Antioxidant and antiplatelet activity by polyphenol‐rich nutrients: focus on extra virgin olive oil and cocoa

Abstract

Cardiovascular disease is the most common cause of death in the Western world. In the last decades nutraceutical approaches have been proposed to counteract atherosclerotic complications. In particular, polyphenols, a class of bio‐active molecules prevalently contained in foods such as cocoa, fruits, vegetables, wine and tea, have been widely studied for their beneficial properties. Several epidemiological and interventional studies have shown that polyphenol‐rich nutrients, as in extra virgin olive oil (EVOO) and cocoa, are associated with a risk reduction of cardiovascular events and/or modulation of cardiovascular risk factors. Definition of the mechanisms accounting for this putative cardio‐protective effect is still elusive. This review focuses on the mechanisms that may be implicated in the beneficial effects of EVOO and cocoa, including down‐regulation of oxidative stress and platelet aggregation, improvement of endothelial function and cardiovascular risk factor such as blood pressure, serum cholesterol and insulin sensitivity.

Introduction

Cardiovascular disease (CVD) (myocardial infarction and stroke) represent the most relevant cause of death in the Western world. Even though the development of new pharmaceutical therapies continues, the prevalence of cardiovascular disease is growing 1.

During the last decades, a ‘nutraceutical’ approach has been proposed to counteract the spreading phenomenon of cardiovascular disease. The term ‘nutraceutical’, derived from the union of the words ‘nutrition’ and ‘pharmaceuticals’, was coined by De Felice in 1989 to point out the beneficial effects on human health of substances contained in foods 2. A nutraceutical approach focuses attention on biologically active products contained in foods. Several epidemiological and interventional studies have analysed nutraceutical properties of polyphenols in the prevention and treatment of cardiovascular disease.

Polyphenols represent a class of natural, synthetic and semi‐synthetic substances characterized by the presence of large multiples of phenol units. The term polyphenols was proposed in 1962 by the phytochemists White, Bate‐Smith, Swain and Haslam 3, 4. They defined polyphenols as ‘water‐soluble phenolic compounds having molecular weights between 500 and 3000 Da. Besides giving the usual phenolic reactions, they have special properties such as the ability to precipitate alkaloids, gelatin and other proteins from solution’ 3, 4. Polyphenols, in the form of flavonoids, are broadly classified into anthocyanidins (e.g., cyanidin, delphinidin, malvidin), flavanols (e.g., catechin, epicatechin), flavonols (e.g., quercetin, fisetin) and flavones (e.g., luteolin) 4, 5. Natural polyphenols are prevalent in cocoa, fruits, vegetables, wine and tea. Flavonoids account for two‐thirds of the total polyphenolic daily intake (approximately 1 g) 4. After oral ingestion, flavonols undergo a biotransformation from the gut microflora, generating a large variety of metabolites 4, 5, 6; the maximum plasma concentration of flavonols rarely exceeds 1 μM 4, 5, 6.

Epidemiological studies demonstrated that polyphenol intake is associated with reduced risk of cardiovascular disease 7, 8, 9, 10. The Mediterranean diet is the golden standard for healthy nutrition. It is particularly rich in polyphenols and is associated with reduced risk of cardiovascular events 11. Thus, the Mediterranean diet is characterized by high intake of fruits, vegetables, cereals, fish and moderate wine consumption, with scarce intake of dairy products and red meat. The health benefits of the Mediterranean diet have been attributed to the high intake of monounsaturated fats, mostly represented by extra virgin olive oil (EVOO) 12, which is rich of polyphenols (total polyphenols: 435 μg/ml of gallic acid equivalents) 13. Thus, recent results from the PREDIMED study showed that EVOO added to the Mediterranean diet reduces the risk of cardiovascular events compared with controls 14.

Cocoa is another polyphenol‐rich nutrient. Cocoa beans have a high content of polyphenols (total polyphenols: 40–84 mg/g of gallic acid equivalents) 15. This content is sensibly reduced by several production processes (e.g. storage, fermentation, drying and roasting of cocoa beans) 15; consequently, commercial products contain lower concentrations of polyphenols (total polyphenols: 4–60 gallic acid equivalents) 16. The beneficial effects of cocoa have been widely described to prevent and treat cardiovascular disease 17.

This review will report on properties of polyphenol‐rich nutrients such as EVOO and cocoa in patients at risk or with cardiovascular disease and the potential mechanisms responsible for their beneficial effects.

