Oligomere Proanthocyandine und deren Wirkung auf die Blutgefäße

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Titel: Oligomere Proanthocyandine und deren Wirkung auf die Blutgefäße
Orginaltitel: Effects of Cocoa Extracts on Endothelium-Dependent Relaxation
Erschienen in: JN The Journal of Nutrition(2000)

Autoren: • Malina Karim, • Kellie McCormick, • C. Tissa Kappagoda



The aim of this study was to examine the effects of procyanidins derived from cocoa on vascular smooth muscle. Two hypotheses were tested: 1) extracts of cocoa, which are rich in procyanidins, cause endothelium-dependent relaxation (EDR), and 2) extracts of cocoa activate endothelial nitric oxide synthase (NOS). The experiments were carried out on aortic rings obtained from New Zealand White rabbits. The polymeric procyanidins (tetramer through decamer of catechin) caused an EDR. In addition, the Ca²+-dependent NOS activity, measured by the L-arginine to L-citrulline conversion assay, was significantly increased in aortic endothelia cells exposed to polymeric procyanidins, whereas monomeric compounds had no such effect. These fndings demonstrate that polymeric procyanidins cause an EDR that is mediated by activation of NOS. J. Nutr. 130: 2105S—2108S, 2000.

Plant favonoids exhibit diverse biological properties that have been demonstrated in a variety of experimental systems(Middleton and Kandaswami 1994). The broad spectrum of the different effects produced by these compounds has engaged the attention of researchers for several decades. One recurring theme in these properties is their antioxidant and free radical scavenging ability(Kandaswami and Middleton 1996) How ever, they also have the capacity to complex metals and to modulate the action of enzymes (Chen et al. 1990, Robak and Gryglewski 1988). When viewed against this background, it would not be surprising if flavonoids influenced the contractile state of vascular smooth muscle. A recent study reported from this laboratory examined the effect of red wine on aortic smooth muscle. It was shown that red wine induced endothelium-dependent relaxation (EDR) in rings of rabbit aorta in vitro (Cishek et al. 1997). Alcohol alone, in comparable concentrations, had no effect on these rings. Because red wine also contains a variety of flavonoids, a parallel series of experiments was carried out to determine whether some of the flavonoids extracted from grape seeds could mimic the effects of red wine. The flavonoids, which were a mixture of polymeric procyanidins, also caused EDR. This response was shown to be mediated by nitric oxide (Cishek et al. 1997). Cocoa is a plant produkt that ia rich in procyanidins such as epicatechin and its polymers. It is not known whether the extracts of cocoa have the same effects on vascular smooth muscle as the extracts from grape seed. The current study was undertaken in rings of the rabbit aorta to test two hypotheses: 1) extracts of cocoa, which are rich in procyanidins, cause EDR and 2) extracts of cocoa activate endothelial nitric oxide (NO) synthase (NOS). This study was approved by the Animal Use and Care Committee of the University of California, Davis.


