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COCOA, CHOCOLATE & CARDIOVASCULAR HEALTH BENEFITS

FROM ANCIENT REMEDY TO MODERN SCIENCE
Chocolate is a food as rich in history as it is in taste. Packed with polyphenols, cocoa has been used by indigenous peoples to improve health for thousands of years. Ancient societies revered the cocoa bean for its sustaining and health-affirming qualities.(1) In all, more than 100 medicinal uses of cacao are listed throughout ancient codices which range from cough suppression to intestinal health to cardiovascular benefits.   Specific cardiovascular benefits noted in these documents include alleviating ‘faint of heart’ or feeling faint, reducing angina (chest pain), generating blood, relieving heart palpitations, strengthening the heart and even prolonging life.(1) With cocoa’s journey from Central America to Europe, much of its connection to health benefits was lost when the Europeans created a more indulgent and diluted chocolate treat by adding milk and sugar. The Kuna Indians (or Amerinds) provide a modern day example of this ancient effect. The Kuna live off the coast of Panama on the San Blas Islands. Their traditional diet is high in sodium yet they show little to no increase in blood pressure as they get older.(2) When they migrate to other areas, however, their blood pressure tends to rise with age similar to other populations. When in their indigenous villages, the Kuna drink an average of five cups of a cocoa-based beverage per day. This phenomena intrigued researchers.

POLYPHENOLS, FLAVANOLS & ANTIOXIDANTS
In 1912, a paper was published in the Journal of the Chemical Society, noting that “the essential oil of cocoa contained 12 distinct compounds.” With the advent of modern chemical analytical technology, more than 700 compounds have been identified as being naturally present in cocoa beans. Cocoa contains several compounds that can act as emollients (pectin, sucrose, starch), analgesics (chlorogenic acid, quercitin, coumarin, ferullic acid), antiseptics (caffeic acid, citric acid, coumaric acid, furfural, kampferol), anesthetics (coumarin, linalool), anti-inflammatory agents (epicatechin, procyanidins) and numerous antioxidants. Still many others may exist in the bean as well.

The compounds receiving the most attention today are the polyphenols – a large class of molecules that includes numerous subcategories. Polyphenols are generally found in plant foods and are grouped according to their similar chemical structure of containing two or more phenolic rings. The word phenol is also used to refer to any compound that contains a six-membered aromatic ring, bonded directly to a hydroxyl group (-OH).  Polyphenols contain two or more phenolic rings. One subcategory, flavonoids, can be further divided into 13 subclasses, including the flavanols (or more precisely flavan-3-ols) found in cocoa. A few plant-based foods including apples, onions, soybeans, blueberries, cranberries, teas (green and black) and red wines are distinguished by their high polyphenolic content.

Many flavonoids can act as antioxidants—molecules capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent creating a “free radical” which starts a series of chain reactions that damage cells, DNA and other biological entities. The antioxidative properties of flavonoids are related to their polyphenolic structure.  Flavonoids generally have two aromatic on the ends bound in the center.  The B-ring allows rapid donation of a hydrogen (electron) for stabilization of free radicals. This hydrogen electron transfer terminates the free radical chain reactions.

Among the flavonoids, flavanols are particularly powerful antioxidants. Flavanols can be found in both monomeric and oligomeric forms. Monomers include catechin and epicatechin. Oligomeric forms are referred to as “proanthocyanins” and can range from dimers to decamers and even larger.

On a gram weight basis, cocoa beans rank among the highest in flavanol concentration compared to other plant-based foods.(3) Further, data from the USDA and independent research sponsored by The Hershey Company indicate that most cocoa-containing products have flavanols. Generally, the higher the concentration of natural (non-alkalized) cocoa, the more flavanols in the product.(4) Among cocoa and chocolate products, natural cocoa has the highest concentration of flavanols, followed by unsweetened baking chocolates, dark chocolate and milk chocolate.(4) The amount of flavanols present in finished food products such as chocolate and cocoa-containing foods depends on numerous factors such as the cocoa bean itself (cultivar type, region of origin), fermentation time, roasting procedures and alkalization.

