Scientists may have discovered more reasons to love chocolate.
A new study by researchers at the University of Illinois suggests that three of the phenolic compounds in cocoa bean shells have powerful effects on the fat and immune cells in mice, potentially reversing the chronic inflammation and insulin resistance associated with obesity.
Visiting scholar in food science Miguel Rebollo-Hernanz and Elvira Gonzalez de Mejia, a professor in the department, found that cocoa shells contain high levels of three beneficial bioactive chemicals also found in cocoa, coffee and green tea – protocatechuic acid, epicatechin and procyanidin B2.
Rebollo-Hernanz, the study’s lead author, created a water-based extract containing these compounds and tested its effects on white fat cells called adipocytes and immune cells called macrophages.
Using computer modeling and bioinformatic techniques, he also examined the impact that each of the phenolics individually had on the cells.
“The objectives of the study were to test whether the bioactive compounds in the cocoa shells were efficacious against macrophages – the inflammatory cells – at eliminating or reducing the biomarkers of inflammation,” said de Mejia, also a director of nutritional sciences.
“We wanted to see if the phenolics in the extract blocked or reduced the damage to fat cells’ mitochondria and prevented insulin resistance.”
Similar to batteries within cells that burn fat and glucose to generate energy, mitochondria can become damaged when high levels of fat, glucose and inflammation occur in the body, de Mejia said.
When the scientists treated adipocytes with the aqueous extract or the three phenolic compounds individually, damaged mitochondria in the cells were repaired and less fat accumulated in the adipocytes, blocking inflammation and restoring the cells’ insulin sensitivity, Rebollo-Hernanz said.
The scientists reported their findings recently in a paper published in the journal Molecular Nutrition and Food Research.
When adipocytes accumulate too much fat, they promote the growth of macrophages.
This initiates a toxic cycle in which the adipocytes and macrophages interact, emitting toxins that inflame fat tissue, de Mejia said.
Over time, this chronic inflammation impairs cells’ ability to take up glucose, leading to insulin resistance and possibly type 2 diabetes as glucose levels in the blood escalate.
To recreate the inflammatory process that occurs in the body when macrophages and adipocytes begin their toxic dance, Rebollo-Hernanz grew adipocytes in a solution in which macrophages had been cultured.
“That’s when we observed that these inflammatory conditions in the solution increased the oxidative damage” to the fat cells’ mitochondria, he said.
Fewer mitochondria were present in the adipocytes that were grown in the solution, and the mitochondria that did exist in these cells were damaged, he found.
When the scientists treated the adipocytes with the phenolics in the extract, however, the adipocytes underwent a process called browning, in which they differentiated – that is, converted – from white adipocytes into another form called beige adipocytes.
Beige adipocytes are a specialized form of fat tissue with greater numbers of mitochondria and enhanced fat-burning efficiency.
“We observed that the extract was able to maintain the mitochondria and their function, modulating the inflammatory process and maintaining the adipocytes’ sensitivity to insulin,” Rebollo-Hernanz said.
“Assuming that these phenolics were the main actors in this extract, we can say that consuming them could prevent mitochondrial dysfunction in adipose tissue.”
Cocoa shells are a waste byproduct that’s generated when cocoa beans are roasted during chocolate production.
About 700,000 tons of the shells are discarded annually, causing environmental contamination if not disposed of responsibly, de Mejia said.
In addition to providing cocoa producers with another potential revenue stream, processing the shells to extract the nutrients would reduce the environmental toxicants generated currently by cocoa shell waste, de Mejia said.
Once extracted from cocoa bean shells, the phenolic compounds could be added to foods or beverages to boost products’ nutritional value, she said.
he three main cultivars of cacao beans are: Criollo, Forastero and Trinitario.
However, with the aim of increasing production and resistance to pests, new varieties have been created from the three original cultivars.
Forastero is the most widespread (mainly in Africa) and used around the world, due to its high adaptability and resistance to pests. Criollo is native from Mexico, Central and South America.
It is considered the most ancient cultivar and is appreciated for its high quality, flavor and aroma.
However, its production represents only 5% of the world cacao beans production, due to its low resistance to pests. Trinitario is a hybrid between the Criollo and Forastero trees that combines good-quality flavor and aroma with pest and disease resistance [1,2].
