OUP user menu

Clinical benefit and preservation of flavonols in dark chocolate manufacturing

Andrew McShea, Emma Ramiro-Puig, Sandra B Munro, Gemma Casadesus, Margarida Castell, Mark A Smith
DOI: http://dx.doi.org/10.1111/j.1753-4887.2008.00114.x 630-641 First published online: 1 November 2008


The consumption of high-cacao-content chocolate has been associated with positive health benefits ascribed to flavonol antioxidants derived from the ground, fermented cocoa seeds of Theobroma cacao. However, flavonols impart a bitter, astringent flavor to foodstuffs, frequently masked in chocolates and confections by aggressive processing and adulteration with other flavors. Recent reports have implied that not all varieties of dark chocolate are created equally, and significant caveats exist regarding its potential health benefits. It is perhaps not surprising that extensive processing, dilution, and the addition of flavor modifiers may improve the palatability of chocolate, but could have negative nutritional and clinical benefits. This article examines the chemical composition of chocolate and the clinical data associated with the consumption of flavonoid-rich cocoa. We review the steps in chocolate manufacturing that directly affect the antioxidant levels in chocolate products, and the caveats associated with claims of health benefits from the consumption of dark chocolate.

  • antioxidant
  • chocolate
  • flavonoid
  • manufacturing


Chocolate is a three millennia old comestible that originated as a frothy, spicy, cocoa-like drink in Central America.1 Between 1847 and 1879, fundamental improvements to the processing of cacao resulted in the more appetizing chocolate bars from Fry's (UK) and Nestle and Lindt (Switzerland). Interest in chocolate confections continued to grow to the point where cacao products have become almost ubiquitous in the Western diet. Consumption of chocolate products in northern climates is significant and estimated at 5–10 kg per person per year, with some geographical variation linked to cultural norms and local climate. Overall, the world demand for cacao remains very high (estimated at around 3.5 million tons for 2008) with chocolate occupying an unusual multifaceted niche as an indulgent luxury food with healthy connotations in the $17 billion US confectionary market.

In ancient times cacao was thought to confer magical properties when consumed, and it was associated with a variety of rituals. Despite the passage of time and advancement of technology, an air of mystery still surrounds chocolate. Indeed, the art and science of chocolate manufacturing has generally remained a covert operation performed “behind closed doors” and laden with trade secrets. Meanwhile, there is a significant body of evidence to suggest that chocolate's “magical” properties are associated with a spectrum of pharmacological effects on the human body, most of which are attributable to Flavan-3-ols.2 There has now been over a decade of articles in scientific journals describing the connections of cocoa with antioxidant activity and a variety of significant clinical phenomena. The trend to highlight the health benefit of high-percentage-cacao-containing products has continued to increase based not only on scientific merit but also for business reasons. Ultimately, the morass of marketing and scientific detail has become challenging to untangle, and the vacillation in the lay press on whether chocolate is ultimately “good” or “bad” has done little to demystify a complex problem for the consumer.

This article details the complexity and inherent variation of the chocolate-making process to give a flavor of just how complex this material remains. In parallel, the article examines significant pharmacological data demonstrating the health benefits of cacao and how this may relate to process methodology in chocolate-making.


Chocolate is derived from finely ground, fermented beans (seeds) of the Theobroma cacao tree, which is generally cultivated in small farms located in a climate belt approximately within 20 degrees of the equator. Cacao beans are harvested biannually using a labor-intensive process from squash-like pods that grow proximal to the trunk of the tree. Generally, three broad varietals of Theobroma cacao are employed in chocolate making: Criollo, Forastero, and Trinitario, with the last being the most common. Short terminal repeat analysis of T. cacao genomes suggests that the number of distinct varieties of the plant exceeds 14,000,3 implying that the genetic diversity of cacao is significant. However, most cacao products currently consumed consist of a mixture of varietals, so genetic differences play a minimal role in the overall taste and character of mass-marketed chocolate products. Recently, smaller chocolate manufacturers have recognized this disparity and “single origin” and “single varietal” chocolate products have begun to appear in a manner similar to varietal and vintage trends in the wine-making industry.

