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New Tyrosinase Inhibitors, (+)-Catechin-Aldehyde Polycondensates 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 August 27, 2003; Revised Manuscript Received December 1, 2003
In this study, new tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates, have been developed. Tyrosinase is a copper-containing enzyme that catalyzes the hydroxylation of a monophenol (monophenolase activity) and the oxidation of an o-diphenol (diphenolase activity). In the measurement of tyrosinase inhibition activity, (+)-catechin acted as substrate and cofactor of tyrosinase. On the other hand, the polycondensates inhibited the tyrosine hydroxylation and L-DOPA oxidation by chelation to the active site of tyrosinase. The UV-visible spectrum of a mixture of tyrosinase and the polycondensate exhibited a characteristic shoulder peak ascribed to the chelation of the polycondensate to the active site of tyrosinase. Furthermore, circular dichroism measurement showed a small red shift of the band due to the interaction between tyrosinase and the polycondensate. These data support that the polycondensate acts as an inhibitor of tyrosinase. Introduction Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes two distinct reactions of melanin synthesis; the hydroxylation of tyrosine by monophenolase action and the oxidation of L-DOPA to o-dopaquinone by diphenolase action. o-Dopaquinone is unstable in aqueous solution and rapidly suffers a nonenzymatic cyclization to leukodopachrome, which is further oxidized nonenzymatically by another molecule of o-dopaquinone to yield dopachrome and one molecule of regenerated L-DOPA.1,2 Tyrosinase widely exists in plants and animals, and is involved in the formation of melanin pigments.3,4 In the food industry, tyrosinase is a very important enzyme in controlling the quality and economics of fruits and vegetables.4,5 Tyrosinase catalyzes the oxidation of phenolic compounds to the corresponding quinones and is responsible for the enzymatic browning of fruits and vegetables. Therefore, development of highperformance tyrosinase inhibitors has strongly been required in agricultural and food fields. Tyrosinase plays an important role in the developmental and defensive functions of insects. Tyrosinase is involved in melanogenesis, wound healing, parasite encapsulation, and sclerotization in insects.6 Recently, the development of tyrosinase inhibitors has become an active alternative approach to control insect pests. In addition, tyrosinase inhibitors have become increasingly important for medicinal and cosmetic products that may be used to prevent or treat pigmentation disorders.7 Tyrosinase may also be a target for * Corresonding authors. Telephone: +81-75-383-2460 (H.U.); +81-75383-2459 (S.K.). FAX: +81-75-383-2461. E-mail:
[email protected]. kyoto-u.ac.jp (H.U.);
[email protected] (S.K.). † Kyoto University. ‡ Bio-oriented Technology Research Advancement Institution.
developing medicines to treat hypopigmentation-related problems, such as albinism.3 In the formation of melanin pigments, three types of tyrosinase (met-, oxy-, and deoxytyrosinases) with different binuclear copper structures of the active site are involved.8-10 Mettyrosinase contains two tetragonal Cu(II) ions antiferromagnetically coupled through an endogenous bridge, although hydroxide exogenous ligands other than peroxide are bound to the copper site. This species can be converted by addition of peroxide to oxytyrosinase, which in turn decays back to mettyrosinase when the peroxide is lost. Oxytyrosinase also consists of two tetragonal Cu(II) atoms, each coordinated by two strong equatorial and one weaker axial NHis ligands. The exogenous oxygen molecule is bound as peroxide and bridges the two Cu centers. Deoxytyrosinase has a bicuprous structure (Cu(I)-Cu(I)). Structural models for their active site have been proposed.10,11 The mechanism for the monophenolase activity of tyrosinase has widely been studied9,12 on the basis of these three forms of the enzyme. Kinetic studies on the steady-state of the pathway show the lower catalytic efficiency of tyrosinase on monophenols than o-diphenols.10,13 The monophenolase activity is typically characterized by a lag time12,14 which is dependent on various factors such as substrate and enzyme concentrations, and presence of a hydrogen donor.2 In the kinetic studies, the lag time is the time required for the resting met form, to be drawn into the active deoxy form by the reducing agent, arising by the action of the small amounts of the oxy form usually accompanying the met form. It was reported that L-DOPA at a very low concentration was the most effective reducing agent for elimination of the lag time.2,14 Flavonoids are one of the most numerous and best-studied group of plant polyphenols. Green tea catechins, belonging
