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Keywords:

  • atherosclerosis;
  • cocoa;
  • oxidative stress;
  • platelets

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References

Summary. Background: Dark chocolate is reported to decrease platelet activation but the underlying mechanism is still undefined. Dark chocolate is rich in polyphenols that could exert an antiplatelet action via inhibition of oxidative stress. The aim of the present study was to assess if dark chocolate inhibits platelet reactive oxidant species (ROS) formation and platelet activation. Methods: Twenty healthy subjects (HS) and 20 smokers were randomly allocated to receive 40 g of dark (cocoa > 85%) or milk chocolate (cocoa < 35%) in a cross-over, single-blind study. There was an interval of 7 days between the two phases of the study. At baseline and 2 h after chocolate ingestion, platelet recruitment (PR), platelet ROS, platelet isoprostane 8-ISO-prostaglandin F2α (8-iso-PGF2α), Thromboxane (TxA2) and platelet activation of NOX2, the catalytic sub-unit of NADPH oxidase, and serum epicatechin were measured. Results: Compared with HS, smokers showed enhanced PR, platelet formation of ROS and eicosanoids and NOX2 activation. After dark chocolate, platelet ROS (−48%, P < 0.001), 8-iso-PGF2α (−10%, P < 0.001) and NOX2 activation (−22%, P < 0.001) significantly decreased; dark chocolate did not affect platelet variables in HS. No effect of milk chocolate was detected in both groups. Serum epicatechin increased after dark chocolate in HS (from 0.454 ± 0.3 nm to 118.3 ± 53.7 nm) and smokers (from 0.5 ± 0.28 nm to 120.9 ± 54.2 nm). Platelet incubation with 0.1–10 μm catechin significantly reduced PR, platelet 8-iso-PGF2α and ROS formation and NOX2 activation only in platelets from smokers. Conclusions: Dark chocolate inhibits platelet function by lowering oxidative stress only in smokers; this effect seems to be dependent on its polyphenolic content.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References

Epidemiologic studies demonstrated that a diet rich in polyphenols reduces cardiovascular events in the general population and in patients at a risk of cardiovascular disease [1–6]. The clinical effects included a reduction in cardiovascular mortality, myocardial infarction and stroke [1–6].

Platelets play a key role in the athero-thrombotic process as shown by a significant reduction in cardiovascular events using antiplatelet drugs in acute and chronic coronary syndromes and in patients with acute and chronic cerebro-vascular disease [7]. Several nutrients containing polyphenols have been investigated in humans to assess if they possess antiplatelet effects. Polyphenol-rich cocoa or dark chocolate consistently showed an acute inhibitory effect on agonist-induced platelet aggregation: thus, among nine interventional trials only one showed no effect on platelet aggregation [8–16]. However, the mechanism accounting for the inhibitory effect of cocoa and dark chocolate on platelet function was not clarified [8–16].

There is a great deal of evidence that show polyphenols possess antioxidant properties that may be implicated in the antiplatelet effect of cocoa or dark chocolate. Thus, polyphenols inhibit NADPH oxidase-dependent platelet reactive oxygen species (ROS) formation or enhance nitric oxide (NO) generation and/or bioactivity [17]; both of these activities may be involved in the inhibition of platelet function [17]. Furthermore, ROS generated by NADPH oxidase are involved in the formation of isoprostane 8-ISO-prostaglandin F2α (8-iso-PGF2α), which plays a pivotal role in the amplification of the platelet response to the agonists via activation of Glicoprotein IIb/IIIa [18]. Therefore, we aimed to investigate if acute ingestion of polyphenol-rich dark chocolate or milk chocolate, that has lower polyphenol content [19,20], differently affected platelet-derived oxidative stress and platelet activation. At this end, we compared the effect of dark or milk chocolate in healthy subjects and in smokers, who are known to have enhanced oxidative stress and platelet activation [21,22]. Furthermore, we performed an in vitro study to evaluate if epicatechin, one of the polyphenols commonly found in the human circulation after dark chocolate administration [19], affected platelet function with an oxidative stress-mediated mechanism.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References

Twenty healthy subjects (HS) and twenty smokers, matched for age and gender, (Table 1) agreed to participate in the study, which was performed between October and December 2010. Each smoker consumed an average number of cigarettes per day of 11 (range 6–28). All study participants did not take any medications. They were randomly allocated to a treatment sequence with 40 g of dark chocolate (≥ 85% cocoa) or milk chocolate (≤ 35% cocoa) in a cross-over, single-blind design. There was a 1-week washout between the two phases of the study.