Chocolate is a highly processed form of cacao, and although many forms of manufacturing process exist, they fundamentally all have the same basic steps: fermentation, roasting, and milling. These processing steps have been developed over centuries to render the cacao seed palatable by altering the chemical and physical properties of the bean to produce the chocolate flavors. Not surprisingly, a cacao seed is quite unpalatable in its native form, so a series of steps is required to break down the cotyledon, the shelled seed or nib, and neutralize the unpleasant flavor. The first step of seed fermentation occurs by the spontaneous inoculation of piles of harvested cacao seeds and lasts for up to 7 days. Yeasts (S. cerevisiae being the most abundant) dominate the fermentation for the first 24 hours, then lactic acid bacteria replace the yeasts, and the sugary pulp that surrounds the cacao seed is converted to ethanol. The piles are manually aerated during the fermentation, and as oxygen penetrates the fermentation, acetic acid bacteria take over, oxidizing ethanol to acetic acid, which persists in significant quantities after fermentation.4 During this time, the temperature rises to about 50°C, with the combined heat and acid resulting in breakdown of the cacao seed. Organic acids (oxalic, phosphoric, succinic, and malic acids) are also generated and likely assist in the chemical breakdown of the bean cotyledons. These and other complex reactions contribute to precursors of the chocolate flavor. The acetic acid dominates the flavor of the fermented bean at this stage and is removed/reacted-off during downstream steps of chocolate manufacturing, as it imparts an unpleasant flavor at high concentrations.

It is perhaps obvious from this description that the fermentation is a relatively uncontrolled, non-sterile process, with a significant number of variables that can affect the final characteristics of the chocolate. For example, colonization of the beans by molds can frequently cause significant problems with the flavor profile and overall yield of usable material. In addition, fermentation is almost always unevenly distributed throughout the beans. Thus, a significant amount of knowledge specific to the cacao-growing region and fermentation practices is required to produce good results. Interestingly, controlled inoculations of large-scale fermentation efforts have proven to be challenging to industrialize. This is likely due to the low level of sterility in the process and difficulties in controlling the auto-thermal reactions driving the fermentation.

Significant changes in the concentrations and composition of organic metabolites such as procyanidins and flavan-3-ols occur during the fermentation process.5 The profiles of these antioxidants are complex in chocolate and the chemical terminology is somewhat redundant (see Figure 1).

Figure 1

Nomenclature of polyphenolic flavonoids. Flavanols (Flavan-3-ols) and proanthocycanin polymers are primary components in cacao products.

Adapted from Murphy et al.28

Generally speaking, under-fermented cacao has a higher antioxidant content and a markedly more astringent flavor profile. Recent trends to seek chocolate products with high antioxidant content have even resulted in some demand for marginally fermented cacao. Note that the fermentation process is not homogeneous throughout a population of cacao beans, so it is possible to separate flavor and antioxidant concentration to some degree. In addition, the type of microbial colonization and length of fermentation phase (i.e., alcohol, lactic, acetic) will also likely affect flavonol content and breakdown. Indeed significant variations in the flavonol content of cacao can be observed between batches and growing regions.

After the fermentation process is complete, a natural or assisted process to lower the moisture content of the beans dries the cacao. This completes the fermentation process and permits more straightforward storage and shipment of the cacao for downstream processing. However, spoilage of this microbiologically active material by molds and water damage is a common problem that requires significant diligence to avoid during transport and storage.

After a cleaning process, the fermented, dried cacao beans are then roasted. This serves as the first sterilization process for the beans, and is used to loosen the shell from the fermented cotyledon. Roasting also generates favorable flavor characteristics through Maillard and caramelization reactions of sugars, amino acids, and peptide oligomers. A wide variety of flavors can be produced, depending on the specific amino acids and sugars available, as well as the pH and temperature of the roasting process. More than 25 diketopiperizine roasting reaction products are readily detectable in cacao nibs, with proline-valine dimers predominating and producing bitter flavor qualities. In addition, characteristically bitter flavors of dark chocolate are imparted by theobromine, caffeine, l-leucine, and catechin flavonoids.6