Bioavailability studies indicate that plasma levels of the two main flavanols – epicatechin and catechin – are elevated one hour after consumption of chocolate or cocoa and are dissipated six hours after consumption.(5)

THE HEART OF CHOCOLATE  
One of the characteristic disease processes associated with cardiovascular disease is atherosclerosis. This hardening of the arteries results from inflammation due to chronic injury to the inner lining of blood vessels, or the endothelium. There are many causes of endothelial injury such as smoking, infections, high blood levels of homocysteine and free radicals.(6) Free radicals are thought to contribute to atherosclerosis through direct action on the endothelium and by oxidizing ‘bad’ low-density lipoprotein cholesterol. When LDL cholesterol becomes oxidized, its potential to cause arterial damage is greatly increased.(7) Following such damage, the endothelium attempts to heal itself by attracting monocytes and other inflammatory molecules and cells. This promotes the formation of the hallmark of cardiovascular disease: atherosclerotic plaques. The injured endothelium tends to constrict in the regions where plaques form, which can lead to blood clots.(6) A large number of recent studies have shown that several biomarkers of cardiovascular risk are influenced by flavonoid-rich foods.(8) The flavonoids subclass has been the subject of much research due to their antioxidant activity. While the flavanols, the flavonoids found abundantly in cocoa, can act as antioxidants, they also have other physiologically important properties.

COCOA IN THE TEST TUBE  
Numerous in vitro studies indicate that cocoa flavanols may mitigate risk factors for cardiovascular disease. The addition of dark chocolate to blood samples inhibited LDL oxidation even at low concentrations in the blood.(9-11) Purified cocoa flavanols reduced platelet activation and aggregation in blood in the presence of an activating agent.(12) Cocoa flavanols also led to a decrease in blood clotting and inflammation, not only through direct effects on platelet activation and aggregation, but also by moderating the production of eicosanoids, precursors of the inflammation process.(13)  

COCOA IN THE BODY  
The strong in vitro evidence of dark chocolate, cocoa and cocoa components in improving cardiovascular disease risk has led to a number of clinical trials with humans volunteers. Risk factors of interest include: platelet activity and eiscosanoid production, high blood pressure, endothelial function/nitric oxide production and blood cholesterol.
Platelet activity: Platelets carry blood clotting factors and are important for wound healing, but in the arterial endothelium, they can aggregate and cause plaques. A decrease in platelet activity is thus favorable to overall cardiovascular health. In one study, after drinking cocoa high in polyphenols, a reduction in platelet surface markers (PAC-1) was found, indicating decreased platelet activation.(14) A separate study showed cocoa consumption decreased the formation of platelet microparticles – which correlates positively with thrombotic, or blood clotting disorders.(14)

“Aspirin-like” effects: Low dose aspirin therapy is often prescribed for cardiovascular disease patients because it reduces the production of prostaglandins involved in inflammation. Pearson et al. compared the effect of natural cocoa and low dose aspirin administration on platelet function. Treatments included low dose (baby) aspirin, 300 ml (~10 fluid oz) of a cocoa beverage and a combination of the two. The cocoa treatment demonstrated an inhibitory effect on platelet activation and function.(15) The effect from the cocoa beverage was slightly less than the baby aspirin treatment, while the combination produced slightly better results, especially related to microparticle formation.(15) This indicates that cocoa is associated with a transitory reduction in platelet reactivity, which in turn may reduce the risk for formation of clots. Currently it is theorized that the primary action of flavanols with respect to platelet formation is to increase levels of the anti-aggegratory prostaglandin prostacyclin and decrease the levels of the pro-aggregatory leukotrienes. Consumption of dark chocolate has also been noted to decrease leukotriene formation.(13) Leukotrienes are eicosaniods naturally produced in the body from arachidonic acid and are involved in inflammatory responses including the production of histamine. A high level of leukotrienes is associated with an increased risk of some disorders such as asthma, ischemia and shock.

