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Biochemical and Molecular Actions of Nutrients

Flavonoids of Cocoa Inhibit Recombinant Human 5-Lipoxygenase¹

Institut für Physiologische Chemie I, Heinrich-Heine-Universität Düsseldorf, D-40001 Düsseldorf, Germany and ^* Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, D-10098 Berlin, Germany

³To whom correspondence should be addressed. E-mail: sies{at}uni-duesseldorf.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
(-)-Epicatechin and its related oligomers, the procyanidins, are present in sizable amounts in some cocoas and chocolates. Intake of flavonoid-rich chocolate in humans has been reported to increase the plasma level of (-)-epicatechin and concomitantly to significantly decrease the plasma level of proinflammatory cysteinyl leukotrienes. Because leukotrienes are formed via the 5-lipoxygenase pathway of arachidonic acid metabolism, we examined whether 5-lipoxygenase is a possible target for the flavonoids of cocoa. Recombinant human 5-lipoxygenase was reacted with arachidonic acid and yielded a mixture of mainly 5-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid (5-HpETE) and hydrolysis products of 5,6-leukotriene A₄ (LTA₄). The formation of these products was significantly inhibited by (-)-epicatechin in a dose-dependent manner with 50% inhibitory concentrations (IC₅₀) of 22 and 50 µmol/L, respectively. Among the procyanidin fractions isolated from the seeds of Theobroma cacao, only the dimer fraction and, to a lesser extent, the trimer through pentamer fractions exhibited comparable effects, whereas the larger procyanidins (hexamer through nonamer) were almost inactive. We conclude that (-)-epicatechin and its low-molecular procyanidins inhibit both dioxygenase and LTA₄ synthase activities of human 5-lipoxygenase and that this action may contribute to a putative anti-inflammatory effect of cocoa products.

KEY WORDS: • arachidonic acid metabolism • chocolate • inflammation • leukotrienes • procyanidins

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Procyanidins are oligomeric flavonoids composed of flavan-3-ol units that occur in some plant foods and beverages such as cocoa, red wine, apples and cranberries (1 ). Recently, these compounds have attracted attention due to their putative health benefits. Thus, a number of biological actions have been reported that are protective for the cardiovascular system, including endothelium-dependent relaxation via activation of nitric oxide synthase (2 ), enhancement of prostacyclin release (3 –5 ), inhibition of oxidative modification of LDL (6 –8 ) and inhibition of platelet aggregation (9 –11 ). Moreover, some anti-inflammatory actions such as scavenging of peroxynitrite (12 ) and modulation of expression and secretion of interleukins (13 ,14 ) have also been described. Ingestion of procyanidin-rich chocolate has been reported to give rise to an increase in the plasma level of (-)-epicatechin and its metabolites (4 ,15 –17 ). Thus, the beneficial effects of procyanidins appear to be mediated at least in part by monomeric (-)-epicatechin and/or its biotransformation products. However, the fate of dietary procyanidins during gastrointestinal passage and absorption is not yet well understood. In an in vitro study, one group reported a cleavage at low pH, thus simulating the gastric environment, to (-)-epicatechin dimers and monomers, which in turn are absorbed in the small intestine (18 ); however, in vivo, very little breakdown of procyanidins to monomers was observed (19 ). Therefore, the biological activities of procyanidin oligomers deserve some attention as well. They may also reach a biological target extracellularly [e.g., peroxynitrite released from cells, see (12 )].

The beneficial effects of procyanidins and (-)-epicatechin have been related mainly to their capability of scavenging reactive oxygen and nitrogen species (20 ). However, inhibitory effects on oxidant enzymes have also been reported (21 ,22 ). In a recent paper, we described the inhibition by (-)-epicatechin and cocoa procyanidins of a mammalian reticulocyte-type 15-lipoxygenase (15-lipoxygenase-1), an important catalyst of enzymatic lipid peroxidation of biomembranes and plasma lipoproteins (23 ). With this enzyme, the higher oligomers were found to be more potent than the monomer and the medium-sized oligomers. Because cocoa flavonoids also inhibited soybean 15-lipoxygenase L-1 and the mammalian 12-lipoxygenases of leukocyte- and platelet-types (23 ), it was tempting to speculate that they may be general lipoxygenase inhibitors. Here we report the inhibition of human 5-lipoxygenase which is a key enzyme of the synthesis of proinflammatory leukotrienes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Flavonoids and other chemicals.