10.1021/bm034320x CCC: $27.50 © 2004 American Chemical Society Published on Web 01/10/2004
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Scheme 1
to the group of flavonoids, exhibit biological and pharmacological effects including antioxidant, antimutagenic, anticarcinogenic, probiotic, antimicrobial, and antiinflammatory properties in numerous human, animal, and in vitro studies.15 It was reported that some flavonoids inhibited tyrosinase activity by active site chelation and others acted as cofactor and/or substrate of tyrosinase.16-18 In the presence of reducing agents (hydrogen donors) known as a cofactor, especially o-diphenol derivatives such as L-DOPA, tyrosinase was activated and the lag time was shortened or abolished.14,16 We have designed polymerized catechins in order to improve their biological and physiological activity. Poly(catechin), which was synthesized by an enzymatic oxidative polymerization, showed much greater superoxide scavenging and inhibition activity of xanthine oxidase than catechin monomer.19 We previously reported the regioselective synthesis and characterization of (+)-catechin-aldehyde polycondensates (1-5) (Scheme 1).20 This study deals with new tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates showing a strong inhibition toward both monophenolase and diphenolase activities. We also demonstrate the mechanism of tyrosinase inhibition activity of these polycondensates. Experimental Section Materials. Mushroom tyrosinase (EC 1.14.18.1) used for the bioassay was purchased from Sigma Chemical Co. and used as received. L-Tyrosine, L-DOPA (L-3,4-dihydroxyphenylalanine), and (+)-catechin were obtained from Tokyo Kasei Kogyo Co. Polycondensates were synthesized according to the literature.20 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. Enzyme Assay. Mushroom tyrosinase used in this study was a mixture of 85% met (resting form) and 15% oxy forms. The polymer samples were first dissolved in DMSO and used at 10 times dilution with water. For the measurement of monophenolase inhibition activity, 0.5 mL of 20 mM tyrosine solution was mixed with 4.3 mL of 0.1 M phosphate buffer (pH 6.8). Then, 0.1 mL of the sample solution (100 unit µM) and 0.1 mL of tyrosinase solution (300 units) were added to the mixture to immediately measure the increase of optical density (OD) at 475 nm for 20 min for detection of dopachrome formed.
For the measurement of diphenolase inhibition activity, 0.1 mL of the sample solution (100 unit µM) and 0.1 mL of tyrosinase solution (300 units) were mixed with 4.3 mL of 0.1 M phosphate buffer (pH 6.8) and preincubated at 25 °C for 10 min. Then, 0.5 mL of 10 mM L-DOPA was added to the mixture and incubated at 25 °C for 10 min. Afterward, the OD was immediately measured at 475 nm. Diphenolase inhibition activity was calculated according to the following formula. diphenolase inhibition activity (%) ) [ODcontrol - (ODsample - ODinactivated)] × 100 ODcontrol where ODcontrol and ODsample represent optical density in the absence and presence of sample, respectively. ODinactivated represents optical density using the inactivated tyrosinase. Electrochemical and Spectrophotometric Methods. Cyclic voltammograms were recorded using Bioanalytical Systems equipment at a 100 A electrochemical analyzer. The electrochemical cell was equipped with an Aurum working electrode, a platinum auxiliary electrode, and a Ag/AgCl reference electrode. The measurement was carried out in 0.1 M phosphate buffer including 3.0 mM of substrate and 0.1 M NaOH at a sweep rate of 50 mV‚s-1. Circular dichroism spectra were recorded on a Jasco J-820 spectropolarimeter at 25 °C in a nitrogen flow rate of 5 mL/min. UV-visible spectra were recorded on a Hitachi U-2001 spectrophometer at 25 °C. Results and Discussion Monophenolase Inhibition Activity. In the tyrosinasecatalyzed oxidation of tyrosine with and without catechin or the polycondensates, the formation of dopachrome was monitored by a UV-visible spectrometer at 475 nm for evaluation of monophenolase inhibition activity. Except catechin, a lag time was observed (Figure 1). The lag time is derived from oxidative hydroxylation of monophenolic substrates.12,14 It was reported that the lag time disappeared in the presence of catechin, in which catechin acted as cofactor and substrate for monophenolase action.16,18 Under the present conditions, tyrosine was oxidized by tyrosinase without the lag time in the presence of catechin, as shown in Figure 1. In addition, the increase of the absorbance at 475 nm by the hydroxylation of tyrosine in the presence of catechin was much larger than that by the oxidation of
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Figure 1. Monophenolase inhibition activities of (+)-catechin and polycondensates against the hydroxylation of tyrosine.