Blood pressure effects: A number of studies have investigated the role of cocoa and chocolate on blood pressure. An epidemiological study, the Zutphen Study (formerly part of the Seven Countries study) found that elderly men who consumed chocolate had significantly lower blood pressure than nonconsumers.(16) In addition, the researchers found that the chocolate consuming men had a ~50% reduction in risk of cardiovascular mortality and all-cause mortality.(16)  A number of intervention studies have demonstrated short-term and long-term reductions in blood pressure with dark chocolate consumption.    One study provided 100g of dark chocolate or a similar amount of white chocolate (as control) to 13 individuals aged 55-64 with untreated isolated systolic hypertension over 14 days in a crossover design. Within 10 days of treatment with the dark chocolate, both systolic and diastolic blood pressures were reduced with no effect from the white chocolate.(17) Another study found similar results after 15 days of feeding 100g of dark chocolate.(18) Two studies have reported blood pressure values for individuals with normal blood pressure fed cocoa or chocolate. One study found that dark chocolate, but not white chocolate, reduced blood pressure, yet still in the normal range.(19) However, the other study noted no change in blood pressure in their healthy study population after ingestion of flavanol-rich cocoa beverage.(20)   A more recent study demonstrated significant reductions in both systolic and diastolic blood pressure with a much smaller quantity of a 50% cacao dark chocolate (6.5g; 30 kcal) when consumed daily for 18 weeks compared to white chocolate.(21)  See Table 1 for chocolate/cocoa and blood pressure studies.

Table 1.  Chocolate/Cocoa and Blood Pressure Studies

Authors	Year	N	Animals/ Patients	Duration	Intervention	Outcome
Taubert et al. (17)	2003	13	Patients	14	Consumption of dark chocolate vs. white chocolate	Consumption of dark chocolate lowers systolic and diastolic BP
Grassi et al. (18)	2005	15	Patients	15 days	Consumption of dark chocolate vs. white chocolate	Dark chocolate decreases blood pressure and improves insulin sensitivity in healthy persons
Grassi et al.  (19)	2005	20	Patients	15 days	Consumption of flavanol-rich dark chocolate	DC decreased BP and serum LDL cholesterol, improved FMD, and ameliorated insulin sensitivity in hypertensives
Fisher et al. (20)	2003	27	Patients	5 days	Consumption of flavonoids-rich beverages	Consumption induced vasodilation via activation of NO system
Taubert et al., (21)	2007	44	Stage 1 hypertensives	18 weeks	Consumption of 6.3g dark chocolate v. white chocolate	Consumption of dark chocolate lowers systolic and diastolic BP

 

Endothelial health/function:

Movement of blood throughout the body is highly dependent upon the health of the cells that line the insides of blood vessels, the endothelial cells.  Endothelial cells produce substances that allow blood vessels to dilate and endothelial dysfunction correlates strongly with cardiovascular disease.(22) A process known as flow-mediated dilation (FMD) can be used to assess endothelial health/function and several studies have shown that high dietary antioxidant intake improves FMD.(23) Cocoa and dark chocolate contain high levels of flavanol antioxidants and studies have found significant improvement in FMD in healthy volunteers following both acute (24-26) and chronic (27, 28) cocoa or dark chocolate consumption.  Improvements in FMD have also been reported following acute cocoa or dark chocolate consumption in individuals at risk for cardiovascular disease (29) and in smokers.(30, 31) Similar improvements have been observed following chronic cocoa or dark chocolate consumption in individuals with hypertension (19, 32) and diabetes.(33) Table 2 provides a list of studies investigating chocolate or cocoa consumption and endothelial health. 

Nitric Oxide (NO):

 

NO is a gas (not to be confused with nitrous oxide; laughing gas) that causes blood vessels to dilate. The discovery of this effect was awarded the 1998 Nobel Prize for Medicine and pharmaceuticals that stimulate NO production are now routinely used to treat blood vessel dysfunction.  Recent studies indicate that cocoa flavanols affect blood pressure and endothelial health by stimulating the production of nitric oxide (NO).(29, 34) Oral administration of (-)epicatechin, a flavanol found in very high concentrations in cocoa, results in acute increases in NO production and vasodilation in healthy men.(35) It has also been demonstrated that the vasodilation observed following the consumption of flavanol-rich cocoa is associated with increases in circulating bioactive NO (24, 30) and that inhibition of NO production prevents cocoa-induced vasodilation.(20, 24, 30)

 

Insulin stimulates NO release (36) and decreased NO production is believed to contribute to elevations in blood pressure seen in insulin-resistant states.(37) Moreover, decreased NO-mediated vasodilation in insulin-resistant states is believed to contribute to impaired glucose uptake in individuals with insulin resistance.(38) In two recent studies, participants with high blood pressure were fed 100g of dark chocolate for two weeks and had significant improvements in FMD and blood pressure.(19, 32) Interestingly, participants also had improved insulin sensitivity.  NO production was not measured in either of the previous study; however it is likely that observed changes were mediated by cocoa flavanol-induced NO production and subsequent improvements in vasodilation and glucose uptake.