(-)-Epicatechin and procyanidin oligomers, isolated from cocoa as described elsewhere (24 ,25 ), were kindly supplied by Mars Incorporated (Cocoapro, Hackettstown, NJ) and used as stock solutions of 100 mEq/L (29.0 g/L) in peroxide-free 2-methoxyethanol. (-)-Epigallocatechin gallate, quercetin and isopropyl ß-D-thiogalactopyranoside were purchased from Sigma-Aldrich (Deisenhofen, Germany), and arachidonic acid, CaCl₂, EDTA, dipalmitoyl phosphatidylcholine and sodium borohydride from Serva (Heidelberg, Germany), respectively. Standards of 5-hydroxyeicosatetraenoic acid (HETE),⁴ 8-HETE, 5S, 6R-dihydroxyeicosatetraenoic acid (diHETE), 5S, 6S-diHETE, 5S,12R-diHETE and 5S, 12S-diHETE were obtained from Cayman Chemical (Ann Arbor, MI, distributed by Alexis, Grünberg, Germany) or Biomol (Hamburg, Germany). HPLC solvents were from Merck (Darmstadt, Germany).

Preparation of recombinant human 5-lipoxygenase.

The enzyme was prepared as described in detail elsewhere (26 ) with minor modifications. Briefly, bacteria (Escherichia coli strain HB101) were transformed with a PKK-233–2–based expression plasmid containing the cDNA of human 5-lipoxygenase. A bacterial culture (1 L) was grown at 37°C overnight in LB medium containing 0.1% ampicillin. The expression of 5-lipoxygenase was induced by addition of 0.5 mmol/L isopropyl ß-D-thiogalactopyranoside (final concentration) and the cells were maintained at 30°C for an additional 24 h. Then the bacteria were centrifuged, washed with PBS, resuspended in 0.1 mol/L Tris buffer, pH 7.5 containing 1 mmol/L EDTA, and sonicated with a tip sonifier three times for 30 s each at the highest level. The cell lysate was centrifuged (10 min, 4000 x g) and 1.5-mL aliquots of the supernatant (13.5 g/L protein) were stored in liquid nitrogen until use.

Reaction of 5-lipoxygenase with arachidonic acid.

Aliquots of the lysate supernatant (10 µL, 135 µg total protein) were added to 215 µL of 50 mmol/L Tris buffer, pH 7.4, and preincubated for 10 min in the presence or absence of flavonoid. Thereafter, 25 µL assay mixture containing 1 mmol/L ATP, 4 mmol/L CaCl₂, 1 mmol/L EDTA, and 13 mg/L dipalmitoyl phosphatidylcholine was added. The lipoxygenase reaction was started after another 5 min by addition of 0.75 µL of 33 mmol/L arachidonic acid in methanol and stopped after a reaction period of 15 min by addition of 250 µL cold methanol. Then 5 µL sodium borohydride (saturated solution in cold ethanol) and 25 µL glacial acetic acid were added. After centrifugation (5 min, 4000 x g), the supernatants were directly subjected to reverse-phase HPLC (see below).

Analytical procedures.