The cacao bean is a fruit widely recognized as one of the main sources of phenolic compounds with the highest flavanols content of all foods on a per weight basis 
The content and profile of bioactive compounds of the cocoa depends on a number of factors such as type and quality of the crop, place of culture, type of process (fermentation, drying and roasting), such that in 6 samples of cocoa liquor around the world, the highest to least quantity of the following components: (-)-epicatechin is presented in samples from Ghana, followed by Mexico and Venezuela; (+)-catechin from Sao Tome and Ghana; caffeic acid derivatives from Venezuela, Ecuador and Ghana; (-)-gallocatechin (GC) from Ecuador, Madagascar and Mexico, without presence in the rest of the samples; (-) – epigallocatechin (EGC) from Madagascar, Ecuador and Sao Tome without the presence of these compounds in Mexico and Venezuela; caffeine from Ecuador, Venezuela and Mexico; and theobromine from Sao Tome, Ghana and Madagascar . About 13 flavanols have been detected and quantified, mainly (-)–epicatechin (0.12–2.83 mg/g), (+)–catechin (0.040-0.090 mg/g), epigallocatechin, epigallocatechin-3-gallate and procyanidins B1(0.035 mg/g), B2 with three different isomers (0.13–0.97 mg/g), B3, B4 with 2 isomers, C1 and D; 7 flavones—luteolin orientin, isoorientin, apigenin, vitexin, ixovitexin; 4 flavanones—naringenin, prunin, hesperidin, eriodyctyol; 4 flavonols—quercetin (0.21–3.25 µg/g), quercetin 3-o-arabinoside (2.1–3.2 µg/g), isoquercitrin (4–4.3 µg/g), and hyperoside; 4 anthocyanidins—cyanidin, and 3 different glycosylated cyanidins; and finally, 8 fenolic acids—vanillic acid, syringic acid, chlorogenic acid, phlorectic acid, coumaric acid, caffeic acid, ferulic acid and phenilacetic acid .
Epidemiological and clinical studies show and confirm that regular intake of cocoa powder and/or dark chocolate (50%–70% cacao) is related to a decrease in systolic blood pressure (SBP) (−3.2 to −5.88 mmHg) and diastolic blood pressure (DBP) (−2.0 to −3.30 mmHg), as well as to an improvement of the vascular endothelial function (measured as a function of endothelium vasodilatation and an increase in the production of nitric oxide) in groups with some type of cardiovascular disease or with multiple risk factors [5,6,7].
Cocoa flavanols reduce the blood pressure by increasing the availability of nitric oxide (increasing the nitric oxide synthase activity and reducing the oxidative stress), consequently vasodilatation increases and finally blood pressure is reduced; or, by inhibition of angiotensin-converting enzyme, interrupting the chain of reactions of angiotensinogen that by the action of renin produces angiotensin I which in turn by action of angiotensin converting enzyme (ACE) is transformed into angiotensin II and finally increases blood pressure .
Metabolic syndrome (MS) is a heterogeneous group of correlated metabolic disorders that occur together and raise the risk for diabetes type II (DM2) and cardiovascular diseases with high rates of morbi-mortality [9,10,11].
To diagnose MS, the International Diabetes Federation (IDF) considers the presence of abdominal obesity (defined as waist circumference with ethnicity specific values) as the main risk factor, plus two additional symptoms: i) high blood pressure (SBP: ≥10 mmHg; DBP: ≥85 mmHg or a specific treatment for hypertension arterial (HTA)); ii) high plasmatic triglycerides (≥150 mg/dL or a treatment specific for this disorder); iii) low HDL-cholesterol (M: <40 mg/dL; W: <50 mg/dL); iv) impaired fasting glycemia (≥100 mg/dL) or diabetes mellitus type 2 (DM2) diagnosis .
Several clinical and epidemiological studies have demonstrated that the intake of flavonoids found in vegetables, fruits and oilseeds reduce the risk of developing several types of non-communicable chronic diseases derived from metabolic disorders [13,14].
Among the vast group of flavonoids found in nature, flavanols are a sub-group of particular interest since it has shown multiple protective effects against diseases associated with metabolic and oxidative stress .
