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Enzymatic Synthesis and Antioxidant Properties of Poly(rutin) Motoichi Kurisawa,†,‡ Joo Eun Chung,†,‡ Hiroshi Uyama,*,† and Shiro Kobayashi*,† Bio-oriented Technology Research Advancement Institution and Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan Received May 5, 2003; Revised Manuscript Received July 1, 2003
Rutin, quercetin-3-rutinoside, is one of the most famous glycosides of flavonoid and widely present in many plants. In this study, we performed an oxidative polymerization of rutin using Myceliophthora laccase as catalyst in a mixture of methanol and buffer to produce a flavonoid polymer and evaluated antioxidant properties of the resultant polymer. Under selected conditions, the polymer with molecular weight of several thousands was obtained in good yields. The resulting polymer was readily soluble in water, DMF, and DMSO, although rutin monomer showed very low water solubility. UV measurement showed that the polymer had broad transition peaks around 255 and 350 nm in water, which were red-shifted in an alkaline solution. Electron spin resonance (ESR) measurement showed the presence of a radical in the polymer. The polymer showed greatly improved superoxide scavenging activity and inhibition effects on human low-density lipoprotein (LDL) oxidation initiated by 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), compared with the rutin monomer. The polymer also protected endothelial cells from oxidative injury induced by AAPH as a radical generator with a much greater effect than the rutin monomer. Introduction Recent interest in flavonoids has increased greatly due to their biological and pharmacological activity including antioxidant, anticarcinogenic, probiotic, antimicrobial, and antiinflammatory properties.1 The flavonoids consist of a large group of low molecular weight polyphenolic substances, naturally occurring in fruits, vegetables, tea, and wine, and are an integral part of the human diet. Rutin (1) is one of the most commonly found flavonol glycosides identified as vitamin P with quercetin and hesperidin (Chart 1) and widely present in many plants, especially the buckwheat plant. Rutin has been reported to have clinically relevant functions, including antioxidant, antihypertensive, antiinflammatory, and antihemorrhagic activity, the strengthing of the capillaries of blood vessels and the regulation of the capillary permeability, and the stabilization of platelets.2 These properties are potentially beneficial in preventing diseases and protecting the stability of the genome. Many of these activities have been related to its antioxidant actions. In general, the activities of flavonoids are known to be limited for only few hours in a body, although the metabolism has not been established. In addition, several flavonoids have been shown to act as prooxidants and generate reactive oxygen species, such as hydrogen peroxide. In contrast, a relatively high molecular fraction of extracted flavonoids has been reported to exhibit enhanced physiological properties, such as antioxidant and anticarcinogenic activity, and a * To whom correspondence should be addressed. Tel: +81-75-753-5638, +81-75-753-5608. Fax: +81-75-753-4911. E-mail addresses: uyama@ mat.polym.kyoto-u.ac.jp,
[email protected]. † Kyoto University. ‡ Bio-oriented Technology Research Advancement Institution.