The analysis of the oxygenated metabolites of arachidonic acid was performed on a Shimadzu HPLC system connected to a Hewlett-Packard diode array detector 1040 (Shimadzu Deutschland, Duisburg, Germany). Reverse phase-HPLC was carried out on a Nucleosil C-18 column (Macherey-Nagel, Düren, Germany; KS-system, 250 x 4 mm, 5-µm particle size) coupled with an appropriate guard-column (30 x 4 mm, 5-µm particle size). For analysis of the oxygenated products of arachidonic acid, a solvent system of methanol/water/acetic acid (75:25:0.1, v/v/v) was used at a flow rate of 1 mL/min. Absorbency was monitored at 235 nm (conjugated dienes) and 270 nm (conjugated trienes). Experimental means were compared with control means by Student’s t test, and differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Human 5-lipoxygenase was expressed in E. coli and the supernatant of the cell lysate was used as the enzyme source for the experiments. The 5-lipoxygenase was reacted with arachidonic acid for 15 min at 20°C in the presence of cofactors necessary for achieving optimal enzymatic activity (Ca²⁺, ATP, phosphatidylcholine) (27 ). Although no conversion of arachidonic acid was observed with lysates from control bacteria (not shown), the lysates from 5-lipoxygenase–transfected bacteria yielded a complex mixture of products (Fig. 1A ). In addition to 5-hydroperoxy-6E,8Z,11Z, 14Z-eicosatetraenoic acid (5-HpETE; analyzed upon reduction by borohydride as the corresponding stable 5S-hydroxy compound, 5-HETE), a small amount of 8-H(p)ETE was formed; this is a minor side product of the primary dioxygenase reaction because this enzyme can also display 8-lipoxygenase activity (28 ). Moreover, four fractions showing a UV spectrum typical of polyenoic fatty acids containing a conjugated E,E,E-triene system were detected. They were identified as diastereomers of 5,12-dihydroxy-eicosatetraenoic acid (5,12-diHETE) and 5,6-dihydroxy-eicosatetraenoic acid (5,6-diHETE; Fig. 1 A). Because these E,E,E-diHETE are known to arise from spontaneous hydrolysis of 5,6-leukotriene A₄ (LTA₄) (27 ), it follows that under the experimental conditions employed, human 5-lipoxygenase catalyzes not only the primary dioxygenation of arachidonic acid at C-5 but also the subsequent conversion of 5-HpETE to LTA₄ by virtue of its intrinsic LTA₄ synthase activity (26 ,27 ,29 ). In Figure 2 are shown the routes of conversion of arachidonic acid catalyzed by the various catalytic activities of 5-lipoxygenase as well as the compounds identified and quantified.

View larger version (24K): [in this window] [in a new window]   FIGURE 1 Reaction of recombinant human 5-lipoxygenase with arachidonic acid and the effect of (-)-epicatechin. Human 5-lipoxygenase was expressed in E. coli, and cell lysate supernatant (see Materials and Methods) was preincubated for 10 min in the presence of 1.25 µL 2-methoxyethanol (A) or 1.25 µL of 10 µmol/L (-)-epicatechin dissolved in 2-methoxyethanol (B) at 20°C. Thereafter, the enzyme preparation was reacted with arachidonic acid and the reaction products were analyzed as described in Materials and Methods. The fractions detected at 235 nm (conjugated dienes) and 270 nm (conjugated trienes) were identified by cochromatography with authentic standards and other investigations (26 ): a, 5S, 12S-diHETE(E,E,E); b, 5S,12R-diHETE(E,E,E); c, 5S,6R-diHETE(E,E,E); d, 5S,6S-diHETE(E,E,E). For formulas of the compounds, see Figure 2 . 5-HETE, 5S-hydroxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid; 8-HETE, 8S-hydroxy-5Z,9E,11Z, 14Z-eicosatetraenoic acid.

View larger version (22K): [in this window] [in a new window]   FIGURE 2 Routes of conversion of arachidonic acid by the various catalytic activities of 5-lipoxygenase under the conditions of the experimental setup. 5-H(p)ETE, 5S-hydro(pero)xy-6E,8Z, 11Z,14Z-eicosatetraenoic acid; 8-H(p)ETE, 8S-hydro(pero)xy-5Z,9E,11Z, 14Z-eicosatetraenoic acid; LTA4, 5,6-leukotriene A4; 5,6-diHETE, 5S, 6R/S-dihydroxy-8E,10E,12E, 14Z-eicosatetraenoic acids; 5,12-diHETE, 5S, 12R/S-dihydroxy-6E,8E,10E, 14Z-eicosatetraenoic acids. Reaction types are written in italics. Abbreviations in boxes indicate the compounds analyzed. After the 5-lipoxygenase reaction, 5-HpETE and 8-HpETE were reduced by sodium borohydride to 5-HETE and 8-HETE, respectively, due to the greater stability of the latter compounds for analysis.