Figure 2. Diphenolase inhibition activities of (+)-catechin and polycondensates against the oxidation of L-DOPA, n ) 5.
catechin without tyrosine (data not shown), supporting that this absorbance increase is mainly due to the oxidation of tyrosine. In the oxidation of tyrosine with polycondensate (1), on the other hand, the lag time became longer than that without an additive and the absorbance at 475 nm was the smallest after 20 min. These data indicate that 1 is the most effective inhibitor for monophenolase. In the case of 2-5, the lag time was somewhat shorter than that in the absence of the inhibitor; however, the formation of dopachrome was much more suppressed, as compared with catechin or without any additives, showing that 2-5 are also good inhibitors of monophenolase. Considering that a cofactor is a reductant or proton donor which can reduce mettyrosinase to active deoxytyrosinase,14,16 polycondensates (2-5) would not act as a cofactor. The shorter lag time for 2-5 may be because the oxidation of low molecular weight fraction of 2-5 or a phenol group in the side chain of 4 or 5 takes place, resulting in the apparent increase of the absorbance at 475 nm. Diphenolase Inhibition Activity. For evaluation of diphenolase inhibition activity, the enzymatic oxidation of L-DOPA to dopachrome in the presence of the inhibitors has been examined (Figure 2). Catechin accelerated the oxidation of L-DOPA, probably due to the activation of diphenolase by the action of catechin as a cofactor. Moreover, catechin itself was oxidized by tyrosinase. On the other hand, all the polycondensates efficiently inhibited the diphenolase activity. Among them, 1 showed the highest inhibition effect, whose
Kim et al.
Figure 3. Lineweaver-Burk plots for the inhibition of tyrosinase by polycondensate (1) with respect to L-DOPA as substrate.
tendency is similar to that of the monophenolase inhibition activity of the polycondensates. Tyrosinase Inhibition Mechanism. To analyze the inhibition type of the present polycondensate for tyrosinase, a steady-state analysis was performed. Linewaever-Burk plots for the inhibition of tyrosinase (diphenolase) by 1 were obtained with variable concentrations of 1 and substrate (Figure 3). The intersection of these lines on the vertical axis indicates that 1 is a competitive inhibitor of tyrosinase with respect to L-DOPA as substrate. The Ki value estimated by a secondary plot of the slopes of Lineweaver-Burk plots vs the concentrations was 68.7 µM. These data strongly suggest that 1 effectively inhibits the tyrosinase activity by binding to the active site. To further elucidate the tyrosinase inhibition and activation mechanism of catechin and the polycondensates, we examined their chelation behaviors with a copper ion and tyrosinase. A monophenolic substrate initially coordinates to an axial position of one of the coppers of oxytyrosinase. Rearrangement through a trigonal bipyramidal intermediate leads to o-hydroxylation of the monophenol by the bound peroxide, loosing H2O, and formation of the mettyrosinasediphenol complex.8 This complex can either render free diphenol as a first step in the diphenolase cycle, or undergo oxidation of the diphenolate intermediate bound to the active center, giving a free quinone and a reduced binuclear cuprous enzyme site (deoxytyrosinase). Mechanism of tyrosinase inhibition by flavonoids is explained as follows.16 Flavonoids form the chelation with copper in the enzyme and then irreversibly inactivate the tyrosinase. In the UV-visible spectrum of catechin, a characteristic shoulder peak was observed at 310 nm by addition of Cu2+, which is probably due to the chelation between the vicinal 3′,4′-dihydroxyl group of catechin and Cu2+ (Figure 4). In the presence of tyrosinase, on the other hand, a broad large peak centered at 450 nm due to the oxidation product (o-quinone) of catechin appeared, indicating that catechin acted as a substrate.18 Polycondensate 1 also showed a characteristic shoulder peak centered at 310 nm in the presence of Cu2+, due to chelation for Cu2+ (Figure 5). A similar shoulder peak (380 nm) due to chelation was observed in the mixture of 1 and tyrosinase,16 whose behavior is much different from that of
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Figure 4. UV-visible spectra of (+)-catechin (100 µM) (A), (+)catechin with Cu2+ (200 µM) (B), and (+)-catechin with tyrosinase (300 units) (C).
