Polyphenols of Cocoa: Inhibition of Mammalian 15 ...

Biol. Chem., Vol. 382, pp. 1687 – 1696, December 2001 · Copyright © by Walter de Gruyter · Berlin · New York

Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase

Tankred Schewe1, Christian Sadik1, Lars-Oliver Klotz1, Tanihiro Yoshimoto2, Hartmut Kühn3 and Helmut Sies1,* 1

Institut für Physiologische Chemie I, Heinrich-HeineUniversität Düsseldorf, P.O. Box 10 10 07, D-40001 Düsseldorf, Germany 2 Department of Pharmacology, Kanazawa University School of Medicine, Kanazawa 920-8640, Japan 3 Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Schumannstr. 20/21, D-10098 Berlin, Germany * Corresponding author

Some cocoas and chocolates are rich in (–)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 µM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 µM) and epicatechin gallate (5 µM) were more potent than (–)-epicatechin (IC50 = 60 µM). (–)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12lipoxygenase (another representative of the 12/15lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 µM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells. Key words: Antioxidants / Chocolate / Flavonoids / Procyanidins.

Introduction Polyphenols, mainly flavonoids and phenolic acids, are abundant in a number of dietary sources such as certain cocoas, tea, wine, fruits and vegetables (Scalbert and Williamson, 2000; Middleton Jr. et al., 2000). It is generally hypothesised that in man dietary polyphenols may reduce the risk of disorders associated with enhanced production of reactive oxygen and nitrogen species, such as coronary heart disease, stroke, inflammatory diseases and cancer (Scalbert and Williamson, 2000; Wang et al., 2000, and references cited therein). Certain cocoas and chocolates contain considerable amounts of monomeric and oligomeric flavanols, the latter also being called procyanidins (Adamson et al., 1999; Hammerstone et al., 2000). For (–)-epicatechin, the major monomeric flavanol of cocoa, and the corresponding procyanidins, a number of beneficial antioxidative actions have been described. Thus, these compounds were found to protect against peroxynitrite-mediated reactions (Arteel and Sies, 1999; Arteel et al., 2000; Schroeder et al., 2001). Moreover, it has been reported recently that in humans the intake of procyanidin-rich chocolate causes an increase in the plasma levels of (–)-epicatechin (Wang et al., 2000) and its metabolites (Baba et al., 2000) and concomitantly a decrease in the leukotriene/prostacyclin ratio (Schramm et al., 2001), which suggests a potential protective action of cocoa flavonoids on the cardiovascular system. Here, we address the issue of whether the beneficial antioxidative capacities of cocoa flavonoids do not only encompass scavenging of reactive oxygen and nitrogen species but also suppression of relevant prooxidative enzymes. For pycnogenol, a procyanidin-rich pine bark extract, inhibition of xanthine oxidase and moderate inhibition of soybean lipoxygenase as well as selective binding to certain proteins have been reported (Moini et al., 2000). Actions of the procyanidins from cocoa on oxidative enzymes have not been studied before. We chose the reticulocytetype 15-lipoxygenase (15-LOX-1) as a potential target. Unlike a variety of other lipoxygenases, this enzyme is capable of dioxygenating not only free arachidonic and linoleic acids but also phospholipids, cholesterol esters, biomembranes and lipoproteins, thus appearing as a general enzymatic prooxidant (Schewe and Kühn, 1991; Kühn and Borngräber, 1999). Oxidative modification of low density lipoprotein (LDL) has been implicated in atherogenesis (Cyrus et al., 1999; Steinberg, 1999). In vitro, the 15-LOX-1 is capable of oxygenating LDL to an atherogenic form and thus, it may be considered as pro-atherogenic enzyme. For the time being the precise role of the 15-LOX-1 is not yet fully understood (Kühn and Chan, 1997; Cornicelli and

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Trivedi, 1999; Cathcart and Folcik, 2000) and even antiatherogenic activities have been reported (Shen et al., 1996). Nevertheless, dietary constituents or drugs that inhibit this enzyme may exhibit anti-atherogenic properties, thus protecting the cardiovascular system. Although some polyphenols such as nordihydroguaiaretic acid and 4-nitrocatechol are known to inhibit this and other lipoxygenases (Spaapen et al., 1980; Schewe et al., 1986; Kemal et al., 1987), the possible role of flavonoids as enzyme inhibitors of mammalian 15-LOX-1 has not been addressed before. The only exception is the recently reported suppression by quercetin and (–)-epicatechin of 15-LOX-1-induced oxidative modification of low-density lipoprotein (LDL) (da Silva et al., 1998, 2000). However, these effects may be assigned to the well-known scavenging of secondary free radicals such as lipid peroxyl radicals occurring in this complex system rather than to direct inhibition of 15-LOX-1 activity, since these flavonoids also strongly inhibit the oxidation of LDL induced by copper ions (Osada et al., 2001). Here we present for the first time evidence that (–)-epicatechin and the related procyanidins are direct inhibitors of 15LOX-1 and other mammalian lipoxygenases independent of their scavenging properties.