relatively longer circulation time in vivo.3 High molecular weight plant polyphenols have also been reported to show no prooxidant effects.4 Many investigations have explored the antioxidant effects of low molecular weight flavonoids, but few have considered polymeric flavonoids. We have designed various polymerized flavonoids or polymeric flavonoid conjugates, in consideration of extension of the amplification of physiological properties of the flavonoids. We reported recently that poly(catechin) as one of the strategic molecular designs was synthesized by the enzyme-catalyzed oxidative coupling using horseradish peroxidase as a catalyst and exhibited great improvement in radical scavenging activity, protection effects against lowdensity lipoprotein (LDL) oxidation, and inhibition effects on xanthine oxidase activity, compared with a catechin monomer.5 In this study, we synthesized poly(rutin)s by the enzymecatalyzed oxidative coupling using laccase as a catalyst to amplify the antioxidant activity of rutin and investigated their scavenging activity against reactive oxygen species and protection effects from peroxidation of LDL and from oxidative injury of endothelial cells. Experimental Section Materials. Rutin (1) was purchased from Tokyo Kasei Co. The purity of rutin is above 98%. A rutin derivative (2, R-G-rutin PS) and laccase (5.7 × 107 units) derived from Myceliophthora were kindly donated by Toyo Sugar Refining Co., Ltd. and Novozymes Japan Ltd., respectively. Lowdensity lipoprotein (LDL) from human plasma was purchased from Sigma. Xanthine, xanthine oxidase (from butter milk), 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), and
10.1021/bm034136b CCC: $25.00 © 2003 American Chemical Society Published on Web 08/14/2003
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Chart 1. Structure of Rutin (1) and Water-Soluble Rutin Derivative (2)
ethylenediamine tetraacetic acid (EDTA) were obtained from Wako Pure Chemical Industries, Japan. 2-Methyl-6-p-methoxyphenylethynylimidazopyrazinone (MPEC) was purchased from Atto Co., Ltd., Japan. Diphenyl-1-pyrenylphosphine (DPPP) was purchased form Dojindo, Japan. Bovine aortic endothelial cells were purchased from Dainippon Pharmaceutical Co., Ltd., Japan. Alamar blue was purchased from Trek Diagnostic Systems Ltd., U.K. Other reagents and solvents are commercially available and used as received. Laccase-Catalyzed Synthesis and Characterization of Poly(rutin)s. In a 50 mL flask, rutin (0.20 g) was dissolved in a mixture of methanol and buffer (20 mL). Laccase solution was added to the mixture, followed by gentle stirring for 24 h at room temperature under air. The reaction solution was dialyzed (cutoff molecular weight 1 × 103) in water. The remaining solution was lyophilized to give the polymer. Polymer structure was analyzed by NMR (Bruker DPX400), FT-IR (Perkin-Elmer Spectrum One equipped with universal ATR sampling accessory), UV-visible (Hitachi U-2001), fluorescence (Hitachi F-2500), and electron spin resonance (ESR) (JEOL JES-TE100) spectrometers. UV-visible spectrum of rutin (1) was measured after dilution of rutin/ methanol stock solution with a quite excess amount of water. Rutin is insoluble in water, but rutin that was once dissolved in methanol does not form any precipitate when the solvent is exchanged into water. Molecular weight was estimated by size exclusion chromatography (SEC, Tosoh GPC-8020 equipped with RI-8020 detector). The SEC analysis was performed with TSKgel R3000 column and DMF containing 0.10 M LiCl eluent at a flow rate of 0.50 mL/min at 60 °C. The calibration curves for SEC analysis were obtained using polystyrene standards. Superoxide Scavenging Activity. Superoxide anion was generated by xanthine/xanthine oxidase (XO) and measured by chemiluminescent superoxide probe method.6 The chemiluminescence (CL) intensity of MPEC triggered by superoxide anion was measured in a 100 mM potassium phosphate buffer solution (pH 7.5) containing 0.05 mM EDTA, 0.04 unit‚mL-1 of XO, MPEC, and a test sample. Light emission was started by the addition of 0.3 mM of xanthine. CL spectra were monitored for 30 s using a Corona microplate photoncounter, MTP-700CL (Corona Electric Ltd. Japan). Superoxide anion scavenging activity was calculated ac-
cording to the following formula. Superoxide scavenging activity (%) ) CLcontrol - CLsample × 100 CLcontrol where CLcontrol and CLsample represents chemiluminescent intensity in the absence and presence of samples, respectively. Determination of LDL Susceptibility to Oxidation. LDL (100 µg/mL) was incubated with 200 µM (final concentration) of DPPP for 5 min at 37 °C under N2 in the dark. The DPPP-labeled LDL was preincubated with samples (0-70 µM) for 1 h at 37 °C in the dark. Oxidation of LDL was carried out by further incubation with AAPH (1mM) for 25 h at 37 °C. Oxidation of DPPP was measured at the indicated times using a 1420 ARVOSX multilabel counter (Wallac). Wavelengths of excitation and emission were set at 355 and 405 nm, respectively. Cell Culture. Bovine aortic endothelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 µg/mL of streptomycin in a humidified atmosphere 5% CO2 incubator. Evaluation of Protection Effects on Radical-Induced Cytotoxicity. Bovine endothelial cells were seeded at a concentration of 3 × 105 cells/mL, 200 µL/well, in 96-well flat-bottomed microassay plates (Falcon 3072 Co., Becton Dickenson, Flanklin Lakes, NJ) for 24 h before adding sample solutions. A desired amount of sample stock solutions was diluted into DMEM without FBS. The sample solution (190 µL) was added into 96-well plates, and preincubated for 1 h at 37 °C. AAPH solution (10 µL, 200 mM) as a radical generator was added into the well, and the cells were cultured for 48 h at 37 °C in a humidified 5% CO2 atmosphere. To evaluate of cell viability, culture media was replaced with RPMI 1640 containing 10% alamar blue, a dye that changes color from blue to red when subjected to reduction by cytochrome c activity, and incubated for 4 h at 37 °C.7 Optical absorbance was read at 560 and 600 nm to obtain a dye reduction amount using a 1420 ARVOSX multilabel counter (Wallac).