Determination of the effect of cocoa flavonoids on vascular tone. Rabbit aortic rings were obtained as previously described (Kappagodaet al. 1991). Fresh aortic rings in Krebs’ buffer were mounted within 20 min; one can expect the rings to be viable in oxygenated Krebs buffer for several hours. The rings were tested initially to establish that the endothelium was viable by exposing them to acetylcholine after precontraction with norepinephrine (1025 mol/L). The rings can be exposed to repeated doses of norepinephrine and acetylcholine and repeated washings and still be viable. Three aortic rings with functional endothelium were precontracted with norepinephrine (1025 mol/L). When the contraction reached a steady state, cumulative doses of cocoa flavonoids (1027 to 1025 mol/L) were added. A fourth precontracted ring was tested with acetylcholine (1027 to 1025 mol/L) and served as a time control. The usual variation from one ring to another of the same size is normally ,10%. All four rings were taken from the same rabbit. The order of addition of cocoa flavonoids was done in increasing dose to obtain a dose-response curve that is the standard method to determine vasodilatory capacity of pharmacological compounds. The curves are obtained as conventional l cumulative dose-response curves, so doses cannot be added in random order (it is not possible to wash the tissues between doses). These experiments evaluated the effects of acute exposure to flavonoids. In selected rings, the effect of Nv-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, on the responses to procyanidins was tested. In three rings, the effect of atropine (1025 mol/L) was examined. Effect of prolonged exposure to procyanidins. To define the effect of prolonged exposure to all three compounds, the rings were each incubated with procyanidin (1025 mol/L) for 30 min, and then the EDR to cumulative doses of acetylcholine was determined. Also, the effect of prior incubation on dose-dependent EDR to procyanidins was examined. Effect of cocoa procyanidins extract on NOS activity. To determine the effects of cocoa procyanidins extract on NOS activity, aortic thoracic segments of a similar size were suspended in Krebs Henseleit buffer in an organ bath maintained at 37°C and under control conditions or exposed to the pentamer and catechin monomer (1025 mol/L). After 2 min, the tissues were removed from the organ bath, and each segment was pinned to a cork board and cut longitudinally. Endothelial cells were obtained by a single scrape of the luminal surface of the aorta with a blade. The cells were placed in Eppendorf tubes containing 70 ml of 50 mmol/L Tris buffer (pH 7.4) and immediately frozen in liquid nitrogen. Before freezing, 5-ml aliquots of each sample were mixed with 5 ml of methylene blue. The stained cells in the suspension were counted in a hemocytometer using a light microscope. Determination of the endothelial NO production was carried out by a modification of a previously described method (Sobey et al. 1995). The samples frozen in liquid nitrogen were alternatively thawed and refrozen five times in a water bath (37°C) to break up intracellular membranes. Duplicate determinations of the conversion of L-arginine to L-citrulline were made on each sample. The cell homogenate (30 ml) was added to the reaction buffer consisting of 4 mCi of [3H]-L-arginine (Amersham, Arlington Heights, IL), 2 mmol/L NADPH, 6mmol/L FAD, 6mmol/L FMN, 15 mmol/L tetrahydrobiopterin, 1 mmol/L calmodulin and 1.25 mmol/L CaCl2. To determine whether citrulline activity was due to NOS activity, a parallel aortic sample boiled at 100°C was analyzed. Samples were incubated for 30 min at 37°C, and the reaction was terminated by adding 1 ml of ice-cold 100 mmol/L HEPES buffer (pH 5.5) containing 10 mmol/L EGTA. The total volume was applied to a 5-ml AG 50W-X8 column (NA1 form) that had been equilibrated with HEPES buffer without EGTA. Citrulline was eluted twice with 1.0 ml of HEPES buffer. The radioactivity of the eluate was counted with a liquid scintillation counter. NOS activity was quantified in terms of the amount of [3H]-L-citrulline produced/min and the mean number of endothelial cells in the sample. The data were expressed in pmol citrulline generated z min21 z 1026 cells.

Extraction of flavonoids from cocoa. The extracts used in the present study were provided by Mars Incorporated (Hackettstown,NJ). Approximately 230 g of cocoa beans was frozen in liquid nitrogen and ground to a fine powder in a laboratory mill. The powder was extracted three times with 750 ml of hexane to remove lipids. The procedure yielded ;100 g of defatted cocoa powder. The defatted cocoa powder was extracted with 1:l of a mixture of acetone and water (70:30 v/v). The aqueous extract was reextracted with hexane to remove residual lipids. The hexane layer was discarded, and the acetone layer was evaporated at 45°C under partial vacuum to a final volume of 200 ml. The resulting solution was freeze-dried to yield ;20 g of acetone extract. Further purification was undertaken with HPLC using methods previously described by Adamson et al. (1999) and Lazarus et al. (1999). The following extracts were used in this study: 1) procyanidin mixture containing monomers, dimers, trimers and tetramers (combination 1), 2) procyanidin mixture containing pentamers through decamers (combination 2) and 3) pure procyanidin (monomer through decamer). Each of the latter compounds had a purity of .90%. Statistical analysis. Relaxations are expressed as a percent of the contraction in response to norepinephrine. All drug concentrations are expressed as the final concentration in the tissue bath fluid. Group data were expressed as means 6 SEM of n experiments. Analysis of covariance was used to compare dose-response curves. A P-value of ,0.05 was considered statistically significant. The Student’st test was carried out on NOS data to establish significance, and the P-value was corrected with a Bonferroni adjustment. FIGURE 1 EDR responses to acetylcholine, combination 1 (procyanidin mixture containing monomers, dimers, trimers and tetramers), combination 2 (procyanidin mixture containing pentamers through decamers) and pure pentamer in rings of rabbit aorta. Abscissa shows the agonist concentration (mol/L). Ordinate shows the relaxation (percent of maximum contraction). Combination 1 has no effect. The maximal effects of the pentamer and combination 2 are similar to that of acetylcholine (n 5 5).


