J Thromb Thrombolysis (2009) 28:482–488 DOI 10.1007/s11239-008-0273-3

Dark : consumption for pleasure or therapy?

Giuseppe Lippi Æ Massimo Franchini Æ Martina Montagnana Æ Emmanuel J. Favaloro Æ Gian Cesare Guidi Æ Giovanni Targher

Published online: 23 September 2008 Ó Springer Science+Business Media, LLC 2008

Abstract Traditional chocolate is derived from the cocoa preferable to consume than the white (milk) bean, which is one of the most concentrated sources of variety. flavanols, a subgroup of the natural antioxidant plant compounds called flavonoids. Accumulating evidence from Keywords Cardiovascular disease Á Cardiovascular risk Á the past 10 years demonstrates that moderate consumption Chocolate Á Cocoa of chocolate, especially dark chocolate, may exert protec- tive effects against the development of cardiovascular disease. Several mechanisms have been proposed to Introduction explain this positive influence, including metabolic, anti- hypertensive, anti-inflammatory, and anti-thrombotic Cardiovascular disease (CVD) is the main cause of death in effects, as well as effects on insulin sensitivity and vascular the Western world. Nutrition plays a key role in the pre- endothelial function. Should these results be confirmed in vention of many chronic diseases including CVD (both randomised, controlled, cross-over, multi-dose trials, then coronary heart disease and stroke), cancers, and degener- the pleasure associated with chocolate consumption might ative brain diseases [1–3]. Evidence is now accumulating also be justified from health and psychological perspec- that antioxidant nutrients and related bioactive compounds tives. However, since dark chocolate has substantially common in some foods might protect against insults to the higher levels of flavonoids than , and milk vascular endothelium associated with vascular diseases proteins may inhibit absorption of flavonoids, it might be such as atherosclerosis. Accordingly, several lines of evi- dence support the role of diets rich in fruits and vegetables in promoting health and attenuating, or delaying, the onset of various diseases, including CVD [4]. G. Lippi Á M. Franchini (&) Á M. Montagnana Á G. C. Guidi Sezione di Chimica Clinica, Dipartimento di Scienze Chocolate and cocoa are different products. Indeed, Biomediche e Morfologiche, Universita` di Verona, Verona, Italy while cocoa is the non-fat component of cocoa liquor e-mail: [email protected] (finely ground cocoa beans), chocolate comprises a com- bination of cocoa, , sugar, etc. into a solid food M. Franchini Servizio di Immunoematologia e Trasfusione, Dipartimento product [5]. The traditional chocolate, as commonly di Patologia e Medicina di Laboratorio, Azienda Ospedaliero- available on the market, is thus derived from the cocoa Universitaria di Parma, Via Gramsci, 13, 43100 Parma, Italy bean, which is one of the most concentrated sources of flavanols, a subgroup of the natural antioxidant plant E. J. Favaloro Department of Haematology, Institute of Clinical Pathology compounds called flavonoids, found in tea, red wine, and Medical Research (ICPMR), Westmead Hospital, tomatoes, fruits, and other foods. Flavonoids belong to a Westmead, Australia class of plant derived compounds called polyphenols [6]. The basic structure of flavonoids is a C6–C3–C6 backbone G. Targher Sezione di Endocrinologia, Dipartimento di Scienze Biomediche with two aromatic rings and varying degrees of hydroxyl- e Chirurgiche, Universita` di Verona, Verona, Italy ation differentiating one flavonoid type from another [7]. 123 Chocolate and cardiovascular disease 483