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Table 1. Laccase-Catalyzed Oxidative Polymerization of Rutin Derivativesa buffer enzyme Mnb buffer content (unit, yield entry monomer pH (%) ×10-5) (%) (×10-3) Mw/Mnb 1 2 3 4 5 6 7 8 9
1 1 1 1 1 1 1 2 2
5 5 5 5 5 7 c 5 5
30 50 50 50 70 50 50 50 100
11 2.9 11 29 11 11 11 11 11
70 39 73 76 81 66 79 75 55
7.9 7.4 7.1 7.1 9.3 6.8 8.3 10 11
1.6 1.6 1.5 1.5 1.7 1.6 1.6 1.6 1.6
a Polymerization of rutin derivative (0.20 g) using laccase catalyst in a mixture of methanol and buffer (pH 7) (total 20 mL) at room temperature for 24 h under air. b Determined by SEC. c Use of distilled water as cosolvent.
Results and Discussion Laccase-Catalyzed Polymerization. Enzymatic oxidation of flavonoid polyphenols is very important in biochemistry because the subsequent coupling reactions are involved in some biosynthetic pathways such as tannin and melanin formation. In the oxidative coupling of catechin by peroxidase or polyphenol oxidase, oligomeric compounds with complicated structure were formed.5,8 Peroxidase was reported to catalyze an oxidation of rutin with use of hydrogen peroxide as an oxidizing agent.9 The reaction monitoring by UV-visible spectroscopy showed the formation of o-quinone intermediate, which may be further polymerized. In a mixture of 1,4-dioxane and buffer, the polymer with molecular weight of 2.3 × 103 was obtained with the peroxidase catalysis in 30% yield.10 Laccase is a protein containing copper in its active site and uses oxygen as an oxidizing agent. So far, laccase has been used as catalysts for polymerization and curing of phenol derivatives, yielding high-performance polymeric materials.11 In recent years, this laccase-catalyzed polymerization has retained the attention due to an environmentally benign process of polymer production in air without the use of hydrogen peroxide as an oxidizing agent. In this study, we used laccase derived from Myceliophthora as a catalyst, which showed high catalytic activity for the oxidative polymerization of syringic acid.12 Because rutin is readily soluble in methanol but insoluble in water, the polymerization was performed in a mixed solvent of methanol and buffer at room temperature for 24 h. Molecular weight of the polymer was estimated by size exclusion chromatography (SEC) with DMF containing 0.10 M LiCl as eluent. Polymerization results are summarized in Table 1. In the enzymatic oxidative polymerization of phenol derivatives in an aqueous organic solvent, powdery polymers were often precipitated during the polymerization.13 However, the present polymerization proceeded in the homogeneous phase without any precipitation. After dialysis of the reaction mixture, a brown powdery polymer with molecular weight of several thousands was obtained by lyophilization. In all cases, a unimodal peak was seen in the SEC trace. In an equivolume mixture of methanol and acetate buffer (pH 5), the polymer yield increased as a function of the
Table 2. Solubility of Rutins and Poly(rutin)sa sample acetone DMF DMSO methanol pyridine toluene water 1 poly(1)b 2 poly(2)c b
-
++ ++ ++ ++
++ ++ ++ ++
++ + ++ (
++ ( ++ (
-
++ ++ ++
a ++, soluble; +, almost soluble; (, partly soluble; -, insoluble. Sample of entry 3. c Sample of entry 8.