Effect of cocoa extracts on rings of rabbit aorta. Acetylcholine invariably caused relaxation of aortic rings, and this relaxation was abolished by the removal of the endothelium. Certain cocoa procyanidins also evoked a relaxation of precontracted aortic rings that was abolished by removal of endothelium. Combination 1 had no effect on the aortic rings, whereas procyanidin combination 2 caused a dose-dependent EDR of the rings similar to acetylcholine (n 5 5) (Fig. 1). The responses to procyanidin combination 2 were abolished by L-NAME (n 5 3). Atropine, which abolished the effect of acetylcholine, had no effect on the responses to procyanidin combination 2 (n = 3). When the individual procyanidins were tested, the monomers, dimers and trimers failed to elicit any EDR (Fig. 2). The tetramers through decamers elicited EDR. The failure of combination 1, which contained a small quantity of the tetramer, to elicit an EDR was probably due to a low concentration of the compound in the mixture. Effect of preincubation of tissues with cocoa extracts. Preincubation of the tissues for 30 min with the pentamer and combination 2 (1025 mol/L) attenuated the relaxations evoked by acetylcholine and the extract (Fig. 3). The maximum relaxation to acetylcholine before incubation was 49 6 5.1%, and after incubation with combination 2, it was 0.83 6 0.8% (n 5 5). The maximum relaxation to the pentamer was 46.5 6 4.5%, and after incubation with the pentamer, the response to acetylcholine was 2.1 6 1.4% (n 5 5). To determine whether L-arginine could reverse this inhibition, the tissues were exposed to L-arginine (1024 mmol/L) for an additional 30 min after the initial incubation with the flavonoids. The tissues were then retested with acetylcholine (Fig. 4) and the extracts. The maximum relaxations observed after arginine were 18.3 6 5.7% for the pentamer and 21.5 6 5.7% for acetylcholine. Similar results were obtained with the responses to combination 2. These observations were similar to those observed previously with an extract of grape seed (Karim et al. 1998) (data not shown). Effect of cocoa extracts on NOS activity in endothelial cells. Endothelial NOS activity was measured in tissues in the basal state and after exposure to the pentamer and monomer (Fig. 5). It was found that endothelial NOS activity was FIGURE 3 Effects of incubation of aortic rings with flavonoids on the EDR responses to acetylcholine. Abscissa shows the acetylcholine concentration (mol/L). Ordinate shows the relaxation (percent of maximum contraction). The three stimulus-response curves were obtained after incubation with the corresponding agent for 30 min. Incubation with combination 1 (procyanidin mixture containing monomers, dimers, trimers and tetramers), had no effect on the response. EDR responses to acetylcholine were significantly diminished after incubation with pentamer and combination 2 (procyanidin mixture containing pentamers through decamers) (n 5 5). FIGURE 4 The effect of incubation of aortic rings with arginine after pentamer incubation (30 min) on the EDR responses to acetylcholine. Abscissa shows the acetylcholine concentration (mol/L). Ordinate shows the relaxation (percent of maximum contraction). After incubation with arginine (30 min), the response to acetylcholine was partially restored (n 5 5). Note that the scale on the y axis is different from that in Fig. 3. increased significantly by exposure to the pentamer (181 6 21 pmol of citrulline z 1 million endothelial cells21 z min21, n 5 6) compared with tissues that were maintained in the basal state (110 6 10 pmol of citrulline z 1 million endothelial cells21 z min21, n 5 9; P , 0.05). In contrast, cells obtained from tissue exposed to the monomer showed an activity that was significantly lower than control (36 6 6 pmol of citrulline z 1 million endothelial cells21 z min21, n 5 3). FIGURE 5 The effect of exposure of endothelial cells to pentamer on NOS activity expressed as pmol of citrulline z 1 million endothelial cells21 z min21. The control (baseline) represents unstimulated cells (n5 9). There is a significant increase in the generation of citrulline during acute exposure of aortic tissue to the pentamer (n 5 6) (**significantly different from control and monomer, P , 0.01). The monomer does not stimulate endothelial cells (n 5 3) (*significantly different from pentamer, P , 0.01; *significantly different from control, P , 0.05).