View larger version (18K): [in this window] [in a new window]   FIGURE 3 Inhibition by (-)-epicatechin of product formation from arachidonic acid by recombinant human 5-lipoxygenase. Conditions as in Figure 1 excepted that the final concentration of (-)-epicatechin varied. The products detected at 235 nm (5S-hydroxy-6E,8Z,11Z, 14Z-eicosatetraenoic acid [5-HETE], squares, and 8S-hydroxy-5Z,9E,11Z, 14Z-eicosatetraenoic acid [8-HETE], circles) and 270 nm (5S,12epi-dihydroxy-6E,8E, 10E,14Z-eicosatetraenoic acids [5,12-diHETE], triangles), were quantified by peak area and related to the means of corresponding controls. The data represent means ± SD from 3–5 analyses of 3 independent series of experiments. The controls quantities were 0.70 ± 0.12 nmol 5-HETE/mg bacterial lysate supernatant protein, 0.10 ± 0.02 nmol 8-HETE/mg protein and 0.44 ± 0.12 nmol 5,12-diHETE isomers/mg protein, (n = 7), as estimated from calibration of the peak areas with authentic standards of 5-HETE and 5,12-diHETE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

It is not easy to decide whether cocoa flavonoids display their 5-lipoxygenase-inhibitory activity also in vivo. Although intake of cocoa drink or chocolate has been reported to markedly elevate the plasma level of (-)-epicatechin (4 ,15 –17 ), in those studies the plasma levels did not reach the concentration range that was inhibitory toward 5-lipoxygenase in our experiments. Moreover, the major part of the plasma level of flavonoids is contributed by secondary metabolites such as glucuronic acid conjugates (15 ,34 –37 ), whose potential lipoxygenase-inhibitory activities have not yet been tested. At first sight, these considerations are seemingly not in line with a biological role of nonmetabolized flavonoids in vivo. However, Walle and co-workers (38 ) recently found using ¹⁴C-quercetin that the biological half-life of quercetin in humans is astonishing long (up to 72 h); the majority of radioactivity was recovered as ¹⁴CO₂ in the expired air, whereas <10% was recovered in urine and feces. The authors proposed the involvement of enterohepatic recirculation. Thus, the glucuronides and other conjugates seem to be transport metabolites in plasma rather than bioactive final compounds and major excretion products. Apparently they are taken up by cells and further metabolized, possibly via free aglycone. Although data on the tissue distribution of flavonoids currently are not available, the existence of a pool of nonmetabolized aglycones in certain lipophilic compartments of the organism such as the lipid bilayer of biomembranes or in adipose tissue may be hypothesized, a pool that may act as a store for nonmetabolized flavonoid. Furthermore, it may well be that (-)-epicatechin accumulates in cells that are competent for leukotriene formation. It has been reported recently that in humans, the intake of procyanidin-rich chocolate consistently decreased the plasma levels of cysteinyl leukotrienes by 30%, which coincided with the peak of the plasma level of (-)-epicatechin (4 ). Given these data, we can assume that the effect observed by Schramm and co-workers (4 ) is due to direct inhibition of 5-lipoxygenase activity by flavonoids as demonstrated here (Figs. 1 and 3) .

In a preceding communication we described the inhibition by flavan-3-ols of other mammalian lipoxygenases, the 15-lipoxygenase-1 and the 12-lipoxygenases of 12-lipoxygenases of platelet– and leukocyte types (23 ). The demonstration of inhibitory effects on 5-lipoxygenase activities identifies this class of compounds as nonselective lipoxygenase inhibitors. The precise mechanism of the lipoxygenase inhibition by flavan-3-ols remains unclear, although some aspects of the putative mode of action were discussed in detail before (23 ). In any case, the lipoxygenase-inhibitory action of flavonoids is clearly distinct from the well-known free radical–scavenging properties of these compounds. The fatty acid radical intermediates formed during the catalytic cycle of lipoxygenases remain tightly bound at the active site and consequently are not accessible to certain radical scavengers, such as 2,6-di-tert-butyl-hydroxytoluene or -tocopherol, which do not inhibit mammalian lipoxygenases. When liberated from the enzyme, however, radical intermediates become accessible to radical scavengers, and under such conditions, the lipoxygenase reaction may be inhibited. The data presented here suggest that both a direct lipoxygenase-inhibitory potency and the general antioxidant properties of dietary flavan-3-ols may contribute to the antileukotriene action reported by Schramm and co-workers (4 ).

The procyanidins of cocoa, except the dimeric fraction, were weaker 5-lipoxygenase inhibitors than the corresponding monomer. Opposite relations have been observed for rabbit reticulocyte 15-lipoxygenase and soybean 15-lipoxygenase L-1. With these enzymes, the fractions of large procyanidins were the most potent inhibitors (23 ). It appears, therefore, that large procyanidins exhibit a certain degree of isoenzyme specificity. Assuming that the inhibition of 15-lipoxygenase activities by large procyanidins also occurs in vivo, which is still unclear (see above), the natural mixture of flavonoids occurring in cocoa products would exhibit a more effective antioxidative capacity toward the various mammalian lipoxygenases than the isolated fractions of flavan-3-ols alone.