Table 1.   Clinical characteristics of smokers and HS
VariablesHS (n = 20)Smokers (n = 20)P-value
  1. *Data are expressed as mean ± SD. HS, healthy subjects; BMI, body mass index.

Mean age (years)*33 ± 1133 ± 110.954
Males/females7/137/131.0
High (cm)*168.7 ± 8.5168.7 ± 10.11.0
Weight (kg)*65.5 ± 9.864.75 ± 9.50.808
BMI*23 ± 2.822.8 ± 3.30.775
Systolic blood pressure (mmHg)*113 ± 10114 ± 120.977
Diastolic blood pressure*68 ± 668 ± 80.983
Cigarettes/day*011 ± 5.2< 0.001

Commercially available chocolate was used for the present study; compared with milk chocolate (40 g), dark chocolate (40 g) has a higher content of fiber (3 vs. 0 g) and a lower content in cholesterol (0 vs. 10 mg), sodium (20 vs. 40 mg) and sugar (5 vs. 20 g). Furthermore, there were no significant differences in calorific content between the dark (Calories 230) and milk (Calories 220) chocolate.

Platelet activation and oxidative stress, as assessed by recruitment, ROS production, soluble NOX2-derived peptide (sNOX2-dp), 8-iso-PGF2α, nitric oxide (NO) generation and thromboxane formation were assessed at baseline, after 24 h’ abstinence from food rich in polyphenols, and 2 h after ingestion of chocolate. Participants were studied after a fasting period of 8 h and, for smokers, after a smoke-free interval of at least 2 h before each experiment.

All subjects underwent a full medical history and physical examination. Subjects were excluded from the study if they had liver insufficiency, serious renal disorders (serum creatinine> 2.8 mg dL−1), acute cerebro-vascular disease, acute myocardial infarction, dyslipidemia, diabetes, hypertension or were taking antioxidants or any drug known to interfere with platelet function.

Randomization and blinding

An individual not involved in the study, assigned codes to the study treatments, randomly allocated the participants to a treatment sequence with dark or milk chocolate, and kept the key in a sealed envelope. The randomization was carried out by a procedure based on a random numeric sequence. The authors and laboratory technicians were unaware of the treatment allocation.

Platelet preparation

To obtain platelet-rich plasma (PRP), samples were centrifuged 15 min at 180 g. To avoid leukocyte contamination, only the top 75% of the PRP was collected. The platelet pellet was suspended in HEPES buffer, pH 7.4 (2 × 108 platelets mL−1, unless otherwise noted). Platelets were activated with or without arachidonic acid (AA) (0.5 mm) for 10 min at 37 °C and supernatant stored at −80° unless otherwise indicated.

Platelet ROS production

The cell suspension was incubated with 2’,7’-dichlorofluorescin diacetate (5 μmol L−1) for 15 min at 37 °C. After incubation, platelets were activated with AA. Platelet ROS production was expressed as mean fluorescence and ROS production in stimulated cells, and was expressed as the stimulation index (mean level of fluorescence in stimulated cells/mean level of fluorescence in unstimulated cells) (SI).

Fluorescence intensity was analyzed on an Epics XL-MCL cytometer (Coulter Electronics; Beckman Coulter, Milan, Italy) equipped with an argon laser at 510–550 nm (green). For every histogram, 50 000 platelets were counted to determine the proportion of positive platelets. The fluorescent signal generated by the probe was expressed as mean fluorescence intensity (SI). Intra-assay coefficient of variation was 5%.

Platelet 8-iso-PGF2α assays

To measure the platelet isoprostane 8-iso-PGF2α, the cell platelet suspension was activated with AA. The supernatant was stored at −80 °C until measurement. Quantification of isoprostanes was performed measuring 8-iso-PGF2α using a previously described and validated EIA assay method [23] and was evaluated in vivo and in vitro studies. Intra-assay and inter-assay coefficients of variation were 5.8% and 5.0%, respectively.