Results Flavanols Inhibit Reticulocyte-Type 15-Lipoxygenase The effects of selected polyphenols on both rabbit 15LOX-1 and soybean lipoxygenase (isoenzyme L1) were examined with potassium linoleate as substrate at pH 7.4. All compounds tested inhibited rabbit 15-LOX-1 in a dose-dependent manner up to more than 90%. The inhibitory effects occurred independently of whether linoleic or arachidonic acids were used as substrate or whether oxygen uptake or formation of conjugated dienes was measured. However, for testing inhibitory compounds we employed the oxygraphic assay using linoleic acid as substrate because of two reasons: (i) at high concentrations the polyphenols interfered with the spectrophotometric assay owing to their UV absorption; (ii) rabbit 15-LOX-1 showed a stronger mechanism-based self-inactivation with arachidonic acid than with linoleic acid as substrate, so that a linear part of the kinetic progress curves cannot be achieved with this fatty acid at room temperature. The respective IC50 values are compiled in Table 1. Interestingly, the flavonol quercetin exhibited a markedly higher inhibitory potency than (–)-epicatechin, despite identical positions of the five hydroxyl groups in these two flavonoids. Thus, it may be concluded that not only the hydroxyl groups but also the spatial structure and the degree of unsaturation of the C ring of the flavonoid (for chemical structures, see e. g. Haslam, 1998; Middleton Jr. et al., 2000; Rice-Evans et al., 1996), which differ between quercetin and catechins, may contribute to the binding affinity of the inhibitors toward the active site of the enzyme. The gallic acid esters of flavanols, which are major constituents of green tea, were

Table 1 Inhibition of Rabbit and Soybean 15-Lipoxygenases by Selected Polyphenols. Compound

IC50 (µM) Rabbit

Soybean

Flavanols Epicatechin Epigallocatechin Epicatechin gallate Epigallocatechin gallate Procyanidin decamer

60 100 5 4 0.8

inactive inactive not tested 1000 not estimated

Other flavonoids Quercetin Baicalein Genistein

4 1 18

5 35 1000

Non-flavonoid polyphenols Nordihydroguaiaretic acid Esculetin Resveratrol Caffeic acid

0.5 4 32 1000

3 100 650 inactive

Rabbit reticulocyte 15-LOX-1 and soybean lipoxygenase L1 were assayed oxygraphically with potassium linoleate as substrate at pH. 7.4 in the presence or absence of varying concentrations of the compounds listed as described in Materials and Methods. The activities of the control samples (1% solvent only) were 0.08 – 0.12 nkat and 0.15 – 0.20 nkat for the rabbit and the soybean enzyme, respectively. The IC50 values were interpolated from the corresponding dose-response curves obtained from at least two independent sets of measurement.

more potent by one order of magnitude than (–)-epicatechin. Their higher inhibitory potency is in line with the strong inhibition of rabbit 15-LOX-1 by medium-chain alkyl gallates as observed previously (Luther et al., 1992). The relatively poor activity of epigallocatechin may be due to its tendency to autoxidise, which is supported by the observation that under our assay conditions a lipoxygenase-independent oxygen uptake with this compound occurred before reaction start with substrate. In contrast to rabbit 15-LOX-1, soybean lipoxygenase L1 was not or only scarcely affected by monomeric flavanols independent of whether the assay was performed in phosphate buffer, pH 7.4, or at the pH optimum of the latter enzyme at pH 9.0 in Tris or borate buffer. Hence, the soybean enzyme turns out to be not a suitable model for the interaction of polyphenols with mammalian 15-lipoxygenases. This observation merits attention, as many earlier studies on 15-lipoxygenase-inhibitory activities of flavonoids were carried out exclusively with the soybean enzyme. Cocoa Procyanidins Inhibit Both Reticulocyte-Type and Soybean 15-Lipoxygenase Fractions of procyanidin oligomers, isolated from the seeds of Theobroma cacao (Adamson et al., 1999), were