Evidence from epidemiological and interventional studies

Epidemiological studies demonstrated that a diet rich in polyphenols reduces cardiovascular events in the general population and in patients at risk of cardiovascular disease. The clinical effects included a reduction of cardiovascular mortality, myocardial infarction and stroke 7, 8, 9, 10.

The beneficial effects of the Mediterranean diet were described for the first time by Ancel Keys in the Seven Countries study in the 1950s–1980s 18. This observational study showed that people living in Greece, Italy and the former Yugoslavia had a lower mortality rate from cardiovascular disease compared to people living in Northern Europe 18.

The first interventional trial that evaluated the effect of the Mediterranean diet on cardiovascular disease was the Lyon Diet Heart Study 19. The study, which was performed in patients affected by myocardial infarction, demonstrated that the Mediterranean diet reduced the risk of any type of new heart attack or CVD complications by 50%. After the publication of the Lyon trial, some misunderstandings regarding the type of Mediterranean diet tested were raised. One misunderstanding concerned the fact that omega‐3‐rich rapeseed oil, olive oil and margarine were used to replace butter and cream in the experimental group 20. However, the findings of the Lyon trial were confirmed in 2003 by Trichopoulou et al. 21, who found a significant inverse association between adherence to the traditional Mediterranean diet and mortality rate 21.

Recently, the PREDIMED study analysed the effect of the Mediterranean diet on cardiovascular events 14. This trial enrolled 7447 persons randomized to the Mediterranean diet supplemented with extra virgin olive oil, the Mediterranean diet supplemented with mixed nuts, or to control diet 14. This study reported that adopting a Mediterranean‐type diet reduced the risk of CVD complications by 30% (hazard ratios: 0.70; 95% confidence interval: 0.54–0.92, and hazard ratios: 0.72; 95% confidence interval: 0.54–0.96 for the Mediterranean diet with extra virgin olive and Mediterranean diet with nuts), over a follow‐up of about 5 years 14. In particular, compared to control, Mediterranean diet supplemented with EVOO significantly reduced the incidence of cardiovascular events, such as stroke, suggesting a role for EVOO in lowering atherosclerotic progression 14.

Several studies evaluated the effect of cocoa on cardiovascular disease. Some studies observed that the Indians of Kuna, a population living on the island off the Panama coast and following a diet characterized by high intake of cocoa, had lower blood pressure and better kidney function compared to other Pan‐American populations 22, 23. This ‘cardiovascular protection’ was lost after migration of the Kuna Indians to an urban city, where they changed their diet 22. Further epidemiological studies, as in the Iowa Women's Health Study, confirmed the inverse relationship between cocoa intake and cardiovascular disease; in particular, this study reported an inverse relationship between chocolate intake and mortality from coronary heart disease in postmenopausal women 24. In addition, the European Prospective Investigation into Cancer and Nutrition 10 analysed the effect of chocolate on cardiovascular mortality; after an 8‐year follow‐up, the study reported a lower rate of myocardial infarction and stroke in the quartile of subjects with the highest chocolate consumption (7.5 g/day). Another prospective study, the Stockholm Heart Epidemiology Program 25, reported a reduction of cardiac mortality in the group with higher chocolate intake in non‐diabetic patients with previous myocardial infarction. However, the lack of interventional randomized trials limits any conclusion. Therefore the effect of polyphenols contained in cocoa on cardiovascular protection needs further evaluation in the future.

Effects on cardiovascular risk factors

The beneficial properties of cocoa and the Mediterranean diet on the cardiovascular system may be due to their capability to exert beneficial effects on the classic cardiovascular risk factors (Figure 1).

Figure 1.

Polyphenols contained in cocoa and Mediterranean diet exert their beneficial properties on health improving classic cardiovascular risk factors and arterial endothelial function, exerting antioxidant activity and reducing platelet activation

Sub‐studies of the PREDIMED trial showed an improvement in blood pressure, total cholesterol and fasting glucose in patients treated with the Mediterranean diet 14, 26. In particular, the study reported a mean reduction of 2.3 mm Hg for systolic blood pressure, 1.2 mm Hg for diastolic blood pressure, 6.1 mg/dl (P = 0.016) for fasting blood glucose, 11.3 mg/dl for total cholesterol 26.

Recently, we analysed the effect of EVOO in a typical Mediterranean meal on blood glucose and serum lipid profile 27. We found that 2 h after eating, subjects who consumed a meal with EVOO had significantly lower blood glucose, DPP‐4 protein and activity, serum cholesterol and higher levels of insulin, GLP‐1 and GIP levels compared with those without EVOO 27. These findings are in keeping with the assumption that improvement of cardiovascular risk factors is among the most important mechanisms accounting for the protection against cardiovascular disease of the Mediterranean diet.