ELISA detection of platelet sNOX2-dp

NOX2-derived peptide, a marker of NADPH oxidase activation, was detected in vivo and in vitro studies using the ELISA method as previously described by Pignatelli et al. [24]. The peptide was recognized by a specific monoclonal antibody against the amino acidic sequence (224–268) of the extra membrane portion of NOX2. To measure sNOX2-dp, the platelet suspension was activated with AA and 100 μL of the supernatant stored at −80 °C until measurement. Values were expressed as pg mL−1, and intra-assay and inter-assay coefficients of variation were 5.2% and 6%, respectively.

Platelet recruitment

Platelet recruitment was performed with a method modified from that described by Krotz et al. [25]. Platelet-rich plasma samples were incubated (30 min at 37 °C) with or without scalar doses of epicatechin (0.1–10 μm), catechin (0.1–10 μm), epicatechin + catechin (0.1–10 μm) or NOX2ds-tat (a specific inhibitor of NADPH oxidase) (0.1–10 μm), before activation with collagen (6 μg mL−1). Solvents were used as controls. In some experiments, catechin and epicatechin were removed from the supernatant after 30 min incubation to evaluate the recovery of platelet reactivity. Collagen-induced platelet aggregation was measured for 10 min. Then, an equal portion of untreated platelets was added to each tube, which increased the density of the solution and hence caused a reduction in light transmission. Aggregation of the newly added platelet portion in the presence of an existing aggregate was then measured for 5 min and expressed as a percentage of the aggregation that had been initially reached [17]. Platelet aggregation was measured according to Born’s method [17] and calculated as light transmission difference (LT%) between PRP and platelet-poor plasma (PPP) as described previously [26].

Platelet NOx

A colorimetric assay kit (Tema Ricerca, Italy) was used to determine the nitric oxide metabolites nitrite and nitrate (NOx) in 100 μL of the unstimulated platelet suspension maintained under stirring conditions for 10 min at 37 °C. Intra-assay and inter-assay coefficients of variation were 2.9% and 1.7%, respectively.

Platelet TxA2

AA (0.5 mm)-activated PRP samples were centrifuged and the supernatant stored at −80 °C. Platelet TxA2 was analyzed as previously described [26,27]. Briefly, platelet TxA2 was measured by evaluating its stable metabolite TxB2 using an EIA commercial kit (Amersham Pharmacia, Biotech, Little Chalfont, UK) and expressed as pg per 108 cells or ng mL−1, respectively. Intra- and inter-assay coefficients of variation for TxB2 EIA Kit were 4.0% and 3.6%, respectively.

Sample preparation and epicatechin evaluation in HPLC

100 μL serum was extracted twice with 0.8 mL followed by 0.5 mL of ethyl acetate by vortexing for 1 min in a 1-mL vial. After centrifuging the mixture for 5 min at 3500 g, the top layers comprising ethyl acetate were removed, pooled and evaporated to dryness under nitrogen [28]. The dried samples were dissolved in methanol (0.1 mL) before analysis using HPLC.

The determination of epicatechin (EC) was carried out using an Agilent 1200 Infinity series HPLC system equipped with an Eclipse plus C18 column (4.6 × 100mm). All determinations were undertaken at 25 °C. An isocratic mobile phase consisting of water–methanol (80:20) was used; running at a flow rate of 1.2 mL min−1 and the UV detection were carried out at 280 nm. The sample injection volume was 20 μL. Chromatographic peaks of analytes were identified by comparing their retention times with those of the standard [29].