Plant flavonoids are widely recognized as having diverse biological effects that could be beneficial in the prevention and treatment of human diseases (Kuhnau 1976). These effects cover a variety of processes, including inflammation (Kandaswami and Middleton 1984), platelet function (Pellegrini et al. 1996), angiogenesis (Fotsis et al. 1998) and the regulation of vascular tone (Duarte et al. 1993, Fitzpatrick et al. 1993). The investigation reported in this paper has focused on the effects of these compounds on EDR. Previous studies reported in the literature relating to the effect of these compounds on blood vessels have yielded conflicting results. For instance, in the rat aorta, Fitzpatrick et al. (1993) observed an EDR while Duarte et al. (1993) observed a relaxation which was not dependent upon the presence of endothelium. These studies were undertaken with both crude extracts and pure monomeric flavonoids, such as quercetin and catechin. Subsequently, Cishek et al. (1997) demonstrated that extracts derived from grape seed elicited EDR in aortic rings of New Zealand White rabbits fed a nonpurified stock diet. The active agent in the extract were the polymeric procyanidins. The monomers derived from grape seeds were inactive, despite their widely recognized antioxidant activity (Cishek et al.1997). The current study is the first investigation that has examined the effects of cocoa procyanidins on EDR in a systematic manner. The main conclusion from these findings is that tetramers and higher polymers of epicatechin induce EDR in rabbit aortic rings in vitro. The monomers, dimers and trimers were not capable of eliciting an EDR. This finding is of particular interest in the context of the antioxidant properties of these compounds. The procyanidins scavenge free radicals, and those derived from cocoa have been shown to protect the oxidation of LDL in vitro through such a mechanism (Kondo et al. 1996). These observations raise the possibility that the EDR observed in the present study was the result of an “NO-sparing” effect of these compounds (secondary to their antioxidant activity). If such a mechanism were the basis for the EDR observed in this study, a relaxant response would have been evident with the monomeric procyanidins as well because their antioxidant capacity is also well documented (Chen et al. 1990). Thus, it is necessary to seek an alternative explanation. This investigation has clearly demonstrated that the polymeric compounds have the capacity to activate endothelial NOS, whereas the monomeric compounds do not. The phenomena associated with this activation are of interest. There is a similarity between the responses observed with grape seed extracts and cocoa extracts. Monomers from both sources did not evoke an EDR, whereas the polymers did so. Polymeric procyanidins from grape seeds increased cGMP activity in aortic rings (Cishek et al. 1997) and activated endothelial NOS (Karim et al., 1999a). L-NAME blocked the responses evoked by both extracts. Thus, it would be reasonable to conclude that the EDR evoked by the polymeric procyanidins in vitro is due to activation of endothelial NOS. The EDR evoked by both extracts was not blocked by atropine, indicating another mode of activation besides the muscarinic receptors in the endothelial cells. These observations raise the intriguing possibility that the effects of the polymeric procyanidins are mediated by a unique receptor mechanism. Alternatively, a non–receptor-mediated mechanism similar to the action of a calcium ionophore could be involved. An antioxidant effect per se appears unlikely to be the explanation because of the failure of monomeric extracts to elicit EDR. Finally, the effect of prolonged exposure of aortic rings to procyanidins merits comments. It was evident that the EDR in these rings was greatly attenuated. This effect was in part due to loss of the substrate arginine and in part due to inactivation of the NOS. It raises the possibility that the EDR elicited by procyanidins in vitro may not be the best indicator of their effects on the circulatory system in vivo. Indeed, there is no evidence at this time that oligomeric procyanidins are absorbed as such when given orally, although plasma catechin concentrations show an increase after the consumption of cocoa beverages containing polymeric procyanidins. In the absence of direct measurements of polymeric procyanidins in blood, it is not possible to extrapolate findings obtained in vitro to the intact circulation. However, recent studies have shown that EDR is preserved in rabbits fed a nonpurified stock diet enriched with grape seed extracts containing polymeric procyanidins (Karim et al. 1999b).

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