More than 200 chemical compounds have been identified in cacao beans, and most of them are stored in the vacuoles of the so-called “polyphenolic cells” .
The polyphenol content makes up about 12%–18% of the whole bean’s dry weight. Approximately 60% of the total polyphenols content in non-fermented cacao beans corresponds to monomeric (catechin and epicatechin) and oligomeric flavanols.
The main monomeric flavanol is (−)-epicatechin (with up to 35% of polyphenol content), followed by (+)-catechin and procyanidin B2 (epicatechin-(4β-8)-epicatechin) .
Flavanols are secondary metabolites that belong to a sub-class of a larger group of plant compounds known as flavonoids.
They share a general chemical structure that includes two rings (A and B) linked through three carbons that form an oxygenated heterocyclic ring (C). As a particular feature, flavanols have multiple hydroxyl groups on the A, B and C rings that have been associated with a decrease in oxidative stress markers. As with all bioactive substances, flavanols and procyanidins mechanisms are largely dependent on their bioavailability at their target tissue [17,18].
Upon ingestion, flavanols bioavailability depends on their absorption, metabolism at the gastrointestinal tract, tissue and cellular distribution, and tissue metabolism.
In vitro study showed that procyanidins are hydrolyzed into oligomers when passing through the gastric lumen, due to the high acidity of the medium (pH 2) .
Given the above, it was hypothesized that gastric de-polymerization of procyanidins favors their absorption by the small intestine.
While the gastric concentrations of epicatechin and procyanidins B2, B5 (dimeric) and C1 (trimeric) did not significantly change over the stomach transit period .
Depolymerization of procyanidins (composed mainly by epicatechin monomers) would have resulted in an increase in the epicatechin gastric concentrations and a change in the ratios of oligomers to epicathechin and catechin to epicatechin.
However, neither hydrolysis of procyanidins nor change in the aforementioned ratios were observed .
Once monomeric and oligomeric flavanols reach the small intestine, they can undergo a series of biotransformations (mainly of phase II) that produce O-methylated, O-sulfated, and O-glucuronidated metabolites, which can be absorbed into the blood stream.
Procyanidins with a high degree of polymerization cannot be absorbed in the small intestine, so they reach the colon to be used for microbial catabolism, which leads to the formation of smaller phenolic compounds capable of reaching the liver and then undergo phase II conjugation [24,25].
This has been demonstrated in previous in vivo absorption kinetic studies, in which the presence of these metabolites was observed in the plasma (in concentrations of macro to nanomoles) 30 to 60 minutes after the ingestion of cocoa-based beverages [16,20,21].
The monomers epicatechin and catechin showed the highest absorption rates (22%–55%), while dimeric and trimeric procyanidins were less absorbed (equal or less than 0.5%) . Using liquid chromatography-mass spectrometry, total concentrations of (-)-epicatechin, (+)-catechin and procyanidin B2 were quantified after 30 and 120 minutes after the intake of 0.375 g of cocoa/kg of body weight in healthy subjects (average intake of 26.4 g of cocoa: 323 mg monomers; 256 mg dimers). At both times, it was observed that the plasma epicatechin levels were higher than those of procyanidin B2 (0.5 h: 2.61 ± 0.46 μmol/L as compared to 16 ± 5 nmol/L and 2 h: 5.92 ± 0.60 μmol/L as compared to 41 ± 4 nmol/L, respectively) .
Table 1 shows the results of different studies on the bioavailability of pure flavanol compounds (catechin, epicatechin and procyanidin B2). As observed, the maximum plasma concentration is reached about 1 h after administration and is dose-dependent
Regarding (-)-epicatechin, their methylated and non-methylated metabolites were produced almost in the same proportion.
On the other hand, catechin produces three more times non-methylated than methylated metabolites.
When catechin and epicatechin are given as a mix, their metabolite profile remains similar to the one observed when both compounds are administered alone. After the oral administration of (+)-catechin, (−)-epicatechin and a mixture of the two, it can be shown that (-)-epicatechin is the flavanol with the highest absorption, even though (+)-catechin is also a monomer and its plasma metabolites are similar to the ones produced by (-)-epicatechin (glucuronides, sulfates and sulfoglucuronides) . Similar results have been reported in bioavailability studies of epicatechin and procyanidin B2, when administered individually.
