Neuroscience and Biobehavioral Reviews 37 (2013) 24452453

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Neuroscience and Biobehavioral Reviews

journal homepage: www.elsevier.com/locate/neubiorev

Review

Chocolate and the brain: Neurobiological impact of cocoa avanols on cognition and behavior
Alexander N. Sokolov a, , Marina A. Pavlova b , Sibylle Klosterhalfen a , Paul Enck a
a Department of Internal Medicine VI: Psychosomatic Medicine and Psychotherapy, Research Division, University of Tbingen Medical School, D 72076 Tbingen, Germany b Developmental Cognitive and Social Neuroscience Unit, Department of Pediatric Neurology and Developmental Medicine, Childrens Hospital, University of Tbingen Medical School, D 72076 Tbingen, Germany

a r t i c l e

i n f o

a b s t r a c t
Cocoa products and chocolate have recently been recognized as a rich source of avonoids, mainly avanols, potent antioxidant and anti-inammatory agents with established benets for cardiovascular health but largely unproven effects on neurocognition and behavior. In this review, we focus on neuromodulatory and neuroprotective actions of cocoa avanols in humans. The absorbed avonoids penetrate and accumulate in the brain regions involved in learning and memory, especially the hippocampus. The neurobiological actions of avanols are believed to occur in two major ways: (i) via direct interactions with cellular cascades yielding expression of neuroprotective and neuromodulatory proteins that promote neurogenesis, neuronal function and brain connectivity, and (ii) via blood-ow improvement and angiogenesis in the brain and sensory systems. Protective effects of long-term avanol consumption on neurocognition and behavior, including age- and disease-related cognitive decline, were shown in animal models of normal aging, dementia, and stroke. A few human observational and intervention studies appear to corroborate these ndings. Evidence on more immediate action of cocoa avanols remains limited and inconclusive, but warrants further research. As an outline for future research on cocoa avanol impact on human cognition, mood, and behavior, we underscore combination of functional neuroimaging with cognitive and behavioral measures of performance. 2013 Published by Elsevier Ltd.

Article history: Received 18 February 2013 Received in revised form 17 June 2013 Accepted 18 June 2013 Keywords: Cocoa avanols Chocolate Antioxidant Anti-inammatory Neurogenesis Angiogenesis Age- and disease-related decline Neurocognition Neuromodulation Neuroprotection

Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cocoa avanols in the brain signaling cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroprotective action of cocoa avanols in aging and neurological disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Human population studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuromodulation of cognition, mood, learning, and memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2445 2446 2447 2447 2448 2448 2449 2449 2449 2450 2452 2452

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1. Introduction Cocoa products and especially, chocolate has taken a special place in our daily life and culture. This food of the gods as tells its Latin name Theobroma cacao given by the noted Swedish nosologist Carl Linnaeus in 1753, has been ennobled in many countries around the globe as a curative drug, a culinary delight, and even a currency for commodity trading, retaining its appeal over the centuries. No

Corresponding author at: Department of Internal Medicine VI: Psychosomatic Medicine and Psychotherapy, Research Division, University of Tbingen Medical School, Osianderstr. 5, D 72076 Tbingen, Germany. Tel.: +49 7071 29 89118; fax: +49 7071 29 4382. E-mail address: alexander.sokolov@uni-tuebingen.de (A.N. Sokolov). 0149-7634/$ see front matter 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.neubiorev.2013.06.013

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other natural product but chocolate has ever been viewed as having a positive effect on a wide variety of health conditions ranging from intestinal and female complaints, fever, and cardiovascular diseases to promotion of strength before military and sexual conquests (Wilson, 2010; Wolfe and Shazzie, 2005). Reports on chocolates health benets are dated back as far as Aztec and Maya medical practice (e.g., Hurst et al., 2002) and ever since, anecdotal evidence has been abundant on chocolate effects on health. Only by the end of the 20th century, however, claims on supposed health benets of chocolate have increasingly drawn a scientic interest in cocoa products and chocolate, which eventually resulted in an approval by the European Food Safety Agency (EFSA, 2012) of a health claim for dark chocolate with high avanol content as to its impact on maintenance of normal endothelium-dependent vasodilation. Most studies so far have been conducted on the effects of chocolate intake on the cardiovascular system, skin, cholesterol concentrations, and the release of neurotransmitters anandamide and serotonin, and on the health-related properties of highquality dark chocolate, containing the stimulants theobromine and caffeine (Lamuela-Ravents et al., 2005; Katz et al., 2011). Dark chocolate also comprises high concentrations of avanols (a avonoid subgroup, mainly epicatechin; Whiting, 2001) known as potent antioxidative agents. While some work has been done on the inuence of theobromine and caffeine on mood and cognition (e.g., Smit and Rogers, 2000; Smit et al., 2004; Smit and Blackburn, 2005; Nehlig, 2010), the impact of cocoa avanols on human cognitive and affective function, executive control and behavior has yet to be determined. In accord with accumulating evidence for enhancing effects of chocolate consumption on cognitive function, Messerli (2012) reports, as an occasional note, a strong positive correlation between chocolate intake per capita and the number of Nobel laureates in various countries. In contrast to potential effects on cognition and behavior, evidence-based benets of cocoa and chocolate consumption for cardiovascular system are well established and include endothelium-dependent vasodilation recently found to contribute to normal blood ow (Engler et al., 2004; Hooper et al., 2012; Kay et al., 2006; Grassi et al., 2005, 2008). Cardiovascular health has been closely linked to cognitive performance (e.g., DeCarli, 2012). Animal studies have shown that the absorbed avonoids directly interact with a number of cellular and molecular targets in the brain, exerting pronounced antioxidative effects and improving brain tissue and function in the regions mainly implicated in learning, memory, and cognition (Andrs-Lacueva et al., 2005; Passamonti et al., 2005; Vauzour et al., 2008). This suggests a potential neuromodulatory and neuroprotective role for cocoa avanols and their signicance for cognitive and affective function, executive control and behavior. However, only few human studies so far have specically addressed neurobiological, cognitive, affective and behavioral effects of avanol-rich cocoa products. The present review focuses on analysis of the existing evidence on potential neuromodulatory and neuroprotective actions of cocoa avanols in humans. Our analysis highlights further ways in investigation of the impact of avanol-rich cocoa products on neurocognition and behavior. 2. Cocoa avanols in the brain signaling cascades The avanol monoisomers epicatechin and catechin are the predominant avonoid compounds in cocoa, with the 2-phenyl-3,4-dihydro-2H-chromen-3-ol as underlying skeleton. These monomers represent the base molecules for concatenated oligomers, the proanthocyanidins. Antioxidant properties of avanols are chemically mediated through oxidation of two aromatic hydroxyl groups to a quinone (Bors and Michel, 2002). In addition, avanols foster antioxidant system through modulation of