Inhibition of Lipoxygenases by Cocoa Polyphenols

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Fig. 1 Inhibition of Rabbit and Soybean 15-Lipoxygenases by Fractions of Procyanidins Isolated from Seeds of Theobroma cacao. The isolated procyanidin fractions were dissolved in peroxide-free 2-methoxyethanol and diluted to a concentration of 2.9 mg/ml [equivalent to 10 mM (–)-epicatechin]. These solutions (4 µl) were added to the assay buffer (400 µl), and the lipoxygenase activities with potassium linoleate as substrate were recorded oxygraphically as described in Materials and Methods. The activities of the control samples (1% solvent only) were 0.12 nkat and 0.15 nkat for the rabbit and the soybean enzyme, respectively. The solvent did not exert significant effects. The data represent mean values ± SD of 4 – 6 data points from at least 3 independent experiments. Left, hatched columns, soybean 15-lipoxygenase L1; right, solid columns, rabbit 15-LOX-1.

comparatively tested on both 15-LOX-1 and soybean lipoxygenase L1. Since as a rule each subunit of the oligomers contains a complete set of the hydroxyl groups of epicatechin, we added a final concentration of 29.0 µg/ml corresponding to 100 µeq/l epicatechin subunits of each fraction to the assays, irrespective of their different molecular masses. The data, compiled in Figure 1, indicate that with 15-LOX-1 the inhibitory potencies of the procyanidins decreased from monomer reaching a minimum at the trimer and tetramer fractions and increased again through the decamer fraction. Figure 2 shows the dose-response curve for the decamer fraction. Its IC50 value was 8 µeq/ml which corresponds to 0.8 µM. Thus, among cocoa flavanols the large procyanidins are the most potent inhibitors of mammalian 15-lipoxygenase. Soybean lipoxygenase L1 was not significantly inhibited by the dimer and trimer fractions; even at 1 meq/l only weak inhibitory effects were observed (data not shown). Marked inhibition of this enzyme occurred with the tetramer fraction which again increased through the decamer fraction (Figure 1). Interestingly, this increase exhibited an approximately linear dependence on the logarithm of the oligomer size (not shown). Collectively, these data reveal different biological activities of small and large procyanidins. The inhibitory effects of (–)-epicatechin and procyanidins were not abolished by prior complexation with Fe3+ in the absence of phosphate, even at a more than 10-fold molar excess (data not shown), which contrasts the behaviour of other 15-lipoxygenase inhibitors such as sali-

Fig. 2 Dose–Response Curve for the Action of the Procyanidin Decamer Fraction from Seeds of Theobroma cacao on Rabbit 15-LOX-1. Conditions were as described in the legend to Table 1. The concentrations are indicated in micromolar concentrations of epicatechin subunits. Data from 3 independent experiments.

cylhydroxamic acid, 4-nitrocatechol (Schewe et al., 1986) and quercetin (Sadik et al., unpublished data). To examine whether the inhibition by flavanols might be due to reduction of hydroperoxy fatty acids that are required for activation of lipoxygenase, we also tested (–)-epicatechin and procyanidins when the 15-LOX-1 was partly inhibited by glutathione plus glutathione peroxidase which con-


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wine (Frankel et al., 1993; Kinsella et al., 1993). Therefore, we studied the effect of (–)-epicatechin and cocoa procyanidins in these systems. With the Cu2+-dependent reaction, we consistently observed a 1.5- to 1.75-fold prolongation of the kinetic lag phase (Cadenas and Sies, 1998) by (–)-epicatechin and the procyanidin dimer and nonamer fractions from cocoa seeds at concentrations as low as 0.33 µeq/l. By contrast, these concentrations of flavanols, which are below those required to inhibit 15-LOX-1 activity (cf. Table 1), did not affect 15-LOX-1-mediated oxidation of LDL under comparable conditions (data not shown). From these observations it is tempting to speculate that the protection by flavanols of LDL from being oxidised is primarily due to scavenging free radicals rather than inhibiting reticulocyte-type lipoxygenases. Fig. 3 Action of Pycnogenol on Rabbit 15-LOX-1 () and Soybean Lipoxygenase L1 (). Conditions as in Table 1. Data from 3 independent experiments.

