May/June 2003
Published by the American Chemical Society
Volume 4, Number 3
© Copyright 2003 by the American Chemical Society
Communications Amplification of Antioxidant Activity and Xanthine Oxidase Inhibition of Catechin by Enzymatic Polymerization Motoichi Kurisawa,†,‡ Joo Eun Chung,†,‡ Young Jin Kim,† Hiroshi Uyama,*,† and Shiro Kobayashi*,† Bio-oriented Technology Research Advancement Institute, Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501, Japan Received January 10, 2003; Revised Manuscript Received February 27, 2003
To amplify the antioxidant activity, we synthesized poly(catechin) by the enzyme-catalyzed oxidative coupling using horseradish peroxidase as a catalyst. The poly(catechin) showed great improvement in antioxidant activity such as radical scavenging activity against the superoxide anion and inhibition effects against free radical induced-oxidation of low-density lipoprotein, compared with a catechin monomer. In addition, poly(catechin) showed very high inhibition effects on xanthine oxidase activity, whereas the catechin monomer showed very less inhibition effects. The amplified activities might offer a high potential as a therapeutic agent for prevention of various free radicals and/or enzyme-related diseases. Hydrogen peroxide, hydroxyl radicals, peroxide anions, and superoxide anions are generally known as reactive oxygen species (ROS) inducing aging and many kinds of diseases such as mutagenesis and carcinogenesis.1 Xanthine oxidase (XO) is not only an important biological source of ROS but also the enzyme responsible for the formation of uric acid associated with gout leading to painful inflammation in the joints.2 Thus, antioxidation and XO inhibition are an important pharmacological action, and antioxidants possessing both ROS scavenging and XO inhibition abilities may be used as protective agents in a number of diseases related to ROS and/or XO. Green tea catechins, belonging to the group of flavonoids, exhibit biological and pharmacological effects including antioxidant, antimutagenic, anticarcinogenic, antimicrobial, and anti-inflammatory properties in numerous human, ani* To whom correspondence should be addressed. Phone: +81-75-7535638; +81-75-753-5608. FAX: +81-75-753-4911. E-mail: uyama@ mat.polym.kyoto-u.ac.jp;
[email protected]. † Kyoto University. ‡ Bio-oriented Technology Research Advancement Institute.
mal, and in vitro studies.3 These properties might potentially be beneficial in preventing disease and protecting the stability of the genome. However, the activities of flavonoids generally persist for limited short periods in vivo. In addition, several flavonoids have been shown to pro-oxidize and generate reactive oxygen species, such as hydrogen peroxide. Tea polyphenol was also reported to have pro-oxidant effects at lower dosages in the aqueous phase.4 In contrast, high molecular weight plant polyphenols such as tannins and procyanidines have been reported to show no pro-oxidant effect. Moreover, a relatively high molecular fraction of extracted flavonoids has been reported to exhibit enhanced physiological properties such as antioxidant and anticarcinogenic activity, as well as a relatively longer circulation time in a body.5 From these perspectives, we have designed polymerized flavonoids in order to improve their biological and physiological activity including antioxidant action and XO inhibition. Polymerized flavonoids might be also expected to prolong the activity for a relatively long period of time because of the high molecular weight.
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Figure 1. Superoxide scavenging activity of poly(catechin), n ) 3.