enzymatic activity (Stevenson and Hurst, 2007; Mann et al., 2009). Flavanols occur in high concentrations in beverages such as green tea and red wine, fruits and berries (e.g., apple skin, grapes, pears, blueberry), vegetables (tomatoes, soy, and olives), and, especially, cocoa (Manach et al., 2004; Neveu et al., 2010; Scalbert et al., 2011; Sies et al., 2012). The avonoid contents in cocoa products and chocolate differ substantially depending on the cocoa variety (in some beans, amounting up to 20%), geographic origin, cultivating, agricultural and postharvest practices, and manufacturing (Wollgast and Anklam, 2000; Niemenak et al., 2006). In the Dutch population, chocolate contributes to about 20% of the total avonoid intake in adults, with an even higher percentage in children (Arts et al., 1999). In American diet, chocolate represents the third top source of antioxidants after fruits and vegetables (255, 233, and 104 mg/day, respectively; Vinson et al., 2006). Also in the French adult population with the total daily dietary polyphenol intake of 1.2 g, including 99 mg catechins, cocoa products account for the third major source of epicatechin (17%) after green tea (28%) and apples (24%; Prez-Jimnez et al., 2011). Yet human and animal studies on neuroprotective properties of avonoids, especially in preventing cognitive decline, have mostly examined plant-derived substances other than cocoa avanols (e.g., Macready et al., 2009). Flavonoid-rich pine extracts such as Gingko biloba are reported to delay the onset of memory loss, dementia, and Alzheimers disease (Weinmann et al., 2010), but the evidence remains controversial (Birks and Grimley Evans, 2009). Animal studies show that avanols and their metabolites can cross the bloodbrain barrier, inducing benecial effects on the brain tissue and function (angio- and neurogenesis, changes in neuron morphology) and stimulating widespread blood circulation in the brain (Vauzour et al., 2008). The most common avanol found in cocoa, epicatechin (Whiting, 2001), is rapidly absorbed in humans and is detectable in blood plasma already 30 min after intake. The epicatechin levels peak 23 h after intake, exhibiting a strong positive correlation with the dose of ingested chocolate (Richelle et al., 1999), and return to baseline by 68 h after consumption. The possibility of avanols and metabolites to penetrate and accumulate in the brain regions mainly related to learning and memory, suggests they may exert a direct positive impact on the brain, including cognition and neuroprotection (Nehlig, 2013). Neurobiological impact of avanols on the brain, learning, memory, and cognition are believed to occur in two major ways (Fig. 1). First, avonoids can specically interact within a number of cellular signaling pathways, primarily with mitogen-activated protein (MAPK), extracellular-signal-regulated (ERK) and phosphoinositide 3-kinase (PI3-kinase/Akt) signaling cascades. These cascades trigger gene expression and protein synthesis for maintaining long-term potentiation (LTP) and establishing long-term memories (Kelleher et al., 2004). Flavonoids modulate the transcription factors engaged in signal transduction through protein-kinase inhibition (Goyarzu et al., 2004), and promote the expression of brain derived neurotrophic factor (BDNF) that is critical for neurogenesis, also in adult animals, synaptic growth and neuron survival, especially in the learning- and memory-related brain regions such as the hippocampus and subventricular zone (Kim et al., 2006; Valente et al., 2009). Second, avonoids facilitate production of the signaling molecule nitric oxide, which inhibits the incidence of atheromatous plaque adhesion molecules causing inammation (Gonzalez-Gallego et al., 2007), and importantly, improves vascular endothelial function by relaxing the smooth muscle tissue of blood vessels (e.g., Heiss et al., 2003; Schroeter et al., 2006). In this way, avanol-rich cocoa can impose vasodilation in a nitric oxidedependent way both at the cardiovascular and peripheral levels. This in turn results in enhanced cerebral blood ow and blood perfusion throughout the central and peripheral nervous system (Fisher et al., 2003, 2006; Hollenberg et al., 2009), affording better

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Cocoa avanols

avonoids may effectively protect against neural and oxidative damage eventually giving rise to neurological disease, cognitive and functional decline. 3.1. Animal studies

MAPK, ERK, PI3 signaling

nitric oxide synthesis

gene expression, protein synthesis

inammaon

expression of BDNF

vascular endothelian funcon, angiogenesis vasodilaon (heart, CNS, sensory systems)

neurogenesis, synapc growth, neuron survival

LTP, memory formaon

oxygen, glucose supply

neurocognive funcons

neuroprotecon

Fig. 1. Schematic of putative neurobiological actions of cocoa avanols; small upand downward inclined arrows show up and down regulation of function, respectively. The absorbed avanols either directly interact with cellular cascades (left, green), yielding expression of neuroprotective and neuromodulatory proteins that promote neurogenesis, neuronal function and brain connectivity, or act by improving central and peripheral blood ow and angiogenesis in the brain and sensory systems (right, yellow). Note the two action pathways may cross-talk, contributing to more than one function. For example, improved neurogenesis, synaptic growth, and neuron survival may promote neuroprotection, while neurocognition may benet from increased oxygen and glucose supply due to vasodilation. LTP, long-term potentiation; mitogen-activated protein (MAPK), extracellular-signalregulated (ERK), and phosphoinositide 3-kinase (PI3-kinase/Akt) cascades; BDNF, brain derived neurotophic factor; CNS, central nervous system. (For interpretation of the references to color in the gures throughout the article, please consult the web version of this article.)

