Cocoa Extract

Cocoa extract (also referred to as cocoa polyphenolics) are derived from cocao seeds as a bitter bulk ingredient for commercial usage and supplementation.^[1] In general, the phrase "cocoa extract" refers to the collection of polyphenols found in dark chocolate which may confer health benefits. Most of the polyphenols in cocoa are flavonoids, specifically the subset known as flavanols,^[1] and so the terms cocoa flavonoids and cocoa flavanols are sometimes used interchangeably with cocoa extract.

Cocoa extract refers to the polyphenolics or 'flavonoids' usually derived from the seeds of the plant which is eventually used to make chocolate. These flavonoids are the molecules which mediate the vast majority of health benefits associated with chocolate consumption.

Cocoa specifically contains (unsweetened cocoa powder unless otherwise specified):

• (-)-epicatechin at 158.30mg/100g^[2] with other sources suggesting a range of approximately 1.5-2.5mg/g.^[3][4] (-)-epicatechin is lower in unsweetened baking chocolate (1-1.2mg/g^[4]), dark chocolate (0.31-0.32mg/g^[4]) semi-sweet baking chips (0.4-0.57mg/g^[4]), milk chocolate (0.02-0.14mg/g^[4]), and chocolate syrup (0.06-0.12mg/g^[4])

• (+)-catechin at 107.75mg/100g^[2] almost always at lesser amounts than the (-)-epicatechin content in all chocolate products^[4]

• Procyanidins of varying chain length (all degrees of polymerization between a dimer and greater than 10 represented, favoring longer chains^[4]) with their quantities correlated to the (-)-epicatechin content, unsweetened cocoa powder having a range of 22-24mg/g^[4] with one source suggesting up to 44mg/g^[3] baking chocolate between 13-16mg/g,^[4] milk chocolate at less than 1mg/g,^[4] and dark chocolate possessing 3-4mg/g total procyanidins^[4]

• (-)-Epicatechin-(2a-7)(4a-8)-epicatechin 3-O-galactoside at 5mg/100g^[2]

• Clovamide at 1.4-2.6mg/kg in cocoa powder from unroasted beans, and 0.6-1.3mg/kg in roasted powder^[5]

• Cinnamtannin A2 at 33.17mg/100g^[2] (catechin tetramer, currently thought to be exclusive to chocolate)^[2]

• Benzoic acid (0.06mg/100g)^[2]

• 3-Methylcatechol, 4-Methylcatechol, and 4-Ethylcatechol (all below 0.1mg/100g)^[2] and catechol at 0.12mg/100g^[2]

• Quercetin (dark chocolate) at 25mg/100g^[2]

• Resveratrol (0.04mg/100g) and its 3,O-glucoside (0.1mg/100g) in dark chocolate^[2]

• Molybdenum at 2.80µg/100g^[6]

• Ferulic acid (dark chocolate) at 24mg/100g^[2]

• Nicotine at 0.2-1.6mcg/kg^[7]

As a general statement, milk chocolate and chocolate syrup have negligible quantities of cocoa bioactives, while standard dark chocolate, dutched chocolate, and semisweet baking chips are comparably good sources of catechins and procyanidins, while unsweetened baking chocolate is better and unsweetened cocoa powder the best dietary source of these bioactives.^[3][4]

Cocoa extract contains polyphenolics^[8] ranging from 8.07 to 484.7mg/g (defatted cocoa powder)^[9], which places it as one of the better dietary sources (alongside select herbs used as spice, dark colored berries, and select vegetables).^[2][8][10] 

In regard to the polyphenolics, cocoa has a large amount of procyanidins (chains of catechin molecules) and a particularly high content of (-)-epicatechin relative to other catechin sources like green tea. Other phenolics such as resveratrol and quercetin seem to be lower than the catechin and procyanidin content and their relevance to cocoa powder is uncertain.

Other components of cocoa extract include:

• Xanthines (caffeine and theobromine)

• Biogenic amines (Phenylethylamine, tyramine)

• Caffeoyl aspartic acid (37mg/100g)^[2]

Cocoa has a low amount of low-weight psychoactive trace amines and xanthines such as caffeine which are not thought to be highly relevant following ingestion of chocolate, excluding potential MAOI interactions due to tyramine.

Cocoa has around a 60/40 epicatechin:catechin ratio due to having higher levels of procyanidin B2 (epicatechin dimer) and procyanidin C1 (epicatechin trimer), with lower relative concentrations of procyanidin B1 (epicatechin-catechin), when compared to other procyanidin sources (Grape seed extract or Pycnogenol).^[11] Natural cocoa products appear to have more total flavonoids than do other dark chocolate or cocoa products, with milk chocolate products having the least amount of flavonoids.^[12]

As a general statement concerning cocoa products and their flavonoid content, processing or manufacturing stages that reduce pure cocoa content will gradually reduce the flavonoid content and total flavonoids due to a reduction in the cocoa percentage of the product. However, some processes reduce the flavonoid content of the powder itself, such as Dutch processing (alkalization) which can reduce the (-)-epicatechin, (+)-catechin, and total flavonoid content by upwards of 60%^[13][14] with losses scaling with processing time.^[15] The conentration of other components like procyanidins and quercetin content are also affected by processing.^[13] Alkalization has been noted to increase the content of (-)-catechin relative to other processing methods,^[14] but this molecule is not normally found in cocoa and is not linked to the health benefits of cocoa products, and is probably an epimerzation product of (-)-epicatechin.^[13]

Processing tends to reduce the flavonoid content of cocoa products. Dutch processing, or alkalization, of cocoa products tends to result in an abnormally rapid loss of the beneficial compounds in cocoa products.

'Dark' chocolate, beyond the color and bitterness (from the xanthine molecules), refers to chocolate products which tend to be 70% cocoa or above by weight and confer a significantly higher concentration of catechins and other bioactives compared to other typically edible forms, although it possesses fewer catechins than cocoa powder and baking chips.^[4]

Dark chocolate is a variant of edible chocolate with a significantly higher cocoa content, and thus more of the beneficial compounds in chocolate, conferring benefits at a much lower oral dose.

A particular brand of cocoa extract known as 'Chocamine', which is patented by RFI, is standardized to theobromine (greater than 12% by weight), and also contains caffeine (less than 0.5%), polyphenolics (greater than 5%) and added tapioca starch and some other spices (ginger, allspice, cinnamon, and vanilla powder in undisclosed amounts) according to their website.^[16]

Chocamine is a patented theobromine-rich cocoa powder.

Nitric oxide is a gasotransmitter involved in relaxing blood vessels, and increasing the activity of this molecule promotes blood flow and in certain instances may also reduce blood pressure.

Cocoa flavanols are known to improve blood flow in a way that is prevented by blocking the endothelial nitric oxide synthase (eNOS) enzyme.^[17] While biomarkers of nitric oxide activity (such as flow-mediated dilation and plasma markers of NO) seem to be increased after flavanol-rich cocoa ingestion,^{[18][19][20][21]} the antioxidant capacity is unaffected^[22][23][24] even in hypertensives.^[25][26] This suggests that flavanols must work by promoting nitric oxide formation but it is not working via antioxidative means (antioxidants such as grape seed extract may preserve nitric oxide availability in instances of a high bodily oxidative state, but such a mechanism would increase antioxidant capacity).^[22][23][24]

The increase NO activity may be traced back to eNOS induction, since (-)-epicatechin in chocolate have been noted to induce eNOS activity in vitro,^[27][28] with isolated (-)-epicatechin being most active at a concentration of 1μM 20-40min after incubation,^[27] similar to the time (-)-epicatechin acts in humans following oral ingestion. This increase in eNOS concentration is associated with increased phosphorylation of Ser-1177 and Ser-633 (dephosphorylation of Thr-495) secondary to calmodulin associated with eNOS due to PI3K activation.^[27] Catechin appears to be 25% as potent as (-)-epicatechin with a mixture of both is less potent than pure epicatechin, which is thought to underlie why cocoa extract may be more potent than other catechin sources such as green tea.^[27]

(-)-epicatechin appears to increase the levels of the eNOS enzyme, which helps make nitric oxide. By increasing the amount of this protein, more nitric oxide is produced, which then act to enhance blood flow.

