Biomacromolecules 2004, 5, 547-552
547
Superoxide Anion Scavenging and Xanthine Oxidase Inhibition of (+)-Catechin-Aldehyde Polycondensates. Amplification of the Antioxidant Property of (+)-Catechin by Polycondensation with Aldehydes Young-Jin Kim,† Joo Eun Chung,†,‡ Motoichi Kurisawa,†,‡ Hiroshi Uyama,*,† and Shiro Kobayashi*,† Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan, and Bio-oriented Technology Research Advancement Institution Received October 2, 2003; Revised Manuscript Received December 1, 2003
In this study, the antioxidant property of (+)-catechin-aldehyde polycondensates has been examined. Superoxide anions are one of the most typical reactive oxygen species (ROS) and generated by xanthine oxidase (XO). The measurements of the superoxide anion scavenging and XO inhibition activity showed that catechin had pro-oxidant properties in lower concentrations and little XO inhibition. On the other hand, the polycondensates exhibited much higher effects compared to the catechin monomer, and their physiological activities were greatly affected by the structure of polycondensates. Steady-state analysis of the inhibition against XO showed that the inhibition type of the polycondensate was uncompetitive. Furthermore, the results of the circular dichroism and UV-visible measurements of a mixture of the polycondensate and XO were in good agreement with that of the steady-state analysis; the spectral changes due to the chelation of the polycondensate onto the Fe/S and/or the FAD center of XO were observed. These data strongly suggest that the polycondensates possess a great potential as antioxidant for various applications. Introduction Reactive oxygen species (ROS) in the forms of hydrogen peroxide, hydroxyl radicals, and superoxide anions are byproducts of the normal metabolism. They attack biological molecules such as lipids, proteins, enzymes, DNA, and RNA, leading to cell or tissue injury associated with degenerative diasease.1 Among them, superoxide anions are one of the most typical ROS, which are formed during the normal aerobic metabolism and by activated phagocytes.2 The reduction of molecular oxygen to superoxide anion by xanthine oxidase (XO), generating hydroxyl radicals and uric acid, is an important physiological pathway.3 However, an excess of superoxide anions are capable of damaging biomacromolecules by both directly and indirectly forming hydrogen peroxide or highly reactive hydroxyl radicals.1,4 Although the organism possesses defense mechanisms to reduce the oxidative damage, such as enzymes (superoxide dismutase (SOD), catalase, and glutathione peroxidase) and antioxidant molecules to diminish the damaging properties of ROS, continuous exposure to chemicals and contaminants may lead to increase of free radicals in the body beyond control, and cause irreversible oxidative damage. Therefore, antioxidants help to protect the body against damages by ROS, and may have relevance as prophylactic and therapeutic * To whom correspondence should be addressed. Phone: +81-75383-2460; +81-75-383-2459. Fax: +81-75-383-2461. E-mail: uyama@ mat.polym.kyoto-u.ac.jp;
[email protected]. † Kyoto University. ‡ Bio-oriented Technology Research Advancement Institution.
agents for diseases in which oxidants or free radicals are implicated.5 Furthermore, fats and oils are easily deteriorated by oxidation. The addition of antioxidants to food is an effective way to prevent the formation of various off-flavors and undesirable compounds that result from lipid peroxidation.5,6 In this respect, the development of high-performance antioxidants is very important for preventive medicine and the food industry. XO is a complex molybdoflavoenzyme, containing a molybdenum, flavin adenine dinucleotide (FAD), and a pair of Fe/S redox centers in each of its two catalytically independent subunits,7 which catalyzes the oxidation of hypoxanthine and xanthine to uric acid accompanying the generation of superoxide anions. The intestinal mucosa and the liver are the richest sources of XO. The complete catalytic reaction of XO consists of two half reactions.8 During the first half of the catalytic reaction, called the reductive half reaction, xanthine binds to the molybdenum active center and transfers two electrons to the enzyme. In this process, the enzyme is reduced and uric acid is formed from xanthine as the oxidation product at the molybdenum active center. During the second half of the catalytic reaction, called the oxidative half reaction, molecular oxygen oxidizes the reduced form of XO, and superoxide anions are formed from oxygen at FAD. Catechin, an ingredient of green tea and wine, belongs to the flavonoids which are one of the most numerous and beststudied groups of plant polyphenols. The flavonoids consist of a large group of low-molecular-weight polyphenolic
10.1021/bm034392o CCC: $27.50 © 2004 American Chemical Society Published on Web 01/10/2004
548
Biomacromolecules, Vol. 5, No. 2, 2004
Kim et al.