supply of oxygen and glucose to the neurons and removal of waste metabolites in the brain and sensory systems (e.g., blood delivery to the retina; Huber et al., 2006; Kalt et al., 2010). In addition, animal models indicate that cocoa avonoid administration stimulates angiogenesis in the hippocampus (van Praag et al., 2007). In a nutshell, the multiple neurobiological actions of cocoa avonoids in enhancing cognitive function and behavior may be attributed to the expression of neuroprotective and neuromodulatory proteins that increase the number and connectivity of neurons, and improve neuronal function. The other class of putative mechanisms is related to the effects on vascular function that through enhanced blood perfusion both in the brain and peripheral nervous system including sensory systems, may improve neuro- and angiogenesis (Williams and Spencer, 2012), cognitive function, and executive control. However, while the long-term actions of cocoa avanols in counteracting oxidative stress, neuroinammation and neurodegeneration appear to be better understood, the mechanisms of more immediate neuromodulatory effects on cognition and behavior remain unclear. 3. Neuroprotective action of cocoa avanols in aging and neurological disease A number of health conditions in typical and atypical aging (Alzheimers disease, vascular dementia, Parkinsons disease) and acute neurological conditions such as stroke have been associated with disturbances of cerebral blood ow and oxidative stress (McGeer and McGeer, 2003; Hirsch et al., 2005). Potent antioxidant action and endothelium-dependent vasodilation capacity of

Several animal models of normal and pathological aging examined the effects of high-avanol cocoa on the onset of age-related cognitive decits. Rozan et al. (2007) assessed the preventive effects of Acticoa powder, a cocoa polyphenolic extract, on free radical production by leucocytes in rats following heat exposure and protective effects of the cocoa powder on subsequent cognitive performance. Either high-avanol cocoa powder (22.9 mg/kg/day) or vitamin E, as the antioxidant reference, was administered orally to rats for 14 days prior to heat exposure. The day after heat exposure, free radical production in rats treated with cocoa powder or vitamin E was signicantly reduced compared to controls. Unlike controls, cocoa powder and vitamin E-treated rats discriminated between active and inactive levers in a light extinction paradigm. Throughout testing, the treated rats also exhibited decreased escape latencies for reaching the hidden platform in the Morris water maze. The results suggest that the daily oral administration of Acticoa powder or vitamin E counteract the overproduction of free radicals, and thereby protect rats from cognitive impairments caused by heat exposure. Bisson et al. (2008) administered orally Acticoa powder to rats (as they grew from 15 to 27 months of age) at the dose of 24 mg/kg/day. In aged rats, high-avanol cocoa improved cognitive performance in light extinction and water maze paradigms, increased lifespan and preserved high urinary free dopamine levels. Acticoa powder appears to retard age-related impairments, including cognitive decline in normal aging and neurodegenerative diseases. Converging evidence comes from animal models of neurodegenerative diseases (such as Alzheimers disease), which make use of polyphenol-enriched diets. Fernndez-Fernndez et al. (2012) examined an ability of a diet rich in polyunsaturated fatty acids and polyphenols from dry fruits and cocoa (called LMN diet, a patented product known to induce hippocampal neurogenesis in adult mice) for counteracting the age-related impairment and neuropathology in wild type and transgenic mice (Tg2576 genotype with over-expression of the human APP gene carrying the Swedish mutation, K670N:M671L), an animal model of Alzheimers disease. At the age of 13 months, once the amyloid plaques (A) were formed, both mice types received LMN diet for further ve months. In the last two months, they performed on a behavioral test battery. Overall, both typically aging wild and, to a greater degree, Tg mice exhibited reduction in sensorimotor reexes, exploratory behavior in the hole board, activity in the elevated plus-maze, ambulation in the home cage during the dark phase, and in spatial learning in the Morris water maze. The diet did not impact the detrimental effects observed in sensorimotor reexes, but did clearly reverse the behavioral effects of both aging and Tg genotype. This behavioral improvement correlated with a 70% increase in cellular proliferation in the subventricular zone of the brain, rather than with a decrease of amyloid plaques. In contrast, LMN diet administered at an age of 10 months (before the plaques occurred) led to a decreasing tendency of soluble and brillar A levels in the hippocampus along with a decrease in A plasma content, suggesting a putative role of the diet in delaying plaque formation. This is the rst evidence that LMN diet can prevent behavioral deterioration caused by aging and Tg genotype, and delay the A plaque formation. The results also highlight the increasing importance of polyphenols from dry fruits and cocoa as human dietary supplements in amelioration of the cognitive and functional impairment during aging and neurological disease. Arendash et al.

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(2007), however, administered a polyphenol-rich omega-3 fatty acid diet of Greenland Eskimos to another type of Tg mice (another animal model of Alzheimers disease), and did not nd any diet benets, except for several behavioral measures such as open eld activity and maze entries. In both studies, Tg mice were similar, with Arendash et al. (2007) using a second-generation cross of the Tg2576 and the 6.2 lines that carried an additional mutation in the presenilin gene 1, PS1. The distinct outcomes were likely due to the difference in either diets or study protocols to which the mice were exposed. In an animal model of stroke, Shah et al. (2010) administered orally mice with 5, 15, or 30 mg/kg epicatechin 90 min before middle cerebral artery occlusion. Epicatechin-treated mice, compared to the control group, exhibited signicantly smaller lesion volumes and improved neurological scores. Mice that were posttreated with 30 mg/kg of epicatechin at 3.5 h after the occlusion had also signicantly smaller infarct volumes and improved neurological scores. Villarreal-Calderon et al. (2010) reported that a treatment with dark chocolate prevents the inammation of the vagus nerve resulting from a 16-month exposure of mice to the polluted air of Mexico City. Mice exposed to polluted air had a signicant imbalance in genes coding for antioxidant defenses, apoptosis and neurodegeneration at the level of the dorsal vagal complex and this imbalance was mitigated by chocolate administration. 3.2. Human population studies Observational human population studies on neuroprotective and neuromodulatory action of avonoids, including high-avanol cocoa, have often been poorly controlled, whereas prospective longitudinal studies remain laborious and costly. To date, therefore, evidence for benecial neuroprotective and anti-inammatory effects of cocoa avanols on cognitive and behavioral decline in aging and neurological disease is rather limited. In a prospective study of a large sample of men aged 6989 years (Kalmijn et al., 1997), risk of cognitive decline (assessed by the Mini-Mental State Examination) tended to inversely relate to avonoid intake, though no association was found between the risk of cognitive decline and vitamin C- or E-intake served as the antioxidant reference to avonoids. In a cross-sectional study of the elderly Norwegian population, Nurk et al. (2009) investigated the association between cognitive performance and avonoid intake from chocolate, wine, and tea. Participants aged 7074 years (n = 2031; 55% females) completed a comprehensive cognitive test battery consisting of the Kendrick Object Learning Test, Trail Making Test (part A), versions of the Digit Symbol Test, Block Design, Mini-Mental State Examination, and Controlled Oral Word Association Test. Habitual food intake was assessed by a self-reported food frequency questionnaire. Chocolate, wine, or tea consumers yielded signicantly better mean test scores and lower prevalence of poor cognitive performance. Those consuming all three items had the best test scores and the lowest risk for poor test performance. The association between intake of the foods and cognition were dose-dependent, with sharp improvements at intakes of 10 g/day chocolate and 75100 ml/day wine, and a linear improvement for tea intake. The effect was most pronounced for wine and modestly weaker for chocolate intake. It appears that in the elderly, a diet high in some avonoid-rich foods is associated with better performance in several cognitive abilities in a dose-dependent manner. Participants of a prospective 10-year Personnes Ages Quid (PAQUID) study (n = 1640; aged 65 years, free from dementia) underwent testing of cognitive function at four consecutive time points (Letenneur et al., 2007). At each visit, the cognitive functions were assessed by test battery including Mini-Mental State Examination, Bentons Visual Retention Test, and Isaacs Set. Flavonoid