enzyme amount, whereas the molecular weight did not depend on the enzyme amount (entries 2-4). The yield and molecular weight of the polymer obtained in the polymerization using the acetate buffer as a cosolvent were relatively close to that in a phosphate buffer of pH 7 (entries 3 and 6). A similar behavior was observed in the laccase-catalyzed polymerization of syringic acid in an aqueous acetone.12 In a mixture of methanol and the acetate buffer, the effect of the buffer content was examined (entries 1, 3, and 5). Although the mixed ratio scarcely affected the polymer yield, using 70% buffer afforded the polymer of the highest molecular weight (entry 5). The polymerization also proceeded in an equivolume mixture of methanol and distilled water to produce the polymer in a high yield (entry 7). In entries 3 and 7, quantitative consumption of rutin was confirmed by SEC analysis of the product before dialysis. The monomer shows a very low solubility in water (less than 0.1% at room temperature), whereas the water solubility of the polymer was very high (larger than 5%) (Table 2). The polymer was soluble in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) besides water. The solubility of the polymer toward pyridine and methanol was inferior to that of the monomer. A rutin derivative (2) soluble in water is commercially available, which consists of 2a and 2b (20:80 mol %) (Chart 1). This derivative was also polymerized by the laccase catalyst; the laccase-catalyzed polymerization in an equivolume mixture of methanol and acetate buffer produced the polymer in a high yield (entry 8). The molecular weight of the polymer reached 1 × 104, which was larger than that from rutin. The polymer from 2 was readily soluble in DMF, DMSO, and water. The polymerization also proceeded in the acetate buffer without use of an organic solvent, although the polymer yield was smaller than that in the aqueous methanol (entry 9). 1 H NMR of rutin and poly(rutin) was measured in DMSOd6. In the spectrum of the polymer, broad peaks were observed at δ 2.5-4.0, 4.0-5.6, and 6.2-8.5, whereas the monomer has sharp peaks in these regions. Figure 1 shows IR spectra of rutin and poly(rutin) (entry 3). The peak pattern of poly(rutin) was similar to that of rutin, although all of the peaks of poly(rutin) became broader. In the spectrum of poly(rutin), a broad peak centered at 3340 cm-1 due to the vibration of O-H linkage of phenolic and hydroxyl groups, a peak at 1650 cm-1 ascribed to the carbonyl vibration, and a peak at 1595 cm-1 ascribed to the CdC vibration of aromatic groups were observed. Peaks at 1521, 1122, 971, and 592 cm-1 became smaller, and characteristic peaks did not newly appear. UV-visible spectra are shown in Figure 2. In water, the monomer has two peaks at 255 and 350 nm due to the π-π*
Synthesis and Properties of Poly(rutin)
Figure 1. FT-IR spectra of (A) rutin and (B) poly(rutin) (entry 3).