The absorption of epicatechin was evaluated after oral administration of different doses of cocoa powder or the pure compound. Results showed that bioavailability of (-)-epicatechin present in cocoa powder was absorbed as efficiently as (-)-epicatechin administered alone .
A study in healthy volunteers analyzed the postprandial profile of (-)-epicatechin plasma metabolite profiles after oral consumption of a cocoa beverage.
Through the combination of different enzymatic hydrolysis (arylsulfatase and β-glucuronidase) and the use of de novo chemically synthesized reference standards, it was possible to identify and measure 8 circulating postprandial (-)-epicatechin metabolites. As reported in previous studies, (-)-epicatechin-3′-β-D-glucuronide was the most abundant metabolite, followed by (-)-epicatechin-3′-sulfate and 3′-O-methyl-(−)-epicatechin-5/7-sulfate. It was also shown that O-sulfonation is a key conjugating reaction in the metabolism of (−)-epicatechin in humans, since the group of (−)-epicatechin sulfates resulted in being the most diverse .
After the intake of 100 g of dark chocolate (70% cacao), have been identified (-)-epicatechin metabolites in human plasma and urine . In this study were identified 10 (-)-epicatechin metabolites, of which (-)-epicatechin-3′-β-D-glucuronide, (-)-epicatechin-3′-sulfate and 3′-O-methyl-(-)-epicatechin 5-sulfate were the major metabolites. Finally, (-)-epicatechin metabolite profile could be divided into three groups (glucuronides, sulfates, and O-methyl sulfates) and their distribution might be modified depending on the amount of (-)-epicatechin ingested, due to enzymatic activity .
Given the above, Figure 1 shows that in both humans and rats a large proportion of (-)-epicatechin (approximately 90%) is absorbed in different conjugated metabolites (glucuronides, sulfates, methylates) that are produced in the intestinal mucous lining. Through portal circulation, these compounds are transported to the liver where they undergo different biotransformation reactions of phase I and II, which produce new O-sulfated, O-glucuronidated and O-methylated forms of (-)-epicatechin.
Afterwards, these new forms can be distributed among tissues or can be excreted from the body via bile or urine. The fraction of polymeric procyanidins unabsorbed in the upper gastrointestinal tract can reach the colon and become available for the local microbiota. Gut flora can produce several low–molecular-weight metabolites that can then be efficiently absorbed [16,20,21,31].
Regarding the absorption of procyanidin B2, it has been reported that it is less efficiently and less rapidly absorbed than epicatechin (5%–10% of the absorbed concentration of (-)-epicatechin), and, therefore, its human and rat plasma concentration is lower (10–40 nM/L) [27,32].
Using liquid chromatography and mass spectrometry, studies of kinetic absorption in humans and rats revealed that procyanidin B2 reachs a maximum plasma concentration at 30 to 60 min after administration of the pure compound .
Another study showed that human plasma level of procyanidin B2 reached the maximum at about 2 h after the consumption of a flavanol-rich cocoa .
However, a subsequent study showed that the maximal concentrations (Cmax) for total (14C) in blood were not attained until 5 to 6 h after oral administration (10.5 and 21 mg/kg) in rats .
Therefore, it was suggested that much of the radioactivity was absorbed from the distal part of the small intestine and/or the colon, whereas plasma concentrations of procyanidin B2 detected 30 min and 2 h after oral administration corresponded to the absorption in the proximal part of the small intestine.
Considering the low levels of procyanidin dimer B2 detected in human plasma and that little was known about its colonic metabolism, the catabolism by human faecal microbiota of (-)-epicatechin and procyanidin B2 was compared using an in vitro culture model.
Results showed that from 10 phenolic acid catabolites common to both substrates, solely five phenolic catabolites were unique to procyanidin B2 .
Although full characterization and further investigation of these catabolites is needed, it has been suggested that they might be of interest with regard to potential biological effects.
Another topic of discussion regarding procyanidin B2 absorption is its possible biotransformations at the gastrointestinal tract.
An in vitro study where human gastric conditions were simulated (gastric juice (pH 2.0) at 37 °C for up to 3.5), showed that oligomeric procyanidins (dimer to hexamer) decompose essentially to epicatechin monomeric and dimeric units.