intake from multiple food items including chocolate (assessed once as the study begun) was associated with both better cognitive performance at baseline and better evolution of performance over time. The most positive evolution was found in participants in the two highest quartiles of avonoid intake compared to those in the lowest quartile. After 10-year follow-up, participants with the lowest avonoid intake had lost on average 2.1 points on the Mini-Mental State Examination, compared to the 1.2-point loss in participants of the highest quartile. The study raises the possibility of an association between dietary avonoid intake and cognitive aging.

3.3. Clinical studies The known benets of avonoids for vascular health (Ried et al., 2012) may represent a promising approach in treating cerebrovascular disorders and protecting cognitive and functional behavior in the elderly. Age- and disease-related disturbances in cerebral blood ow are thought to be commonly accompanied by cognitive and behavioral decline. Dietary high-avanol cocoa intake is associated with an increased cerebral blood ow velocity in the middle cerebral artery, suggesting a promising role for high-avanol cocoa consumption in the treatment of cerebrovascular ischemic syndromes such as dementia and stroke. For instance, Sorond et al. (2008) report that following two weeks of high-avanol cocoa intake, in 34 healthy elderly volunteers (aged 72 6 years; 16 males), mean blood ow velocity in the middle cerebral artery measured by transcranial Doppler ultrasound signicantly increases by 8% at one week and 10% at two weeks. Potent antioxidative and anti-inammatory properties of avonoids have been proposed to play a role in preventing mild cognitive impairment, a precursor of dementia, and Alzheimers disease. In Alzheimers disease, an increased production and accretion of A-peptides activates microglia, resulting in release of inammatory mediators that further enhance A production, giving rise to neuronal dysfunction and cellular death. While and -secretase facilitate A production, -secretase inhibits it. Recent work in cultured human neuroblastoma cells shows that low concentrations of nitric oxide up-regulate the expression of secretase and down-regulate that of -secretase. This suggests the cerebrovascular nitric oxide might inhibit A production (McCarty, 2006; Pak et al., 2005). Cocoa avanols, especially epicatechin, act directly on the endothelium of brain vessels, stimulating activity of the endothelial nitric oxide synthase that in turn induces vasodilation and improves cerebrovascular perfusion (Fisher et al., 2006; Schroeter et al., 2006; Patel et al., 2008). So far, there does not seem to be any proven association between intake of antioxidants and vitamins and Alzheimers disease (see Luchsinger and Mayeux, 2004 for review), however, several studies did report a diminished cerebral blood ow in dementia patients (Ruitenberg et al., 2005; Nagahama et al., 2003). Cerebrovascular atrophy is also known to lead to mild cognitive impairment and subsequently to Alzheimers disease. It is therefore conceivable that benecial properties of cocoa avanols may slow down the transition from mild cognitive impairment to Alzheimers disease (Nagahama et al., 2003). Commenges et al. (2000) conducted a clinical trial with 1367 participants aged over 65 years, 66 from which developed dementia. The relative risk of developing dementia adjusted for age for the two highest consumptions of avonoids was 0.55 (95% condence interval, 0.340.90). After further adjustment for sex, education level, weight and vitamin C intake, the relative risk decreased to 0.49 (95% condence interval, 0.260.92). Thus, it appears that antioxidant avonoid intake is inversely related to the risk of dementia. However, in this study avonoids came mainly from fruits, vegetables, wine and tea rather than cocoa.