trols lipoxygenase activities by lowering the hydroperoxide tone (Hatzelmann et al., 1989; Weitzel and Wendel, 1993). No significant influence on the inhibitory actions was observed (Sadik et al., unpublished work). Pycnogenol, a procyanidin-rich pine bark extract which contains a complex mixture of polyphenols of different chemical classes, inhibited under our experimental conditions both rabbit 15-LOX-1 and soybean lipoxygenase L1 in a dose-dependent manner (Figure 3). On a weight basis, the inhibitory potency of pycnogenol towards 15-LOX-1 was approximately in the range of those of (–)-epicatechin and the cocoa procyanidin fractions of medium oligomer size, but was less active by one order of magnitude than the procyanidin decamer fraction. Soybean lipoxygenase L1 proved to be approximately equally sensitive towards pycnogenol as rabbit 15-LOX1, which is in contrast to the results obtained with the flavanols from cocoa. As can be seen from Figure 3, the IC50 for soybean lipoxygenase L1 was about 10 µg/ml at pH 7.4 and thus considerably lower than that observed by Moini et al. (2000) at pH 9.0 (approximately 40 µg/ml). Action of Cocoa Procyanidins on Oxidative Modification of Low-Density Lipoprotein Oxidative modification of low-density lipoprotein (LDL) is a crucial step in the development and progression of atherosclerosis. It can be brought about by either free radical-induced lipid peroxidation or enzymatic processes or a combination of both. As mentioned above, by virtue of its direct dioxygenation of cholesterol esters in LDL, 15LOX-1 is believed to play a pivotal role in the formation of atherosclerotic lesions (Kühn and Chan, 1997; Cornicelli and Trivedi, 1999; Steinberg, 1999; Cathcart and Folcik, 2000). The interaction of 15-LOX-1 with LDL has been investigated in detail (Lass et al., 1996; Belkner et al., 1998). Inhibition of copper-catalysed oxidation of LDL has been observed earlier for the phenolic fraction of red

Action of Epicatechin and Procyanidins on the Conversion of Arachidonic Acid in LipoxygenaseTransfected Cells The results presented above provide ample evidence that both (–)-epicatechin and procyanidins directly inhibit 15LOX-1 activities at the level of the isolated enzyme. This observation does not imply, however, that these compounds suppress the activity of reticulocyte-type lipoxygenases also in intact cells. Therefore, we conducted experiments using lipoxygenase-transfected cell lines. The macrophage cell line J774A.1 was transfected with the gene for porcine leukocyte 12/15-lipoxygenase which is closely related to rabbit or human 15-lipoxygenase-1 and is therefore regarded as a species isoform of the latter enzymes despite different regiospecificities of the dioxygenation of arachidonic acid (Kühn and Thiele, 1999). These cells were allowed to react with arachidonic acid after pre-treatment with (–)-epicatechin or (–)-epigallocatechin gallate or various procyanidin oligomer fractions from Theobroma cacao. After reaction stop, the hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) formed were analysed by reversephase HPLC. In several independent experiments, we did not observe sizeable inhibition of cellular lipoxygenase activity with any of these flavonoids even at concentrations as high as 1 mM. In some experiments, (–)-epicatechin caused feeble inhibitory effects, whereas the procyanidin oligomers stimulated the formation of lipoxygenase products by an unknown mechanism. In contrast, sonication of the cells before addition of the flavanols rendered their lipoxygenase activity sensitive to these compounds (Table 2; Figure 4). Strongest inhibition was observed with (–)-epigallocatechin gallate which was also the most potent inhibitor of isolated rabbit 15-LOX-1. In another set of experiments we used rabbit smooth muscle cells transfected with human 15-lipoxygenase-1 and observed significant inhibitory effects by (–)-epicatechin and procyanidin nonamer without sonication of the cells (Table 3). Together, the data may reveal different susceptibility of the reticulocyte-type lipoxygenases towards flavanols in different cell types.



Table 2 Effect of Selected Flavanols on the Formation of 12(S)-Hydroxy-5Z,8Z,10E,14Z-Eicosatetraenoic acid (12-HETE) in Intact and Sonically Disintegrated Murine J774A.1 Cells Transfected with Porcine Leukocyte 12/15-Lipoxygenase. Expt. no.