 

 

Table 2. Endothelial Function Studies with Cocoa & Chocolate

 

Authors	Year	n	Animals/ Patients	Duration	Intervention	Outcome
Karim et al. (39)	2000	5	Aortic rings from rats	Immediately	Procyanidins derived from cocoa	Endothelium-derived relaxation mediated by activation of NOS
Fisher et al. (20)	2003	27	Healthy People	5d	Flavanol-rich cocoa (821mg/d)	Peripheral vasodilation, improvement in vasodilator response to ischemia assessed by pulse-wave amplitude on the finger
Heiss et al. (29)	2003	26	Patients with at least 1 cardiovascular risk factor	2h (crossover)	Flavanol-rich cocoa	Improvement in flow-mediated dilation, increase in levels of nitrosated and nitrosylated species
Engler et al. (27)	2004	21	Healthy Subjects	2w	High-flavonoid chocolate (213 mg procyanidins, 46mg epicatechin) v low flavonoid chocolate	Improvement in flow-mediated dilatation, increase in blood epicatechin concentrations
Grassi et al. (19)	2005	20	Patients with untreated essential hypertension	15d (cross-over)	100g dark chocolate (22mg catechin, 66mg epicatechins) v flavanol free white chocolate	Increase in flow-mediated dilation, decrease in BP and LDL cholesterol, increase in insulin sensitivity
Heiss et al. (30)	2005	11	Smokers	2h (cross-over)	100mL cocoa drink with high (176-18mg) or low (<1mg) flavanol content	Increase in flow-mediated dilation and circulating NO pool, increase in flavanol metabolites
Schroeter et al. (24)	2006	16	Healthy subjects, isolated rabbit rings		Drink with high flavonoid content	Improvement in flow-mediated dilation paralleled the plasma levels of flavanols; pure epicatechin mimic vascular effects of cocoa; high-flavanol diet associated with high urinary excretion of NO metabolites
Hermann et al. (31)	2006	20	Healthy smokers	2h	40g dark chocolate v white chocolate	Increase in flow-mediated dilation, improvement in antioxidant status and platelet function
Faridi et al. (25)	2008	45	Moderately overweight, otherwise healthy subjects	2h	75g dark chocolate (825mg flavanol); 22g  cocoa (sugar-free/sugar sweetened) (805mg flavanol) v placebo bar and beverage	Increase in flow-mediated dilation and reduction of BP (dark chocolate, sugar-free cocoa and sugar-sweetened cocoa)
Flammer et al. (40)	2007	22	Heart transplant recipients	2h	40 g dark chocolate v flavonoid-free placebo chocolate	Coronary vasodilation, improvement in coronary endothelial function and platelet function
Balzer et al. (33)	2008	41	Diabetes	4w	Drink with high flavanol content	Improvement in flow-mediated vasodilatation (acute and chronic)
Grassi et al. (32)	2008	19	Hypertensives, impaired glucose tolerance patients	2w	Flavanol-rich dark chocolate	Improvement in flow-mediated vasodilatation, insulin sensitivity, beta-cell function, and blood pressure
Shiina et al. (41)	2008	39	Healthy subjects	2w	45g dark chocolate v white chocolate	Improvement in coronary circulation as measured by coronary velocity flow reserve

 

 

Proposed Cocoa Vasodilation/Blood Pressure Mechanism

 

Cholesterol: One factor of concern during clinical studies is whether ingestion of dark chocolate, which contains saturated fat, would lead to negative effects on blood cholesterol. However, the fat from cocoa butter is comprised mainly (about 1/3) of stearic acid. A number of studies have shown that stearic acid (and cocoa butter) does not elevate blood cholesterol.(42-44) Both cocoa and dark chocolate have shown promising results on blood cholesterol levels. Consumption of a cocoa-based beverage for two to four weeks has increased the lag time for oxidation of LDL cholesterol.(11,45-47) In a four-week study when combining cocoa and dark chocolate, significant improvements were found for blood levels of ‘good’ HDL-cholesterol and oxidized LDL cholesterol.(11) Another recent study found improvements in total cholesterol and LDL-cholesterol levels following the consumption of 100g of dark chocolate.(19)