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Most recently, Desideri et al. (2012) studied 90 elderly individuals with mild cognitive impairment (mean age, 71 years; 43 males) who consumed once daily for eight weeks a drink containing either 990 mg (high), 520 mg (moderate), or 45 mg (low-avanol) of cocoa avanols. Cognitive function was assessed by the MiniMental State Examination, Trail Making Test A and B, and verbal uency test. At the end of the follow-up period, Mini-Mental State Examination scores were similar in the treatment groups. In the high- and moderate-avanol compared to the low-avanol groups, time required to complete both Trail Making Tests was signicantly lower. Verbal uency score was signicantly better in the highcompared to low-avanol group. The high- and moderate-avanol groups also exhibited decreased insulin resistance, blood pressure, and lipid peroxidation. This appears to be the rst dietary intervention study to demonstrate the efcacy of regular consumption of cocoa avanols for improving cognitive function in the elderly with mild cognitive impairment. The ability of avonoids to improve and maintain vascular function offers a further possibility to investigate the relationship between cocoa avanol intake and neuronal and functional loss after stroke (for recent animal data, see, e.g., Shah et al., 2010; Section 3.1). In the meta-analysis of three human studies comprising 114,009 participants, Buitrago-Lopez et al. (2011) reported a 29% reduction of stroke risk in high chocolate consumers compared to low consumers. Buijsse et al. (2010) found an even stronger inverse correlation between chocolate consumption and stroke risk than for myocardial infarction. Rautiainen et al. (2012) examined the association between the total antioxidant capacity (including fruits, vegetables, tea, coffee, and chocolate) and stroke risk in women, aged 49 to 83 years, from the Swedish Mammography cohort. The study included 5680 women with and 31,035 women without a history of cardiovascular disease. Diet was assessed with a self-reported food frequency questionnaire, and dietary total antioxidant capacity calculated using oxygen radical absorbance capacity values. Stroke cases were subdivided into cerebral infarctions, hemorrhagic and unspecied strokes. Using multivariate analyses with hazard ratios, the dietary antioxidant capacity was found to be inversely associated with total stroke in disease-free women (17% risk reduction) and hemorrhagic stroke in women with disease history (45% risk reduction). In Parkinsons disease, abnormal action of the neuromodulator adenosine, which fails to suppress unwanted motor activity in the basal ganglia via striatopallidal neurons, has been linked to impaired motor function (Jankovic, 2008). Adenosine antagonists, including caffeine and chocolate, have therefore been considered for ameliorating parkinsonian motor dysfunction. Parkinsons patients do report an increased chocolate consumption, independent of concomitant depressive symptoms (Wolz et al., 2009). However, in an investigator-blinded, placebo-controlled, crossover trial in 26 patients with moderate Parkinsons disease (Wolz et al., 2012), a single acute dose of dark chocolate failed to improve motor function (assessed by Unied Parkinsons Disease Rating Scale motor score) over white avanol-free white chocolate. The outcome might likely be due to lacking patient blindfolding, the dose of chocolate used, its avanol content, and the time frame of treatment. To the most part, the neuroprotective potential described above is attributable to cocoa and chocolate avanols rather than other ingredients such as caffeine that has been widely implicated in counteracting age- and disease-related cognitive decline such as in Alzheimers and Parkinsons disease (Costa et al., 2010; Santos et al., 2010). Unlike in coffee, tea and soft drinks, the caffeine (and theobromine) concentration in chocolate and cocoa products is much lower than that of avanols to account for potential effects of chocolate (Benton, 2004).

4. Neuromodulation of cognition, mood, learning, and memory 4.1. Animal studies Flavonoids are believed to trigger expression of neuromodulatory proteins in the brain regions implicated in learning, memory, and cognition, suggesting cocoa avanols can exhibit immediate and short-term action on neurocognition, mood, and behavior. Surprisingly, only a few animal studies have addressed these issues. Mice treated with one of the major chocolate avanols, epicatechin, at the dose of 500 g/g (daily supply of 2.5 mg) showed pronounced angiogenesis in the hippocampus (van Praag et al., 2007). Epicatechin treatment combined with exercise (running a wheel) improved retention of spatial memory and increased dendritic spine density in the dentate gyrus of the hippocampus. Moreover, epicatechin treatment facilitated gene expression associated with learning in the hippocampus but did not affect hippocampal adult neurogenesis. Yamada et al. (2009) compared the effects of short-term versus long-term (two-week) oral administration of cocoa mass in large amounts (100 mg/100 g body weight) in the rat elevated T-maze test, an animal model of anxiety. Shortterm administration signicantly abolished avoidance behavior during immediate test performance, suggesting a reduced fear conditioning. Long-term administration enhanced brain concentration of emotion-related neurotransmitter serotonin and its turnover. The ndings indicate short-term cocoa intake shows an anxiolytic effect, whereas long-term intake affects brain monoamine metabolism. This suggests avanol impacts on the amygdala underpinning regulation of anxiety and encoding of affective valence (e.g., Morrison and Salzman, 2010). Flavanol action therefore may occur in the brain regions outside the hippocampus and subventricular zone, in which it has already been established. 4.2. Human studies In humans, several studies have aimed to identify immediate and short-term action of cocoa avanols on mood and cognitive performance with as yet inconclusive outcome. Crews et al. (2008) had healthy older adults (n = 101; 41 males; age, 60 years) to consume daily for 6 weeks either a 37-g bar of dark chocolate and 8 ounces (237 ml) of articially sweetened cocoa beverage or similar placebo products. Participants underwent hematological, blood pressure, and pulse rate measurements, and accomplished several cognitive tests: Selective Reminding, Wechsler Memory Scale-III Faces I and Faces II subtests, Trail Making Test, Stroop Test, Wechsler Adult Intelligence Scale-III Digit Symbol-Coding subtest, and General Activation subscale of the Activation-Deactivation Adjective Check List (A-DACL). The only effect observed was a signicantly higher, compared to the placebo group, pulse rate, with no effects found on blood pressure, hematological, and cognitive variables. In a randomized, double-blind, controlled, balanced, three period crossover study, 30 healthy young adults (mean age, 22 years; 13 males) consumed high-avanol (520 mg and 994 mg) cocoa drinks and a matched control drink, with a three-day washout between drinks (Scholey et al., 2010). Over a 1 h testing period, participants repeatedly performed 10-min cycles of a Cognitive Demand Battery (two serial subtraction tasks, Serial Threes and Serial Sevens), a Rapid Visual Information Processing (RVIP) task, and a mental fatigue scale. High-avanol cocoa intake improved Serial Threes performance. The 994-mg beverage yielded speeded RVIP responses, but also more errors during Serial Sevens. Only the 520-mg beverage attenuated self-reported mental fatigue. This is the rst report on immediate improvements of cognitive function following high-avanol cocoa consumption in healthy young adults.