Addition

12-HETE (nmol/106 cells)

Percent of control

I

Intact cells, control (–)-Epicatechin Nonamer

4.74 ± 1.17 3.81 ± 1.00 4.60 ± 0.89

100 80 97

Sonicated, control (–)-Epicatechin Nonamer

4.23 ± 0.26 2.27 ± 0.15 0.68 ± 0.04

100 54 16

Intact cells, control EGCG Hexamer

1.13 ± 0.16 1.90 ± 0.12 1.74 ± 0.74

100 168 154

Sonicated, control EGCG Hexamer

1.51 ± 0.08 0.034 ± 0.004 0.33 ± 0.02

100 2 22

II

Cells (4.35 × 105 or 104 µg protein in experiment I; 6.98 × 105 or 113 µg protein in experiment II, respectively) were obtained as described in Materials and Methods and divided in two parts, one used directly and the other part after sonication. Aliquots were treated with a final concentration of 400 µM flavanol monomer [EGCG = (–)-epigallocatechin gallate] at or 400 µeq/l, procyanidin oligomer, respectively, and 100 µM arachidonic acid as described in Materials and Methods. The formation of 12HETE within 15 min was quantified by peak area related to a standard of authentic 12-HETE. The data are mean values ± SD of independent samples (n = 3). The higher deviations in the samples with intact cells as compared to those with sonicated cells were due to the formation of cell aggregates in the cell suspension.

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(–)-Epicatechin and Procyanidin Decamer Also Inhibit Platelet 12-Lipoxygenase To examine whether the flavanols also inhibit mammalian lipoxygenases other than those of the reticulocyte-type, we studied the effect of (–)-epicatechin and procyanidin decamer on recombinant human platelet 12-lipoxygenase. As shown in Figure 5, (–)-epicatechin inhibited this enzyme dose-dependently with an approximate IC50 of

Table 3 Effect of (–)-Epicatechin and Procyanidin Nonamer on the Formation of 15(S)-Hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic Acid (15-HETE) in Intact Rabbit Smooth Muscle Cells Transfected with Human 15-Lipoxygenase-1. Expt. No. Addition

15-HETE Percent of (nmol/106 cells) control

I

Vehicle (–)-Epicatechin, 33 µM Nonamer, 10 µM Nonamer, 100 µM

3.00 ± 0.06 1.54 2.14 0.71

100 52 77 24

II

Vehicle 3.15 ± 1.3 (–)-Epicatechin, 400 µM 0.19 ± 0.39

100 6

The cells were harvested by scraping off without trypsin and suspended in PBS. The cells (7 × 104 or 75.4 µg protein in experiment I; 1.75 × 104 or 28.5 µg protein in experiment II, respectively) were treated with the flavanols and arachidonic acid as described in Materials and Methods. The formation of 15-HETE within 15 min was quantified by peak area related to a standard of authentic 15-HETE and corrected for blank (autoxidation of arachidonic acid in the absence of cells). The data are mean values ± SD (n = 3).

Fig. 4 Effect of Selected Flavanols on the Formation of Dioxygenated Arachidonic Acid Products with Sonicated 12/15-Lipoxygenase-Transfected Murine J774A.1 Cells. Cells were obtained as described in Materials and Methods. Aliquots corresponding to 7 × 105 cells containing 75.5 µg protein in 250 µl PBS were mixed with 1.0 µl of 100 mM flavanol in 2-methoxyethanol at room temperature as indicated. After 5 min the lipoxygenase reaction was started with 100 µM arachidonic acid and stopped after 15 min by addition of equal volume of cold methanol. Further analysis by reversed-phase HPLC is described in Materials and Methods. Representative patterns from triplicate experiments are shown.


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Fig. 5 Action of (–)-Epicatechin (EC) and Procyanidin Decamer on Recombinant Human Platelet 12-Lipoxygenase. The reactions were followed oxygraphically as indicated in Materials and Methods. Sodium arachidonate (50 µM) was used as substrate. Numbers denote residual activities in percent of the control corrected for baseline drift.

15 µM. With 10 µM procyanidin decamer an inhibition of 54% was observed. However, considering the fact that the decamer consists of 10 epicatechin subunits per mole, it follows that contrary to the results with rabbit 15LOX-1, the decamer proved to be less active than the monomer.

Discussion Direct Inhibition of Mammalian 15-Lipoxygenase-1 by Flavanols and Its Consequence In a number of previous studies the effects of flavonoids on the soybean lipoxygenase isoenzyme L1 have been investigated (Baumann et al., 1980; Alcaraz and Hoult, 1985; Ratty et al., 1988; Robak et al., 1988; Malterud and Rydland, 2000). Despite its similar product pattern with arachidonic and linoleic acids as substrates, the use of soybean lipoxygenase as model for mammalian 15-LOX-1, however, appears to be limited, as can be seen from the pronounced dissociation of the inhibitory effects of flavanols and cocoa procyanidins on the two enzymes (Table 1, Figure 1). Such differences are plausible, since the genetic relatedness of lipoxygenases does not correspond to their positional specificity toward arachidonic acid and, therefore, their classification solely according to this property is not correct (Kühn and Thiele,1999). In this report, we present evidence for the first time that procyanidins and flavanols are direct inhibitors of mammalian 15-lipoxygenase-1. The inhibitory effects of cocoa flavanols were detected independent of the kind of substrate and of the principle of assay applied. Although flavonoids and other polyphenols have been reported to inhibit a number of other enzymes (for review, see e. g. Middleton Jr. et al., 2000),