Figure 5. UV-visible spectra of polycondensate (1) (100 µM) (A), 1 with Cu2+ (200 µM) (B), and 1 with tyrosinase (300 units) (C). Table 1. Electrochemical Characteristics of (+)-Catechin and Polycondensatesa
(+)-catechin 1 2 3 4 5
Ep,a (mV)
Ep,c (mV)
Ip,a (µA)
377 470 399 421 409 416
117 120 119 119 113 122
10.90 6.65 10.50 6.74 10.10 7.87
a Electrochemical characteristics were measured by cyclic voltammetry (CV) with 3 mM of (+)-catechin and polycondensates in 0.1 M phosphate buffer including 0.1 M NaOH at a sweep rate of 50 mV‚s-1.
catechin and tyrosinase. These data strongly suggest the monophenolase and diphenolase inhibition actions of 1 by the chelation to the active site of tyrosinase. To elucidate the oxidation reactivity of catechin and the polycondensates, the electrochemical analysis was carried out by cyclic voltammetry (CV) (Table 1). For all the compounds, a single oxidation peak was observed. In the reverse sweep, there were distinct reduction peaks, showing that catechin and polycondensates were reversibly oxidized. The anodic oxidation peak potential (Epa) and cathodic reduction peak potential (Epc) of catechin were observed at 377 and 117 mV, respectively, and this redox behavior is caused by catechol moiety of catechin. Interestingly, all the
Figure 6. CD spectra of tyrosinase (A) and tyrosinase with polycondensate (1) of 100 µM (B), both in 0.1 M phosphate buffer (pH 6.8).
polycondensates showed higher Epa and lower anodic oxidation peak current (Ipa) than catechin. Among them, 1 showed the highest Epa and lowest Ipa. These data are closely related to tyrosinase inhibition activity of 1-5; compounds with the lower oxidation reactivity derived from higher Epa and lower Ipa showed larger inhibition effects of monophenolase and diphenolase. Furthermore, circular dichroism (CD) of a mixture of 1 and tyrosinase was measured to elucidate the chelation behaviors by the change of conformation. CD measurements will give more information on the conformation difference of tyrosinase and the polymer based on the chelation. The spectra of tyrosinase and its mixture with 1 are shown in Figure 6. In the spectrum of tyrosinase, a strong negative band was observed at 240 nm in the region corresponding to the peptide backbone absorbance (helical structure) and the shoulder band was observed at 265 nm in the region of aromatic side chain absorbance.21 In addition, the characteristic band due to the copper center of the oxy form of tyrosinase was observed at 325 nm.11 In the case of a mixture of 1 and tyrosinase, the intensity of the negative band centered at 240 nm decreased and the small red shift to 245 nm was observed. The shoulder band due to the aromatic side chain disappeared. On the other hand, the band of the copper center became larger by the addition of 1. These spectral changes may be attributable to the formation of 1-tyrosinase complex, in which the ordered structure of tyrosinase is partially destructed by the chelation of 1 to the active site of the oxy form of tyrosinase.22 In the CD spectrum of a mixture of tyrosinase and catechin, characteristic negative bands centered at 380 and 475 nm were observed (data not shown): the former due to charge transfer from catechin to Cu(II)11 and the latter due to the formation of oxidation product of catechin. This means that the OH f Cu(II) charge-transfer transition and the reduction of the Cu(II) ion of tyrosinase to the Cu(I) state occur, and consequently, tyrosinase is activated.11,14 Tyrosinase has two separate binding sites in its active center, one for substrate and another for reductant.23 In this respect, catechin acted as substrate and cofactor. On the other hand, the tyrosinase inhibition by 1 is caused by the chelation of 1 to tyrosinase, presumably in close proximity to the active site, and accordingly, tyrosinase is
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polycondensates inhibited the action of the oxy form of tyrosinase. This is the same result as that of the tyrosinase inhibition by kojic acid, which affected only oxytyrosinase activity.25 We preliminarily found that the present polycondensates showed much greater superoxide scavenging and inhibition activity of xanthine oxidase than catechin monomer. Further investigations on enzyme inhibition activities as well as antioxidant properties of the present polycondensates for applications in medical, food, and cosmetic fields are under way in our laboratory.