We report, herein, the horseradish peroxidase (HRP)catalyzed polymerization6 of green tea catechin and demonstrate the amplification of antioxidant and XO inhibition activities of the resultant polymer. Enzymatic oxidation of flavonoid is very important in biochemistry, because the subsequent coupling reactions are involved in some biosynthetic pathways, such as tannin and melanin formation. The oxidative coupling of catechin using peroxidase or polyphenol oxidase as catalyst was reported to form oligomeric compounds with complicated structures.7-9 In this study, the catechin polymer was synthesized by an oxidative reaction catalyzed by HRP.10 The number-average molecular weight of the catechin polymer obtained was determined to be 1.4 × 104 (index ) 2.4) by SEC measurement. In UVvisible measurements, catechin showed one peak at 280 nm due to the π-π* transitions of the aromatic fragment. In the case of poly(catechin), an additional broader peak was seen at 380 nm which may be attributed to conjugation in the polymer; the aromatic carbon-carbon linkage between catechin molecules is formed via the oxidative coupling of catechin. Superoxide anions are well-known to be generated by a xanthine/xanthine oxidase (XO) system. They could damage the biomacromolecules both directly and indirectly by forming hydrogen peroxide or the reactive hydroxyl radical.11 The antioxidant activity of poly(catechin) was evaluated in terms of the superoxide anion scavenging ability.12 The superoxide anion was generated by xanthine/XO and measured by the chemiluminescent superoxide probe method.13 Poly(catechin) greatly scavenged superoxide anions in a concentration dependent manner, and almost completely scavenged at 200 µM of a catechin unit concentration (Figure 1). Conversely, catechin showed pro-oxidant property in lower concentrations than 300 µM. This pro-oxidant property is consistent with some investigations often reported for tea polyphenol at lower dosages in the aqueous phase.4 Figure 2 showed XO inhibition activity assessed by evaluating uric acid formation from XO.14 The XO inhibition effect by poly(catechin) was increased as an increasing concentration of catechin units. In contrast, catechin showed a low XO inhibition effect less than about 10% inhibition over a range of tested concentrations. This highly amplified XO inhibition ability of poly(catechin) was considered to be due to the effective multivalent interaction between XO and the polymeric chains of poly(catechin). The XO inhibition effect might partly contribute to the results shown in Figure 1. The XO inhibition and superoxide anion scavenging
Communications
Figure 2. XO inhibition activity of poly(catechin).
Figure 3. Inhibition activity of poly(catechin) against LDL oxidation.
activity of poly(catechin) at 200 µM were 34% (Figure 2) and 90% (Figure 1), respectively. Therefore, the great inhibition effect of poly(catechin) on the chemiluminescence growth resulted predominantly from superoxide anion scavenging rather than from XO inhibition. These results demonstrated that the poly(catechin) possessed much higher potential for both superoxide anion scavenging and XO inhibition, compared with catechin. Oxidation of low-density lipoprotein (LDL) leads to its enhanced uptake by macrophages; this is believed to lead to foam cell formation, which is one of the first stages of atherogenesis. Therefore, antioxidants that protect LDL against oxidation are potentially anti-atherogenic compounds. Although the mechanism for in vivo oxidation of LDL has not been established, free radical autoxidation may be a factor. Incubation of 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), a radical generator, with LDL generates peroxyl radicals, leading to a chain reaction which produces peroxidation products such as hydroperoxides and aldehydes.15 To evaluate the extent of LDL oxidation, LDL was labeled with diphenyl-1-pyrenylphosphine (DPPP), a fluorescent probe sensing hydroperoxide produced by the lipid oxidation. DPPP, a nonfluorescent molecule, reacts stoichiometrically with hydroperoxide to give diphenyl-1-pyrenylphosphine oxide (DPPPdO), which is strongly fluorescent.16 AAPH-induced oxidation of DPPP-labeled LDL which was preincubated with catechin or poly(catechin) was evaluated by measurement of fluorescence intensity of DPPPdO (Figure 3). Poly(catechin) showed much greater inhibition activity against LDL oxidation in a concentration dependent manner, compared to a catechin monomer. These
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data imply that the structure of poly(catechin) is much more capable of inhibiting oxidation of LDL than that of the monomer. In conclusion, the catechin polymer was synthesized by an oxidative enzymatic polymerization using HRP as the catalyst under a mild reaction condition. Poly(catechin) showed much greater superoxide scavenging and XO inhibitory activity than the catechin monomer. For LDL oxidation induced by AAPH, poly(catechin) also showed greatly amplified antioxidant activity, compared with the monomer. These results implied that poly(catechin) would be much more potent both to scavenge free radicals and to inhibit XO than the monomer. We believe that poly(catechin) is useful for a therapeutic agent to offer protection against a wide range of free radical-induced and/or enzyme-related diseases. Acknowledgment. This work was supported by Program for the Promotion of Basic Research Activities for Innovative Bioscience.