the inhibition of 15-LOX-1 merits particular attention, as it may constitute an additional pharmacological quality that contributes to the beneficial cardiovascular actions of procyanidins and other dietary polyphenols suggested. These actions are believed to be in large part due to their contribution to plasma antioxidant capacity (Ursini et al., 1999). Moreover, vasorelaxant activity, angiotensin-converting enzyme inhibitory activity as well as improvement of microcirculation by increasing capillary permeability have been reported for the procyanidin-rich pine bark extract pycnogenol (reviewed by Packer et al., 1999), which was found by us to inhibit the 15-LOX-1 as well (Figure 3). The inhibition of 15-LOX-1 activity by procyanidins from various sources as demonstrated here may also have some importance because of the potential pro-atherogenic role of the enzyme (Cyrus et al., 1999; Steinberg, 1999). Recently, it has been reported that procyanidins of cocoa (Schramm et al., 2001) and wine (Facino et al., 1999) cause significant changes in eicosanoid levels in a direction favourable for the cardiovascular system. Thus, intake of high-procyanidin chocolate led to a significant decrease in the ratio of the plasma levels of leukotriene and prostacyclin metabolites in volunteers, and analogous changes were observed upon treatment of vascular endothelial cells with cocoa procyanidins (Schramm et al., 2001). Our observation that (–)-epicatechin and its related procyanidins occurring in cocoa inhibit rabbit and human 15-LOX-1, porcine leukocyte 12/15-lipoxygenase and recombinant human platelet 12-lipoxygenase suggests that these compounds are general inhibitors of mammalian lipoxygenases. This assumption is further supported by earlier reports that certain flavonoids inhibit mammalian 5-lipoxygenase (Yoshimoto et al., 1983; Laughton et al., 1991). Thus, it may be expected that this action should control the formation of leukotrienes in inflammatory cells, diminishing the leukotriene/prostacyclin ratio. In parallel, the scavenging of reactive oxygen and nitrogen species leading to a lowered hydroperoxide tone may protect the prostacyclin synthase from inactivation by hydroperoxides which occurs already at normal physiological hydroperoxide concentrations in plasma (Warso and Lands, 1984) and by low concentrations of peroxynitrite (Zou and Ullrich, 1996). Further studies are required to better understand how the data obtained with the isolated fractions from Theobroma cacao could be extrapolated to the situation in vivo. The data in Tables 2 and 3 appear to indicate the presence of a permeation barrier for flavanols and procyanidins in a macrophage cell line but not in smooth muscle cells. This observation may imply some selectivity. In case that the 15-LOX-1 activity is inhibited only in certain cell types or only upon damage to cells, the flavanols may selectively suppress the pathologic reactions of 15-LOX-1 without affecting its physiologic functions. Thus, the conditions under which the 15-LOX-1-mediated oxidative modification of LDL occurs are far from clear; it may include damage to cells expressing 15-LOX1 and/or release of this enzyme.