Flavonoids can be divided into various subclasses, the most mechanisms by which dietary flavonoids from chocolate important of which are flavones, flavonols, flavanones, may be protective against development of CVD; these catechins, anthocyanidins, and isoflavones. Cocoa is par- include metabolic, antihypertensive, anti-inflammatory, and ticularly rich in the flavanols, epicatechin, catechin, and anti-thrombotic effects, as well as effects on insulin sensi- procyanidins (polymers of catechins and epicatechins) [8]. tivity and vascular endothelial function (Fig. 1). In dark chocolate, the biologic effects of flavonoids are greater than in white (milk) chocolate, because of the rel- Metabolic effects ative amount of these compounds (52.2 ± 20.2 lmol/g vs. 126 ± 7.4 lmol/g respectively) [9] and furthermore since A key pathogenic mechanism of atherogenesis is the oxi- the intestinal absorption of flavanoids is reduced in white dation of low-density lipoprotein cholesterol. The chemical chocolate due to the interference of milk proteins [10]. structure of flavonoids gives these compounds free radical Also relevant is the fact that the fat in the chocolate scavenging capacity, which means that flavonoids may comes from cocoa butter and consists of approximately have anti-oxidant effects [20]. Flavanoids in chocolate equal amounts of oleic acid (a monounstaurated, heart- have been shown to exert potent antioxidant effects using healthy, fatty acid) and stearic and palmitic acids (both in vitro assays under artificial oxidative stress [21–25]as saturated fats). Although saturated fats are ordinarily linked well as to increase plasma total antioxidant capacity in to a higher risk of CVD, there is large body of evidence various short-term randomized chocolate feeding trials suggesting that stearic acid may be cholesterol-neutral [11]. [25–30]. An ex vivo study shows that epicatechin, the Palmitic acid, on the other hand, does not significantly major polyphenol in chocolate and chocolate extracts, is a affect cholesterol levels, but represents only one-third of powerful inhibitor of plasma lipid oxidation due to poly- fat-related calories in chocolate. phenols’ ability to bind to lower density lipoproteins (LDLs) [31]. In an animal model of atherosclerosis, cocoa significantly inhibited atherosclerosis, lowered total cho- Cocoa intake and cardiovascular diseases. Possible lesterol, LDL, triglycerides and protected the lower density anti-atherothrombogenic mechanisms of chocolate lipoproteins from oxidation [32]. Previous studies have also shown that ingestion of cocoa or chocolate may inhibit Cocoa was believed to strengthen the heart and reduce LDL oxidation ex vivo [24], thereby lowering lower LDL angina pectoris as early as the eighteenth century [12], but cholesterol [33], and improving insulin sensitivity as esti- these presumptive benefits were not based on scientific mated by an homeostasis model assessment [34, 35]. evidence. Successively, a large number of studies have Accordingly, the regular consumption of cocoa flavanol- since investigated the effects of chocolate and cocoa intake containing chocolate bars with added plant sterols can on CVD. Although these results are promising, most significantly lower serum total and LDL-cholesterol levels studies used chocolate or cocoa that contained much higher [36]. Recent evidence also suggests that long-term con- amounts of flavan-3-ols than commercially available sumption of cocoa polyphenols increases the antioxidative products [13]. Interestingly, the Kuna Indians, an indige- capacity of plasma and inhibits LDL oxidation ex vivo [26, nous population in Panama with a regular consumption of a 27, 29]. Moreover, because lipid-soluble flavonoids may cocoa [14, 15], have been shown to have a very low inci- intercalate into the membranes of lipoprotein particles, dence of age-related hypertension [14] as well as a low rate some studies have shown flavonoids to decrease lipid of CVD disease mortality [16]. A recent meta-analysis also peroxidation of biological membranes [37]. indicates that the flavonoid content of chocolate may sig- That dietary flavonols may protect human lymphocytes nificantly lower the risk of coronary heart disease mortality from oxidative damage in vivo has also been demonstrated (RR = 0.81 (95% CI: 0.71–0.92) comparing highest and in a randomized clinical trial [38]. Recent studies also lowest tertiles [11]. show that polyphenolic substances derived from cocoa Since observational studies of stearic acid consumption powder not only contribute to suppress oxidized LDL and provided inconclusive associations with CVD [11], the reduce LDL cholesterol, but also produce an elevation in beneficial effect of dark chocolate on CVD is probably high-density lipoprotein (HDL) cholesterol [39–41]. Mul- sustained by an increase in both the total antioxidant tiple cocoa feeding trials have also found chocolate to capacity and the flavonoids content (especially catechins, increase HDL cholesterol [26, 27, 41]. epicatechins, and procyanidins) within blood. It is estimated that chocolate is a leading source of procyanidin intake in Effects on blood pressure Western nations (18–20%) and chocolate flavonoids have been shown to provide a good dose-response bioavailability Consumption of chocolate or cocoa drinks rich in flavan-3- in humans [11, 17–19]. There are several potential ols lowers blood pressure (BP) [34, 35, 42–44]. In the study 123 484 G. Lippi et al.