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In healthy young adults (n = 30, eight males; age range, 1825 years) who in a crossover, order counterbalanced design consumed once either 35 g dark chocolate (with 720 mg of high-avanol cocoa) or a matched quantity of avanol-free white chocolate, high-avanol cocoa improved visual contrast sensitivity (assessed by reading numbers varying in their luminance relative to background), working memory for location, choice reaction time, and the time required to detect direction of coherent motion (Field et al., 2011). This outcome extends the range of cognitive functions affected by high-avanol cocoa consumption and provides the rst evidence on the immediate effects of high-avanol cocoa on visual functions. In 72 healthy participants aged 4065 years, Pase et al. (2013) examined immediate and sub-chronic effects of cocoa avanols on mood and cognition. Three separate groups were assigned an Acticoa dark chocolate drink mix containing 500, 250, or 0 mg (placebo) of polyphenols once daily for 30 days. At baseline, at 1, 2.5, and 4 h after a single acute dose, and again after 30-day treatment, performance on cognitive tasks (Simple and Choice Reaction Time, Digit Vigilance, Tracking, Spatial and Numeric Working Memory, Immediate and Delayed Word Recall, Delayed Word and Picture Recognition) was assessed with the Cognitive Drug Research battery (Kennedy et al., 2002) and self-reported mood with the Bond-Lader Visual Analogue Scale (Bond and Lader, 1974). While mood remained unaffected by the acute treatment, no effects on cognition occurred at all time points. At 30 days, the high-dose treatment signicantly improved self-rated calmness and contentedness compared to placebo. This is the rst evidence on the effects of cocoa polyphenols on mood in healthy participants. The outcome suggests a possibility for cocoa polyphenols for ameliorating the symptoms associated with clinical anxiety and depression. Although some recent studies do indicate a relationship between chocolate consumption and depressive symptoms (e.g., Rose et al., 2010), the nature of the relationship remains to be claried. Latest developments in modern non-invasive neuroimaging techniques such as functional magnetic resonance imaging (fMRI), electroencephalography, and magnetoencephalography (EEG/MEG), have made it possible to explore modulatory effects of food constituents on neural activity in the human brain and its association with behavior. Yet, with the exception of some work done on the modulation of brain reward system by chocolate intake (e.g., Small et al., 2001; Stice et al., 2008; Valentin et al., 2007), only a few neuroimaging studies have examined the neural networks selectively activated by cocoa avanols. Francis et al. (2006) used a paired letter-digit task in healthy young female participants (n = 16; age range, 1830 years) while recording the blood oxygenation level-dependent (BOLD) responses in an fMRI protocol following 5-day ingestion of 150 mg of cocoa avanols. Participants had to press a key, making either one judgment in the no switch condition (is the letter a vowel or consonant, is the digit even or odd) or two judgments in the switch condition (responding both to the letter and digit). Although an overall BOLD signal increased during the cognitive task, no effects were found in response time, switch cost (the difference in response time between switch and no-switch conditions), and heart rate after consumption of this moderate dose of cocoa avanols. Ingestion of a single acute dose (450 mg of cocoa avanols) yielded an increased cerebral blood ow (Fig. 2), conrming a potential of cocoa avanols for treatment of vascular impairment such as dementia and stroke (see Section 3). Over a 30-day period, Cameld et al. (2012) administered a daily chocolate drink (250 mg or 500 mg cocoa avanols versus lowavanol cocoa as placebo) to 63 volunteers (aged 4065 years). Neurocognitive changes associated with avanol supplementation during performance of a spatial working memory task at baseline

Fig. 2. Increased blood ow response (SEM, standard error of mean) in the gray matter of four female participants following ingestion of an acute dose of high compared to low avanol drink. From Francis et al., 2006. The effect of avanol-rich cocoa on the fMRI response to a cognitive task in healthy young people. Journal of Cardiovascular Pharmacology 47, Suppl. 2, S21520. Copyright 2006 Lippincott Williams & Wilkins with permission of Wolters Kluwer Health/Lippincott Williams & Wilkins.

and at the end of the treatment were assessed using EEG (steady state visually evoked potentials, SSVP). Behavioral measures of accuracy and response time did not differ in a dose-dependent manner, whereas average amplitude and phase of the evoked potentials at a number of posterior parietal and centro-frontal sites signicantly differed between the groups during memory encoding, the memory hold period, and retrieval (Fig. 3). The authors assume the differences in brain activation, even in the absence of behavioral effects, point to an increased neural efciency in spatial working memory associated with chronic cocoa avanol consumption. Table 1 summarizes the results of research on the neuroprotective action of avanols in aging and neurological diseases on the one hand, and the neuromodulatory effects on cognition, mood, learning, and memory on the other hand. As seen from the table, basic animal intervention studies on mechanisms of action of avanols still outnumber human and clinical studies demonstrating that these effects are of physiological and clinical relevance. In summary, recent research has yielded encouraging albeit as yet inconclusive outcome. For exploring possible effects of highavanol cocoa on human behavior, cognitive and brain functions, standardized psychometric tasks and neuroimaging protocols are required along with administering proper high-avanol cocoa dosages. To date, there is still limited evidence for high-avanol cocoa effects on human cognitive and affective function, and behavior. Moreover, it remains unclear how high-avanol cocoa modulates the brain networks underlying neural cognitive processing. 5. Concluding remarks and future directions Flavonoids, potent antioxidant and anti-inammatory agents, represent up to 20% of compounds found in cocoa beans. Flavanols, and especially epicatechin, are the most common cocoa avonoids. The avanol contents in cocoa products and chocolate vary greatly depending on the bean variety and origin, agricultural and processing practices. In part, the variability of avanol contents in cocoa and chocolate may be responsible for the mixed outcomes presently observed in research on the effects of cocoa avanols on neurocognitive and affective function, executive control, and behavior. This might be due to the fact that to date, only few especially, human intervention studies have directly examined exposure to cocoa avonoids, while the most data comes from

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Fig. 3. Topographic differences in the average steady state visually evoked potentials SSVEP amplitudes at baseline and retest for a spatial working memory task (encoding, hold interval, and retrieval) in the low, medium and high cocoa avanol (CF) groups. Warm and cool colors show SSVP amplitude decreases and increases, respectively, post-treatment compared to baseline. (For interpretation of the references to color in the gures throughout the article, please consult the web version of this article.) From Cameld et al., 2012. Steady state visually evoked potential (SSVEP) topography changes associated with cocoa avanol consumption. Physiology and Behavior 105, 94857. Copyright 2011 Elsevier Inc. with permission of Elsevier Ltd.