Polyphenol‐rich foods such as cocoa reduces blood pressure as shown by a decrease in systolic and diastolic blood pressure after dark chocolate intake 28. The underlying mechanism seems to be related to enhanced circulating levels of bioactive nitric oxide species, S‐nitrosoglutathione 28, which possess vasodilating properties, so lowering blood pressure and vascular tone 20. A meta‐analysis of randomized controlled trials confirmed the hypotensive action of polyphenols contained in cocoa 29; however, considering the small sample size of these trials and the great differences in the content of flavonols in chocolate, larger interventional studies are needed to better evaluate the real anti‐hypertensive efficacy.

Polyphenol‐rich cocoa has a positive effect not only on blood pressure but also on lipid profile. Thus, interventional studies demonstrated an increase in HDL cholesterol 30, and a reduction of LDL cholesterol 31, 32, but these findings should be treated with caution because of the small sample size.

Cocoa also has positive effects on glycaemic profile as it improves insulin sensitivity in humans with cardiovascular risk factors such as hypertension 33 and in elderly individuals 34. These findings have been confirmed by a meta‐analysis by Shrime et al. that showed that cocoa polyphenols decreased insulin resistance 35.

Effect on oxidative stress, endothelial function and platelet aggregation

The oxidative stress theory of atherosclerosis is based on the assumption that plaque rupture with ensuing platelet deposition and thrombus growth is dependent upon artery inflammation and consequent accumulation of foam cells 36. Oxidative stress, which is a consequence of an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense, seems to play a key role as demonstrated by experimental study showing that vitamin E administration is able to reduce cholesterol uptake in the macrophages of human atherosclerotic plaque 37, 38, 39.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a key enzyme in the atherosclerotic process; this enzyme is the major cellular ROS source in humans and its activity regulates platelet activation and endothelial function 36.

Cocoa polyphenols and Mediterranean diet exert antioxidant activity that helps counteract the atherosclerotic process (Figure 1). Compared to chocolate with a low content of cocoa (<35%), high content cocoa (>85%) chocolate reduced platelet NADPH oxidase activation, platelet formation of ROS and eicosanoids, and improved artery dilatation in smokers 40. Similar findings have been detected in patients with established atherosclerotic disease, i.e. patients with peripheral artery disease, in whom administration of dark chocolate was associated with NOX2‐mediated oxidative stress down‐regulation and enhanced artery dilatation and maximal walking distance 41, 42. Flammer et al. studied the effect of dark chocolate on coronary vascular function, platelet function and oxidative stress in 22 heart transplant recipients who were scheduled for a regular coronary angiogram 43. The authors showed that dark chocolate induced coronary vasodilation, improved coronary vascular function, and decreased platelet adhesion and oxidative stress 2 hours after chocolate intake 43. Inhibition of LDL oxidation is another mechanism accounting for the cocoa antioxidant property and its ability to retard atherosclerotic progression 32, 44. Thus, ox‐LDL accumulation in the arterial wall is considered a key event in the atherosclerotic process via favouring monocyte recruitment into early atherosclerotic lesions 45. A recent randomized controlled trial in patients with cardiovascular risk factors showed that chronic consumption of cocoa reduced ox‐LDL and increased HDL cholesterol 30.

The Mediterranean diet, and in particular EVOO, also exerts antioxidant activity (Figure 1). In a cross‐over study, we evaluated the effect of EVOO added to a typical Mediterranean lunch (10 g) compared to a control diet with corn oil 13. A significant increase in platelet ROS, 8‐iso‐PGF2α‐III, NOX2 activity, the catalytic sub‐unit of NADPH oxidase, sE‐selectin, sVCAM1 and a decrease in serum vitamin E were detected in controls but not when EVOO was included in the Mediterranean diet 13, suggesting that EVOO protects against post‐prandial oxidative stress 13. Further support for the antioxidant property of the Mediterranean diet has been provided by a prospective, observational study showing that adherence to the Mediterranean diet was associated with lower urinary excretion of 8 iso‐PGF2α, a reliable marker of oxidative stress in vivo 46. The Mediterranean diet also has beneficial effects on platelet function. Thus, in a prospective study performed in patients with atrial fibrillation, adherence to the Mediterranean diet was associated with a significant reduction in 11‐dehydro‐thromboxane A2 (TXA2) urinary excretion, which is a marker of COX1 activation in vivo 47. Of note, EVOO was the only nutrient of the Mediterranean diet which was significantly associated with 11‐dehydro‐TXA2 urinary excretion lowering 47. However, the data regarding polyphenols and platelet activation provided equivocal findings 40, 43, 48, 49, 50, 51. Several studies analysed the effect of cocoa polyphenols on platelet activation (Table 1): ADP‐ or collagen‐induced aggregation, ADP‐ or collagen‐induced ATP release and p‐Selectin expression or release, PFA 100, platelet adhesion and recruitment. Compared to controls, cocoa intake provided divergent and inconclusive results; small sample size, different methodologies and populations (Table 1) could be responsible for these uncertain findings. Further studies with a large sample size are therefore needed to assess the effect of cocoa on platelet function.