After the roasting step, the woody husk of the cacao bean is removed in a violent vibratory process called winnowing. This separation step also breaks down the cacao bean into small 2–5 mm irregular-shaped fragments known as cacao nibs. Depending on the process being applied, cacao nibs can also be roasted a second time to further develop the flavor qualities of the cacao. At this stage, roasted cacao nibs represent the first truly palatable product generated in the chocolate-making process. The roasting process subjects the cacao to significant heat (120–150°C), so changes in the flavonoid biochemistry would be expected during this processing step. Catechin antioxidants are thermally labile to some degree, based on studies of thermal stability in tea;7 however, the exact stability of epicatechin in chocolate will likely depend on stabilizing interactions with other components of the cacao nib. Indeed, studies suggest that the interaction between flavonoids and other small molecules in the cacao nib do determine the flavor as well as the final antioxidant activity. Thus, flavonoid degradation likely occurs but this depends on the starting material and the specific roasting process applied.5

Similar to coffee, cacao must be ground in order to develop the flavor further. However, unlike coffee, the cacao bean is milled to a much finer grain size and is consumed in its entirety, rather than having the flavors extracted. Furthermore, cacao contains between 50% and 56% cocoa butter, an unsaturated triglyceride, which results in the ground material becoming liquefied in the elevated temperature of the high-friction milling equipment. Environmental, genetic, and upstream processing differences in the cacao result in variations in the fat and water content of the cacao, so grinding conditions vary somewhat between processes and raw starting material.

A number of milling methods exist depending on whether it is desirable to separate the cocoa butter from the cocoa solids. Significant changes can be made to the flavor profile and chemical composition of the material depending on the process; however, in all methods, efforts are made to keep the milling temperature in check to avoid overheating the product and further altering the chemistry. Overall, the goal of milling is to reach a relatively homogeneous particle size of below 30 microns. Taste perception of grain sizes below 50 microns is indistinct, i.e., “grittiness” is not perceived; however, the final particle size and conformation has a significant impact on the flavor and “mouth-feel” of the final product. Thus, significant attention must be applied to the milling process to generate a suitable size distribution. Depending on the manufacturing process, the milled liquor may continue down the rest of the chocolate manufacturing process or be solidified into large blocks for transportation to secondary processing plants.

In the next stage, a mixing process combines the milled cocoa liquor with sugar. The quantities of ingredients vary very significantly, with dark chocolate monikers being given to products containing cocoa solids as low as 40% and as high as 100%. In the case of milk chocolate, a significant quantity of milk powder is also added. As chocolate viscosity increases rapidly with water content, the addition of powders or low moisture forms of milk (e.g., dried condensed milk) are required. Most industrialized processes also add an emulsifier such as soy lecithin to the mix to improve viscosity characteristics during manufacturing and to maintain a lower overall cost of production. Though dilution of cacao liquor per se does not directly result in chemical changes to the flavonols, it does greatly complicate the issue of potential beneficial pharmacological effects of cacao ingestion. A significant issue was whether the addition of milk to chocolate would impair the uptake of cacao-derived flavonols. However, recent studies indicate that the uptake of flavonols is not significantly inhibited by milk proteins8,9 and, by inference, milk chocolate should retain the antioxidant activity proportional to its cacao flavonol content. However, the contribution of saturated fat to the product is likely undesirable in terms of clinical benefit and there is no doubt that, in general, high-fat, high-sugar diets are undesirable. Indeed, the lax naming conventions for dark chocolate are frequently exploited to imply increased health benefits, which has been a point of significant controversy10 and has been highlighted to the public by the media.

After the addition of sugar, milk powder, and other additives (note: at what point does chocolate cease to be chocolate?) the chocolate mass is subjected to further rounds of refining to ensure the homogeneity of particle sizes within the product, and enhance the characteristics and dispersion of the particles and fat. Although the material now has properties and an approximate taste profile similar to chocolate, a significant quantity of acetic acid remains from the upstream fermentation imparting an unpleasant tartness to the product.

A multi-day heat treatment step, referred to as conching, is typically applied with gentle grinding to improve the flavor characteristics and reduce the concentration of free acids and other volatile by-products from the cacao bean. This is a delicate manufacturing step, as aggressive processing at this stage can result in significant loss of volatile small molecules that positively influence flavor. An alternative process to conching relies on neutralization of acetic acid with mild base in a process termed “Dutching”. Antioxidant flavonols are significantly reduced during the Dutching process, making this process undesirable for maintaining the flavonol activity in chocolate products.11

At this point, the material resembles molten chocolate in look and taste. Prior to molding into bars or other shapes, the material is tempered to set a compact crystal structure within the triglycerides so that the final product has a glossy finish, hard consistency, and is resistant to blooming or cosmetic discoloration due to separation of fats within the final product.