It was also suggested that the latter were the major components for absorption via the small intestine .
In a following study, the perfusion of isolated small intestine with cocoa procyanidin dimers B2 and B5 (50 mM) showed that both forms are transferred to the serosal side of enterocytes in a lesser extent than the monomer subunits (<1%). Instead, it was observed that unconjugated (-)-epicatechin was the most abundant bioavailable form of procyanidin B2 in plasma (95.8%).
These observations provide an explanation for the high ratio of epicatechin to catechin observed . In addition, the latter confirmed the presence of modest concentrations of procyanidin dimers (<1%) in human plasma after the intake of a flavanol-rich cocoa beverage . Small amounts of methylated B2 dimer have also been detected in plasma (3.2%) after ex vivo perfusion of a rat small intestine .
Using an in situ rat small intestinal perfusion model it was shown that the presence of tetrameric procyanidins enhanced the absorption of procyanidin B2.
This suggests that further studies are needed to fully understand the synergy between procyanidins with different degrees of polymerization, particularly when considering that these coexist naturally in foodstuffs.
Hepatic glucuronidation, sulfation and methylation of procyanidin B2 have also been assessed using mice microsomal incubations.
Unlike (-)-epicatechin, it has been shown that procyanidin B2 remains mostly unmetabolized.
Only a small percentage was converted to four minor glucuronide products, although formation mechanisms have not yet been determined due to their low concentrations.
A wide range of phenolic acid microbial metabolites (high and low molecular weight) derived from (-)-epicatechin and procyanidin B2 biotransformations have been detected in urine samples collected after consumption of cocoa in humans and rats.
It is noteworthy that the variations in the urinary excretion profiles in humans and rats may be influenced by the differences in the ingested dose of cocoa and to the different microbiota present in the intestine of each species .
After cocoa consumption, the major microbial metabolites found in human urine samples were caffeic acid, ferulic acid, 3-hydroxyphenylacetic acid, vanillic acid, 3-hydroxybenzoic acid, hippuric acid, 4-hydroxyhippuric acid, (-)-epicatechin and procyanidin B2.
On the other hand, the major metabolites in rat urine samples were 3,4-dihydroxyphenylpropionic acid, cumaric acid, 3-hydroxyphenylacetic acid, protocatechuic acid, vanillic acid and (-)-epicatechin .
There are few studies regarding the colonic metabolism of phenolic compounds, and even fewer regarding oligomeric flavanols. However, it is now known that colonic microbiota has a large catalytic potential for enzymatic degradation of flavonoids, which results in a huge array of new metabolites with several biological and health-promoting properties [20,23,35,36].
Cacao Flavanols and Their Health Effects
After ingestion, flavanols can undergo significant modifications that result in several bioactive molecules with beneficial effects in chronic diseases related to metabolic disorders and oxidative stress.
The mechanisms that have been proposed to explain the biological actions of flavanols are based on their capacity to act as antioxidants and to interact with signaling proteins, enzymes, DNA and membranes. According to the concentrations achieved in their target tissues, their mechanisms have been classified as direct (high concentration) or indirect (low concentration).
Until now, the most studied direct effects of cacao flavanols are related to their antioxidant capacity. It is well documented that the latter depends on their aromatic rings with hydroxyl substituents, which give flavanols an adequate configuration to act as electron donors (e−) and thus stabilize free radicals .
On the other hand, the degree of polymerization, partition coefficient and number and distribution of hydroxyl groups will influence the type of interactions that occur between flavanols and the cell membrane.
For example, flavanols can partition in the hydrophobic core of membranes or form hydrogen bonds with the polar headgroups of membrane lipids .
Given the above, it has been proposed that flavanols may protect the integrity and function of the cell membrane by modulating changes in its fluidity and permeability produced by molecules with oxidation potential [37,38].
It is well known that when membrane fluidity decreases it is more prone to be oxidized. Instead, when fluidity increases membrane lipids are less exposed to oxidation.
The effects of cocoa procyanidins on bilayer fluidity and susceptibility to oxidation have been studied using predominantly Jurkat T cells and liposomes. Cocoa derived dimers showed to protect Jurkat T cells from AMVN (2,2’-azobis (2,4-dimethylvaleronitrile)-mediated oxidation and to increase membrane fluidity, measured by a decrease in 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization.