Table 1 Cocoa avonoid effects on the brain, cognition, and behavior overview. Brain region/function Whole brain Improved cerebral blood ow (general improvement of neural function due to enhanced oxygen and glucose delivery) Hippocampus Neurogenesis Improved neuronal connectivity Angiogenesis Subventricular zone of the brain Neurogenesis Vision Improved blood delivery to the retina Rapid visual processing Visual contrast sensitivity Coherent motion direction Performance Choice reaction time Cognition, learning, and memory; mood General 22 Mental calculation Working memory for location 29 Fear conditioning (anxiety) Calmness, contentedness Aging Reduced cognitive decline Neurological disease Alzheimer (reduced cognitive decline) Stroke (reduced risk) 1, 6 Animal models 15 Human studies 8, 9, 10, 19, 24, 25

15, 26 27 27 6, 26 14

28

11 23 7 7 7 16 23 7 20 13, 16, 18, 25 4, 5 2, 3, 12, 17, 21

Key to references: 1: Bisson et al. (2008), 2: Buijsse et al. (2010), 3: Buitrago-Lopez et al. (2011), 4: Commenges et al. (2000), 5: Desideri et al. (2012), 6: FernndezFernndez et al. (2012), 7: Field et al. (2011), 8: Fisher et al. (2003), 9: Fisher et al. (2006), 10: Francis et al. (2006), 11: Huber et al. (2006), 12: Janszky et al. (2009), 13: Kalmijn et al. (1997), 14: Kalt et al. (2010), 15: Kim et al. (2006), 16: Letenneur et al. (2007), 17: Mink et al. (2007), 18: Nurk et al. (2009), 19: Patel et al. (2008), 20: Pase et al. (2013), 21: Rautiainen et al. (2012), 22: Rozan et al. (2007), 23: Scholey et al. (2010), 24: Schroeter et al. (2006), 25: Sorond et al. (2008), 26: Valente et al. (2009), 27: van Praag et al. (2007), 28: Vauzour et al. (2008), 29: Yamada et al. (2009).

research using dark chocolate as avanol supply. Despite multiplicity of avanol effects in the brain, neurobiological actions of avanols are believed to occur in two major ways: (i) via direct interactions with cellular and molecular signaling cascades, especially in the brain regions dedicated to learning and memory, and (ii) via central and peripheral blood-ow improvement and angiogenesis, including the brain and sensory systems. Overall, evidence on the persistence of neuroprotective and neuromodulatory actions of cocoa avanols, albeit as yet limited and inconclusive, suggests that cocoa avanols may exert both long lasting and immediate effects on neurocognition and behavior. The immediate effects can be attained with a single acute or subchronic (for several weeks) administration of cocoa avanols in appropriate dosages. The lasting effects likely require chronic intake of avanolrich cocoa products over an extended time frame. In both instances, more controlled follow-up studies should be in place to determine the effects duration. While long lasting neuroprotective properties of avonoid intake for neurodegenerative and neuroinammatory diseases of the nervous system have been relatively well documented (Katz et al., 2011; although more research specically on cocoa avanols and greater, e.g. population-based, participant samples is required), more immediate action on cognitive and affective function, executive control, and behavior remains largely unknown, as are sex differences in both long-term and immediate actions (see Sokolov et al., 2013 for a companion review). Sex-related variability in data is likely to account for the lack of signicant effects of cocoa avanols on the brain responses and cognitive performance observed in some of the studies. In females, distinct phases of the menstrual cycle and the perimenopause may also add to data variability. The main issues to resolve in the studies to come include (i) blindfolding both the experimenter and participants, which is important for reducing the well-known reward value of chocolate intake, (ii) determining an appropriate (immediate vs. short-term vs. long-term) time frame and dose of avanol administration, (iii) choice of simple tests of cognitive, executive and affective function and performance that possess sufcient sensitivity and specicity (Macready et al., 2009), and (iv) for examining neuroprotective properties of avanol consumption, the onset and duration of consumption. Another important issue to consider is a so called