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References and Notes (1) (a) Breimer, L. H. Mol. Carcinog. 1990, 3, 188. (b) Frenkel, K. Pharmacol. Ther. 1992, 53, 127. (2) (a) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1968, 243, 5753. (b) Chiang, H. C.; Lo, Y. J.; Lu, F. J. J. Enzyme Inhibition 1994, 8, 61. (3) (a) Wang, Z. Y.; Cheng, S. J.; Zhou, Z. C.; Athar, M.; Khan, W. A.; Bichers, D. R.; Mukhtar, H. Mutat. Res. 1989, 223, 273. (b) Valcic, S.; Timmermann, B. N.; Alberts, D. S.; Wachter, G. A.; Krutzsch, M.; Wymer, J.; Guillen, J. M. Anticancer Drugs 1996, 7, 461. (c) Roeding-Penman, M.; Gordon, H. J. Agric. Food Chem. 1997, 45, 4267. (d) Nanjo, F.; Mori, M.; Goto, K.; Hara, Y. Biosci., Biotechnol., Biochem. 1999, 63, 1621. (e) Fujimura, Y.; Tachibana, F.; Yamada, K. J. Agric. Food Chem. 2001, 49, 2527. (4) (a) Yen, G. C.; Chen, Y.; Peng, H. H. J. Agric. Food Chem. 1997, 45, 30. (b) Yamanaka, N.; Oda, O.; Nagao, S. FEBS Lett. 1997, 401, 230. (5) (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. (c) Hayakawa, S.; Kimura, T.; Saeki, K.; Koyama, Y.; Aoyagi, Y.; Noro, T.; Nakamura, Y.; Isemura, M. Biosci. Biotechnol. Biochem. 2001, 65, 459. (6) (a) Kobayashi, S.; Shoda, S.; Uyama, H. AdV. Polym. Sci. 1995, 121, 1. (b) Ayyagari, M.; Akkara, J. A.; Kaplan, D. L. Acta Polym. 1996,
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(15) (16) (17)
47, 193. (c) Gross, R. A.; Kaplan, D. L.; Swift, G. (Eds.) ACS. Symp. Ser. 1998, 684. (d) Kobayashi, S.; Uyama, H.; Kimura, S. Chem. ReV. 2001, 101, 3793. (a) Guyot, S.; Vercauteren, J.; Cheynier, V. Phytochem. 1996, 42, 1279. (b) Mejias, L.; Reihmann, M. H.; Sepulveda-Boza, S.; Ritter, H. Macromol. Biosci. 2001, 2, 24. Poly(catechin) prepared by HRP-catalyzed oxidative polymerization is estimated to be composed of C-C-linkage units at the 2′ and 5′ positions of catechin,7 whose structure is different from that of procyanidine, an oligomer of catechin linked at 4 and 8 positions of catechin. So far, antioxidant activity and its related properties of polymerized catechin have not been evaluated to our knowledge. Catechin (1.45 g, 5.0 mmol) and HRP (5 mg) in a mixture of 9 mL of methanol and 21 mL of 0.1 M phosphate buffer solution (pH 7) were placed in a 50 mL flask. The polymerization reaction was carried out for 24 h at room temperature under air with dropwise addition of hydrogen peroxide (5% aqueous solution, 3.4 mL) to the mixture. After reaction, a soluble part of the product was lyophilized (yield 93%). The molecular weight was estimated by size exclusion chromatography (SEC, Tosoh GPC-8020 equipped with RI-8020 detector). The SEC analysis was performed with TSKgel R 3000 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. Floyd, R. A. FASEB J. 1990, 4, 2587. The chemiluminescence (CL) intensity of 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone (MPEC) triggered by the 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 from butter milk, MPEC (10 µM, ATTO Co. Ltd. Japan), 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 according to the following formula: Superoxide scavenging activity (%) ) [(CLcontrol - CLsample)/CLcontrol] × 100, where CLcontrol and CLsample represent the chemiluminescent intensity in the absence and presence of samples, respectively. Shimomura, O.; Wu, C.; Murai, A.; Nakamura, H. Anal. Biochem. 1998, 258, 230. The activity of XO was measured spectrophotometrically by monitoring the formation of uric acid from xanthine at 295 nm for 20 min by a UV-visible spectrometer (Hitachi U-2001, Japan).17 The assay was carried out at the same condition as that of the superoxide anion assay mentioned above, and the percentage of activity was calculated. Niki, E. Methods Enzymol. 1990, 186, 100. Akasaka, K.; Suzuki, T.; Ohrui, H.; Meguro, H. Anal. Lett. 1987, 20, 797. Noro, T.; Oda, Y.; Miyase, T.; Ueno, A.; Fukushima, S. Chem. Pharm. Bull. 1983, 31, 2708.
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