The potencies of (-)-epicatechin to inhibit 5- and 15-lipoxygenases are surpassed by one order of magnitude by those of (-)-epigallocatechin gallate (Table 2) , which is likely due to the presence of the gallic ester moiety. As reported previously, aliphatic gallic esters such as octylgallate are strong inhibitors of 5-HETE and LTB₄ formation in human neutrophils (39 ). The flavonol quercetin differs structurally from catechins by the presence of an additional 2,3-double bond in the C-ring and a carbonyl group at C-4, whereas the number and positions of hydroxyl groups are identical [for chemical structures see (20 ,40 )]. Hence, the structural peculiarities of the C-ring of quercetin obviously render this compound more potent as an inhibitor of 5- and 15-lipoxygenases (Table 2) .

The much higher inhibitory potency of quercetin than of flavan-3-ols also raises the issue of its relevance for the beneficial dietary effect of cocoa products. The total amount of quercetin (free and conjugated) and other flavonols in cocoa has been reported to be as high as 30 mg/100 g (41 ). This corresponds to about one tenth of the content of (-)-epicatechin and procyanidins (1 ,25 ). However, we found higher inhibitory potencies of quercetin than (-)-epicatechin toward both 5- and 15-lipoxygenase activities by more than one order of magnitude (Table 2) . Therefore, quercetin and other flavonols may contribute to the antilipoxygenase effect of the whole flavonoid mixture of cocoa.

Collectively, our data lend support to the assumption that not only general antioxidant effects but also inhibition of lipoxygenase activities contribute to the beneficial effects of dietary flavonoids of various structures and origins.

	ACKNOWLEDGMENTS

 
The authors thank Christa Gerth for her excellent assistance with the bacterial culture and enzyme isolation.

	FOOTNOTES

 
¹ Supported by Mars Incorporated (Hackettstown, NJ).

² H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD.

⁴ Abbreviations used: diHETE, dihydroxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; HpETE, hydroperoxyeicosatetraenoic acid; LTA₄ (B₄, C₄, D₄), leukotriene A₄ (B₄, C₄, D₄).

Manuscript received 14 February 2002. Initial review completed 11 March 2002. Revision accepted 25 March 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Hammerstone, J. F., Lazarus, S. A. & Schmitz, H. H. (2000) Procyanidin content and variation in some commonly consumed foods. J. Nutr. 130:2086S-2092S.[Abstract/Free Full Text]

2. Karim, M., McCormick, K. & Kappagoda, C. T. (2000) Effects of cocoa extracts on endothelium-dependent relaxation. J. Nutr. 130:2105S-2108S.[Abstract/Free Full Text]

3. Schramm, D. D., Pearson, D. A. & German, J. B. (1997) Endothelial cell basal PGI₂ release is stimulated by wine in vitro: one mechanism that may mediate the vasoprotective effect of wine. J. Nutr. Biochem 8:647-651.

4. Schramm, D. D., Wang, J. F., Holt, R. R., Ensunsa, J. L., Gonsalves, J. L., Lazarus, S. A., Schmitz, H. H., German, J. B. & Keen, C. L. (2001) Chocolate procyanidins decrease the leukotriene-prostacyclin ratio in humans and human aortic endothelial cells. Am. J. Clin. Nutr. 73:36-40.[Abstract/Free Full Text]

5. Facino, R. M., Carini, M., Aldini, G., Berti, F., Rossoni, G., Bombardelli, E. & Morazzoni, P. (1999) Diet enriched with procyanidins enhances antioxidant activity and reduces myocardial post-ischaemic damage in rats. Life Sci. 64:627-642.[Medline]

6. Mazur, A., Bayle, D., Lab, C., Rock, E. & Rayssiguier, Y. (1999) Inhibitory effect of procyanidin-rich extracts on LDL oxidation in vitro. Atherosclerosis 145:421-422.[Medline]

7. Ivanov, V., Carr, A. C. & Frei, B. (2001) Red wine antioxidants bind to human lipoproteins and protect them from metal ion–dependent and –independent oxidation. J. Agric. Food Chem. 49:4442-4449.[Medline]