Chocolate is an unusual composite material (Table 1). It contains a significant concentration of astringent-tasting polyphenols embedded in a triglyceride matrix. Polyphenols (proanthocyanidins) are oligomeric and polymeric end products of the flavonoid biosynthetic pathway. They impart astringency to beverages such as wine, fruit juices, and teas, and are increasingly highlighted for their potential beneficial effects on human health. Astringent-tasting polyphenols can be isolated from the bark, leaves, fruit, and seeds of many plants, where they are thought to provide protection against predation.

View this table:
Table 1

Chemical components of fermented, dried, and roasted seed fragments (nibs) from Theobroma cacao that impart flavor associated with chocolate. *

Chemical componentµmol/kg
Bitter-tasting compounds
Sour-tasting compounds
 Citric acid30974.9
 Acetic acid16717.7
 Lactic acid9260.7
 Malic acid3581.2
 Oxalic acid2810.5
 Succinic acid1725.0
Umami-like tasting compounds
 Glutamic acid1781.8
 Aspartic acid1357.9
Bitter and astringent tasting compounds
 Procyanidin B22082.8
 Procyanidin C11628.0
 Procyanidin B5791.5
 Gamma-aminobutyric acid5011.4
 N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid1615.6
 Beta-aminoisobutyric acid1349.3
 N-[4′-hydroxy-(E)-cinnamoyl]-L-aspartic acid551.6
Sweet-tasting compounds
  • * Composition continues to change during downstream processing (refining/conching) resulting in a reduction of acetic acids and some additional changes in flavor components.

  • Adapted from Stark et al.6

Significant antioxidant activity has been associated with polyphenols, particularly the monomeric subunits catechin and epicatechin in the case of cacao products. It is likely no coincidence therefore that dark chocolate bars have a remarkably long shelf life when stored appropriately. This characteristic is likely attributable to the combination of a high level of antioxidants and a low oxygen permeation rate due to the high concentration of lipids.

Both the concentration and activity of antioxidant polyphenols can be detected at physiologically relevant concentrations in certain forms of chocolate containing high percentages of cacao. As a rough estimate, the dosage consumed during nominal consumption of antioxidant-rich dark chocolate corresponds to a dose of up to 2.8 grams of (−)-epicatechin per 40 gram serving (or 40 mg/kg for an average human). Of course truly “raw” chocolate that was under-fermented and under-roasted with very high cacao solid percentages (90–100%) may contain even higher levels of antioxidant polyphenols but would likely be exceedingly unpalatable and have a high microbiological burden from the fermentation process. More studies are required to correlate chocolate processing methodology with antioxidant activity.

Note that the antioxidant activity of chocolate is exceedingly high on a percentage dry weight basis even when compared with traditionally healthy berries, beans, and fruits. For instance, in comparison to the Acai berry (Euterpe oleracea), currently recognized as a highly antioxidant-rich natural foodstuff,12 dark chocolate and cacao powder have approximately 20% and 75% of the antioxidant activity per gram based on measurements in oxygen radical absorbance capacity (ORAC) assays.11

In addition to polyphenols, chocolate also contains the structurally related methylxanthines theobromine and small amounts of caffeine. Theobromine concentrations are significant in chocolate, ranging from 1.5% to 3% for theobromine versus 0.2% to 0.4% for caffeine; however, theobromine has relatively mild vasodilative and diuretic effects. The methylxanthines do, however, contribute a characteristically astringent and bitter quality to the chocolate. Chocolate also contains a complex mixture of amino acids and dipeptide derivatives in various states depending on the amount of fermentation, roasting, and thermal treatment that has been applied to the cacao. As with many roasted foodstuffs there are complex mixtures of reaction products (e.g., diketopiperazines) produced by Maillard and associated reactions that impart a strong flavor to the chocolate.

A variety of simple acids are present in cacao as a byproduct of the fermentation process; however, these are usually significantly reduced or neutralized in most cacao products. Naturally occurring sugars are also present in cacao, but frequently very significant quantities (25–90%) of sucrose are added to make the flavor palatable to a broad consumer base.

Naturally, the concentrations of all of these components can vary considerably depending on the origin and processing of the cacao, and the breadth of variance is currently not well understood.