RECENT ARTICLES
Two recent articles have further bolstered the connection between cocoa/chocolate intake and cardiovascular risk:

Janszky and colleagues from Sweden report in the September 2009 edition of the Journal of Internal Medicine that chocolate consumption is associated with a lower cardiac mortality in a dose dependent manner in non-diabetic patients survicing their first acute myocardial infarction .  They followed 1169 individuals who had confirmed  acute myocardial infarction for 8 years and found a strong inverse association with chocolate consumption and cardiac mortality among this population.  This association did not hold for consumption of other sweets.(48)

Hooper and colleagues in the United Kingdom published a meta-analysis of intervention studies of flavonoid-rich foods and cardiovascular risk in the American Journal of Clinical Nutrition in 2008.  This review included not only chocolate, but other flavonoid-rich foods such as soy protein, black and green tea and red wine and considered multiple cardiovascular risk factors such as blood pressure, blood cholesterol and endothelial function measures .  They report that chocolate and cocoa had significant effects on acute and chronic flow mediated dilation and systolic and diastolic blood pressure, whereas soy protein was shown effective only in reducing diastolic blood pressure.  Black tea consumption increased both systolic and diastolic blood pressure (acutely) and green tea reduced LDL cholesterol.(49)

EVERYTHING IN MODERATION  
Consumption of cocoa may convey protective effects in healthy people and those with risk factors for cardiovascular disease. At this time, long-term effects of cocoa and chocolate on cardiovascular protection have not been determined and deserve further investigation. Of course, cocoa and chocolate products provide fat and calories and need to be consumed in moderation and as part of a balanced diet, but there has never been more evidence that chocolate is not only good to eat – it also may be good for you as well.

 

REFERENCES

1.            Dillinger TL, Barriga P, Escarcega S, Jimenez M, Salazar Lowe D, Grivetti LE. Food of the gods: cure for humanity? A cultural history of the medicinal and ritual use of chocolate. J Nutr 2000;130:2057S-72S.

2.            Hollenberg NK, Martinez G, McCullough M et al. Aging, acculturation, salt intake, and hypertension in the Kuna of Panama. Hypertension 1997;29:171-6.

3.            Lazarus SA, Hammerstone JF, Schmitz HH. Chocolate contains additional flavonoids not found in tea. Lancet 1999;354:1825.

4.            Hurst WJ, et al. The determination of polyphenols and the correlation to cocoa content in selected US confectionary products. Functional foods, bioactive compounds and human health conference 2005; (abstr).

5.            Baba S, Osakabe N, Yasuda A et al. Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers. Free Radical Res 2000;33:635-41.

6.            Hannum SM, Schmitz HH, Keen CL. Chocolate: A Heart-healthy Food? Show Me the Science! Nutrition Today 2002;37:103-9.

7.            Ginter E. [Effect of free radicals and antioxidant on the vascular wall]. Vnitr Lek 2000;46:354-9.

8.            Manach C, Mazur A, Scalbert A. Polyphenols and prevention of cardiovascular diseases. Cur Opin Lipidology 2005;16:77.

9.            Pearson DA, Schmitz HH, Lazarus SA, Keen CL. Inhibition of in vitro low-density lipoprotein oxidation by oligomeric procyanidins present in chocolate and cocoas. Methods Enzymol 2001;335:350-60.

10.        Vinson JA, Proch J, Zubik L. Phenol antioxidant quantity and quality in foods: cocoa, dark chocolate, and milk chocolate. J Agric Food Chem 1999;47:4821-4.

11.        Wan Y, Vinson JA, Etherton TD, Proch J, Lazarus SA, Kris-Etherton PM. Effects of cocoa powder and dark chocolate on LDL oxidative susceptibility and prostaglandin concentrations in humans. Am J Clin Nutr 2001;74:596-602.

12.        Kris-Etherton PM, Keen CL. Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Cur Opin Lipidology 2002;13:41.

13.        Schramm DD, Wang JF, Holt RR et al. Chocolate procyanidins decrease the leukotriene-prostacyclin ratio in humans and human aortic endothelial cells. Am J Clin Nutr 2001;73:36-40.