Scheme 1
substances, naturally occurring in fruits, vegetables, tea, and red wine, and are an integral part of the human nutrition. Their biological and pharmacological effects, including antioxidant, anti-mutagenic, anti-carcinogenic, antiviral, and anti-inflammatory properties, have been demonstrated in numerous human, animal, and in vitro studies.9 These properties are potentially beneficial for the prevention of diseases and protection of the stability of the genome. Many of these effects have been related to the antioxidant effects of catechin.10 In general, the activities of flavonoids are known to be limited to only a few hours in the body, although metabolism of these compounds remains elusive. In addition, several flavonoids have been proven to act as pro-oxidants and to generate ROS, such as hydrogen peroxide.11 In contrast, the high-molecular-weight plant polyphenols have been reported to exhibit excellent biological and physiological properties such as antioxidative and anti-carcinogenic activity without any pro-oxidant effects.12 Many investigations have been done to assess the antioxidant effects of lowmolecular-weight flavonoids, and only a few have investigated the antioxidant properties of the polymeric flavonoids, most of which have concentrated to condensed tannins, a high-molecular fraction of extracted polyphenols from natural plants.12 We have designed and synthesized a polymerized catechin in order to improve its biological and physiological activities including antioxidative and XO inhibition effects.13 Poly(catechin), which was produced by an enzymatic oxidative polymerization, showed much greater superoxide anion scavenging and inhibition activity of XO than the catechin monomer.13a In aging of wine, it is well-known that catechinacetaldehyde condensed oligomers are formed involving oxidation of ethanol. We previously reported the regioselective synthesis and characterization of (+)-catechin-aldehyde polycondensates (1-5) (Scheme 1),14 which can be regarded as model of this natural oligomer. These polymers showed excellent inhibition effects against tyrosinase.15 In the present study, antioxidant properties, the scavenging activity of the superoxide anion, and the inhibition effect of XO of (+)-catechin-aldehyde polycondensates with different molecular structures were investigated. Experimental Section Materials. Xanthine oxidase (XO from buttermilk, EC 1.1.3.22) and xanthine used for the bioassay were purchased from Wako Pure Chemical Industries and used as received.
(+)-Catechin and flavin adenine dinucleotide (FAD) were obtained from Tokyo Kasei Kogyo Co., and 2-methyl-6-pmethoxy-phenylethynylimidazopyrazinone (MPEC) was purchased from ATTO Co. Ltd. Polycondensates were synthesized according to the literature.14 Their molecular weights (Mn) were 3700 (1), 2300 (2), 2300 (3), 1700 (4), and 2000 (5). Other reagents and solvents were commercially available and used as received. Superoxide Anion Scavenging Activity. Superoxide anions were generated by xanthine/XO and measured by the chemiluminescent superoxide anion probe method.16 Catechin and the polymer samples were first dissolved in DMSO and used at 10 times dilution with water. A test sample was mixed using a 0.1 M potassium phosphate buffer (pH 7.5) containing 0.05 mM EDTA, XO (40 milliunits/mL), and MPEC (10 µM) at 25 °C. Light emission was started by the addition of xanthine (300 µM). The changes of the chemiluminescence (CL) intensity triggered by the evolution of superoxide anions was monitored for 30 s using a Corona Microplate Photoncounter, MTP-700CL. The superoxide anion scavenging activity was calculated according to the following formula: superoxide anion scavenging activity (%) ) CLcontrol - CLsample × 100 CLcontrol where CLcontrol and CLsample represent the chemiluminescence intensity in the absence and presence of samples, respectively. XO Inhibition Activity. The XO inhibition activities of catechin and polycondensates were evaluated by measuring the amount of the formed uric acid. The assay mixture, containing XO (40 milliunits/mL) and sample in 0.1 M potassium phosphate buffer (pH 7.5) with 0.05 mM EDTA, was preincubated at 25 °C for 5 min. Then, xanthine (300 µM) was added to the mixture, and the increase of the optical density (OD) was immediately measured at 295 nm for 20 min in order to detect the amount of uric acid. The XO inhibition activities were calculated according to the following formula: XO inhibition activity (%) )
ODcontrol - ODsample × 100 ODcontrol
where ODcontrol and ODsample represent the optical density in the absence and presence of samples, respectively.
Scavenging and Inhibition of (+)-Catechin-Aldehyde
Figure 1. Superoxide anion scavenging activities of (+)-catechin and polycondensates, n ) 3.