Figure 7. UV-visible spectra of tyrosinase (300 units) (A), oxytyrosinase (300 units) (B), polycondensate (1) (100 µM) (C), and 1-oxytyrosinase complex (D). Tyrosinase is a mixture of mettyrosinase (85%) and oxytyrosinase (15%). Oxytyrosinase is generated by reaction of excess H2O2 with tyrosinase in the presence of O2.
inactivated.24 This is in good agreement with the data of UV-visible spectra; the chelation of 1 to the active site of tyrosinase is clearly demonstrated by the changes of UVvisible spectra and CD bands induced by the addition of 1. We confirmed the chelation of 1 to the active site of oxytyrosinase to elucidate the monophenolase inhibition mechanism. When tyrosinase reacted with H2O2 in the presence of O2, mettyrosinase was converted to oxytyrosinase; the characteristic absorption peak of tyrosinase (a mixture of mettyrosinase (85%) and oxytyrosinase (15%)) centered at 205 nm was shifted to 210 nm, and the absorbance became larger by the addition of an excess of H2O2 to tyrosinase (Figure 7B). In a spectrum of a mixture of 1 and oxytyrosinase, the characteristic peaks of 1 (235 nm) and oxytyrosinase (210 nm) were shifted to 240 nm by the formation of a 1-oxytyrosinase complex (Figure 7D). Moreover, like the case of tyrosinase, the characteristic shoulder peak centered at 380 nm was observed. These data suggest that 1 truly acts as an inhibitor of oxytyrosinase, not of mettyrosinase. In the CD measurement of oxytyrosinase, the negative band due to the helical structure was shifted from 235 to 240 nm by the addition of 1 (data not shown). Furthermore, the band due to the copper center became larger and shifted from 330 to 320 nm, supporting specific chelation of 1 to oxytyrosinase. Conclusions The discovery and characterization of new tyrosinase inhibitors are useful for their potential applications in improving food quality and nutritional value, controlling insect pests, and preventing pigmentation disorders and other melanin-related health problems in human beings. However, only a few antimelanogenic reagents, typically kojic acid and arbutin, are currently commercially available. Furthermore, they have some concerns, e.g., high toxicity toward cells, low stability toward oxygen and water, resulting in their limited applications. In the present study, we have found that (+)-catechin-aldehyde polycondensates exhibited high inhibition effects on tyrosinase activity and the inhibition type of polycondensate (1) was a competitive. Interestingly, the