Having established that significant concentrations of antioxidants can persist in a variety of chocolate products,11 the next issue to consider is their bioavailability. Given that thermal processing of cacao products can affect the final profile of polyphenols in chocolate and given the harsh environment of the digestive system, it is reasonable to question whether antioxidant polyphenols can transit to a site of uptake in the gut without being significantly degraded en route.

A study by Rios et al.13 addressed this issue and focused attention on the stability of polyphenols and flavin-3-ols in gastric juices of the stomach. Surprisingly, cacao polyphenols introduced into human subjects through gastric feeder tubes for up to 40 minutes and then recovered showed no change in polymerization state by HPLC. This is significant, as in vitro treatment with acid and thermal treatment can cause changes in the polymerization state of polyphenols. Thus, it seems likely that the gastric environment has minimal effect on polyphenols and that they transit with minimal modification to the small intestine.

Both catechin and epicatechin are readily adsorbed by the gut into the bloodstream. However, there are differences in the uptake of the catechin enantiomers.14 This is significant for the consumption of cacao, since the primary catechin enantiomer present in cacao is of the (−) form that is poorly adsorbed. Thus, it would seem that the most bioavailable antioxidant of significance in cacao is (−)-epicatechin.15,16 Absorption of (−)-epicatechin in humans is efficient, and its metabolite (−)-epicatechin-glucuronide can be identified in plasma at a mean concentration of 625.7 ± 198.3 nmol/L 2 hours after consumption of a cocoa beverage containing 54.4 mg of (−)-epicatechin.17 Epicatechin metabolites, (−)-epicatechin-glucuronide and (−)-epicatechin-sulfates can be detected in urine 6–12 hours after consumption.1519

Interestingly, current data on uptake and bioavailability suggests that cocoa flavonols can be readily adsorbed regardless of the background of other foodstuffs. Mixing cacao products with common chocolate-making materials such as milk powder does not seem to prevent the uptake of epicatechin.8,9 Further studies need to be carried out to understand the effects of dietary antioxidants as food additives, as well as on foodstuffs exhibiting other pharmacological effects, e.g., high dietary caffeine, etc. One intriguing study suggests that certain antioxidants can compete with each other, suggesting that the interplay and intestinal transport systems are complex.16

Some studies have shown the access of flavonoids in brain tissue after their oral intake. Abd el Mohsen et al.20 demonstrated for the first time that epicatechin metabolites, including glucuronide and its 3′-O-methylated form, were able to cross the blood brain barrier after a single oral administration of epicatechin (100 mg/kg) in rats. In addition, quercetin was detected in brain tissue from rats fed long-term with a diet containing 0.1% quercetin.21 It is important to highlight that in both studies flavonoid concentrations were corrected, taking into account the presence of residual blood in the tissue.

The mechanisms by which flavonoids gain entry to the brain remain to be clarified. Flavonoids could enter the central nervous system (CNS) by diffusion, carrier, or receptor-mediated transport across the blood brain barrier.22 Moreover, we cannot discard the possibility that epicatechin glucuronides could be hydrolyzed by tissue glucuronidases, resulting in free epicatechin penetration into the blood brain barrier and subsequent conjugation with glucuronic acid by CNS UDP-glucuronosyltransferase.


Chocolate has been popularized as being able to satiate cravings. There is a wealth of both anecdotal and scientific evidence indicating that chocolate induces gender- and individual-specific responses when consumed. However, persistent evidence of a “smoking gun” for the psychoactive (even purportedly aphrodisiac-like) substances in chocolate remains controversial.23 Indeed, in the absence of candidate drugs in chocolate that could provoke this type of physiological response, it has even been suggested that certain craving responses to chocolate may occur as a result of gut bacteria producing addictive secondary metabolites in certain hosts.24 This remains an elusive problem.