Figure 2. UV-visible spectra of (A) rutin in water, (B) poly(rutin) (entry 7) in water, and (C) poly(rutin) in 1.0 × 10-3 N NaOH solution.
transitions of the aromatic fragment (Figure 2A). In the case of poly(rutin) (entry 3), the former peak was also seen, and the latter became much broader (Figure 2B), which may be attributed to conjugation in the polymer; the aromatic C-C linkage between the monomers is formed via the oxidative coupling of rutin.9,14 In the peroxidase-catalyzed oxidation of rutin, a small shoulder peak was observed at 440 nm,15 which was not detected in the present polymer. In an alkaline solution, both peaks were red-shifted and the peak intensity became larger than that in water (Figure 2C), whereas the peak in an acidic solution was almost the same as that in water (data not shown). Fluorescence spectra of rutin and poly(rutin) were measured with excitation at 350 nm in water. The monomer exhibited an emission spectrum with intense peaks at 417 and 438 nm, whereas such a peak was not observed in poly(rutin). ESR spectroscopy of rutin and poly(rutin) was recorded in the solid state at 25 °C (data not shown). In the range between 1.96 and 2.02 G, no peak was detected in the spectrum of rutin. On the other hand, poly(rutin) (entry 7) possessed a clear singlet peak with g ) 1.984, indicating the presence of a radical species in poly(rutin). This is probably a hydroxyl radical formed during the oxidative polymerization and entrapped in the polymer matrix. A similar radical peak was reported to be found in lignin, a natural phenolic polymer; however, there was no ESR peak in tannin, an oligomeric natural product from flavonoids.16 Superoxide Scavenging Activity. Reduction of molecular oxygen to superoxide anion by xanthine oxidase (XO), generating hydroxyl radicals and uric acid, is an important
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Figure 3. Superoxide scavenging activity of poly(rutin)s: (O) poly(rutin) (entry 7); (b) rutin; (0) poly(rutin) (entry 8); (9) R-G-rutin. Values are the mean ( SD of three independent experiments.
physiological pathway.17 However, superoxide anion damages biomacromolecules both directly and indirectly by forming hydrogen peroxide or highly reactive hydroxyl radicals.18 The antioxidant activity of poly(rutin)s was evaluated in terms of superoxide anion scavenging activity (Figure 3). Poly(rutin) (entry 7) and poly(rutin derivative) (entry 8) greatly scavenged superoxide anions in a concentration-dependent manner without prooxidation. Poly(rutin) almost completely scavenged in 300 µM of a rutin unit concentration. Conversely, rutin and G-rutin showed prooxidant property in lower concentrations. This prooxidant property is consistent with some investigations often reported for tea polyphenol at lower dosages in the aqueous phase.4 Because compounds capable of scavenging superoxide anion can also affect XO inhibition, samples were investigated for their effects on this process. The inhibition effects of poly(rutin)s and the monomer were almost same in a range of tested concentrations (data not shown). Also, a control experiment revealed that chemiluminescence quenching of poly(rutin) was very negligible in the tested concentrations. These indicate that the activity amplification of the poly(rutin)s in this measurement resulted primarily from increase in scavenging activity against superoxide anion, rather than in the inhibition effect on XO. For monomeric flavonoids, the ability to act as antioxidants is dependent on extended conjugation, number and arrangement of phenolic substitutents, and molecular weight.19 For oligomeric flavonoids occurring in nature, the degree of polymerization is proportional to the ability to scavenge peroxyl radicals.20 Hagerman et al. demonstrated that high molecular weight and the proximity of many aromatic rings and hydroxyl groups are more important for free radical scavenging by tannins than specific functional groups.3a The much higher scavenging activity of the poly(rutin) against free radicals, despite the presence of radical species in the polymer matrix, could be attributed to creation of high concentration of phenolic moieties in the molecules. Inhibition against LDL Oxidation. Oxidation of LDL leads to its enhanced uptake by macrophages, which is believed subsequently to result in foam cell formation, one of the first stages of atherogenesis. Therefore, antioxidants that protect LDL against oxidation are potentially antiatherogenic compounds. Although the mechanism for in vivo oxidation of LDL has not been established, free radical
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particular, in the high concentration of 400 µM, the polymer exhibited further raised protection related to a concentration increase. In contrast, the monomer induced fatal cytotoxicity by itself at the same concentration. Because the poly(rutin) is a highly water-soluble, it might not be very soluble in the biomembrane of cells. Therefore, these results imply that the poly(rutin) is a more potent chain-breaking antioxidant when scavenging free radicals in an aqueous system than the monomer. This fact might be one of reasons for poly(rutin) to exhibit very low cytotoxicity by itself.