The activation of eNOS from (-)-epicatechin is known to be calcium-independent^[29][30] (since increasing intracellular calcium can inhernetly activate eNOS^[31]) but may act at an unknown on the cell membrane itself, since anchoring dextran to (-)-epicatechin (to restrict it from entering the cell^[32]) does not prevent its actions in increasing nitric oxide signalling at a low concentration (100-500nM) or from activating PI3K/Akt and PDK1.^[30] Additional evidence for a membrane receptor for (-)-epicatechin comes from the fact that (+)-catechin behaves as a parial agonist in the presence of (-)-epicatechin based on the response curve.^[30] 

Elsewhere, Akt has been noted to associated with heat shock protein 90 (HSP90) to form a complex which then binds with eNOS.^[29]

It should also be noted that (-)-epicatechin can also activate eNOS in a calcium-dependent manner as well as through calcium-independent means.^[33] 

(-)-Epicatechin appears to act at the cell membrane via PI3K to then activates eNOS through heat shock proteins, but the specific receptor that it acts on in the cellular membrane is currently not known.

Incubation of human aortic endothelial cells with a cocoa extract (49% procyanidins) at 2µg/mL resulted in decreases in leukotrienes (LTC₄, LTD₄, and LTE₄) with a doubling of PGI₂.^[34] A similar effect was replicated in vivo through oral ingestion of a chocolate product with a relatively high procyanidin content (0.4%) which acutely increased plasma prostacyclin by 32% and reduce total leukotrienes by 29% relative to a low procyanidin content (0.009%) forumulation; there were also differences in (-)-epicatechin content between the two formulations.^[34]

Changes in eicosonoid levels like those seen above may underlie the anti-asthmatic actions of cocoa extract seen in rodents,^[35] since pharmaceuticals that act similar to prostacyclin (such as iloprost^[36]) and agents that reduce leukotriene activity (like pranlukast and zileuton^[37]) confer anti-asthmatic activity, although the theophylline content of cocoa may also have an minor anti-asthmatic role^[38] although it is underdosed (relative to its maximum efficacy) in cocoa products.

Cocoa polyphenolics (monomers such as (-)-epicatechin as well as procyanidins up to five monomers in length) appear to be structurally stable over the course of one hour while travelling through the acidic medium of the stomach to be absorbed in the intestines.^[39] Administration of famotidine (an H2 receptor antagonist which reduces stomach acidity) does not alter absorption rates of cocoa flavanols.^[40]

The stomach does not appear to destroy or digest active cocoa molecules, so enteric capsules are probably not needed for supplementation.

It is known that the absorption of some dietary polyphenolics can be affected by dietary fats^[41][42] and there may be species differences.^[42] As cocoa polyphenolics are often consumed via food products, their interactions with dairy products has been investigated.

Ingestion of milk (250mL) alongside cocoa polyphenolics (70mg (-)-epicatechin) in healthy human subjects does not appear to reduce absorption when measured after two hours, as the reduction of serum (-)-epicatechin glucuronide from 330+/-150nM in the water control to 273+/-138nM was not significant.^[43] However, a rat study found that whole milk and heavy cream successfully lowered absorption relative to skim milk or water.^[44] Also, another human study which inferred absorption through examining the excretion of metabolites after ingestion of a relatively low-flavanol commercial cocoa powder found that 250mL milk may affect absorption, although the magnitude, suggesting that at lower flavanol concentrations, milk may indeed affect absorption to some degree.^[45]

One study has noted that coingestion of cocoa flavanols (via 125mg/kg cocoa powder) alongside about dietary carbohydrate (about 1g/kg carbohydrate via bread or sugar products) increased their bioavailability and subsequent AUC to 140% of cocoa alone (measured over 2.5 hours) while dietary fats and protein had no significant influence.^[40]

Cocoa that contains low concentrations of flavanols may have their absorption inhibited by milk, while higher concentrations of flavanols seem unaffected. Carbohydrates, on the other hand, may aid in flavanol absorption.

Oral ingestion of drinks (with some additional macronutrients) containing cocoa was noted to increase serum epicatechin when measured at 120 hours post ingestion to approximately 500ng/mL (2g cocoa), 1,200ng/mL (5g), 3,500ng/mL (13g), and 8,000ng/mL (26g) (data inferred from graphs).^[22]

Twice daily dosing of a cocoa drink (450mg flavanols at each dose) has failed to increase steady state catechin concentrations, thought to be related to its short 3.6 hour half-life.^[46]

Consumption of dark chocolate (40 grams) can lead to increased epicatechin in the blood stream two hours after consumption,^[47][48][49] which closely tracks peak improvements in blood flow.^[21] The (-)-epicatechin content appears to reach a concentration of around 118-121nM (0.11-0.12μM) in serum at this time point.^[49] Despite differences in effects between healthy controls and those with prooxidative conditions (hypertension, smoking) there do not appear to be differences in overall serum exposure to (-)-epicatechin.^[48][49] Increasing the flavanol content in solid chocolate products causes dose-dependent increases in serum (-)-epicatechin.^[50]

Chronic consumption of thrice daily dark chocolate ingestion (200 mg flavanols; (-)-epicatechin content not disclosed) resulted in increased serum (-)-epicatechin at week six (78.28+/-64.35ng/mL), nine (46.23+/-41.52ng/mL), and twelve (57.68+/-46.54ng/mL) with very high variability.^[51]

Consumption of low doses of dark chocolate (85% cocoa or greater) appears to result in low micromolar serum concentrations of (-)-epicatechin.

In regard to procyanidins specifically, consumption of chocolate (0.375g/kg) containing 2.8% procyanidins (thus 10.5mg/kg procyanidins) has been noted to increased blood levels of procyanidin B2 to 100nM after two hours (C_max) with approximately 50nM at both the one and four hour marks, and returning to baseline after eight hours.^[52] A study elsewhere using the same chocolate product noted lower serum concentrations of 16+/-5nM after 30 minutes and a peak of 41+/-4nM after two hours.^[53]

Oral ingestion of (-)-epicatechin in mice at 125mg/kg bodyweight for 13 days has been noted to reach brain tissue, reaching a wet weight of 4.3ng/mg (-)-epicatechin and 1.5ng/mg 3'-O-methyl(-)-epicatechin; increasing the oral dose 10-fold resulted in brain concentrations of 7.3ng/mg and 16ng/mg respectively with all aforementioned numbers collectively referring to both free and conjugate forms.^[54] The presence of these two compounds has been confirmed elsewhere in the rat brain with shorter dosing periods.^[55] There is no detectable 4'-O-methyl(-)-epicatechin in the brain of mice following (-)-epicatechin ingestion.^[54] 