It was proposed that this effect could be mediated through complex interactions of dimers with membrane proteins, rather than lipids .
This was confirmed when the interaction of flavanols and procyanidins (dimers to hexamers) with liposomes (composed of phosphatidylcholine and phosphatidylserin) did not influence its membrane fluidity or lipid lateral phase separation .
Membrane lipid oxidation induces the formation of pores that allow the leakage of certain molecules.
The increase in membrane permeability due to lipid oxidation has also been studied in liposomes oxidized with AMVN or ferrous iron. An in vitro study showed that preincubation with procyanidins significantly reduces the effect of ferrous iron on liposome permeability .
Taking into consideration flavanols’ interactions with cell membrane, lipid peroxidation has been widely used to study flavanols’ effects against oxidative stress.
Breaking initiation and propagation reactions are considered as the most important antioxidant strategy of flavanols for inhibiting or retarding lipid oxidation.
During initiation, free radicals (generally HO and O2−) substract a hydrogen (H) atom from membrane polyunsaturated fatty acids (PUFA) methylene groups .
The unpaired electron on the carbon is stabilized by a molecular rearrangement of the double bonds to form a conjugated diene, which then combines with oxygen to form a peroxyl radical (LOO).
The latter has the potential to extend the damage by reacting with other polyunsaturated fatty acids to produce lipoperoxides (LOOH) that can subtract hydrogen atoms from another polyunsaturated fatty acids (PUFA) (propagation reaction).
Additionally, there has been a growing interest in the ability of flavanols to chelate redox-active metals (iron and copper).
In the presence of hydrogen peroxide (H2O2), redox active metal ions such as Fe2+ or Cu+ that are covalently bound to the nucleotide bases of DNA react with it to form highly reactive hydroxyl radical (OH).
The latter abstracts a hydrogen atom from the deoxyribose sugar backbone, which in turn promotes the phosphodiester backbone cleavage and strand scission.
Together, nucleotide bases damage (oxidation) and strand breakage have been associated to genetic mutations, cancer and cell death.
Flavanols metal chelating properties reside in the presence of a catechol group (B ring) and hydroxyl substituents, since they are centers of high affinity for metal ions.
Mechanisms underlying flavanols direct effects have been mainly assessed by in vitro studies. However, in vivo studies (biological systems) have been useful for evaluating in vitro evidence.
The beneficial effect of (-)-epicatechin on lipid peroxidation was evaluated in ApoE knockout rats. Administration of epicatechin (64 mg/kg body weight) during 20 weeks significantly reduced aortic F2-isoprostanes, vascular superoxide and endothelin-1 production (p < 0.05 versus control ApoE(−/−) mice) [43,44].
Considering that direct effects require the presence of high concentrations of flavanols, it is thought that in living organisms these effects can be observed just in the gastrointestinal tract, since their absorption has shown to be limited .
Cocoa polyphenols are expected to activate Nrf2, which induces the transcription of antioxidant enzymes such as glutathione peroxidase, superoxide dismutase, and heme oxygenase 1, thus blocking the production of reactive oxygen species (ROS) and nitric oxide synthase (NOS), and attenuating oxidative stress, as well as a number of cellular kinases, including the mitogenactivated protein kinases (MAPKs) [45,46].
In Zucker diabetic fatty (ZDF) rats, the ingestion of a cocoa-rich diet (10%) for 9 weeks attenuated hyperglycemia, improved insulin sensitivity, and increased β-cell mass and function. At molecular level, cocoa intake prevented β-cell apoptosis by increasing antiapoptotic proteins (Bcl-xL) and decreasing proapoptotic proteins (Bax and caspase-3 activity) .
More information: Miguel Rebollo‐Hernanz et al, Cocoa Shell Aqueous Phenolic Extract Preserves Mitochondrial Function and Insulin Sensitivity by Attenuating Inflammation between Macrophages and Adipocytes In Vitro, Molecular Nutrition & Food Research (2019). DOI: 10.1002/mnfr.201801413
Journal information:Molecular Nutrition and Food Research,Molecular Nutrition & Food Research