Figure 4. Inhibition effects of rutin and poly(rutin) against LDL oxidation induced by AAPH: (O) negative control; (9) poly(rutin) (entry 7); (0) rutin. Values are the mean ( SD of eight independent experiments.
Figure 5. Protection effects of rutin and poly(rutin) (entry 7) against cell death induced by AAPH. Values are the mean ( SD of eight independent experiments.
autoxidation may be a factor. To evaluate antioxidant effect against peroxidation of LDL, LDL was labeled with diphenyl-1-pyrenylphosphine (DPPP), a fluorescent probe sensing hydroperoxide produced by lipid oxidation. The labeled LDL was preincubated with a sample of antioxidant, prior to oxidation by addition of AAPH. Incubation of AAPH with LDL generates peroxyl radicals, leading to a chain reaction that produces peroxidation products such as hydroperoxides and aldehydes.21 DPPP, a nonfluorescent molecule, reacts stoichiometrically with hydroperoxide to give diphenyl-1-pyrenylphosphine oxide (DPPPdO), which is strongly fluorescent.22 The inhibition effect of poly(rutin) (entry 7) against the AAPH-induced oxidation more effectively lasted for long-term oxidation, compared with that of the monomer (Figure 4). The inhibition effect was concentration-dependent (data not shown). Inhibition of Radical-Induced Cytotoxicity. Reactive radical species result in various cell damage including oxidative deterioration of lipids, protein, and DNA, inducing cell death such as apoptosis and necrosis.23 Protection effects of rutin and poly(rutin) (entry 7) against endothelial cell damage caused by AAPH, a radical generator were examined (Figure 5). AAPH induces peroxidation only in lipid membrane of cells during the first step.24 The addition of AAPH caused significant cell death due to oxidative injury. However, poly(rutin) enhanced cell viability with higher protection effects against the oxidative damage than that of the rutin monomer in a concentration of 100 µM. In
Conclusions Synthetic flavonoid polymers were enzymatically synthesized by an oxidative polymerization of rutin derivatives (1 and 2). Both monomers were polymerized by laccase catalyst under mild reaction conditions to give water-soluble polymers with molecular weight of ca. 1 × 104. To our best knowledge, this is the first example of the production of flavonoid polymers through the laccase catalysis. ESR analysis showed the presence of a radical species in the polymer. The polymer exhibited greatly amplified superoxide scavenging activity compared with the rutin monomer, although possessing radical species in the polymer matrix. The polymer was a more potent protector from LDL oxidation and cell injury induced by radicals than the monomer. In addition, the poly(rutin) might provide the potential for controlled biodistribution due to the high molecular weight in vivo. Further studies on the enzymatic synthesis of flavonoid polymers and their applications for a therapeutic agent to offer protection against a wide range of free-radical-induced diseases are under way in our laboratory. Acknowledgment. This work was supported by Program for Promotion of Basic Research Activities for Innovative Bioscience. We acknowledge the gift of laccase and a modified rutin (2) from Novozymes Japan Ltd. and Toyo Sugar Refining Co., Ltd. We are grateful to Professor Yasuhiro Aoyama for the use of the fluorescence spectrometer. References and Notes (1) Jankun, J.; Selman, S. H.; Swiercz, R.; Skrzypezak-Jankan, E. Nature 1997, 387, 561. (2) (a) Hertog, M. G. L.; Hollman, P. C. H.; Kantan, M. B.; Kromhout, D. Nutr Cancer 1993, 20, 21. (b) Formica, J. V.; Regelson, W. Food Chem. Toxicol. 1995, 33, 1061. (3) (a) Hagerman, A. E.; Riedl, K. M.; Alexander, J. G.; Sovik, K. N.; Ritchard, N. T.; Hartzfeld, P. W.; Riechel, T. L. J. Agric. Food Chem. 1998, 46, 1887. (b) Saito, M.; Hosoyama, H.; Ariga, T.; Kataoka, S.; Yamaji, N. J. Agric. Food Chem. 1998, 46, 1460. (4) Li, C.; Xie, B. J. Agric. Food Chem. 2000, 48, 6362. (5) Kurisawa, M.; Chung, J. E.; Kim, Y. J.; Uyama, H.; Kobayashi, S. Biomacromolecules 2003, 4, 469. (6) Shimomura, O.; Wu, C.; Murai, A.; Nakamura, H. Anal. Biochem. 1998, 258, 230. (7) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1998, 48, 589. (8) (a) Guyot, S.; Vercauteren, J.; Cheynier, V. Phytochemistry 1996, 42, 1279. (b) Hamada, S.; Kontani, M.; Hosono, H.; Ono, H.; Tanaka, T.; Ooshima, T.; Mitsunaga, T.; Abe, I. FEMS Microbiol. Lett. 1996, 143, 35.