Spectrophotometric Methods. Circular dichroism (CD) spectra were recorded on a Jasco J-820 spectropolarimeter at 25 °C maintaining a nitrogen flow rate of 5 mL/min. The CD spectra of the sample (100 µM) and a mixture with XO in 0.1 M potassium phosphate buffer (pH 7.5) were collected with a bandwidth of 1.0 nm and a resolution of 0.2 nm as a scan speed of 100 nm/min. For the measurement of the UVvisible spectra, the sample mixture containing Fe2+ or FAD was incubated at 25 °C for 10 min, and then the absorption spectra were recorded on a Hitachi U-2001 spectrometer. Results and Discussion Superoxide Anion Scavenging Activity. Superoxide anions and hydrogen peroxide can react in vitro to form the hydroxyl radicals, which can attack and destroy almost all known biomolecules. It has been suggested that most of the toxic effects of superoxide anions are due to the formation of hydroxyl radicals in vivo.12e In this study, the antioxidant activity of polycondensates with different molecular structures was evaluated by measuring the superoxide anion scavenging activity (Figure 1). A mixture of xanthine and XO generated superoxide anions which were reacted with MPEC to give light emission by chemiluminescence (CL).16 We found that the polycondensates showed remarkably higher scavenging activity on the basis of a monomeric repeating unit.13c The scavenging activities of polycondensates were dependent on the molecular structures; 2 and 5 exhibited the greatest scavenging activity and almost completely scavenged the superoxide anions at 100 µM. On the other hand, catechin showed pro-oxidant properties at lower concentrations, which is consistent with other investigations on flavonoids at lower dosages in aqueous phase.11 These results indicate that the polycondensate has a more potent scavenging activity toward superoxide anion than that of a monomeric catechin.13 XO Inhibition Activity. XO is not only an important biological source of reactive oxygen species (ROS) but also the enzyme responsible for the formation of uric acid associated with gout leading to painful inflammation in the joints.17 Figure 2 shows the XO inhibition activity assessed by determination of the uric acid formation. The inhibition
Biomacromolecules, Vol. 5, No. 2, 2004 549
Figure 2. XO inhibition activities of (+)-catechin and polycondensates, n ) 4.
effect of catechin was not exhibited up to the concentration of 300 µM. It was reported earlier that catechin derivatives such as (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin do not inhibit the XO activity up to the high concentration.18 Under the present conditions, however, all of the polycondensates efficiently inhibited XO activity.13c Moreover, the inhibition effects increased with increasing the concentration of the repeating catechin unit. Among them, 5 showed the highest inhibition activity. This may be due to the interaction of a phenol group of the side chain of 5 and XO by enhanced hydrogen bonds and electrostatic interactions.19 This highly amplified XO inhibition activity might partly contribute to the result of superoxide anion scavenging activity as shown Figure 1. The XO inhibition and superoxide anion scavenging activities of 5 at 100 µM were 48% and 95%, respectively. Therefore, the great inhibition effect of the polycondensate on the increase of the CL intensity resulted from the superoxide anion scavenging and XO inhibition. These results demonstrated that the polycondensates possessed a much higher potential for both the superoxide anion scavenging and XO inhibition, compared with catechin on molar basis of a monomeric repeating unit. Mechanism of Inhibition Actions of Polycondensate against XO. To further characterize the XO inhibition activity of the polycondensate, a steady-state analysis was performed in which the concentrations of xanthine and 1 were varied systematically. The lines of the LineweaverBurk plots were parallel at different concentrations of 1 (Figure 3). This result indicates that 1 is an uncompetitive inhibitor of XO with respect to xanthine as substrate, suggesting that 1 can inhibit the enzyme activity only by binding 1 to the enzyme-substrate complex but not by binding 1 to the active center of the enzyme because the substrate is bound to the active center.20 In the presence of 1, the maximum velocity of the reaction (Vmax) and Michaelis-Menten constant (Km) were lower than those in the absence of 1; that is, a decrease of Vmax from 13.8 to 6.2 µM/min and a decrease of Km from 65.7 to 42.9 µM were found (Table 1). To prove the inhibition mechanism of catechin and polycondensates onto XO, we examined their chelation behavior with ferreous ions, FAD, molybdenum ions, and XO. Allopurinol, a powerful inhibitor of XO, is structurally
550
Biomacromolecules, Vol. 5, No. 2, 2004
Kim et al.