Another issue is the fact that the flavanols are metabolised in the gastrointestinal tract and in liver to a number of metabolites (Donovan et al., 1999; Baba et al., 2000; Li et al., 2000). Some of these metabolites may exhibit biological activities surpassing those of the parent compounds. Therefore, future research will be devoted to lipoxygenase-inhibitory activities of epicatechin metabolites. Moreover, flavonoids may be converted to other compounds by reactive metabolites formed specifically under the conditions of oxidative stress. Thus, it should be emphasised that a chemical conversion to other bioactive compounds has been observed for the interaction of (–)-epicatechin with peroxynitrite (Schroeder et al., 2001, and unpublished results) and for that of quercetin with hypochlorous acid (Binsack et al., 2001). Putative Mode of Action of Flavanols on 15-Lipoxygenase-1 The protective actions of procyanidins and other flavonoids on the cardiovascular system are generally believed to be mainly connected with their free radicalscavenging activities. Such a mode of action can be excluded, however, for the inhibition of lipoxygenases by polyphenols. Although during the catalytic cycle of the lipoxygenase reaction intermediate free radicals may actually be formed, they are sequestered tightly bound at the active site of the enzyme and are not accessible for free radical scavengers. For this reason, the reactions of 15-LOX-1 with various substrates are not inhibited by the well-known antioxidants 2,6-di-t-butyl-4-hydroxytoluene (BHT) and probucol (Schnurr et al., 1995). Nordihydroguaiaretic acid (NDGA) is both a scavenger for lipophilic free radicals and a universal lipoxygenase inhibitor. For soybean lipoxygenase, it has been demonstrated that NDGA rapidly reduces the active ferric species of the enzyme to its inactive ferrous form, thus causing interruption of the catalytic cycle (Kemal et al., 1987). An alternative mechanism for the action of polyphenols is a strong complexation of the ferric iron moiety of the lipoxygenase, thus preventing its reduction via the catalytic cycle as proposed for the action of 4-nitrocatechol on the soybean lipoxygenase isoenzyme L1 (Spaapen et al., 1980). A third conceivable mode of action of polyphenols is the effective reduction of hydroperoxides that are essential activators of the lipoxygenase via conversion of the enzymatically silent ferrous species to the active ferric form. Our observation that lowering of the hydroperoxide tone by glutathione plus glutathione peroxidase did not modulate the inhibitory effects of flavanols on 15-LOX-1 does not support the latter possibility. The second observation that complexation of the flavanols with Fe3+ did not abolish the inhibitory effect may rule out a direct complexation of the iron moiety in ferric lipoxygenase by these catechol compounds. Recently, it has been demonstrated for the interaction of 4-nitrocatechol with the soybean lipoxygenase isoenzyme L3 that the ligand sphere of the iron in the enzyme is drastically

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changed upon interaction with the catechol without direct complexation of the iron (Pham et al., 1998). If a similar mode of action holds for the interaction of flavanols with mammalian lipoxygenases, the corresponding ironpolyphenol complexes may retain their lipoxygenase-inhibitory effect. The much higher inhibitory potency of large procyanidin oligomers towards 15-LOX-1 as compared with the monomer may imply a prominent role of the reducing capacity of the flavanols which is expected to increase with the number of hydroxyl or catechol groups per molecule, as the formation of a permanent complex between the enzyme and such voluminous molecules as the procyanidin decamer is difficult to imagine. We found considerably higher inhibitory potencies on 15-LOX-1 for the gallic acid esters of flavanols than for the non-esterified compounds (Table 1), whereas free epigallocatechin showed the weakest effect. Interestingly, similar structure-activity relations have been recently reported for both cutaneous photoprotection from ultraviolet injury (Elmets et al., 2001) and copper-catalysed oxidation of LDL (Osada et al., 2001). Since the protective actions in the latter systems are believed to include scavenging of free radicals, it is reasonable to assume that similar structural requirements occur for the flavanols with respect to both 15-lipoxygenase-inhibitory activity and scavenging of free radicals.

Materials and Methods Enzymes and Chemicals 15-Lipoxygenase from rabbit reticulocytes was purchased from Oxford Biomedical Research (Oxford, MI, USA) and exhibited, under our assay conditions, a turnover number of approximately 2.2 s-1. For selected experiments, 15-lipoxygenase from rabbit reticulocytes with a turnover number of 38 s-1 was purified as described elsewhere (Belkner et al., 1993). Soybean lipoxygenase L1, prepared by affinity chromatography, was obtained from Sigma-Aldrich (Deisenhofen, Germany), recombinant human platelet 12-lipoxygenase from Calbiochem (Bad Soden, Germany), and bovine erythrocyte glutathione peroxidase from Boehringer Mannheim, Germany. Low-density lipoprotein was isolated according to Kleinveld et al. (1992). Procyanidin oligomers, isolated from cocoa as described elsewhere (Adamson et al., 1999; Hammerstone et al., 1999), were kindly supplied by Mars Inc. (Cocoapro, Hackettstown, NJ, USA) and used as stock solutions of 100 meq/l (29.0 mg/ml) in peroxide-free 2-methoxyethanol. Pycnogenol was a kind gift of Dr. L. Packer (Los Angeles, CA, USA). The other flavonoids were purchased from Sigma-Aldrich and used as 0.1 M stock solutions in 2-methoxyethanol. Lipoxygenase Assays Unless stated otherwise, all lipoxygenase measurements were performed in 0.1 M air-equilibrated potassium phosphate (KPi), pH 7.4, containing 0.1 mM diethylenetriamine pentaacetic acid at 20 °C. The activity of rabbit 15-LOX-1 was measured oxygraphically (Oxygenmeter 781; Strathkelvin Instruments, Glasgow, UK) as described elsewhere (Schewe et al., 1987). Briefly, 20 µl enzyme (diluted 1:10 in 0.01 M KPi, pH 6.0) and 4 µl test compound diluted in 2-methoxyethanol were added to the assay