Ingestion of catechin from cocoa is subject to methylation to produce either 3'-O-methylcatechin or 4'-O-methylcatechin.^[46]

Consumption of any food deemed palatable is able to increase opioidergic activity via a hypothalamic release of β-endorphin,^[56][57] and one study noted a reduction (lessening) of a negative mood state associated with palatable chocolate but not unpalatable chocolate, which the authors speculate may involve the opioidergic system.^[58]

In a mouse study assessing the actions of (-)-epicatechin (125-750mg/kg) on memory, despite an increase in memory formation being noted there did not appear to be any enhancement of newborn cellular survival in the hippocampus.^[54]

It was noted that (-)-epicatechin increased vascularity of the DG subregion of the hippocampus, with no significant influence on CA1 or CA3 areas and without affecting the overall size of any area.^[54] This effect was traced back to increased spine density in DG granule cells, and a few genes involved in learning were upregulated including synaptosomal-associated protein 25 (SNAP-25^[59]) and kinesin family member 17 (Kif17^[60]) alongside some involved in angiogenesis and some downregulation of inflammatory genes.^[54]

It appears that the scent of dark chocolate (relative to no inhalation of aromatics) is enough to potentially reduce appetite in women.^[61]

In otherwise healthy young adults subject to a cognitive task, five days supplementation of 172mg cocoa flavanols (final dose 90 minutes before testing) increases blood oxygenation level-dependent (BOLD) contrast indicating increased cerebral oxygenation specifically in the prefrontal cortex, anterior cingulate cortex (ACC), and parietal cortex;^[62] but even though increased activity of the ACC is thought to result in increased reaction times,^[63] this effect was not seen in this study.^[62] These changes were accompanied by an increase in cerbral blood flow that peaked two hours after supplementation (40% increase) and returned to baseline within six hours.^[62]

In otherwise healthy youth, cocoa flavanols appear to enhance blood flow to the brain alongside an increase in cerebral oxygenation.

In a cohort of 37,103 Swedish men followed for 10.2 years, a decreased risk of stroke was associated with the highest quartile (25%) of chocolate consumption, with a median intake of 62.9g weekly, having an 0.83 relative risk compared to the no chocolate intake (95% CI: 0.70-0.99).^[64]

Epidemiological research a potential protective effect of cocoa flavanol ingestion against strokes when consumed in the diet.

50g of dark chocolate (125mg (-)-epicatechin) given two hours prior to a psychosocial stressor in otherwise healthy men attenuated the rise of salivary cortisol and adrenaline in a manner correlating with serum (-)-epicatechin relative to placebo chocolate, with no influence on noradrenaline or ACTH.^[65]

Alterations in activity of the enzyme indoleamine 2,3-dioxygenase (IDO) activity has been linked to mood disorders such as seasonal affective disorder^[66] and depression.^[67] Activity of this enzyme could affect mood by mediating the conversion of L-tryptophan to L-kynurenine via the L-kynurenine pathway,^[68] which, while beneficial for pathogen defense, may deplete L-tryptophan and thus reduce the amount available for serotonin biosynthesis.^[69] A possible mechanism for cocoa extract in interacting with mood may be its ability to prevent increases in the activity of IDO during cellular inflammation as seen in vitro with concentrations that can be biologically relevant in the gut;^[70] the gut contains a high proportion of the body's serotonin stores (upwards of 95%^[71]) and, while the concentration of cacao flavanols required to inhibit IDO may be too high for serum activity, it may directly affect the serotonin balance of the gut and thus overall serotonin levels.^[70]

It is possible that cocoa components can exert an antiinflammatory effect on immune cells (macrophages and PMBCs) in the gut which, quite indirectly, could exert a mood elevating state related to serotonin. While this is mechanistically possible, the relevance of this signalling pathway to chocolate's effects on mood has not been demonstrated directly.

Consumption of chocolate alongside water has been noted to reduce (lessen) a negative mood state in response to a film without affecting positive or neutral mood states.^[58] A later experiment noted that this was due to the palatability of chocolate, as a chocolate product that was not deemed palatable failed to replicate the effects.^[58]

Palatability per se is known to influence mood via opioid signalling through a release of β-endorphin from the hypothalamus, and can be mimicked in rats by feeding them sucrose.^[56][57]

Once series of experiments found that chocolate could reduce negative mood states, but this effect was associated solely with how much users like the taste of chocolate, since consumption of sweets that the consumer enjoys can per se improve mood state. It is likely that any treat that the consumer likes can have similar effects, and that the mood-boosting effects were not due to the specific ingredients in cocoa.

In older adults (40-65yrs), supplementation of 500mg cocoa polyphenols for 30 days was associated with an improved mood state mainly around calmness and contentness; 250mg was ineffective.^[72]

One study conducted on elderly people with mild cognitive decline noted that cocoa flavanols were able to improve cognitive performance at both 520mg and 990mg (but not 45mg) daily, as assessed by Trail Making Tests and verbal fluency, although Mini Mental State Examination scores were unaffected.^[73] Elsewhere, 250-500mg cocoa polyphenols for 30 days failed to improve attention (measured by a computerized assessment that measured the power and continuity of attention) in healthy, middle-aged people.^[72]

Preliminary human evidence suggests that high doses of cocoa flavanols my improve cognitive function in the elderly with mild cognitive problems, but does not affect the cognition of healthy people.

A study in mice found that ingestion of (-)-epicatechin at 500μg/g (0.05%; 125mg/kg in reference to body weight) of the diet for 14 days resulted in improvements in memory retention;^[54] a dose-response effect was seen up to 3 times the dose, but no effect was seen at six-fold the oral dose (750mg/kg).^[54]

250-500mg of cocoa flavonols daily for 30 days in otherwise healthy middle-aged adults has failed to improve quality of working memory or secondary memory.^[72] In another study, supplementation of 37g of 60% dark chocolate (400mg procyanidins) with 8 ounces of chocolate beverage containing a similar procyanidin content failed to improve working, moderate, or long-term memory as assessed by a battery of tests.^[74]

In another 3-month randomized and controlled trial, healthy but sedentary 50-69 year olds were assgined to a high-flavanol group who consumed 900mg cocoa flavanols (containing 138mg of (−)-epicatechin) per day and a low-flavanol group (who consumed 10mg cocoa flavanols containing less than 2mg (−)-epicatechin daily).^[75] The high- and low-flavanol groups were further split into two groups: those who exercised aerobically 1 hour a day for 4 days per week, and those who did not.^[75] The study found a 630ms improvement in a computerized test of visual memory recognition in the high-flavanol group compared to the low-flavanol group, with exercise having no significant effect; this improvement corresponded to an increase in dentate gyrus function.^[75]

The human evidence to date concerning cocoa's effects on memory or learning are mixed, with a possible effect on visual memory for high doses of cocoa flavanols taken for 3 months.

In vitro evidence suggests that cocoa flavanols may inhibit fat absorption by inhibiting the activity of two key enzymes in that process.^[76] Cocoa extracts inhibited pancreatic lipase with an IC₅₀ of 47-172.4μg/mL with strength of inhibition proportional to the flavanol content of the type of cocoa used. Phospholipase A₂ was even more sensitive to inhibition and exhibited a similar relationship between potency and flavanolic content, with an IC₅₀ ranging from 8.5-19.7μg/mL, with Dutch-processed cocoa being much less potent, only inhibiting the enzyme by 30% at concentrations up to 200μg/mL.^[76]

In vitro evidence suggeests that the procyanidins found in cocoa may be able to inhibit some fat absorption when coingested, although the practical significance of this information is not known.