14.        Rein D, Paglieroni TG, Wun T et al. Cocoa inhibits platelet activation and function. Am J Clin Nutr 2000;72:30-5.

15.        Pearson DA, Paglieroni TG, Rein D et al. The effects of flavanol-rich cocoa and aspirin on ex vivo platelet function. Thrombosis Research 2002;106:191-7.

16.        Buijsse D, Feskens EJ, Kok FJ, Kromhout D.  Cocoa intake, blood pressure, and cardiovascular mortality: the Zutphen Elderly Study.  Arch Intern Med 2006;166:191-7.

17.        Taubert D, Berkels R, Roesen R, Klaus W. Chocolate and blood pressure in elderly individuals with isolated systolic hypertension. JAMA 2003;290:1029-30.

18.        Grassi D, Lippi C, Necozione S, Desideri G, Ferri C. Short-term administration of dark chocolate is followed by a significant increase in insulin sensitivity and a decrease in blood pressure in healthy persons. Am J Clin Nutr 2005;81:611-4.

19.        Grassi D, Necozione S, Lippi C et al. Cocoa reduces blood pressure and insulin resistance and improves endothelium-dependent vasodilation in hypertensives. Hypertension 2005;46:398-405.

20.        Fisher NDL, Hughes M, Gerhard-Herman M, Hollenberg NK. Flavanol-rich cocoa induces nitric-oxide-dependent vasodilation in healthy humans. J Hypertension 2003;21:2281.

21.        Taubert D, Roesin R, Lehmann C, Jung N, Schomig E.  Effects of low habitual cocoa intake on blood pressuew and bioactive nitric oxide.  JAMA 2007;298:49-60.

22.        Vita JA, Keaney JF, Jr. Endothelial function: a barometer for cardiovascular risk? Circulation 2002;106:640-2.

23.        Vita J.A. Polyphenols and cardiovascular disease: effects on endothelial and platelet function. Am J Clin Nutr 2005; 81:292S-297S.

24.        Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK, Sies H, Kwik-Uribe C, Schmitz HH, Kelm, M. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. Proceedings of the National Academy of Sciences 2006;103:1024-1029.

25.        Faridi Z, Njike VY, Dutta S, Ali A, Katz DL. Acute dark chocolate and cocoa ingestion and endothelial function: a randomized controlled crossover trial. Am J Clin Nutr 2008; 88:58-63.

26.        Berry NM, Davison K, Coates AM, Buckley JD, Howe PRC. Impact of cocoa flavanol consumption on blood pressure responsiveness to exercise. British Journal of Nutrition 2010; 1-5.

27.        Engler MB, Engler MM, Chen CY, Malloy MJ, Browne A, Chiu EY, Kwak HK, Milbury P, Paul SM, Blumberg J, Mietus-Snyder ML. Flavonoid-rich dark chocolate improves endothelial function and increases plasma epicatechin concentrations in healthy adults. Journal of American College of Nutrition 2004; 23:197-204.

28.        Njike VY, Faridi Z, Shuval K, Dutta S, Kay CD, West SG, Kris-Etherton PM, Katz D.L. Effects of sugar-sweetened and sugar-free cocoa on endothelial function in overweight adults. International Journal of Cardiology 2009; in press.

29.        Heiss C, Dejam A, Kleinbongard P, Schewe T, Sies H, Kelm M. Vascular effects of cocoa rich in flavan-3-ols. Journal of American Medical Association 2003; 290:1030-1.

30.        Heiss C, Kleinbongard P, Dejam A, Perre S, Schroeter H, Sies H, Kelm, M. Acute consumption of flavanol-rich cocoa and the reversal of endothelial dysfunction in smokers. Journal of the American College of Cardiology 2005; 46:1276-1283.

31.        Hermann F, Spieker LE, Ruschitzka F, Sudano I, Hermann M, Binggeli C, Luscher TF, Riesen W, Noll G, Corti R. Dark chocolate improves endothelial and platelet function. Heart 2006; 92:119-20.

32.        Grassi D, Desideri G, Necozione S, Lippi C, Casale R, Properzi G, Blumberg JB, Ferri C. Blood pressure is reduced and insulin sensitivity increased in glucose-intolerant, hypertensive subjects after 15 days of consuming high-polyphenol dark chocolate. Journal of Nutrition 2008; 138:1671-76.