Figure 3. Lineweaver-Burk plots for the inhibition of XO by polycondensate (1) with respect to xanthine as substrate. Table 1. Kinetic Parameters for XO Inhibition by Polycondensate (1)a concentration (µM)b
Vmax (µM/min)
Km (µM)
0 25 50 100 200
13.8 12.8 11.6 9.3 6.2
65.7 63.7 61.0 53.7 42.9
Figure 4. Double-difference CD spectra of (+)-catechin (100 µM)XO (A) and polycondensate (1) (100 µM)-XO (B) complexes in 0.1 M potassium phosphate buffer (pH 7.5). Each spectrum was obtained by subtracting the spectrum of catechin or 1 in the absence of XO and the spectrum of XO from that of catechin-XO or the 1-XO complex, respectively.
a Kinetic parameters were determined by the vertical axis intercept (Vmax) and extrapolation to horizontal axis (Km) of the Lineweaver-Burk plots in the absence and presence of the polycondensate (1). b Concentration of a monomeric repeating unit of the polycondensate (1).
similar to xanthine and is oxidized by the enzyme to give oxyallopurinol, which binds tightly to the active center of XO and therefore causes strong inhibition.7 It is interesting to note that the chemical structure of the A ring of flavonols has many analogies to the structure of xanthine and uric acid. Therefore, like allopurinol, they bind to the active center in the enzyme and then inactivate XO by the reduction of the enzyme.7,18 On the other hand, some flavonoids are known to inhibit the reduction of the electron acceptor such as molecular oxygen.20 The conformational change of XO caused by the addition of catechin or the polycondensate (1) was first measured with circular dichroism (CD). XO showed a rather intense CD spectrum, which strongly resembles that of spinach ferredoxin and other 2Fe/2S proteins;8 the spectrum of XO exhibited a positive band at 440 nm corresponding to the Fe/S and FAD absorbance, a negative band at 535 nm corresponding to other Fe/S absorbance, and a small negative band at 630 nm due to the molybdenum center (data not shown).8,21 For a mixture of catechin and XO, the change of the CD band was not observed (Figure 4). However, in the CD spectrum of a mixture of 1 and XO, the intensity of the positive band due to the Fe/S and FAD centers of XO decreased. In addition, the characteristic band due to the additional Fe/S center of XO became larger. These spectral changes may be attributable to the reduction of the Fe/S and/ or the FAD center by the formation of 1-XO.21 A very similar behavior was observed in the reduction of the Fe/S centers of XO by sodium dithionite titration. This means the 1 f Fe/S and/or the 1 f FAD charge-transfer transition, resulting in the reduction of the Fe/S and/or the FAD center
Figure 5. Difference UV absorption spectra of (+)-catechin (100 µM)-Fe2+ (50 µM) (A) and polycondensate (1) (100 µM)-Fe2+ (50 µM) (B) complexes. Each spectrum was obtained by subtracting the spectrum of catechin or 1 in the absence of Fe2+ from that in the presence of Fe2+.
of XO. On the other hand, the spectral change due to the chelation to the molybdenum active center was not observed in the present conditions. This is in good agreement with the result of the steady-state analysis; an uncompetitive inhibitor cannot bind to the active center of the enzyme. The UV-visible spectrum of a mixture of catechin or 1 with Fe2+ was measured to elucidate the chelation behaviors. In the UV-visible spectrum, 1 showed a characteristic shoulder peak centered at 420 nm in the presence of Fe2+, due to the chelation with Fe2+,21 whereas the peak was not exhibited by the addition of catechin (Figure 5). Like the case of the CD measurement, neither of the spectral changes of catechin and polycondensate in the presence of Mo (VI) was observed (data not shown). We also examined the complex formation of catechin or polycondensate with FAD by UV-visible measurements. The change of the UV absorption spectra of FAD was observed in the presence and absence of 1 in potassium phosphate buffer (pH 7.5). By adding 1 to the buffer solution containing FAD, the specific peaks around 350 and 445 nm increased, due to the chelation with FAD (Figure 6). However, these spectral changes caused by the addition of
Scavenging and Inhibition of (+)-Catechin-Aldehyde
Biomacromolecules, Vol. 5, No. 2, 2004 551
inhibition activity of the present polycondensates for applications in medicinal and pharmacological fields are under way in our laboratory. Acknowledgment. This work was partly supported by Program for Promotion of Basic Research Activities for Innovative Bioscience and by the 21st Century COE Program, COE for a United Approach to New Materials Science. References and Notes
Figure 6. Double-difference UV spectra of (+)-catechin (100 µM)FAD (50 µM) (A) and polycondensate (1) (100 µM)-FAD (50 µM) (B) complexes. Each spectrum was obtained by subtracting the spectrum of catechin or 1 in the absence of FAD and the spectrum of FAD from that of the catechin-FAD or the 1-FAD complex, respectively.