Table 1.

Cocoa and platelet activation

Studies	Platelet activation analysis	Sample size cocoa group	Sample size control group	Cocoa/ dosage	Control/ dosage	Δ cocoa group (%)	Δ control group (%)	Comparison between groups after treatment (%)	Time	Subjects
Murphy et al. 48	ADP or collagen‐induced Aggregation ADP 3 μmol ADP 10 μmol‐ induced P selectin expression ADP or collagen‐induced ATP release	13	15	Active tablet (39 mg cocoa flavonols) 6 tablets/d	Placebo tablet (<1 mg cocoa flavanols) 6 tablets/d	−28 (ADP) −16 (collagen) −8.2 (ADP 3 μmol) −2.7 (ADP 10 μmol) −3.5 (ADP) −11 (collagen)	0 ADP −8.3 (collagen) −3.3 (ADP 3 μmol) −3.1 8ADP 10 μmol) −14.2 (ADP) −20 (collagen)	−28.6 (ADP) −9 (collagen) +1.3 (ADP 3 μmol) +0.66 (ADP 10 μmol) +1.8 (ADP) +36 (collagen)	28 days	Healthy, non‐smokers
Wang‐Polagruto et al. 49	PFA 100 s‐P selectin release	16	16	High‐flavanol cocoa beverage (12.4 mg total CFs/g and 446 mg of total flavanols) 36 g powder in 240 ml of water	Low‐flavanol cocoa beverage (1.2 mg total CFs/g and 43 mg of total flavanols) 36 g powder in 240 ml of water	+13.7 +13	+2.8 —	−0.3 —	6 weeks	Hypercholesterolemic postmenopausal women
Hermann et al. 50	Platelet adhesion	10	10	Dark chocolate (74% cocoa) 40 g	White chocolate (4% cocoa) 40 g	−36	−6.25	−53	2h	Healthy smokers
Flammer et al. 43	Platelet adhesion	11	11	Dark chocolate (70% cocoa) 40 g	Control chocolate 40 g	−22.4	−6.8	−7.3	2h	Heart transplant
Flammer et al. 51	Platelet adhesion	10	10	Flavonol‐rich chocolate (70% cocoa) 40 g	Control chocolate 40 g	−22.7 +5.4	+5.7 −0.9	−16.72 +19.6	2h, 1 bar 4 weeks, 2 bars daily	Congestive heart failure
Carnevale et al. 40	Recruitment	20	20	Dark chocolate (≥85% cocoa) 40 g	Milk chocolate (≤35%cocoa) 40 g	−27% (smokers) −14.9% (non‐smokers)	−1.4% (smokers) +2.15 (non‐smokers)	−26% (smokers) −16.6% (non‐smokers)	2h	Healthy subjects vs. smokers

Δ expressed by difference of values between before and after treatment

Conclusions

The data reported here indicate that EVOO and cocoa intake is associated with antioxidant and antiplatelet properties, suggesting that these effects may account for the ability of both nutrients to retard atherosclerotic progression. Interventional studies, overall with EVOO, are consistent with a beneficial impact of this polyphenol‐rich nutrient on cardiovascular disease. Interventional studies with cocoa are needed to support its potential benefit on cardiovascular disease.

Competing Interests

All the authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare no support from any organization for the submitted paper. No conflict of interest exists for this paper.

Loffredo, L. , Perri, L. , Nocella, C. , and Violi, F. (2017) Antioxidant and antiplatelet activity by polyphenol‐rich nutrients: focus on extra virgin olive oil and cocoa. Br J Clin Pharmacol, 83: 96–102. doi: 10.1111/bcp.12923.