(-)-Epicatechin given to mice at 1mg/kg twice daily for 15 days increased mitochondrial proteins of the electron transport chain and two markers of the mitochondrial membrane (porin and mitofilin) above control mice, and both in the presence and absence of exercise.^[77]

Rodent studies suggest benefical effects of (-)-epicatechin ingestion on heart tissue by promoting its energetic capacities at a relatively low human-equivalent dose (0.08mg/kg twice daily).

Epidemiological research suggests an association between higher chocolate intake and lower risk for cardiovascular disease, as well as a protective effect seen in surrogate markers for cardiovascular disease (such as blood pressure).^{[78][79][80][81]}

One study looking at coronary circulation in healthy men given chocolate products for two weeks noted that dark chocolate (550mg polyphenols) reported an increase in coronary flow velocity reserve (CFVR) by 26%, which did not occur in those ingesting white chocolate as a control; this change was independent on changes in oxidation status of the blood or blood pressure.^[82]

An increase in coronary blood flow has been noted following ingestion of dark chocolate in otherwise healthy men.

One study in overweight adults has noted transient increases in blood pressure to a mild degree (4mmHg) when chocolate products are acutely ingested, which do not appear to be due to increases in fasting blood pressure,^[83] although acute ingestion of chocolate was not seen to cause this in pregnant women.^[84] It is possible that the small xanthine content of chocolate (caffeine and its metabolites theobromine and theophylline) accounts for this transient increase secondary to increased cardiac output (known to result from acute ingestion of xanthines^[85]).

Chocolate products have the potential to acutely increase blood pressure, which may be due to the xanthine content rather than the flavanol content. This acute increase does not appear to result in a long-term increase in resting blood pressure.

Various polyphenols from cocoa appear to dose-dependently (2.5-40µM) protect red blood cells from lysis against oxidative stressors, with longer chain procyanidins being more effective than monomers such as catechin at lower concentrations; 1mg/mL of the mixed (acetone) extract slightly outperformed Vitamin C as a reference in vitro.^[86]

Oral ingestion of 100mg of cocoa flavanols in rats (500-666mg/kg) appears to confer protection to red blood cells against AAPH (an oxidative stressor).^[86] A study in humans given chocolate at 0.25, 0.375, and 0.50g/kg bodyweight (2.8% procyanidins and 1.2% monomers) noted that all doses protected red blood cells from free radical-induced hemolysis with the middle dose being most effective.^[52]

Aortic pulse wave velocity (PWV) is a measure used to assess aortic stiffness, a hardening of the aorta from calcification^[87] which is the long-term target of Vitamin K for cardiovascular health^[88] and is a good independent predictor of all-cause mortality at all ages.^[89]

When testing cocoa in otherwise healthy adults on this parameter, 10g of 75% dark chocolate daily for a month has resulted in a decrease in PWV of 5% (6.13+/-0.41m/s to 5.83+/-0.53m/s)^[90] which is an effect not observed with acute usage of a higher dose of cocoa (100 grams) in the same demographic.^[23]

It is possible that prolonged ingestion of cocoa products can result in a reduction in arterial calcification.

In vitro, it appears that cocoa polyphenolics are able to inhibit LDL and vLDL oxidation^[91][92] with similar or lesser potency to a similar concentration of green tea catechins.^[93] Despite the potency in vitro, studies assessing oral intake of cocoa flavanols have noted that diets containing the procyanidins (466mg) have caused only mild increases in the lag time of LDL oxidation by 8%^[94] or have no effect on LDL oxidation at all.^[95][82] Large acute doses of flavanols (1,095mg) have also failed to appreciably influence LDL oxidation rates.^[22]

While epicatechin and the procyanidins found in cocoa can reduce LDL oxidation rates due to their antioxidant properties in vitro, this effect does not appear to occur when cocoa is orally ingested.

Some studies have indicated that cocoa may increase nitric oxide production. Ingestion of dark chocolate (30g of 70% cocoa) in prehypertensive subjects can increase the amount of nitric oxide in serum within 15 days by 54% as assessed by serum biomarkers.^[96] One study has also noted a reduction in vascular arginase activity, the enzyme that degrades arginine, thought to result in an increase in L-arginine availability and hence an greater capacity to synthesize nitric oxide.^[97]

It is unclear if this is related to the antioxidant properties of cocoa (which are thought to be the mechanism by which agents like grape seed extract or vitamin C aid nitric oxide production), as at least one study has noted increased blood flow independent of changes in oxidation of LDL (biomarker of oxidation).^[22]

In regard to blood flow and circulatory health, cocoa flavanols can improve the production of nitric oxide.

One study using oral ingestion of cocoa (450mg polyphenols and 87mg (-)-epicatechin) twice daily for two weeks in hypertensives noted an increase in insulin-induced artery width associated with supplementation^[46] and improvements in blood pressure seen in hypertensives seem to coexist with improvements in insulin sensitivity^[25][26] and β-cell function.^[26] A study that assessed blood vessel diameter under resting conditions without insulin stimulation found no significant interaction between cocoa flavanols and vessel diameter, however.^[98]

In type II diabetics on medication, daily ingestion of dark chocolate (963mg flavanols with 203mg (-)-epicatechin, taken in three divided doses) appears to cause a baseline increase in blood flow by 30%^[21] with further spikes two hours after each oral dose lasting for four hours; this occured without improvements in glycemic control or blood pressure.^[21]

An increase in insulin-mediated vasodilation may also occur and contribute to the effects of chocolate on blood flow.

In otherwise healthy young subjects, ingestion of a flavanol-rich dark chocolate (100g) acutely increased arterial diameter in both resting and hyperemic states resulting in an increase in both flow-mediated vasodilation (FMD; 1.43%) and a decrease in the aortic augmentation index (ALX; 7.8%); these benefits occurred without alterations in plasma antioxidant status,^[23] and elsewhere have been noted to extend to an improvement in coronary flow velocity reserve (CFVR).^[82] These changes can be attributed to the flavanol content of the chocolate, as chocolate with a low flavanol content fails to have benefit.^[95][82] Longer-term ingestion also had an effect in healthy young adults; ingestion of 10 grams of dark chocolate (75% cocoa) daily for a month increasing FMD 9.31% relative to baseline (with no change in control).^[90]

Similar acute effects have been seen in otherwise healthy older individuals as well, where drinks containing 5-26g (but not 2g) cocoa (65-1,095mg total flavanols) were able to increase blood flow 120 minutes post-ingestion as assessed by FMD in a dose-dependent and linear manner; these effects were correlated with serum polyphenolics, namely (-)-epicatechin.^[22] This specific isomer is biologically active^[99] and works by increasing nitric oxide synthase activity ^[27] Oral ingestion of isolated (-)-epicatechin in humans appears to mimick the effects seen with cocoa polyphenols.^[100]

Dark chocolate has elsewhere been noted to be improve vascular function in diabetics,^[21] smokers,^[19][101] and those at risk for cardiovascular disease.^{[102][20][103]} This topic has been subject to meta-analysis assessing the effects on various demographics, which found an average increase in blood flow assessed via FMD reaching 2% (95% CI or 1.6-2.39%) with comparable increases with a single dose (2.25%) or daily dosing for up to 12 weeks (1.76%), but there was slightly more benefit in those with one or more risk factors for cardiovascular disease (CVD) (2.36%) than in healthy people (1.53%).^[104] 

It should be noted that some trials have failed to find improvements in blood flow, specifically in those with high risk of CVD; one which found no effect of flavanol-rich cocoa on those with coronary artery disease,^[105] and one study which, while finding some degree of improved blood flow in hypertensive subjects given 75g dark chocolate daily over a week, found not effect in those with worse Framingham risk score and reactive hyperemeia index.^[103]

Cocoa appears to promote circulation in a dose-dependent manner, which correlates very well with serum (-)-epicatechin both acutely and over a period of time in both healthy and less healthy individuals, although there exists some conflicting evidence regarding the latter population.