Fig. 1 Cardiovascular effects lipid oxidation of dark chocolate oxidized LDL MetabolicMetabolic LDL-C effectseeffectsffects HDL-C insulin sensitivity

AntiAnti-hypertensivehypertensive Nitric Oxide (NO) Angiotensin effectseffects Converting Enzyme (ACE)

Nrf2 EndotheliaEnndodotthellialial CHOCOLATE Prostacyclin CHOCOLATE functffunctionunctionon Leukotrienes

Inhibition of T cells Anti-InflInflammatoryory IL-2, IL-5, TGF-beta effectseffects TNF-alpha

Antnti-thromomboticbotic Antiplatelet activity fibrinolytic activity effeffectcts of Buijsse et al., the usual daily cocoa intake was inversely angiotensin-converting enzyme [52]. Randomized trials related to blood pressure and in prospective analysis, cocoa have shown that consumption of high-flavanoid dark intake was associated with a 45–50% lower risk of cardio- chocolate is also associated with a significant increase in vascular and all-cause death [13]. One hundred grams of brachial artery flow mediated vasodilation [53–55], most dark chocolate consumed daily appears to reduce systolic likely mediated by chocolate flavonoids increasing the blood pressure by 5.1 mm Hg and diastolic blood pressure local production of nitric oxide [54, 55]. by 1.8 mm Hg; similar reductions in blood pressure other- wise correspond to a reduction in CVD events of 21% (14– Effects on endothelial function 27%) [45]. Importantly, these effects were observed without an adverse effect on body weight, supporting the concept Endothelial dysfunction at the site of the atherosclerotic that the inclusion of these types of specially formulated plaque rupture is strongly implicated in the development of foods into a balanced diet may help to support cardiovas- atherosclerosis. The consumption of flavanol-rich dark cular health without adverse effects on weight [36]. chocolate has an acute and short-term beneficial effect on One of the suggested pathways is that the cocoa flava- endothelial function and arterial wave reflections [35, 54, nols can enhance the formation of endothelial nitric oxide 56, 57]. The study of Vlachopoulos and colleagues shows (NO) leading to vasodilation and thereby lowering BP [46]. that dark chocolate acutely dilates muscular arteries and The unstable nitric oxide reacts with thiol groups to form decreases wave reflections [57]. A different study found stable S-nitrosothiols [47] that have been suggested to that dark chocolate intake significantly improved coronary contribute to BP regulation [48]. S-nitrosothiol-albumin, circulation in healthy adults, independent of changes in the main constituent of the circulating S-nitrosothiols pool, oxidative stress parameters, blood pressure, and lipid pro- is thought to provide a reservoir of nitric oxide bioactivity, file, whereas non-flavonoid had no such whereas low-mass S-nitrosothiols have been proposed to effects [58]. Moreover, the production of NO and reactive transduce the vasodilative and hypotensive activity [48]. oxygen radicals, triggered by isoflavones and other poly- Accordingly, Taubert et al. found that the decrease in phenols contained in chocolate, mediate dietary polyphenol systolic and diastolic BP is associated with an increase in induced activation of nuclear factor erythroid 2-related circulating levels of vasodilative S-nitrosoglutathione [43]. factor (Nrf2), a redox sensitive basic leucine-zipper tran- Insulin-mediated cell signaling could be one mechanism, scription factor involved in antioxidant response element, because insulin can modulate several signaling molecules which in turn triggers antioxidant response element driven involved in NO-synthase regulation [49]. A second mech- transcription of phase II detoxifying and antioxidant anism could be an oxidant-mediated cell signaling, because defense enzymes in vascular cells [59]. flavanols can modulate oxidative stress and the cell redox Chocolate also increases the levels of vasodilatory state, which in turn defines NO availability and NO-syn- prostacyclin and decreases leukotrienes [60] through the thase activity [50]. A third mechanism could involve the modulation of the endothelial cell eicosanoid system renin-angiotensin system [51] through the inhibition of the pathway by inhibiting 5-lipoxygenase [61, 62]. The 123 Chocolate and cardiovascular disease 485 reduction in the plasma leukotriene/prostacyclin ratio, a inhibit platelet activation in response to epinephrine, and measure of the proinflammatory–antinflammatory eicosa- cocoa consumption was found to suppress unstimulated and noid balance, indicates that cocoa may inhibit platelet stimulated platelet activation in whole blood [28]. Simi- aggregation (see below) and exert anti-inflammatory larly, Pearson and colleagues [81] observed that flavonol- effects [63]. Finally, consumption of chocolate is associ- rich cocoa inhibited epinephrine-stimulated human platelet ated with reduced levels of circulating intercellular activation (measured using the platelet function