catechin were negligibly small. These UV-visible data are in good agreement with those obtained by CD; the chelation of 1 to the Fe/S and/or the FAD center of XO is clearly proved by the changes of the UV-visible spectra and the CD bands induced. These data strongly suggest that the XO inhibition by the polycondensates is caused by reducing the Fe/S and/or the FAD center of XO. The oxidation of xanthine takes place at the molybdenum center. After gaining two electrons from the substrate, the formed Mo (IV) passes two electrons sequentially, by way of the Fe/S centers, to the FAD center which then reduces molecular oxygen to generate superoxide anions.22 Moreover, the molybdenum center deprotonates upon reoxidation to Mo (VI) transferring two electrons to the Fe/S centers. On the other hand, the removal of FAD eliminates both the XO activity and the reactivity of the reduced enzyme with molecular oxygen. In this respect, 1 truly acted as an inhibitor of XO by chelation to the Fe/S and/or the FAD center. This chelation inhibits the intramolecular electron transfer processes from the molybdenum center to the Fe/S and FAD centers and, consequently, interrupts the reoxidation of the molybdenum center. Conclusions Due to their potential applications in the treatment of neurodegenerative disorders, chronic condition, and inflammatory processes in human beings, the discovery of new antioxidants has attracted much interest in recent years. A number of synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have already extensively been used as additives to foodstuffs. However, their use has begun to be questioned because of their toxicity. In the present study, we have found that (+)catechin-aldehyde polycondensates exhibited much greater superoxide anion scavenging and XO inhibition activity than the catechin monomer, resulting in the attenuation of the process involving ROS. Interestingly, the polycondensate binds to the enzyme-substrate complex only but not to the active center of enzyme. Further investigations on the enzyme
(1) (a) Yen, G.-C.; Chen, H.-Y. J. Agric. Food Chem. 1995, 43, 27. (b) Pietta, P.; Simonetti, P.; Mauri, P. J. Agric. Food Chem. 1998, 46, 4487. (2) Fantone, J. C.; Ward, P. A. Hum. Pathol. 1985, 16, 973. (3) Halliwell, B.; Gutteridge, J. M. C. In Free radicals in biology and medicine, 2nd ed.; Clarendon Press: Oxford, U.K., 1989. (4) (a) Floyd, R. A. FASEB J. 1990, 4, 2587. (b) Halliwell, B.; Gutteridge, J. M. C.; Cross, C. E. J. Lab. Clin. Med. 1992, 119, 598. (c) Gyamfi, M. A.; Yonamine, M.; Aniya, Y. Gen. Pharmacol. 1999, 32, 661. (d) Miyake, Y.; Shimoi, K.; Kumazawa, S.; Yamamoto, K.; Kinae, N.; Osawa, T. J. Agric. Food Chem. 2000, 48, 3217. (e) Pulido, R.; Bravo, L.; Saura-Calixto, F. J. Agric. Food Chem. 2000, 48, 3396. (5) (a) Soares, J. R.; Dinis, T. C. P.; Cunha, A. P.; Almeida, L. M. Free Radical Res. 1997, 26, 469. (b) Tseng, T.-H.; Kao, E.-S.; Chu, C.Y.; Chou, F.-P.; Lin Wu, H.-W.; Wang, C.-J. Food Chem. Toxicol. 