One of the mechanisms by which cocoa flavanols may reduce blood pressure is through inhibition of angiotension converting enzyme (ACE), ^[106] although its interactions with nitric oxide are also relevant to blood pressure (being the major mechanism related to blood flow),^[107] potentially related to how an insulin-mediated interaction with nitric oxide can widen blood vessels after cocoa ingestion.^[46]

Cocoa flavanols may reduce blood pressure through two mechanisms: by increasing blood vessel width by increasing nitric oxide and by inhibiting ACE.

Short term studies have noted minor transient increases in blood pressure (alongside an increase in blood flow)^[83][22] or reductions in blood pressure,^[73] while in hypertensives there is similar variability, with one study noting a decrease in ambulatory blood pressure associated with dark chocolate consumption (100 grams for two weeks)^[25] while a similar study administering cocoa beverages to hypertensives failed to find any effect.^[46] But in healthy individuals (young adult soccer players), one study found that consumption of a low dose of flavanols (168mg) via chocolate for one week was associated with minor reductions in systolic (5%), diastolic (7%) and mean (6%) blood pressure relative to cream chocolate control.^[108] 100g of dark chocolate per day for 3 days was also seen to protect against blood pressure increases induced by an oral glucose tolerance test in healthy individuals when compared to a white chocolate control.^[109] When an increase in blood pressure occurs, it appears to be transient and mild (4mmHg^[83]) and does not appear to be associated with an increase in basal or resting blood pressure.^[83]

One meta-analysis of 20 studies lasting up to 18 weeks ^[110] concluded that consumption of cocoa products (usually dark chocolate or cocoa supplementation) is associated with a small 2-3mmHg reduction of systolic blood pressure. An earlier meta-analysis of 13 studies reached a similar conclusion, and noted that cocoa products were most effective in hypertensives and prehypertensives, with no effect on normotensive individuals.^[111]A recent randomized, controlled, double-masked trial further suggested a lack effect on blood pressure in otherwise healthy individuals, where 1000-2000mg/day cocoa flavanols for 6-12 weeks failed to cause significant changes in blood pressure.^[112]

While benefits to blood pressure appear to be more variable than benefits to blood flow, there may be a minor reduction in blood pressure with chronic consumption of cocoa flavanols up to 18 weeks. Longer-term trials have yet to be performed.

(-)-Epicatechin exerts a protective effect on fibrinogen against the free radical known as peroxynitrate (ONOO^-) in vitro at 1-100μM.^[113] This protective effect occurs via (-)-Epicatechin-mediated sequestration of ONOO^-,^[114] which in excess concentrations can damage proteins by modifying their structure. Epicatechin can reduce this modification to proteins in general^[113] and when it does this for fibrinogen, the result is preserved blood clotting.^[115]

In vitro epicatechin can prevent oxidative changes to a major protein involved in blood clotting, which may result in cocoa flavonoids being able to preserve the clotting response; this mechanism is not yet tested in humans.

In otherwise healthy men who consume dark chocolate (50g) two hours prior to a psychosocial test known to promote blood clotting,^[116][117] consumption of the dark chocolate was associated with lower levels of D-dimer (a biomarker of clotting) than white chocolate not containing flavonoids.^[118] This was not related to changes in catecholamines such as adrenaline, which were unaffected.^[118] 

40 grams of dark (85% cocoa) chocolate reduced platelet viscosity in smokers when measured two hours after consumption, an effect accompanied by reduction in oxidation (48% reduction in ROS), the prostaglandin 8-iso-PGF2α (10%), and NOX2 activity (22%),^[49] and unique to smokers as it did not occur in nonsmoking controls;^[49] this may be related to a known higher baseline oxidation and NOX2 activity in smokers.^[49][48] Elsewhere in healthy nonsmoking individuals given 700mg cocoa flavonoids daily for a week^[119] or 900mg total polyphenolics acutely,^[120][121] the platelets removed from subjects appear to be resistant to aggregation when stimulated by clotting factors such as arachidonic acid, ADP, or adrenaline with some differences between the two sexes (although both find overall anticlotting actions).^[122] In contrast, another study examining the effects of cocoa extract on healthy subjects failed to note any differeences in clotting time before or after taking 500 to 2000 mg of cocoa flavanols.^[112] Thus, reports on the effect of cocoa extract on platelet function in healthy individuals are mixed and may depend how platelet function is assessed.

The concentration of (-)-epicatechin required to increased nitric oxide concentration in a platelet, approximately 0.1μM (100nM),^[48] appears to be feasible in the blood following ingestion of 40g dark chocolate.^[48][49] Comparatively speaking, the effects seem to be similar to aspirin in concept although 900mg cocoa polyphenolics (total (-)-epicatechin and procyanidins) is less potent than a baby aspirin (81mg) when both are taken acutely.^[121]

The increased aggregative potential of platelets seen in prooxidative states (studies assessing smokers) seems to be reduced by the ingestion of dark chocolate. Reports on the effects of dark chocolate on platelets and platelet function in otherwise healthy subjects tend to be mixed,however. While cocoa extract may reduce clotting during acute stress in healthy individual, other studies in non-stressed healthy individuals have failed find any difference in clotting time. When present, the anti-clotting effects of cocoa extract are less potent than a baby aspirin.

One meta-analysis looking at interventions using cocoa products of variable time lengths (2-12 weeks) noted that overall there was no significant reduction of triglycerides (95% CI ranging between a reduction of -13.45mg/dL to an increase of 3.32mg/dL);^[123] the studies assessed that had the largest observed reductions^{[108][26][25]} tended to also have nonsignificant results.

The evidence to date suggests that cocoa products do not effect triglyceride levels.

A small meta-analysis on the ingestion of cocoa products for 2 to 12 weeks^[123] assessing five studies on healthy persons^{[74][124][108][95][94]} with the other five being on hypertensive,^[46][25] overweight or obese,^[125][98] or diabetic subjects^[21] found that ingestion of variable doses of cocoa flavanols (88-963mg) or procyanidins (213-754mg) was indicative of a reduction in LDL cholesterol 5.90mg/dL (95% CI of a 1.32-10.47mg/dL reduction) whereas HDL-C and total cholesterol did not appear to be significantly affected overall.^[123]

Cocoa products may reduce LDL levels, but seem to have no effect on HDL and total cholesterol.