In vitro study

In vitro study was performed in blood taken from five healthy subjects and five smokers matched for age and gender (six males and four females, mean age 32 ± 4 years) who had not taken dark or milk chocolate. We analyzed the effect of epicatechin, catechin, epicatechin plus catechin or an inhibitor of NADPH oxidase (NOX2ds-tat) on platelet ROS production, 8-iso-PGF2α formation, platelet recruitment and sNOX2-dp after platelet activation. Platelets were incubated for 10 min at 37 °C with 0.1–10 μm epicatechin, 0.1–10 μm catechin, 0.1–10 μm epicatechin plus 0.1–10 μm catechin or 0.1–10 μm NOX2ds-tat before stimulation with the agonists.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References

Sample size determination

We computed the minimum sample size with respect to a two-tailed one-sample Student’s t-test with Welch’s correction, considering (i) a difference in platelet sNOX2-dp variation in smokers to be detected between dark and milk chocolate treatments |δ|≥ 10, (ii) standard deviation of the paired differences SD = 5 and (iii) a type I error probability α = 0.05 and power 1-β = 0.90.

This resulted in n = 12 patients, which was increased to n = 20.

Statistical methods

Continuous variables are reported as means ± SD unless otherwise indicated. Comparisons between smokers and HS were carried out using the Student’s t-test and were replicated as appropriate with non-parametric tests (Kolmogorov–Smirnov [z] test in case of non-homogeneous variances as verified by Levene’s test).

The cross-over study data were analyzed for the assessment of treatment and period effects, by performing a split-plot anova with one between-subject factor (treatment sequence) and two within-subject factors (period 1 vs. 2; pre- vs. post-treatment) [30]. The full model was considered, allowing for the assessment of all main effects and two- and three-way interactions. Pair-wise comparisons were corrected using Bonferroni’s correction. In vitro experiments were analyzed by anova. A value of P < 0.05 was considered statistically significant. All analyzes were carried out with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References

Interventional study

Clinical characteristics of smokers and HS are reported in Table 1. There were no differences between the two groups with the exception of smoking habit.

At baseline, compared with HS, smokers had a higher platelet production of ROS, sNOX2-dp and 8-iso-PGF2α (Fig. 1A–C) and lower platelet NOx (Fig. 1, Panels E); platelet TxB2 production was weakly increased in smokers with borderline significance (P = 0.048) (Fig. 1, Panel D). Furthermore, platelet recruitment was higher in smokers compared with HS (Fig. 1, Panel F); epicatechin serum levels did not differ between the groups (Fig. 2).

image

Figure 1.  Platelet reactive oxygen species (ROS) (Panel A), platelet sNOX2-dp (Panel B), platelets 8-iso-PGF2α (Panel C), TxB2 (Panel D), platelet NOx levels (Panel E) and platelet recruitment (Panel F) before and 2 h after the intake of dark or milk chocolate in smokers and healthy subjects (HS). Data are presented as means + standard error (SE). (*P < 0.05).

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image

Figure 2.  Serum levels of epicatechin before and 2 h after intake of milk (Panel A) or dark (Panel B) chocolate in smokers and healthy subjects (HS). Data are presented as means + SE. (*P < 0.05).

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After dark chocolate intake, platelet oxidative stress changed in smokers but not in HS. Thus, 2 h after dark chocolate intake, smokers showed lower platelet ROS and NOX2 activation and enhanced platelet NOx (Fig. 1, Panels A, B and E) compared with baseline. Platelet production of eicosanoids was differently modified by dark chocolate as platelet 8-iso-PGF2α decreased whereas platelet TxB2 did not change (Fig. 1, Panels C and D). Dark chocolate administration reduced platelet recruitment in smokers but not in HS (Fig. 1, Panel F). Milk chocolate did not affect platelet oxidative stress, eicosanoid production and platelet function in either smokers or HS (Fig. 1).

The blood epicatechin concentration was measured at baseline and 2 h after dark or milk chocolate administration. Blood epicatechin increased significantly only after dark chocolate in either smokers or HS (Fig. 2).