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A.N. Sokolov et al. / Neuroscience and Biobehavioral Reviews 37 (2013) 24452453 DeCarli, C., 2012. Cerebrovascular disease: assessing the brain as an end-organ of vascular disease. Nat. Rev. Cardiol. 9, 435436. Desideri, G., Kwik-Uribe, C., Grassi, D., Necozione, S., Ghiadoni, L., Mastroiacovo, D., Raffaele, A., Ferri, L., Bocale, R., Lechiara, M.C., Marini, C., Ferri, C., 2012. Benets in cognitive function, blood pressure, and insulin resistance through cocoa avanol consumption in elderly subjects with mild cognitive impairment: the Cocoa, Cognition, and Aging (CoCoA) study. Hypertension 60, 794801. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2012. Scientic Opinion on the substantiation of a health claim related to cocoa avanols and maintenance of normal endothelium-dependent vasodilation pursuant to Article 13(5) of Regulation (EC) No 1924/2006. EFSA J. 10, 2809. Engler, M.B., Engler, M.M., Chen, C.Y., Malloy, M.J., Browne, A., Chiu, E.Y., Kwak, H.K., Milbury, P., Paul, S.M., Blumberg, J., Mietus-Snyder, M.L., 2004. Flavonoid-rich dark chocolate improves endothelial function and increases plasma epicatechin concentrations in healthy adults. J. Am. Coll. Nutr. 23, 197204. Fernndez-Fernndez, L., Comes, G., Bolea, I., Valente, T., Ruiz, J., Murtra, P., Ramirez, B., Angls, N., Reguant, J., Morell, J.R., Boada, M., Hidalgo, J., Escorihuela, R.M., Unzeta, M., 2012. LMN diet, rich in polyphenols and polyunsaturated fatty acids, improves mouse cognitive decline associated with aging and Alzheimers disease. Behav. Brain Res. 228, 261271. Field, D.T., Williams, C.M., Butler, L.T., 2011. Consumption of cocoa avanols results in an acute improvement in visual and cognitive functions. Physiol. Behav. 103, 255260. Fisher, N.D., Hughes, M., Gerhard-Herman, M., Hollenberg, N.K., 2003. Flavanolrich cocoa induces nitric oxide-dependent vasodilation in healthy humans. J. Hypertens. 21, 22812286. Fisher, N.D., Sorond, F.A., Hollenberg, N.K., 2006. Cocoa avanols and brain perfusion. J. Cardiovasc. Pharmacol. 47 (Suppl. 2), S210S214. Francis, S.T., Head, K., Morris, P.G., Macdonald, I.A., 2006. The effect of avanolrich cocoa on the fMRI response to a cognitive task in healthy young people. J. Cardiovasc. Pharmacol. 47 (Suppl 2), S215S220. Gonzalez-Gallego, J., Sanchez-Campos, S., Tunon, M.J., 2007. Antiinammatory properties of dietary avonoids. Nutr. Hospital. 22, 287293. Goyarzu, P., Malin, D.H., Lau, F.C., Taglialatela, G., Moon, W.D., Jennings, R., Moy, E., Moy, D., Lippold, S., Shukitt-Hale, B., Joseph, J.A., 2004. Blueberry supplemented diet: effects on object recognition memory and nuclear factor-kappa B levels in aged rats. Nutr. Neurosci. 7, 7583. Grassi, D., Desideri, G., Necozione, S., Lippi, C., Casale, R., Properzi, G., Blumberg, J.B., Ferri, C., 2008. Blood pressure is reduced and insulin sensitivity increased in glucose-intolerant, hypertensive subjects after 15 days of consuming highpolyphenol dark chocolate. J. Nutr. 138, 16711676. Grassi, D., Necozione, S., Lippi, C., Croce, G., Valeri, L., Pasqualetti, P., Desideri, G., Blumberg, J.B., Ferri, C., 2005. Cocoa reduces blood pressure and insulin resistance and improves endothelium-dependent vasodilation in hypertensives. Hypertension 46, 398405. Heiss, C., Dejam, A., Kleinbongard, P., Schewe, T., Sies, H., Kelm, M., 2003. Vascular effects of cocoa rich in avan-3-ols. J. Am. Med. Assoc. 290, 10301031. Hirsch, E.C., Hunot, S., Hartmann, A., 2005. Neuroinammatory processes in Parkinsons disease. Parkinson. Relat. Disord. 11 (Suppl. 1), S9S15. Hollenberg, N.K., Fisher, N.D., McCullough, M.L., 2009. Flavanols, the Kuna, cocoa consumption, and nitric oxide. J. Am. Soc. Hypertens. 3, 105112. Hooper, L., Kay, C., Abdelhamid, A., Kroon, P.A., Cohn, J.S., Rimm, E.B., Cassidy, A., 2012. Effects of chocolate, cocoa, and avan-3-ols on cardiovascular health: a systematic review and meta-analysis of randomized trials. Am. J. Clin. Nutr. 95, 740751. Huber, K.K., Adams, H., Remky, A., Arend, K.O., 2006. Retrobulbar haemodynamics and contrast sensitivity improvements after CO2 breathing. Acta Ophthalmol. Scand. 84, 481487. Hurst, W.J., Tarka Jr., S.M., Powis, T.G., Valdez Jr., F., Hester, T.R., 2002. Cacao usage by the earliest Maya civilization. Nature 418, 289290. Jankovic, J., 2008. Are adenosine antagonists, such as istradefylline, caffeine, and chocolate, useful in the treatment of Parkinsons disease? Ann. Neurol. 63, 267269. Janszky, I., Mukamal, K.J., Ljung, R., Ahnve, S., Ahlbom, A., Hallqvist, J., 2009. Chocolate consumption and mortality following a rst acute myocardial infarction: the Stockholm Heart Epidemiology Program. J. Intern. Med. 266, 248257. Kalmijn, S., Feskens, E.J., Launer, L.J., Kromhout, D., 1997. Polyunsaturated fatty acids, antioxidants, and cognitive function in very old men. Am. J. Epidemiol. 145, 3341. Kalt, W., Hanneken, A., Milbury, P., Tremblay, F., 2010. Recent research on polyphenolics in vision and eye health. J. Agric. Food Chem. 58, 40014007. Katz, D.L., Doughty, K., Ali, A., 2011. Cocoa and chocolate in human health and disease. Antioxid. Redox Signal. 15, 27792811. Kay, C.D., Kris-Etherton, P.M., West, S.G., 2006. Effects of antioxidant-rich foods on vascular reactivity: review of the clinical evidence. Curr. Atheroscler. Rep. 8, 510522. Kelleher III, R.J., Govindarajan, A., Jung, H.Y., Kang, H., Tonegawa, S., 2004. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 116, 467479. Kennedy, D.O., Scholey, A.B., Wesnes, K.A., 2002. Modulation of cognition and mood following administration of single doses of Ginkgo biloba, ginseng, and a ginkgo/ginseng combination to healthy young adults. Physiol. Behav. 75, 739751. Kim, D.H., Jeon, S.J., Son, K.H., Jung, J.W., Lee, S., Yoon, B.H., Choi, J.W., Cheong, J.H., Ko, K.H., Ryu, J.H., 2006. Effect of the avonoid, oroxylin A, on transient

food matrix, or food composition, in which cocoa avonoids appear in food (e.g., Lamuela-Ravents et al., 2005; Manach et al., 2004). For example, ingestion of 100 g dark chocolate along with 200 ml milk results in a substantial reduction of both total antioxidant capacity and (-)epicatechin content of human plasma, compared to ingestion of 100 g pure dark chocolate, and the reduction is even greater after ingestion of 200 g milk chocolate (Serani et al., 2003). Finally, other constituents of cocoa and plants rich in avanols, such as their relatively high content of tryptophan, a precursor of neurotransmitter serotonin (e.g., Bertazzo et al., 2011), may either contribute to the neurobiological effects of avanols or, depending on the constituents bioavailability (e.g., due to the food matrix; Smit, 2011), exert different actions through different pathways. Forthcoming studies should explore whether these actions are complementary, antagonistic or synergistic. In conclusion, future research has to combine functional neuroimaging techniques such as fMRI, EEG and MEG with neurocognitive and behavioral correlates to uncover long lasting and immediate effects of chocolate consumption on human cognition, mood, and behavior. Acknowledgements We thank Otmar and Edelgard Knoll and the Willy Robert Pitzer Foundation for support. MAP was supported by the Else Krner Fresenius Foundation (Grants P2010 92 and P2013 127), the Reinhold Beitlich Foundation, the Berthold Leibinger Foundation, and by the Heidehof Foundation. References
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