A very small study involving 5 patients with diabetes and heart failure found that consuming dark chocolate and a beverage containing approximately 100 mg of (-)-epicatechin daily for 3 months failed to change hemoglobin A1C levels.^[126]

Ingestion of 100g chocolate in otherwise healthy individuals does not appear to increase insulin secretion in response to an oral glucose tolerance test.^[109]

One study using oral ingestion of cocoa (450mg polyphenols and 87mg (-)-epicatechin) twice daily for two weeks in hypertensives noted an increase in insulin-induced artery width associated with supplementation.^[46]

Insulin is known to promote an increase in Nitric Oxide synthesis and release, which then acts to augment insulin-mediated glucose uptake by increasing blood flow to skeletal muscle.^[127][128] Notably, NO-mediated increases in blood flow to skeletal muscle have been noted to be responsible for as much as 40% of increased glucose uptake in response to insulin stimulation.^[129] Due to the ability of (-)-epicatechin in dark chocolate to increase nitric oxide bioavailability, which is known to drive insulin-mediated vasodilation,^[46] it's influence on glucose uptake and insulin signaling has been investigated.

Healthy adults given 100 grams of dark chocolate (500mg total flavanols) daily for 15 days, relative to white chocolate control, have been noted to experience an increase in insulin sensitivity (HOMA, QUICKI, and ISI) when subject to a glucose load; the average insulin sensitivity index (ISI) appeared to be near doubled with dark chocolate (15.18+/-7.69) relative to white chocolate control (7.4+/-3.5).^[124] An improvement in insulin sensitivity (assessed via HOMA2) has also been noted with overweight adults given 451mg flavanols daily for 12 weeks in a manner that did not interact with modest physical exercise.^[98] Longer term supplementation may be required for augmented insulin signaling, however, as three days supplementation of 100g dark chocolate in otherwise healthy adults in one study failed to have an appreciable effect on sensitivity.^[109]

Elsewhere, essential hypertensives given a cocoa beverage with 450mg flavanols (87mg (-)-epicatechin) for two weeks failed to see any improvement in insulin sensitivity during a hyperinsulinemic isoglycemic glucose clamp despite an increase in insulin-mediated vasodilation.^[46] In contrast, the same (-)-epicatechin dose via 100 grams of dark chocolate in a similar population improved insulin sensitivity during an oral glucose tolerance test alongside improvements in blood flow and pressure in both those with unimpaired insulin sensitivity^[25] and insulin resistant hypertensives (1009mg total polyphenolics with 111mg epicatechin daily for 15 days).^[26]

Flavanols in dark chocolate promote increased insulin sensitivity by improving blood flow to skeletal muscle via increased nitric oxide signaling.

Short-term ingestion of dark chocolate (100g of 70% cocoa) in normal weight obese women (women with high body fat but normal BMI) for one week had no effect on circulating interleukins IL-1α, IL-1β, or IL-6 although it did significantly reduce interleukin 1 receptor antagonist (IL-1Ra) concentrations by 33+/-4%.^[130] IL-1Ra is a potent antiinflammatory agent, antagonizing the the actions of IL-1α and IL-1β^[131] which are secreted in high amounts from visceral fat.^[132] Because IL-1Ra is increased in obesity and has also been shown to antagonize leptin signaling at the level of the hypothalamus in rodents^[133] it is considered a possible biomarker for leptin resistance.^[134] Due to a reduction in waist circumference seen in this sample of women,^[130] the IL-1Ra reduction was thought to be indicative of leptin resensitivity rather than a proinflammatory mechanism per se.

Dark chocolate may influence leptin metabolism in subjects with visceral fat, since it has been seen to influence a biomarker of leptin sensitivity, but no studies have currently assessed leptin levels or sensitivity with prolonged cocoa ingestion.

Fat oxidation rates during modest exercise in overweight adults who consumed 451mg cocoa flavanols for 12 weeks (alongside an exercise program) were not affected relative to either exercise without the flavanols, flavanols without exercise, or neither.^[98]

Studies in overweight or obese adults that incorporate dark chocolate into their dietary regimen have failed to note any inherent weight loss effect of 50g dark chocolate (2135mg polyphenolics) over four weeks despite benefits to blood flow.^[135]

Myostatin, a myokine and potent antagonist of growth in skeletal muscle, increases with aging and muscle wasting diseases.^[136] In contrast, follistatin counteracts the growth-limiting effects of myostatin.^[136][137] Thus, the follistatin to myostatin ratio is an important determinant of the anabolic state of muscle tissue, with greater ratios favoring muscle growth.

A pilot study using six middle-aged subjects (41+/-5 years) of average weight given 1mg/kg (-)-epicatechin twice a day for seven days noted that follistatin to myostatin ratio increased by 49.2+/-16.6%.^[138] Importantly, the increase in follistatin to myostatin ratio also correlated with a bilateral increase in hand strength of around 7%.^[138] Moreover, when (-)-epicatechin is fed orally to mice at 2mg/kg daily (in two divided doses of 1mg/kg) for two weeks, the 18% increase in myostatin and 30% decrease in follistatin which normally occur with aging were prevented. Young mice given the same dose of (-)-epicatechin showed a 15% reduction of myostatin with no influence on follistatin.^[138]

The (-)-epicatechin found in dark chocolate has been shown to promote muscle anabolism in both aging mice and middle-aged humans by increasing follistatin to myostatin ratio in skeletal muscle. This suggests that cocoa extract supplementation may be helpful for conditions associated with muscle loss, such as during aging (sarcopenia) and muscle wasting diseases.

Cocoa powder (6.43% (-)-epicatechin content and 3.54% procyanidin B2) at 50mg/kg daily in mice for two weeks has been noted to reduce blood glucose, which was thought to be secondary to an increase in fat oxidation in and mitochondrial biogenesis in skeletal muscle.^[139] This hypothesis was supported by an increase in resting energy expenditure (RER) as well as increases in the expression of CPT2 in skeletal muscle and UCP1 in brown adipose tissue. This occured independent of any changes in locomotor activity.^[139] 

Enhanced mitochondrial bioenergetics were also noted in a preliminary study of five humans with impaired mitochondrial function (type II diabetic and with heart failure) who were given 100 mg (-)-epicatechin daily for three months.^[126] An improvement of mitochondria cristae has been noted both in mice with poor oxidative metabolism in skeletal muscle^[140] as well as the aforementioned human study.^[126]

(-)-Epicatechin in cocoa powder has been shown to enhance oxidative metabolism and mitochondrial function in addition to promoting mitochondrial biogenesis in animal models. Preliminary evidence suggests this may also occur in humans.

Consumption of 100g dark chocolate (70% cocoa) has been noted to nonsignificantly increase plasma non-esterified fatty acids (NEFA) when consumed two hours prior to cycling exercise relative to milk chocolate.^[141] Serum free fatty acids also increased when 40g was consumed before a 90 minute cycle,^[142] with no observable changes in total triglyceride content or exercise performance.^[142] 

It is known that consumption of fatty acids per se can increase total free fatty acids in serum during exercise even without changes in performance^[143] although the differing effects between dark chocolate and cocoa-free control with a similar fatty acid profile^[142] suggest a role for the catechins.

Dark chocolate may enhance fatty acid mobilization, although it has not been shown to increase exercise performance.