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medium in the oxygraphic measuring chamber. After 2 min preincubation the reaction was started by addition of 20 µl 5.3 mM potassium linoleate in 0.1 M KPi, pH 7.4, containing 4% (w/v) sodium cholate. The linear part of the oxygen uptake trace (in most cases the period from approximately 15 s up to 2 min after start) was evaluated, which was the maximal rate after the lag period was overcome by autoactivation and before suicide inactivation of 15-LOX-1 gave rise to a decline of the activity. The activity of soybean lipoxygenase was measured in a similar way, except for the absence of sodium cholate. For measurement of 12-lipoxygenase activity, the reaction was started with potassium arachidonate (final concentration 50 µM) in the absence of sodium cholate, and was followed for a longer period as the mechanism-based self-inactivation of this enzyme is quite less pronounced. In selected experiments, the 15-lipoxygenase activities were also measured by recording the formation of conjugated dienes at 234 nm (UV/VIS spectrometer Lambda 2, Perkin-Elmer, Norwalk, CT, USA) and yielded similar results. Conversion of Exogenous Arachidonic Acid in Lipoxygenase-Transfected Cells The murine macrophage-like cell line J774A.1 was transfected with porcine leukocyte 12/15-lipoxygenase as described elsewhere (Sakashita et al., 1999). The cells were cultured at 37 °C with 5% (v/v) CO2 in Dulbecco’s minimal essential medium supplemented in a humidified atmosphere with 10% (v/v) foetal calf serum. The cells were sub-cultured every 3-4 days using a standard trypsinisation protocol. After having reached confluence, the cells were harvested by scraping off without trypsin treatment, washed with 10 ml phosphate-buffered saline (PBS), centrifuged and re-suspended in 1 ml PBS. The cell suspension contained about 90% viable cells as judged from trypan blue exclusion test. Before reaction with substrate, the cells (4-8 × 105) were preincubated in 250 µl PBS with the flavanol or procyanidin at room temperature for 5 min. Then 0.75 µl 33 mM arachidonic acid in methanol were added. After 15 min the reaction was stopped by addition of 250 µl cold methanol and 5 µl glacial acetic acid. For reduction of hydroperoxy fatty acids, 5 µl of a saturated solution of sodium borohydride in cold ethanol were added. The samples were separated from denatured proteins by centrifugation at 15 000 g for 5 min. The supernatants were subjected to HPLC separation. To examine the influence of cell integrity on the effects of the flavanols, the cell suspension was sonicated with a probe sonifier (4 × 20 impulses of 0.5 s each) on ice. After this treatment the percentage of intact cells was lowered to less than 1% as judged by microscopy. Rabbit smooth muscle cells transfected with human 15lipoxygenase-1 were a kind gift of Dr. S. Ylä-Herttuala (Kuopio, Finland) and were treated in an identical way with the exception that 70 000 cells per reaction sample or less were sufficient because of the high expression of 15-LOX-1 in these cells. Moreover, the sonicated cells proved to be inactive under these conditions; therefore sonication was carried out immediately after addition of flavanol and arachidonic acid. 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. Reverse phaseHPLC was carried out on a Nucleosil C-18 column (MachereyNagel, KS-system, 250 × 4 mm, 5 µm particle size) coupled with an appropriate guard-column (30 × 4 mm, 5 µm particle size). For analysis of the mono-oxygenated fatty acids (HETE- and HpETE-isomers) a solvent system of methanol/water/acetic acid (80/20/0.1, v/v/v) was used at a flow rate of 1 ml/min. Absorbance was monitored at 235 nm.

Oxidative Modification of Low-Density Lipoprotein (LDL) The oxidative modification of LDL induced by copper or rabbit 15-LOX-1 was studied according to published experimental strategies (Kleinveld et al., 1992; Lass et al., 1996; Mazur et al., 1999). The reaction mixture contained 0.1 µM LDL (as estimated by determination of total cholesterol assuming for LDL a molecular mass of 2.5 MDa and a cholesterol content of 31.5%) in PBS and either 1 µM pure rabbit 15-LOX-1 or 10 µM CuSO4. The reactions were followed continuously at 234 nm (formation of conjugated dienes) at 20 °C.

Acknowledgements This work was supported by Mars Inc. (Hackettstown, NJ, USA) which is gratefully acknowledged. H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD, USA

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