The vasoactive effects of chocolate are somewhat better understood. In general, these effects can be linked to the consumption and bioavailability of (−)-epicatechin in chocolate.25 Consumption of chocolate results in both acute modulations of blood pressure26 as well as positive changes in cholesterol biochemistry.27 Cocoa intake reduces cardiovascular disease risk due to a combination of different effects including the improvement of antioxidant status (shown by the decrease of oxidative stress biomarkers, including thiobarbituric acid reactive substances and low-density lipoprotein oxidation), vasodilation, and inhibition of platelet activation and aggregation.28,29

Epidemiological studies of long-term cocoa ingestion seem to tally with the shorter-term clinical studies, suggesting that the consumption of certain cacao products have significant clinical benefit.30 Significantly, the doses used in the Taubert study30 were exceedingly low (6.3 grams of chocolate per day). This demonstrated that the ingestion of flavonoids, through the medium of chocolate, had minimal impact on calorific intake (30 kcal per day) but produced statistically significant decreases in systolic and diastolic blood pressure and an increase in S-nitrosoglutathione. Furthermore, cacao flavonoids also seem to mediate very significant anti-inflammatory effects that can be measured as reductions in platelet and endothelial cell activation31 and the expression of inflammatory mediators.32 Of note, a negative effect on bone density in mature women who consume chocolate has recently been identified.33

In addition to the possible protection against coronary heart disease, cocoa intake improves cerebral blood flow in humans34,35 and this may have a positive impact in aging and cerebrovascular diseases, such as stroke and dementia, in which endothelial function is impaired. Cocoa effects on brain perfusion seem to be attributed to NO-dependent vasodilatation mediated by flavonoids.34 Although short-term cocoa intake increases blood oxygenation levels, it does not affect the reaction time in task-switching.35 This lack of cognitive improvement is not surprising since this study was performed in young subjects with high cognitive abilities. Hence, using people with low cognitive performances (due to fatigue, aging, or cerebrovascular disease) is more likely to provide an effect in cognitive tasks. In this regard, long-term administration of cocoa extract (24 mg/kg p.o.) is capable of delaying the onset of age-related cognitive deficits in rats, as shown by the improved cognitive performances in light extinction and water maze paradigms.36

In addition, several in vivo studies demonstrate the benefits of cocoa flavonoids including catechin, epigallocatechin-3 gallate (EGCG) and quercetin in cerebral ischemia and hypoxia models.3743 For instance, flavonoids improve spatial memory39 and decrease brain edema, neuronal death in the hippocampus,39 and MDA42 and NO40 levels induced by ischemia. However, in almost all these studies, high doses of flavonoids were administered as intraperitoneal injections; therefore, the observed effects are not representative of oral consumption. Epidemiological studies suggest that the consumption of products with high flavonoid content delays the onset of dementia and Alzheimer's disease (AD), particularly among those who are at high risk.44,45 Therefore, further studies using moderate doses administered orally should be performed to confirm the relevancy of dietary cocoa flavonoids in the improvement of cerebral ischemia.

Neurodegenerative diseases such as AD and Parkinson's are prompted by multiple factors including oxidative stress, inflammation, reduced expression of trophic factors, and accumulation of iron and protein aggregates, among others, which lead to neuronal loss.4653 One important feature of neurodegenerative diseases is the abnormal accumulation of iron in dying neurons, which enhances the production of reactive oxygen radicals and induces the aggregation of neurotoxic peptides including amyloid-β and α-synuclein (as reviewed by Mandel et al.54). Therefore, the combination of iron chelating and antioxidant therapies may be a useful strategy for neuroprotection.

Cocoa has recently attracted attention as a potentially neuroprotective natural product due to its antioxidant and potent iron-chelating activities.5457 Some studies suggest there are beneficial effects of cocoa on neurodegenerative disorders such as AD and Parkinson's disease. In this sense, cocoa extract, epicatechin, and catechin reduce the toxic effects of amyloid-β, a constituent of senile plaques in AD, on a neuronal cell line through membrane and mitochondrial protective mechanisms.58 Moreover, EGCG decreases amyloid precursor protein (APP) levels and consequently amyloid-β formation in both in vitro and in vivo studies.59,60 These effects seem to be mediated by the iron chelating capacity of EGCG since APP is post-transcriptionally regulated by iron regulatory proteins (IRPs).61 In addition, oral administration of cocoa extract (100 mg/kg/day) has been recently shown to attenuate nigrostriatal dopaminergic cell loss in a murine model of Parkinson's disease induced by the infusion of the neurotoxin 6-hydroxidopamine.62

The mechanisms by which cocoa flavonoids exert neuroprotective and neurorescue activities seem to be beyond the simple radical scavenging activity. Accumulating evidence suggest that cocoa flavonoids interact selectively with mitogen-activated protein kinase signaling (MAPK) cascades that are involved in neuronal survival, regeneration, and cell death. Thus, epicatechin and its in vivo metabolite 3′O-methyl-epicatechin are potent intracellular inhibitors of oxidized LDL-induced neuronal cell death by counteracting JNK, c-Jun, and caspase-3 activation.63 It is worth highlighting that although 3′O-methyl-epicatechin has a lower antioxidant activity than its precursor, it is as effective as epicatechin in protecting neurons against oxidized LDL; this provides evidence for the non-antioxidant mechanisms of cocoa flavonoids.