Oral ingestion of 1mg/kg (-)-epicatechin twice daily in one year-old mice (C57BL/6N) for 15 days alongside exercise caused improvements in duration and distance until failure relative to exercise alone.^[77] (-)-Epicatechin at this dose without exercise failed to have any effect when compared to control.^[77] Since physical performance tends to decrease in mice over one year in age,^[144] these results suggest that (-)-epicatechin may have some potential to delay physical decline during aging when combined with exercise. In rats that are bred for low endurance performance (LCR rats^[145][146] with defects in aerobic metabolism^[147]) the same dose of (-)-epicatechin for a month increased mitochondrial cristae, angiogenesis, and mitochondrial biogenesis which are thought to underlie improvements in physical performance (although this was not directly measured in the study^[140]). This study also noted a 40% increase in expression of VEGF-A, an angiogenic factor, which was normalized 15 days after (-)-epicatechin cessation.^[140] 

(-)-epicatechin is further thought to be relevant to glycolytic muscle (such as the plantaris muscle in rats) since this muscle type differs in these rats selectively bred for low performance relative to those bred for high performance.^[148] One study in mice specifically looking at detraining noted that administration of 1mg/kg twice daily (-)-epicatechin for 15 days of rest in trained mice prevented improvements in exercise performance from being lost during prolonged rest. This was associated with preservation of exercise-induced increases in mitochondrial complex III and IV levels normally lost during periods of reduced activity.^[149]

In soccer players, consumption of chocolate (containing 168mg flavanols; 39mg (-)-epicatechin) once daily for a week was associated with improvements in various measures of oxidative stress, although this failed to influence their performance via self-report during the week-long intervention.^[108]

(-)-Epicatechin at a dose where it improved endurance performance in mice after 15 days (1mg/kg twice daily) failed to have any influence on force output or acute contractility (when tested ex vivo).^[77] In spite of this, mice were resistant to fatigue from repeated contractions in this study.^[77] 

In another study in sedentary middle aged adults (42 years of age) 1mg/kg (-)-epicatechin twice daily increased grip strength by 7% relative to baseline, although this was not compared to a placebo control.^[138]

Dark chocolate (100g of 70% cocoa) given to otherwise healthy men acutely before exercise caused an increase in plasma (-)-epicatechin that was associated with an increase in blood total antioxidant capacity immediately before and after exercise. This failed to affect IL-6 concentrations or oxidative burst in neutrophils, however.^[141]

In a study assessing the effects of cocoa extract on vascular function in overweight men, 70g high flavanol chocolate (1078mg flavanols containing 349mg (-)-epicatechin) was compared to 70g of standard dark chocolate (58% cocoa containing 259mg flavanols and 97mg (-)-epicatechin). While standard cocoa dark chocolate caused a slight acute increase sICAM-1 levels, both high flavanol chocolate and standard dark chocolate decreased sICAM-1 and sICAM-3 levels after four weeks of supplementation when measured in a fasted state or after a high-fat meal.^[50] These changes are consistent with lowered risk of cardiovascular disease due to a decreased risk of atherosclerotic plaque formation.^[150]

Four weeks dark chocolate consumption has been shown in one study to reduce leukocyte adhesion factor levels, which could help to delay or even prevent the formation atherosclerotic plaques in blood vessels.

Cocoa procyanidins do not affect IL-5 secretion in PMBCs at rest, but do augment IL-5 increases in PHA-stimulated PBMCs.^[151]

Cocoa extracts have been noted in vitro to downregulate inflammatory cytokines from macrophages including MCP-1, TNF-α, and IL-6 with a potency greater than a similar concentration of pure (-)-epicatechin^[152] (with similar antiinflammatory effects noted in whole blood^[153]). Other studies have noted the isolated cocoa constituent clovamide has similar effects in LPS-stimulated macrophages.^[154] In the absence of LPS or other pro-inflammatory stimuli, procyanidins from cocoa have been noted to possess the ability to increase the secretion of IL-1, IL-6, and TNF-α in PMBCs,^[155][156] where the longer chain flavanols seem to be more potent.^[156]

It has been suggested^[157] that cocoa constituents may suppress macrophage activity via their intrinsic antioxidant action, since inflammatory pathways tend to be REDOX-sensitive, and antioxidants in general have been shown to suppress the activation of macrophages. Indeed, NF-kB, which plays a prominent role in macrophage activation, is suppressed by antioxidants.^[158] Moreover, (-)-epicatechin has been shown to suppress NF-kB.^[153].

(-)-epicatechin and other flavanols in cocoa may limit macrophage activation via their intrinsic antioxidant activity.

The indoleamine 2,3-dioxygenase (IDO) enzyme mediates the breakdown of L-tryptophan into L-kyurenine and various metabolites (known as the kynurenine pathway^[68]), and is induced by IFN-γ in T cells (as well as macrophages, but less so in B cells and HUVECs^[159][160]) during inflammation and appears to have a role in viral and bacterial infections.^[161][162] IDO is also thought to be a potential link between depression and inflammation as it hypothetically depletes L-tryptophan stores which could have otherwise been used to synthesize serotonin.^[69] Moreover, IDO activity (assessed by neopterin as a proxy measure) is thought to increase with oxidation as the two are correlated in immune cells^[163] and in vivo.^[164]

In regards to cocoa extract, the degradation of L-tryptophan via IDO in PMBCs was almost completely suppressed at a concentration of 5µg/mL cocoa extract in the context of mitogen activation. Monocytic THP-1 cells were not affected.^[70] Notably, IFN-γ production was suppressed by cacao in PHA-stimulated PBMCs. Because IFN-gamma is a cytokine released by T-cells, and monocytic THP-1 cells were not affected,^[70] this indicated that IDO-mediated degradation of L-tryptophan occurred in T-cells.

Many of the same immunological changes that cause symptoms of allergies (watery eyes, sneezing, etc) cause allergic asthma, the most common type of asthma disorder.^[165] In a study designed to evaluate the effects of unsweetened cocoa on allergic asthma, guinea pigs sensitized to an antigen (ovalbumin) were given cocoa at one of two doses (300 of 600mg/kg) for 35 days with the final dose an hour before antigen challenge. Notably, cocoa supplementation demonstrated a dose-dependent anti-asthmatic action relative to water control.^[35]

Alhough anti-retroviral therapy has shown some efficacy in limiting the neurological symptoms of HIV infection,^[166] conventional therapies have failed to completely eliminate the cognitive disorders associated with this disease.^[167] Two HIV proteins in particular, gp120 and Tat, are known to cause mitochondrial dysfunction that leads to overproduction of ROS, a major cause neurodegenerative disease and cognitive disorders.^[168][166]

Given the connection between HIV and oxidative-stress driven neuronal dysfunction, a group of investigators designed a high throughput in vitro assay to screen for compounds that might suppress oxidative stress caused by the HIV proteins gp120 and Tat.^[169] This assay revealed that molecules with a structure similar to epicatechin found in chocolate (also one of the main four Green Tea Catechins) normalized increases in apoptosis-inducing proBDNF and decreases in BDNF induced by the HIV Tat protein. This resulted in neuroprotective effects against HIV with greater potency than Resveratrol.^[169]

In vitro studies have shown that epicatechin, a flavanol found in chocolate, has potential to limit neuronal dysfunction associated with HIV infection. While promising, more studies are needed to determine if this could be used to help HIV patients.