In vitro study

The basal values of platelet ROS production (mean fluorescence 1.3 ± 0.2 vs. 3.6 ± 0.3, P = 0.004, respectively) (Fig. 3, Panels A and B), sNOX2-dp levels (8.8 ± 1.9 pg mL−1 vs. 14.5 ± 4.6 pg mL−1, P = 0.02, respectively) (Fig. 3, Panels C and D), 8-iso-PGF2α (14.1 ± 4.9 pmol L−1 vs. 20.1 ± 6.1 pmol L−1, P = 0.03, respectively) (Fig. 3, Panels E and F) and platelet recruitment (4.15 ± 1.17% vs. 7.1 ± 1.5%, P = 0.01, respectively) (Fig. 3, Panels G and H) were significantly higher in smokers as compared with HS.

image

Figure 3.  Effect of scalar doses of epicatechin (0.1–10 μm), catechin (0.1–10 μm), epicatechin plus catechin (0.1–10 μm) or NOX2ds-tat (0.1–10 μm) on platelet reactive oxygen species (ROS) (Panel A and B), platelet sNOX2-dp (Panel C and D), platelet 8-iso-PGF2α formation (Panel E and F) and platelet recruitment (Panel G and H). The experiments were performed in blood taken from five smokers and five healthy subjects (HS). *P < 0.001.

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AA-stimulated platelet incubated with epicatechin, epicatechin plus catechin or NOX2ds-tat resulted in a significant inhibition of platelet oxidative stress and function including platelet ROS, NOX2 activation, 8-iso-PGF2α formation and platelet recruitment. Epicatechin or catechin alone dose-dependently inhibited oxidative stress and platelet function with an effect which was higher when used in combination. The inhibitory effect of polyphenols was observed in platelets from smokers but not in platelets from HS (Fig. 3). Conversely inhibition of oxidative stress and platelet function by NOX2ds-tat was detected in both smokers and non-smokers.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References

The present study provides evidence that in smokers dark chocolate inhibits platelet 8-iso-PGF2α via down-regulation of platelet NOX2-induced ROS generation.

ROS serves as a second messenger of platelet activation via modulation of several intra-signaling mechanisms including activation of PLA2, inactivation of nitric oxide (NO) and the formation of 8-iso-PGF2α [17,18,26,31]. Isoprostanes are chemically stable eicosanoids that are derived from ROS interactions with AA [32]; they are pro-aggregating molecules that favor propagation of platelet aggregation via activation of Glycoprotein IIb/IIIa [18]. NOX2 plays a key role in platelet isoprostane formation as suggested by low 8-iso-PGF2α production in platelets taken from patients with hereditary deficiency of NOX2 [33]. The first novel finding of the present study is the up-regulation of NOX2 in platelets from smokers reinforcing the concept that smoking is associated with oxidative stress [21]. The increase of NOX2 was coincident with enhanced ROS, 8-iso-PGF2α formation and platelet recruitment suggesting a link between ROS generated by NOX2 and 8-iso-PGF2α -induced platelet activation. Smokers had a weak increase of platelet TxB2, which is apparently in contrast with a previous study showing no difference in serum TxB2 between smokers and non-smokers [34]. However, the difference between smokers and non-smokers was of borderline significance (P = 0.05), therefore we are not certain that such a weak COX-1 up-regulation is of biologic relevance.

In the present study, we tested the hypothesis that a polyphenol-rich nutrient down-regulates platelet NOX2 and in turn reduces platelet activation via lowering 8-iso-PGF2α formation. The present study hypothesis was supported by the results of the interventional study as dark chocolate but not milk chocolate was associated with down-regulation of ROS generated by platelet NOX2, platelet 8-iso-PGF2α formation and platelet recruitment. The reduction in oxidative stress probably accounted for the enhanced platelet NO generation as oxygen free radicals are known to inactivate NO or reduce NO synthase activation [17]. The antioxidant and antiplatelet effects were observed in smokers but not in non-smokers suggesting that baseline generation of ROS is a prerequisite to lower oxidative stress by compounds with antioxidant property. This finding is apparently in contrast with a previous report from our groups showing that Vitamin E is an antioxidant molecule that inhibits the formation of lipid peroxides [35] and platelet aggregation in healthy subjects [35]; however, vitamin E possesses other properties that may affect platelet function with mechanisms independent from its antioxidant action [36].

In contrast to 8-iso-PGF2α, dark chocolate had hardly any influence on platelet TxB2 indicating that COX1 activation was not affected by a polyphenol-rich nutrient. This is consistent with previous data showing that oxidative stress has a neutral action on COX1 activation [18].