Acknowledgment. This work was partly supported by the 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 (1) (a) Garcı´a-Ca´novas, F.; Garcı´a-Carmona, F.; Vera. J.; Iborra, J. L.; Lozano, J. A. J. Biol. Chem. 1982, 257, 8738. (b) Rodrı´guez-Lo´pez, J. N.; Tudela, J.; Varo´n, R.; Garcı´a-Ca´novas, F. Biochim. Biophys. Acta 1991, 1076, 379. (2) Cooksey, C. J.; Garratt, P. J.; Land, E. J.; Pavel, S.; Ramsden, C. A.; Riley, P. A.; Smit, N. P. M. J. Biol. Chem. 1997, 272, 26226. (3) Pawelek, J. M.; Ko¨rner, A. M. AMSCA 1982, 70, 136. (4) (a) Mayer, A. M. Phytochemistry 1987, 26, 11. (b) Whitaker, J. R. In Food Enzymes, Structure and Mechanism; Wong, D. W. S.; Ed.; Champman & Hall: New York, 1995; p 271. (5) Friedman, M. J. Agric. Food Chem. 1996, 44, 631. (6) (a) Barrett, F. M. Can. J. Zool. 1984, 62, 834. (b) Sugumaran, M. AdV. Insect Physiol. 1988, 21, 179. (c) Lee, S.-E.; Kim, M.-K.; Lee, S.-G.; Ahn, Y.-J.; Lee, H.-S. Food Sci. Biotechnol. 2000, 9, 330. (7) (a) Mosher, A. M.; Pathak, M. A.; Fitzpatrick, T. B. In Dermatology in General Medicine; Fitzpatrick, T. B.; Eisen, A. Z.; Wolff, K.; Freedberg, I. M.; Austern, K. F., Eds.; Mc-Graw-Hill: New York, 1983; p 205. (b) Maeda, K.; Fukuda, M. J. Soc. Cosmet. Chem. 1991, 42, 361. (8) Lerch, K. In Metal Ions in Biological Systems; Sigel, H., Ed.; Marcel Dekker: New York, 1981; p 143. (9) Wilcox, D. E.; Porras, A. G.; Hwang, Y. T.; Lerch, K.; Winkler, M. E.; Solomon, E. I. J. Am. Chem. Soc. 1985, 107, 4015. (10) Sa´nchez-Ferrer, A Ä .; Rodrı´guez-Lo´pez, J. N.; Garcı´a-Ca´novas, F.; Garcı´a-Carmona, F. Biochim. Biophys. Acta 1995, 1247, 1. (11) (a) Himmelwright, R. S.; Eichman, N. C.; Lu Bien, C. D.; Lerch, K.; Solomon, E. I. J. Am. Chem. Soc. 1980, 102, 7339. (b) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. ReV. 1996, 96, 2563. (12) (a) Burton, S. G. Catal. Today 1994, 22, 459. (b) Sanjust, E.; Cecchini, G.; Sollai, F.; Curreli, N.; Rescigno, A. Arch. Biochem. Biophys. 2003, 412, 272. (13) (a) Laskin, J. D.; Piccinini, L. A. J. Biol. Chem. 1986, 261, 16626. (b) Cabanes, J.; Garcı´a-Ca´novas, F.; Lozano, J. A.; Garcı´a-Carmona, F. Biochim. Biophys. Acta 1987, 923, 187. (14) (a) Ros, J. R.; Rodrı´guez-Lo´pez, J. N.; Garcı´a-Ca´novas, F. Biochim. Biophys. Acta 1993, 1163, 303. (b) Fenoll, L. G.; Rodrı´guez-Lo´pez, J. N.; Garcı´a-Sevilla, F.; Garcı´a-Ruiz, P. A.; Varo´n, R.; Garcı´aCa´novas, F.; Tudela, J. Biochim. Biophys. Acta 2001, 1548, 1. (15) (a) Jankun, J.; Selman, S. H.; Swiercz, R.; Skrzypczak-Jankun, E. Nature (London) 1997, 387, 561. (b) Roeding-Penman, M.; Gordon, H. J. Agric. Food Chem. 1997, 45, 4267. (c) Bodoni, A.; Hrelia, S.; Angeloni, C.; Giordano, E.; Guarnier, C.; Caldarera, C. M.; Biagi, P. L. J. Nutr. Biochem. 2002, 13, 103. (16) (a) Kubo, I.; Kinst-Hori, I. J. Agric. Food Chem. 1999, 47, 4121. (b) Kubo, I.; Kinsy-Hori, I.; Chaudhuri, S. K.; Kubo, Y.; Sa´nchez, Y.; Ogura, T. Bioorg. Med. Chem. 2000, 8, 1749. (17) (a) No, J. K.; Soung, D. Y.; Kim, Y. J.; Shim, K. H.; Jun, Y. S.; Rhee, S. H.; Yokozawa, T.; Chung, H. Y. Life Sci. 1999, 65, 241. (b) Go´mez-Cordove´s, C.; Bartolome´, B.; Vieira, W.; Virador, V. M. J. Agric. Food Chem. 2001, 49, 1620.
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