1997, 35, 1159. (6) Jung, H. A.; Park, J. C.; Chung, H. Y.; Kim, J.; Choi, J. S. Arch. Pharm. Res. 1999, 22, 213. (7) Lin, J. K.; Chen, P. C.; Ho, C. T.; Lin-Shiau, S. Y. J. Agric. Food Chem. 2000, 48, 2736. (8) (a) Hille, R.; George, G. N.; Eidsness, M. K.; Cramer, S. P. Inorg. Chem. 1989, 28, 4018. (b) Sau, A. K.; Mondal, M. S.; Mitra, S. J. Chem. Soc., Dalton Trans. 1998, 4173. (9) (a) Jankun, J.; Selman, S. H.; Swiercz, R.; Skrzypezak-Jankun, E. Nature 1997, 387, 561. (b) Bodoni, A.; Hrelia, S.; Angeloni, C.; Giordano, E.; Guarnier, C.; Caldarera, C. M.; Biagi, P. L. J. Nutr. Biochem. 2002, 13, 103. (10) (a) Jovanovic, S. V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic, M. G. J. Am. Chem. Soc. 1994, 116, 4846. (b) Zhao, J.; Wang, J.; Chen, Y.; Agarwal, R. Carcinogenesis 1999, 20, 1737. (11) (a) Laughton, M. J.; Halliwell, B.; Evans, P. J.; Robin, J.; Hoult, S. Biochem. Pharmacol. 1989, 38, 2859. (b) Roedig-Penman, A.; Gordon, M. H. J. Agric. Food Chem. 1997, 45, 4267. (c) Yamanaka, N.; Oda, O.; Nagao, S. FEBS Lett. 1997, 401, 230. (d) Yen, G.-C.; Chen, H.-Y.; Peng, H.-H. J. Agric. Food Chem. 1997, 45, 30. (12) (a) Ariga, T.; Hamano, M. Agric. Biol. Chem. 1990, 54, 2499. (b) Hagerman, A. E.; Riedl, K. M.; Jones, G. A.; Sovik, K. N.; Ritchard, N. T.; Hartzfeld, P. W.; Riechel, T. L. J. Agric. Food Chem. 1998, 46, 1887. (c) Saito, M.; Hosoyama, H.; Ariga, T.; Kataoka, S.; Yamaji, N. J. Agric. Food Chem. 1998, 46, 1460. (d) Duweler, K. G.; Rohdewald, P. Pharmazie 2000, 55, 364. (e) Li, C.; Xie, B. J. Agric. Food Chem. 2000, 48, 6362. (13) (a) Kurisawa, M.; Chung, J. E.; Kim, Y. J.; Uyama, H.; Kobayashi, S. Biomacromolecules 2003, 4, 469. (b) Kurisawa, M.; Chung, J. E.; Uyama, H.; Kobayashi, S. Biomacromolecules 2003, 4, 1394. (c) Chung, J. E.; Kurisawa, M.; Kim, Y. J.; Uyama, H.; Kobayashi, S. Biomacromolecules 2004, 5, 113-118. (14) Kim, Y. J.; Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Macromol. Chem. Phys. 2003, 204, 1863. (15) Kim, Y. J.; Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Biomacromolecules in press. (16) Shinomura, O.; Wu, C.; Murai, A.; Nakamura, H. Anal. Biochem. 1998, 258, 230. (17) (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. (18) (a) Chang, P.; Lee, Y.; Lu, F.; Chiang, H. Anticancer Res. 1993, 12, 2165. (b) Cos, P.; Ying, L.; Calomme, M.; Hu, J. P.; Cimanga, K.; Poel, B. V.; Pieters, L.; Vlietinck, A. J.; Berghe, D. V. J. Nat. Prod. 1998, 61, 71. (19) Lin, C. M.; Chen, C. S.; Chen, C. T.; Liang, Y. C.; Lin, J. K. Biochem. Biophys. Res. Commun. 2002, 294, 167. (20) (a) Saito, T.; Nishino, T. J. Biol. Chem. 1989, 264, 10015. (b) Moini, H.; Guo, Q.; Packer, L. J. Agric. Food Chem. 2000, 48, 5630.
552
Biomacromolecules, Vol. 5, No. 2, 2004
(21) (a) Davis, M. D.; Olson, J. S.; Palmer, G. J. Biol. Chem. 1982, 257, 14730. (b) Porras, A. G.; Palmer, G. J. Biol. Chem. 1982, 257, 11617. (c) Davis, M. D.; Olson, J. S.; Palmer, G. J. Biol. Chem. 1984, 259, 3526. (d) Hille, R.; Hagen, W. R.; Dunham, W. R. J. Biol. Chem. 1985, 260, 10569. (e) Ryan, M. G.; Ratnam, K.; Hille, R. J. Biol. Chem. 1995, 270, 19209.
Kim et al. (22) (a) Komai, H.; Massey, V.; Palmer, G. J. Biol. Chem. 1969, 244, 1692. (b) Tai, L. A.; Hwang, K. C. Angew. Chem., Int. Ed. 2000, 39, 3886. (c) Hille, R.; Anderson, R. F. J. Biol. Chem. 2001, 276, 31193.
BM034392O