8. Osakabe, N., Baba, S., Yasuda, A., Iwamoto, T., Kamiyama, M., Takizawa, T., Itakura, H. & Kondo, K. (2001) Daily cocoa intake reduces the susceptibility of low-density lipoprotein to oxidation as demonstrated in healthy human volunteers. Free Radic. Res. 34:93-99.[Medline]

9. Chang, W. C. & Hsu, F. L. (1989) Inhibition of platelet aggregation and arachidonate metabolism in platelets by procyanidins. Prostaglandins Leukot. Essent. Fatty Acids 38:181-188.[Medline]

10. Rein, D., Paglieroni, T. G., Pearson, D. A., Wun, D., Schmitz, H. H., Gosselin, R. & Keen, C. L. (2000) Cocoa and wine polyphenols modulate platelet activation and function. J. Nutr. 130:2120S-2126S.[Abstract/Free Full Text]

11. Rein, D., Paglieroni, T. G., Wun, D., Pearson, D. A., Schmitz, H. H., Gosselin, R. & Keen, C. L. (2000) Cocoa inhibits platelet activation and function. Am. J. Clin. Nutr. 72:30-35.[Abstract/Free Full Text]

12. Arteel, G. E. & Sies, H. (1999) Protection against peroxynitrite by cocoa polyphenol oligomers. FEBS Lett. 462:167-170.[Medline]

13. Sanbongi, C., Suzuki, N. & Sakane, T. (1997) Polyphenols in chocolate, which have antioxidant activity, modulate immune functions in humans in vitro. Cell. Immunol. 177:129-136.[Medline]

14. Mao, T., Van de Water, J., Keen, C. L., Schmitz, H. H. & Gershwin, M. E. (2000) Cocoa procyanidins and human cytokine transcription and secretion. J. Nutr. 130:2093S-2099S.[Abstract/Free Full Text]

15. Baba, S., Osakabe, N., Yasuda, A., Natsume, M., Takizawa, T., Nakamura, T. & Terao, J. (2000) Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers. Free Radic. Res. 33:635-641.[Medline]

16. Rein, D., Lotito, S., Holt, R. R., Keen, C. L., Schmitz, H. H. & Fraga, C. G. (2000) Epicatechin in human plasma: in vivo determination and the effect of chocolate consumption on plasma oxidation status. J. Nutr. 130:2109S-2114S.[Abstract/Free Full Text]

17. Wang, J. F., Schramm, D. D., Holt, R. R., Ensunsa, J. L., Fraga, C. G., Schmitz, H. H. & Keen, C. L. (2000) Dose-response effect from chocolate consumption on plasma epicatechin and oxidative damage. J. Nutr. 130:2115S-2119S.[Abstract/Free Full Text]

18. Spencer, J. P., Chaudry, F., Pannala, A. S., Srai, S. K., Debnam, E. & Rice-Evans, C. (2000) Decomposition of cocoa procyanidins in the gastric milieu. Biochem. Biophys. Res. Commun. 272:236-241.[Medline]

19. Rios, L. Y., Bennett, R. N., Lazarus, S. A., Remesy, C., Scalbert, A. & Williamson, G. (2002) Cocoa procyanidins are stable during gastric transit in humans. Am. J. Clin. Nutr. 75(in press).

20. Rice-Evans, C. A., Miller, N. J. & Paganga, G. (1996) Structure–antioxidant activity relationship of flavonoids and phenolic acids. Free Radic. Biol. Med. 20:933-956.[Medline]

21. Moini, H., Guo, Q. & Packer, L. (2000) Enzyme inhibition and protein-binding action of the pine bark extract pycnogenol: effect on xanthine oxidase. J. Agric. Food Chem. 48:5630-5639.[Medline]

22. Middleton, E., Jr., Kandaswami, C. & Theoharides, T. C. (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52:673-751.[Abstract/Free Full Text]

23. Schewe, T., Sadik, C., Klotz, L.-O., Yoshimoto, T., Kühn, H. & Sies, H. (2001) Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase. Biol. Chem. 382:1687-1696.[Medline]

24. Hammerstone, J. F., Lazarus, S. A., Mitchell, A. E., Rucker, R. & Schmitz, H. H. (1999) Identification of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry. J. Agric. Food Chem. 47:490-496.[Medline]