To explore the mechanism trough which dark chocolate inhibited 8-iso-PGF2α formation and platelet activation, in vitro experiments were conducted by incubating platelets with epicatechin. Thus, after dark chocolate epicatechin was detected in the peripheral circulation of both smokers and non-smokers at a concentration similar to that previously detected after administration of dark chocolate or cocoa [15]. Conversely, blood epicatechin did not increase after milk chocolate probably because its polyphenol content is much lower compared with dark chocolate [20] or that the antioxidant effect of cocoa is attenuated if milk is added.

The experiments demonstrated that epicatechin, at a concentration found in the peripheral blood after dark chocolate administration, possess an antioxidant property as its incubation with platelets these patients resulted in lowered NOX2 activation and ROS generation. This effect was associated with reduced 8-iso-PGF2α formation and platelet recruitment suggesting down-regulation of platelet ROS as a mechanism potentially impairing activation of platelets via reduced 8-iso-PGF2α production. Of note, such changes were observed only in platelets from smokers while no effect of epicatechin was detected in platelets from non smokers. This would imply that the rate of intracellular ROS generation could represent a ‘conditio sine qua non’ to lower oxidative stress by molecules with antioxidant properties. However, it is interesting to note that platelet incubation with an inhibitor of NOX2 resulted in down-regulation of oxidative stress and platelet activation also in healthy subjects. Therefore, it cannot be excluded that another pharmacologic approach with polyphenol-rich nutrients may results in oxidative stress and platelet activation inhibition also in healthy subjects.

The present study corroborates and extends previous reports showing that polyphenol-rich nutrients such as dark chocolate exert an antiplatelet effect with an antioxidant effect. Herewith we show for the first time that such antiplatelet activity is mediated by NOX2 down-regulation and ultimately lowered 8-iso-PGF2α formation [15]. The inhibition of NOX2 activation by epicatechin is consistent with other studies showing that polyphenols possess antioxidant effects via inhibition of ROS generated by NOX2 [37]. Even if epicatechin per se may account for the inhibitory effect of dark chocolate, it is possible that a synergism among polyphenols contributes to the antiplatelet effect in vivo. Thus, an in vitro combination of epicatechin with catechin, another polyphenol contained in dark chocolate, potentiated the antioxidant and antiplatelet effect achieved by a single polyphenol. This is in accordance with our previous studies showing that polyphenols synergize in inhibiting platelet NADPH oxidase [17] and would imply that the antiplatelet effect of dark chocolate could be attributed not only to epicathechin but also to other polyphenols contained in dark chocolate.

The present study has implications and limitations that should be acknowledged. In spite of the intrinsic difficulties in performing a double-blind study with dark and milk chocolate, we tried to mitigate this bias by performing blind laboratory analyzes. We did not analyze how long the antiplatelet affect of dark chocolate lasted, therefore, a further study is necessary to explore this issue. Hence, the present study should be considered a proof-of-concept study with no implication for clinical purpose but suggests analyze if the antiplatelet effect is detectable also after chronic administration of dark chocolate. The lack of antiplatelet effect in non-smokers reinforces the concept that nutrients with antioxidant properties are unlikely to counteract oxidative stress in healthy subjects and should not be used in the general population [38].

In conclusion, the present study demonstrates that dark chocolate down-regulates platelet ROS generated by NOX2 and ultimately inhibits platelet activation via inhibition of platelet 8-iso-PGF2α. Such an effect seems to be related to the antioxidant property of its polyphenol content as platelet incubation with epicatechin and catechin reproduced the findings achieved in vivo. The results warrant further study to see if a similar effect can be obtained with chronic administration of dark chocolate.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References

R. Carnevale study design, paper preparation and study coordination; L. Loffredo study design, paper preparation; P. Pignatelli paper preparation, data elaboration laboratory experimental procedures; C. Nocella laboratory experimental procedures; S. Bartimoccia laboratory experimental procedures; S. Di Santo laboratory experimental procedures; F. Martino patients’ recruitment; E. Catasca patients’ recruitment; L, Perri clinical data collection; F. Violi study design and paper preparation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. Addendum
  9. Disclosure of Conflict of Interest
  10. References
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