The antiinflammatory effects of cortisol are reduced by oxidative stress, and the antioxidant abilities of (-)-epicatechin (1-50µM) can preserve the actions of cortisol in isolated monocytes.^[170]

A diet high in cocoa flavanols (494mg with 89mg epicatechin and 21mg catechin) daily for four weeks has been noted to increase the bacterial count of bifidobacterial, enterococcus, and lactobacilli strains in the intestines while decreasing clostridia compared to a low-flavanol control as assessed by fecal examination .^[171] This study also noted decreases in blood pressure and C-reactive protein, with the latter correlated to changes in lactobacilli.^[171] The suppression of Clostridium histolyticum noted with cocoa flavanols has also been observed with isolated (+)-catechin^[172] and other green tea catechins.^[173]

In subjects with liver cirrhosis, eating a meal can result in low blood pressure due to a phenomenon known as the hepatic venous pressure gradient (HPVG); due to hypertension in the liver, more blood is redirected to the liver to accommodate food intake resulting in a reduction in peripheral blood pressure.^[174][175] This is likely due to decreased nitric oxide bioavailability in the liver and is exacerbated by excess oxidation, with both nitric oxide donors^[176] and vitamin C (due to its antioxidant properties) being therapeutic.^[177] 

In those with portal hypertension due to cirrhosis, a liquid meal containing dark chocolate (85% cocoa) was associated with less of a post-meal increase in the HPVG relative to a meal without flavonoids.^[178] This was thought to be related to improve liver circulation, although no differences in portal vein blood flow, hepatic artery blood flow, or total blood flow were noted between groups.^[178] In spite of this, the peripheral gradient was about halved with dark chocolate and peripheral arterial pressure increased without changes in heart rate.^[178]

The post meal reduction in blood pressure that subjects with cirrhosis experience can be attenuated by ingestion of dark chocolate products containing polyphenols.

In diabetic rats, injections of 15-30mg/kg (-)-epicatechin for 35 days appeared to dose-dependently attenuate lipid peroxidation relative to diabetic control; there was no effect of (-)-epicatechin on rats without diabetes.^[179] These effects were also associated with a preservation of superoxide dismutase (SOD) activity.^[179]

In otherwise healthy subjects given 1g/kg dark chocolate (70% cocoa) and measured two hours later, supplementation increased the oxygenation of medullary tissue in the kidneys compared to the control of white chocolate; this effect was correlated with the serum (-)-epicatechin content.^[180]

Acute ingestion of 40g dark chocolate in glaucoma patients and normal controls failed to influence blood pressure and intraocular pressure (IOP) any more than white chocolate control.^[181] While there appeared to be increased venule dilation in response to a flicker test, this was only present in controls (without glaucoma) given dark chocolate.^[181]

Oral administration of 1mg/kg (-)-epicatechin to aged mice appears to attenuate some of the age-related changes in antioxidant proteins and some proteins involved in mitochondrial biogenesis (sirtuins). The senescence-associated biomarker β-galactosidase was also reduced compared to aged control.^[182]

In otherwise healthy women, ten grams of chocolate with a flavanol content of 200mg thrice daily for twelve weeks failed to enhance photoprotection as assessed by minimum erythema dose (MED) compared to a control chocolate with low flavanol content.^[51] The same study found a slightly increased skin elasticity on the temple, but not arms, relative to control chocolate. There were no observed changes in skin hydration status between groups, however.^[51]

Although 12 weeks cocoa flavanol supplementation failed to have an effect on photoprotection in the aforementioned study, evidence has been mixed. An earlier study using an increased dosage of flavanols noted significant improvements in photoprotection. In this double-blinded 12 week study on 24 female subjects, MED was first established at baseline, to determine how much UV radiation would be used for each individual. Subjects were then given either a high cocoa flavanol drink (326mg per day flavanols) or a low flavanol drink (27mg/day cocoa flavanols) for the duration of the study. In the high flavanol group, UV-induced skin damage (1.25x MED) was reduced by 15% and 25% after 6 weeks and 12 weeks respective supplementation. High flavanol cocoa also increased blood flow to skin and subcutaneous tissues and increased skin hydration.^[183] 

A more recent double-blind, placebo controlled trial further suggests that cocoa flavanols may have a protective effect on skin. In this study moderately photo-aged Korean women with visible facial wrinkles were given a placebo or 320mg cocoa flavanols/day. After 24 weeks supplementation, the cocoa flavanol group had significantly smoother skin (as assessed in the study by ‘roughness value’) and improved skin elasticity, suggesting that long term cocoa flavanol supplementation may help to reduce photoaging.^[184]

There are mixed reports that cocoa flavanols may confer a degree of protection from skin photodamage in response to UV exposure. While two studies have shown that cocoa flavanol supplementation in the range of 300-326mg over a 6-24 week time period may have a modest protective effect against UV-induced skin damage, another study using high flavanol chocolate (providing 600mg cocoa flavanols) failed to note an effect. More research is needed to determine which extraction methods may yield cocoa flavanols with photoprotective properties.

In adult men with a history of acne given either chocolate (in the form of unsweetened capsules) or a visually identical placebo in varying doses (1-6oz) for one week, the number of lesions relative to placebo was increased.^[185] This effect may be limited to the acne prone, as a pilot study using 39g chocolate bars over a week only noted a third of subjects increased lesion count^[186] with another pilot study using higher doses (340g or 12oz of milk chocolate) over week in acne prone men noting a similar 4-7 day time course for exacerbation of acne lesions.^[187]

NOX-2 is a catalytic subunit of NADPH oxidase, which produces free radicals and can cause a reduction of NO signalling as the oxidation promotes conversion nitric oxide into peroxynitrate, reducing nitric oxide (NO) availability.^[188] Dark chocolate is known to suppress NOX2 activity and increase blood flow in instances of low blood flow associated with oxidation,^[48][49] which also characterizes periperal artery disease (PAD).^[189] A mixture of catechins (but not the catechins on their own) from cocoa extract has also been moted to increase NO by reducing the activity of NOX-2 in vitro, suggesting possible therapeutic potential for patients with PAD.^[47]

This hypothesis was tested in people diagnosed with PAD; ingestion of a single dose of dark chocolate (40 grams of 85% cocoa) but not milk chocolate (40g of 35% cocoa) resulted in an increase in serum epicatechin and other catechins, which was thought to underlie the benefits seen in walking distance (11% increase) and walking time (15% increase) along with increases in serum NO as measured by the nitrite/nitrate ratio.^[47]

In contrast, ingestion of 50 grams of dark chocolate in PAD sufferers without a walking test has failed to influence blood flow at rest and failed to influence any measured circulatory/microcirculatory parameters relative to baseline. White chocolate, which was used as a control, is this study, was also ineffective.^[190]

Dark chocolate is known to acutely benefit any condition where blood flow is impaired alongside elevated oxidation, including peripheral artery disease. Human experiments testing the effects of dark chocolate on those with PAD have had mixed results, however.

It is known that aging is related to a reduction in blood flow to the brain.^[191][192] This may explain in part the associations between dietary flavonoids and reduced risk of cognitive decline seen in both epidemiological research on dementia^[193] and rodent interventions and neural susceptability to damage.^[194] The potential benefit of flavonoids is similar to what is hypothesized for dietary nitrate intake; by increasing cerebral blood flow (-)-epicatechin and the dark chocolate it is derived from^[62] hold promise as a therapeutic to protect against dementia.^[195] At least two of the brain regions to which (-)-epicatechin increases blood flow, the prefrontal and parietal cortices,^[62] exhibit reduced blood during Alzheimer's disease.^[196][197] The hypothesis that cocoa flavonoids protect against Alzheimer's disease in humans, however, has not been directly tested to date.

An in vitro study with human umbilical vascular endothelial cells stimulated with endothelial growth factor found that a combination of (-)-epicatechin, EGCG, and catechin at concetrations of 0.1-10μM (similar to the levels found in the serum after ingesting 40 grams of dark chocolate) lead to increased NO generation, whereas no effect was observed when each was used on its own. This is suggestive of additive or synergistic effects of catechin constituents in chocolate relative to (-)-epicatechin alone.^[47]