Synthesis and Properties of Poly(rutin) (9) (a) Loth, H. Arch. Pharm. 1961, 294, 498. (b) Loth, H. Arch. Pharm. 1962, 295, 161. (10) Mejias, L.; Reihmann, M. H.; Sepulveda-Boza, S.; Ritter, H. Macromol. Biosci. 2002, 2, 24. (11) (a) Kobayashi, S.; Uyama, H.; Kimura, S. Chem. ReV. 2001, 101, 3793. (b) Kobayashi, S.; Uyama, H.; Ohmae, M. Bull. Chem. Soc. Jpn. 2001, 74, 613. (c) Gross, R. A.; Kumar, A.; Kalra, B. Chem. ReV. 2001, 101, 2097. (12) Ikeda, R.; Sugihara, J.; Uyama, H.; Kobayashi, S. Polym. Int. 1998, 47, 295. (13) (a) Oguchi, T.; Tawaki, S.; Uyama, H.; Kobayashi, S. Macromol. Rapid Commun. 1999, 20, 401. (b) Uyama, H.; Maruichi, N.; Tonami, H.; Kobayashi, S. Biomacromolecules 2002, 3, 187. (14) Poly(rutin) prepared by a peroxidase-catalyzed oxidative polymerization in an aqueous 1,4-dioxane is estimated to be composed of C-C-linkage unit.10 (15) Takahama, U. Biochim. Biophys. Acta 1986, 882, 445. (16) Sakagami, H.; Oh-hara, T.; Kohda, K.; Kawazoe, Y. Chem. Pharm. Bull. 1991, 39, 950.
Biomacromolecules, Vol. 4, No. 5, 2003 1399 (17) Halliwell, B., Gutteridge, J. M. C., Eds. Free radicals in biology and medicine, 2nd ed.; Clarendon Press: Oxford, U.K., 1989. (18) (a) Floyd, R. A. FASEB J. 1990, J4, 2587. (b) Kitagawa, M.; Tokiwa, Y. Chem. Lett. 1998, 281. (19) (a) Hodnick, W. F.; Milosavljevic, E. B.; Nelson, J. H.; Pardini, R. S. Biochem. Pharmacol. 1988, 37, 2607. (b) Van Acker, S. A. B. E.; Van Den Berg, D.-J.; Tromp, M. N. J. L.; Griffioen, D. H.; Van Bennekom, W. P.; Van Der Vijgh, W. J. F.; Bast, A. Free Radical Biol. Med. 1996, 20, 331. (20) Ariga, T.; Hamano, M. Agric. Biol. Chem. 1990, 54, 2499. (21) Niki, E. Methods Enzymol. 1990, 186, 100. (22) Akasaka, K.; Suzuki, T.; Ohrui, H.; Meguro, H. Anal. Lett. 1987, 20, 797. (23) Halliwell, B. Lacent 1994, 344, 721. (24) Takahashi, M.; Shibata, M.; Niki, E. Free Radical Biol. Med. 2001, 31, 164.
BM034136B