In serum-deprived neurons, EGCG (0.1–1 µM) modulates the expression of specific proteins associated with neurite outgrowth, cell survival, and iron chelation.64 Thus, EGCG increases the level of cytoskeletal and structural proteins (e.g., the actin binding protein tropomyosin 3, beta-tubulin IV)64 that are associated with the neurite outgrowth activity.65 Moreover, EGCG enhances cell survival, in part, through the inhibition of caspase-3 pathway66 and the upregulation of the binding protein 14-3-3 gamma, which blocks the apoptotic protein Bad.64 EGCG also interferes in the iron-oxygen sensors of hypoxia-inducible factor (HIF)-1 alpha pathway. Recent findings have pointed out the importance of this pathway in iron regulation and its potential applicability in the treatment of neurodegenerative diseases.67

In addition to intracellular signaling modulation, we cannot discard the interaction of cocoa flavonoids with cell surface receptors, as described in cancer and immune cells.6870 In this regard, the specific binding of EGCG to the 67 kDa laminin receptor has been shown to be involved in anticancer and antiallergic effects.68,70 This laminin receptor is also present in neuronal cells and its expression is associated with tumor malignancy and metastasis.71

A summary of in vivo work to date on CNS effects of cacao is shown in Table 2.

View this table:
Table 2

Effects of cocoa and related flavonoids on central nervous system.