25. Adamson, G. E., Lazarus, S. A., Mitchell, A. E., Prior, R. L., Cao, G., Jacobs, P. H., Kremers, B. G., Hammerstone, J. F., Rucker, R. B., Ritter, K. A. & Schmitz, H. H. (1999) HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity. J. Agric. Food Chem. 47:4184-4188.[Medline]

26. Schwarz, K., Gerth, C., Anton, M. & Kühn, H. (2000) Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity. Biochemistry 39:14515-14521.[Medline]

27. Denis, D., Falguyeret, J. P., Riendeau, D. & Abramovitz, M. (1991) Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors. J. Biol. Chem. 266:5072-5079.[Abstract/Free Full Text]

28. Zhang, Y. Y., Radmark, O. & Samuelsson, B. (1992) Mutagenesis of some conserved residues in human 5-lipoxygenase: effect on enzyme activity. Proc. Natl. Acad. Sci. USA 89:485-489.[Abstract/Free Full Text]

29. Rouzer, C. A., Matsumoto, P. & Samuelsson, B. (1986) Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A₄ synthase activities. Proc. Natl. Acad. Sci. USA 83:857-861.[Abstract/Free Full Text]

30. Dongmo, A. B., Kamanyi, A., Anchang, M. S., Chungag-Anye Nkeh, B., Njamen, D., Nguelefack, T. B., Nole, T. & Wagner, H. (2001) Anti-inflammatory and analgesic properties of the stem bark extracts of Erythrophleum suaveolens (Caesalpiniaceae), Guillemin & Perrottet. Ethnopharmacol. 77:137-141.

31. Yoshimoto, T., Furukawa, M., Yamamoto, S., Horie, T. & Watanabe-Kohno, S. (1983) Flavonoids: potent inhibitors of arachidonate 5-lipoxygenase. Biochem. Biophys. Res. Commun. 116:612-618.[Medline]

32. Silverman, E. S. & Drazen, J. M. (1999) The biology of 5-lipoxygenase: function, structure, and regulatory mechanism. Proc. Assoc. Am. Physicians 111:525-536.[Medline]

33. Samuelsson, B., Dahlén, S. E., Lindgren, J. A., Rouzer, C. A. & Serhan, C. N. (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science (Wash. DC) 237:1171-1176.[Abstract/Free Full Text]

34. Donovan, J. L., Bell, J. R., Kasim-Karakas, S., German, J. B., Walzem, R. L., Hansen, R. J. & Waterhouse, A. L. (1999) Catechin is present as metabolites in human plasma after consumption of red wine. J. Nutr. 129:1662-1668.[Abstract/Free Full Text]

35. Li, C., Lee, M.-J., Sheng, S., Meng, X., Prabhu, S., Winnik, B., Huang, B., Chung, J. Y., Yan, S., Ho, C.-T. & Yang, C. S. (2000) Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion. Chem. Res. Toxicol. 13:177-184.[Medline]

36. Walle, T., Otake, Y., Walle, U. K. & Wilson, F. K. (2000) Quercetin glucosides are completely hydrolyzed in ileostomy patients before absorption. J. Nutr. 130:2658-2661.[Abstract/Free Full Text]

37. Graefe, E. U., Wittig, J., Mueller, S., Riethling, A.-K., Uehleke, B., Drewelow, B., Pforte, H., Jacobasch, G., Derendorf, H. & Veit, M. (2001) Pharmacokinetics and bioavailability of quercetin glycosides in humans. J. Clin. Pharmacol. 41:492-499.[Abstract]

38. Walle, T., Walle, U. K. & Halushka, P. V. (2001) Carbon dioxide is the major metabolite of quercetin in humans. J. Nutr. 131:2648-2652.[Abstract/Free Full Text]

39. Christow, S., Luther, H., Ludwig, P., Gruner, S. & Schewe, T. (1991) Action of gallic esters on the arachidonic acid metabolism of human polymorphonuclear leukocytes. Pharmazie 46:282-283.[Medline]

40. Haslam, E. (1998) Practical Polyphenolics. From Structure to Molecular Recognition and Physiological Action 1998 Cambridge University Press Cambridge, U.K. .

41. Lamuela-Raventós, R. M., Andrés-Lacueva, C., Permanyer, J. & Izquierdo-Pulido, M. (2001) More antioxidants in cocoa. J. Nutr. 131:834.[Free Full Text]

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