analyzer adhesion molecule (ICAM), the target ligand for integrins PFA-100) with an effect similar to the effect of aspirin. In a expressed by white blood cells [64]. recent in vitro and ex vivo study, Heptinstall and col- leagues [82] observed that the use of a flavonol-rich cocoa Effects on inflammation resulted in a significant inhibition of platelet aggregation and platelet–leukocyte conjugate formation. Another study Triggers and mechanisms leading to inflammation may vary supporting the findings of the suppressive effect of flavo- between clinical conditions but they share many common noid-rich food on platelet activation observed that cocoa mediators, including specific patterns of eicosanoid and consumption decreased the number of platelet microparti- cytokine production. Chronic inflammation is recognized as cles, which are generated during physiologic platelet a key mechanism in athero-thrombogenesis and several activation [79]. Taken together, the above results are con- proinflammatory markers independently predict the risk of sistent with the concept that platelet reactivity is reduced incident CVD [65–69]. In addition to anti-inflammatory after the consumption of flavonol-rich cocoa. The exact effects on the lipoxygenase pathway, chocolate flavonoids mechanisms by which flavanols inhibit platelet activity is have also been shown to decrease inflammation via several not yet well understood, although several possibilities have mechanisms, including inhibition of mitogen-induced acti- been proposed, including changes in membrane fluidity, vation of T cells, polyclonal activation of B cells and ligand receptor affinity and intracellular signaling pathways reduced secretion of interleukin-2 (IL-2) by T cells [70]. [83]. For example, some in vivo and in vitro studies have Researchers have also determined that chocolate procy- found that flavanols-rich foods negatively modulate dif- anidins can modulate a variety of other cytokines, including ferent pathways of eicosanoid metabolism within platelets, interleukin-5 (IL-5), tumor necrosis factor-alpha (TNF- including products of both cyclooxygenase (prostacyclin alpha) and transforming growth factor beta (TGF-beta), and thromboxane) and lipoxygenase (leukotrienes) [80, 84, thus reducing their inflammatory effects [71–75]. It is also 85]. Flavonols may also mediate their antiplatelet effects important to mention, however, that other studies have through antioxidant and nitric oxide-related pathways. failed to identify any significant effect of chocolate on Indeed, they have been shown to reduce platelet aggrega- urinary F(2) isoprostane levels or on markers of inflam- tion and hydrogen peroxide production in vitro and to mation including the whole-blood cytokines, interleukin-1 attenuate oxidant stress-induced platelet hyperactivity beta, interleukin-6, TNF-alpha, high sensitivity C-reactive in vivo [86]. Furthermore, an increased production of the protein, and P-selectin [27]. Since the evidence for benefi- endothelium-derived nitric oxide, which helps to maintain cial effects of chocolate flavanols in providing meaningful platelet quiescence, has been observed after flavonol-rich anti-inflammatory actions has been gathered predominantly cocoa consumption [56]. Other investigators have reported from in vitro experimentation, additional research in well- that flavonols can also modulate platelet activation by designed human clinical experiments, using cocoa properly interfering with membrane fluidity, which can in turn result characterized in terms of flavanol content, would provide in changes in membrane receptor function, enzymatic supportive evidence and remove ambiguity as to whether activity and intracellular signaling [87]. In addition to the this benefit truly exists [76]. antiplatelet effect, there is evidence that flavonols increase fibrinolytic activity by increasing plasminogen activators Anti-thrombotic effects [88]. In conclusion, the antiplatelet and fibrinolytic effects of flavonol-rich cocoa, along with its antioxidant proper- The most striking effect of cocoa flavonoids on haemostasis ties, could account for the cardioprotective effects of this is their antiplatelet activity [77–80]. A study conducted by food observed from various epidemiological studies [89]. Rein and colleagues found that cocoa modulated both platelet activation and function. Thus, the in vitro addition of cocoa procyanidin trimers to whole blood was found to Conclusions increase the binding of PAC-1 (which recognizes the acti- vated conformation of the fibrinogen-binding receptor Waterhouse et al. first described the antioxidant activity of GPIIb-IIIa) and the expression of P-selectin (both markers cocoa polyphenols against LDL oxidation in 1996 [25]. of platelet activation) in unstimulated platelets, as well as to Over a decade since this original report, chocolate 123 486 G. Lippi et al.

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