SubjectsInterventionFlavonoid intake/dayMain outcome
Brain perfusion and cognition
 Healthy patients (>50 years old)34920 mL cocoa beverage/5 days821 mgPeripheral vasodilatation dependent on NO synthase
1000 mL cocoa beverage/7 days900 mg↑ Cerebral blood flow
 Female patients (n = 16; 18–30 years)35High-flavonol cocoa beverage/5 days172 mg↑ Blood oxygenation
No effect on cognition
 Wistar rats (15 mo)36Cocoa flavonoid extract/1 year24 mg/kg↑ Cognition
↑ Lifespan
Preservation of high urinary free dopamine levels
Cerebral ischemia models
 ModelInduction of damageFlavonoid treatmentMain outcome
 Repeated cerebral ischemia (Wistar rats)3910 m artery occlusion × 2Quercetin (50 mg/kg) 30 min before occlusion↑ Spatial memory
↓ Neuronal cell death in the hippocampal CA1
 Excitoxic neuronal damage (rat primary hippocampal neurons)4210 µM NMDA; 10 µM AMPA; 20 µM KainateEGCG (10 µM); at time of occlusion↑ Cell survival
↓ MDA production
 Unilateral cerebral ischemia (gerbils)4230, 60, or 90 min carotid artery occlusionEGCG (50 mg/kg) 30 min before occlusion and after ischemia↓Brain edema
↓Infarct volume
↓MDA production
 Transient forebrain ischemia (gerbils)413 min carotid arteries occlusionEGCG (50 mg/kg) 1–3 h after occlusion↓ Hippocampal damage
↓ Polyamine (putrescine) levels in brain
 Bilateral ischemia (Wistar rats)4010 min carotid/vertebral arteries occlusionEGCG (50 mg/kg i.p.) before occlusion↓ NO currents in the hippocampus
 Transient ischemia (gerbils)37Ischemia/reperfusionCatechin for 2 weeks before ischemia↓ Neuronal cell death in hippocampal CA1
↑ Superoxide scavenging activity
 Hypoxia (Wistar rats)43Altitude chamber 10,000 m for 4 hEGCG (25–50 mg/kg i.p.)↑ Cell survival
Neurodegenerative disease
Alzheimer's disease
 Type of modelInduction of damageFlavonoid treatmentMain outcome
 Rat PC12 cells5825–35 (25 µM, 24 h)Cocoa extract (one or both: 7–45 µg/mL or 10–100 µM epicatechin and catechin) 10 min before cell damage↓ Aβ cytoxicity preservation of membrane integrity and mitochondrial function
 Human neuroblastoma SHSY5Y59EGCG (1–10 µM) for 2 h↑ sAAPα mediated via an increase of α-secretase activity
 Rat PC12 cells5925–38, Aβ1–42, Aβ1–40 (10 µM 48 h)EGCG (1 µM) 30 min before cell damage↓ Aβ cytoxicity ↑mitochondrial function
↓ Apoptosis
 Rat PC12 cells5925-35 (10 µM 48 h)EGCG (0.1–1 µM) 2 h afterNeurorescue
 C57/Bl mice59NOEGCG (2 mg/kg/day p.o.) for 7 and 14 days↓ Holo-APP levels in the hippocampus
↑ sAPP; PKC activation
 Human neuroblastoma SHSY5Y60Serum withdrawalEGCG (1–10 µM) for 48 h↑ Transferring receptor
↓Holo-APP by suppressing APP translation
↑ sAPPα/holo-APP
 CHO/ΔNL (sweAPP)60NOEGCG (1–10 µM) for 48 h↓ Aβ secretion
↓ Holo-APP
Parkinson's disease
 Sprague-Dawley rats62Neurotoxin 6-OHDA 12 µgCocoa rich in procyanidins (100 mg/kg/d) 4 days before↑ Dopaminergic neuron survival
Neurodegenerative disease and aging
 Rat PC12 cells64Serum withdrawalEGCG (0.1–10 µM) for up to 3 days after serum withdrawal↑ Cell survival
↑ Neurite outgrowth
↑ PKC activation
 Human neuroblastoma SHSY5Y64Serum withdrawal (5 days)EGCG (0.1–1 µM) for 48 h 3 days after serum withdrawal↑ Cytoskeletal and structural proteins
↓ Metabolic enzymes
↓ Heat shock proteins
 Rat PC12 cells72Sodium nitroprusside (NO donor)EGCG (100–300 µM) 30 min before cell damage↓ Cytotoxicity
↓ Released cytochrome C
↓ Apoptotic changes
↓Bax/Bcl2, caspase 9, –8, and –3
 Mouse primary striatal neurons63Oxidized LDL (12.5 µg of protein/mL) for up to 24 hEpicatechin and 3′-O-methylepicatechin (30 µM) up to 18 h before↓Cytotoxicity
↓ JNK and c-Jun activation
↓Caspase-3 activation


Dietary modification and exercise is a cheap, simple, and lower risk method of attempting to modulate or prevent the onset of disease. Clearly, the trend in the clinical and scientific data suggests that the consumption of dietary flavonols can produce mostly positive clinical benefits. The benefit to the vascular system seems clear and a number of studies suggest there may also be significant benefit to the cerebrovascular system and elements of brain function in neurodegenerative disease. However, the context in which flavonols are realistically consumed outside of controlled scenarios is unclear. More work needs to be done to determine the context in which dietary intervention would be most clinically efficient and result in the highest level of patient compliance. Given the broad popularity of chocolate, clearly significant potential exists here. However, the monikers of “chocolate” and even “dark chocolate” are applied to a very wide range of foodstuffs and processing techniques. This is due, in part, to the highly complex manufacturing processes that have developed but also to the wide and bewildering range of chocolate consumer products in a market in which the pressure to very aggressively differentiate and promote products is significant.

Indeed, the positive data regarding the consumption of cacao also has significant caveats, given that no one actually consumes raw, lightly fermented cacao beans anymore; note that this was, however, more like what was consumed by the Olmec/Aztec/Mayan populations millennia ago. So the health benefits of chocolate must be regarded in the context of the other ingredients in chocolate bars, an area that is still unclear. Certainly chocolate can contain very high concentrations of refined sugar and have a very high saturated fat content derived from the cacao bean itself, added milk powder, or the addition of vegetable oils to chocolate products. Thus, the net benefit of cacao must be weighed against the other components in chocolate and reformulated if necessary. In the meantime, strict adherence to labeling standards, additional consumer education and more openness in the chocolate industry can also help improve consumer understanding of the issues.


Declaration of interest.  AM and SBM are employees of Theo Chocolate (http://www.theochocolate.com).


View Abstract