33.        Balzer J, Rassaf T, Heiss C, Kleinbongard P, Lauer T, Merx M, Heussen N, Gross HB, Keen CL, Schroeter H. Sustained benefits in vascular function through flavanol-containing cocoa in medicated diabetic patients: a double-masked, randomized, controlled trial. Journal of the American College of Cardiology 2008; 51:2141.

34.        Balzer J, Heiss C, Schroeter H, Brouzos P, Kleinbongard P, Matern S, Lauer T, Rassaf T, Kelm M. Flavanols and Cardiovascular Health: Effects on the circulating NO Pool in Humans. Journal of Cardiovascular Pharmacology 2006;47:S122.

35.        Loke WM, Hodgson JM, Proudfoot JM, McKinley AJ, Puddey IB, Croft KD. Pure dietary flavonoids quercetin and (-)-epicatechin augment nitric oxide products and reduce endothelin-1 acutely in healthy men. Am J Clin Nutr 2008;88:1018.

36.        Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, Quon MJ. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 2000; 101:1539.

37.        Scherrer U, Sartori C. Defective nitric oxide synthesis: a link between metabolic insulin resistance, sympathetic overactivity and cardiovascular morbidity. Eur J Endocrinol 2000;142:315.

38.        Laakso M, Edelman SV, Brechtel G, Baron AD. Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. Journal of Clinical Investigation 1990;85:1844.

39.        Karim M, McCormick K,  Kappagoda CT. Effects of cocoa extracts on endothelium-dependent relaxation. J Nutr. 2000;130:2105S-2108S.

40.        Flammer AJ, Hermann F, Sudano I, Spieker L, Hermann M, Cooper KA, Serafini M, Luscher TF, Ruschitzka F, Noll G, Corti R. Dark chocolate improves coronary vasomotion and reduces platelet reactivity. Circulation. 2007;116:2376-2382.

41.        Shiina Y, Funabashi N, Lee K, Murayama T, Nakamura K, Wakatsuki Y, Daimon M, Komuro I. Acute effect of oral flavonoid-rich dark chocolate intake on coronary circulation, as compared with non-flavonoid white chocolate, by transthoracic Doppler echocardiography in healthy adults. Int J Cardiol. 2009;131:424-429.

42.        Weisburger JH. Chemopreventive effects of cocoa polyphenols on chronic diseases. Exp Biol Med. 2001;891-7.

43.        Kris-Etherton PM, Mustad VA. Chocolate feeding studies: a novel approach for evaluating the plasma lipid effects of stearic acid. Am J Clin Nutr 1994;60:1029S-36S.

44.        Yu S, Derr J, Etherton TD, Kris-Etherton PM. Plasma cholesterol-predictive equations demonstrate that stearic acid is neutral and monounsaturated fatty acids are hypocholesterolemic. Am J Clin Nutr 1995;61:1129-39.

45.        Kondo K, Hirano R, Matsumoto A, Igarashi O, Itakura H. Inhibition of LDL oxidation by cocoa. Lancet 1996;348:1514.

46.        Osakabe N, Yasuda A, Natsume M, Takizawa T, Terao J, Kondo K. Catechins and their oligomers linked by C4 --> C8 bonds are major cacao polyphenols and protect low-density lipoprotein from oxidation in vitro. 2002;51-6.

47.        Osakabe N, Baba S, Yasuda A et al. Daily cocoa intake reduces the susceptibility of low-density lipoprotein to oxidation as demonstrated in healthy human volunteers. 2001;93-9.

48.        Janszky I, Mukamal KJ, Ljung R, Ahnve S, Ahlbom A, Hallqvist J.  Chocolate consumption and mortality following a first acute myocardial infarction: the Stockholm Heart Epidemiology Program.  J Intern Med 2009;266:248-57.

49.        Hooper L, Kroon PA, Rimm EB, Cohn JS, Harvey I, Le Cornu KA, Ryder JJ, Hall WL, Cassidy A.  Flavonoids, flavanoid-rich foods, and cardiovascular risk: a meta-analysis of randomized controlled trials.  Am J Clin Nutr 2008;88:38-50.