogle Scholar
  19. 19.
    Bronnikov, G. E., Kulagina, T. P., and Aripovsky, A. V. (2009) Addition of dihydroquercetin to old mice diet results in retention of activity of skeletal muscle mitochondrial enzymes, Biol. Membr. (Moscow), 26, 387–393.Google Scholar
  20. 20.
    Middleton, E., Jr., Kandaswami, C., and Theoharides, T. C. (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer, Pharmacol. Rev., 52, 673–751.PubMedGoogle Scholar
  21. 21.
    Mizutani, K., Toyoda, M., Sagara, K., Takahashi, N., Sato, A., Kamitaka, Y., Tsujimura, S., Nakanishi, Y., Sugiura, T., Yamaguchi, S., Kano, K., and Mikami, B. (2010) X-Ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 — resolution using a twinned crystal, Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 66, 765–770.CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Guo, J., Liang, X. X., Mo, P. S., and Li, G. X. (1991) Purification and properties of bilirubin oxidase from Myrothecium verrucaria, Appl. Biochem. Biotechnol., 31, 135–143.CrossRefPubMedGoogle Scholar
  23. 23.
    Miliauskas, G., Venskutonis, P. R., and van Beek, T. A. (2004) Screening of radical scavenging activity of some medicinal and aromatic plant extracts, Food Chem., 85, 231–237.CrossRefGoogle Scholar
  24. 24.
    Aquino, R., Caceres, A., Morelli, S., and Rastrelli, L. (2002) An extract of Tagetes lucida and its phenolic constituents as antioxidants, J. Nat. Prod., 65, 1773–1776.CrossRefPubMedGoogle Scholar
  25. 25.
    Saija, A., Trombetta, D., Tomaino, A., Cascio, R. L., Princi, P., Uccella, N., Bonina, F., and Castelli, F. (1998) “In vitro” evaluation of the antioxidant activity and biomembrane interaction of the plant phenols oleuropein and hydroxytyrosol, Int. J. Pharm., 166, 123–133.CrossRefGoogle Scholar
  26. 26.
    Bruno, F. F., Trotta, A., Fossey, S., Nagarajan, S., Nagarajan, R., Samuelson, L. A., and Kumar, J. (2010) Enzymatic synthesis and characterization of polyquercetin, J. Macromol. Sci. Pure Appl. Chem., 47, 1191–1196.CrossRefGoogle Scholar
  27. 27.
    Park, S. Y., Kim, Y. H., Won, K., and Song, B. K. (2009) Enzymatic synthesis and curing of polycardol from renewable resources, J. Mol. Catal. B: Enzymatic, 57, 312–316.CrossRefGoogle Scholar
  28. 28.
    Mejias, L., Reihmann, M. H., Sepulveda-Boza, S., and Ritter, H. (2002) New polymers from natural phenols using horseradish or soybean peroxidase, Macromol. Biosci., 2, 24–32.CrossRefGoogle Scholar
  29. 29.
    Won, K., Kim, Y. H., An, E. S., Lee, Y. S., and Song, B. K. (2004) Horseradish peroxidase-catalyzed polymerization of cardanol in the presence of redox mediators, Biomacromolecules, 5, 1–4.CrossRefPubMedGoogle Scholar
  30. 30.
    Shimanko, N. A., and Shishkina, M. V. (1987) Atlas of the Absorption Spectra of Aromatic Esters in IR and UV Regions [in Russian], Nauka, Moscow.Google Scholar
  31. 31.
    Kolesnik, Y. A., Titova, E. V., Chertkov, V. A., Tashlitskiy, V. N., Tichonov, V. P., and Shmatkov, D. A. (2011) Stereoisomeric composition of two bioflavonoids from Larix sibirica, Planta Med., 77, 1266.CrossRefGoogle Scholar
  32. 32.
    Sinha, R., Joshi, A., Joshi, U. J., Srivastava, S., and Govil, G. (2014) Localization and interaction of hydroxyflavones with lipid bilayer model membranes: a study using DSC and multinuclear NMR, Eur. J. Med. Chem., 80, 285–294.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • M. E. Khlupova
    • 1
  • I. S. Vasil’eva
    • 1
  • G. P. Shumakovich
    • 1
  • O. V. Morozova
    • 1
  • V. A. Chertkov
    • 2
  • A. K. Shestakov
    • 3
  • A. V. Kisin
    • 3
  • A. I. Yaropolov
    • 1
    Email author
  1. 1.A. N. Bach Institute of BiochemistryRussian Academy of SciencesMoscowRussia
  2. 2.Faculty of ChemistryLomonosov Moscow State UniversityMoscowRussia
  3. 3.State Research Institute of Chemistry and Technology of Organoelement CompoundsMoscowRussia

Personalised recommendations