Cocoa-enriched Diet Research Articles

Diet enriched with high-phenolic cocoa potentiates hippocampal brain-derived neurotrophic factor expression and neurogenesis in healthy adult micewith subtle effects on memory.

Cocoa is widely known for its health benefits, but its neurocognitive impact remains underexplored. This preclinical study aimed to investigate the effects of cocoa and cocoa polyphenols on hippocampal neuroplasticity, cognitive function and emotional behavior. Seventy young-adult C57BL/6JRj male and female mice were fed either a standard diet (CTR) or a diet enriched with 10% high-phenolic content cocoa (HPC) or low-phenolic content cocoa (LPC) for at least four weeks. In a first experiment, behavioral tests assessing exploratory behavior, emotional responses and hippocampal-dependent memory were conducted four weeks into the diet, followed by animal sacrifice a week later. Adult hippocampal neurogenesis and brain-derived neurotrophic factor (BDNF) expression in the hippocampus and prefrontal cortex were evaluated using immunohistochemistry and western blot. In a different experiment, hippocampal synaptic response, long-term potentiation and presynaptic-dependent short-term plasticity were studied by electrophysiology. Cocoa-enriched diets had minimal effects on exploratory activity and anxiety-like behavior, except for reduced locomotion in the LPC group. Only the HPC diet enhanced object recognition memory, while place recognition memory and spatial navigation remained unaffected. The HPC diet also increased adult hippocampal neurogenesis, boosting the proliferation, survival and number of young adult-born neurons. However, both cocoa-enriched diets increased immobility in the forced swimming test and hippocampal BDNF expression. Hippocampal electrophysiology revealed no alterations in neuroplasticity among diets. The results were mostly unaffected by sex. Overall, the HPC diet demonstrated greater potential regarding cognitive and neuroplastic benefits, suggesting a key role of cocoa flavanols in dietary interventions aimed at enhancing brain health.

A Cocoa Diet Can Partially Attenuate the Alterations in Microbiota and Mucosal Immunity Induced by a Single Session of Intensive Exercise in Rats.

BackgroundFollowing intensive sports events, a higher rate of upper respiratory tract infections and the appearance of gastrointestinal symptomatology have been reported. We aimed to evaluate the effect of a cocoa-enriched diet on the cecal microbiota and mucosal immune system of rats submitted to high-intensity acute exercise, as well as to elucidate the involvement of cocoa fiber in such effects.MethodsWistar rats were fed either a standard diet, a diet containing 10% cocoa providing 5% fiber and a diet containing only 5% cocoa fiber. After 25 days, half of the rats of each diet performed an exhaustion running test. Sixteen hours later, samples were obtained to assess, among others, the cecal microbiota and short chain fatty acids (SCFAs) composition, mesenteric lymph nodes (MLNs) and Peyer’s patches (PPs) lymphocyte composition, and immunoglobulin (Ig) content in salivary glands.ResultsThe intake of cocoa, partially due to its fiber content, improved the SCFA production, prevented some changes in PPs and in MLNs lymphocyte composition and also decreased the production of proinflammatory cytokines. Cocoa diet, contrary to cocoa fiber, did not prevent the lower salivary IgM induced by exercise.ConclusionA cocoa dietary intake can partially attenuate the alterations in microbiota and mucosal immunity induced by a single session of intensive exercise.

Protective Effect of a Cocoa-Enriched Diet on Oxidative Stress Induced by Intensive Acute Exercise in Rats.

Intensive acute exercise can induce oxidative stress, leading to muscle damage and immune function impairment. Cocoa diet could prevent this oxidative stress and its consequences on immunity. Our aim was to assess the effect of a cocoa-enriched diet on the reactive oxygen species (ROS) production by peritoneal macrophages, blood immunoglobulin (Ig) levels, leukocyte counts, and the physical performance of rats submitted to an intensive acute exercise, as well as to elucidate the involvement of cocoa fiber in such effects. For this purpose, Wistar rats were fed either a standard diet, i.e., a diet containing 10% cocoa (C10), or a diet containing 5% cocoa fiber (CF) for 25 days. Then, half of the rats of each diet ran on a treadmill until exhaustion, and 16 h later, the samples were obtained. Both C10 and CF diets significantly prevented the increase in ROS production. However, neither the cocoa diet or the cocoa fiber-enriched diet prevented the decrease in serum IgG induced by acute exercise. Therefore, although the cocoa-enriched diet was able to prevent the excessive oxidative stress induced by intensive exercise, this was not enough to avoid the immune function impairment due to exercise.

Theobromine Is Responsible for the Effects of Cocoa on the Antibody Immune Status of Rats

A 10% cocoa-enriched diet influences immune system functionality including the prevention of the antibody response and the induction of lower immunoglobulin (Ig) concentrations. However, neither cocoa polyphenols nor cocoa fiber can totally explain these immunoregulatory properties. This study aimed to establish the influence of cocoa theobromine in systemic and intestinal Ig concentrations and to determine the effect of cocoa or theobromine feeding on lymphoid tissue lymphocyte composition. Three-week-old female Lewis rats were fed either a standard diet (AIN-93M; RF group), a 10% cocoa diet (CC group), or a 0.25% theobromine diet (the same amount provided by the cocoa diet; TB group) in 2 separate experiments that lasted 19 (experiment 1) or 8 (experiment 2) d. Serum IgG, IgM, IgA, and intestinal secretory IgA (sIgA) concentrations were determined. In addition, at the end of experiment 2, thymus, mesenteric lymph node (MLN), and spleen lymphocyte populations were analyzed. Both CC and TB groups in experiments 1 and 2 showed similar serum IgG, IgM, and IgA and intestinal sIgA concentrations, which were lower than those in the RF group (46-98% lower in experiment 1 and 23-91% lower in experiment 2; P<0.05). In addition, in experiment 2, the cocoa and theobromine diets similarly changed the thymocyte composition by increasing CD4-CD8- (+133%) and CD4+CD8- (+53%) proportions (P<0.01), changed the MLN composition by decreasing the percentage of T-helper (Th) lymphocytes (-3%) (P=0.015), and changed the spleen composition by increasing the proportion of Th lymphocytes (+9%) (P<0.001) after 1 wk of diet treatment. The theobromine in cocoa plays an immunoregulatory role that is responsible for cocoa's influence on both systemic and intestinal antibody concentrations and also for modifying lymphoid tissue lymphocyte composition in young healthy Lewis rats. The majority of these changes are observed after a single week of being fed a diet containing 0.25% theobromine.

Cocoa Diet and Antibody Immune Response in Preclinical Studies.

The ability of cocoa to interact with the immune system in vitro and in vivo has been described. In the latter context, a cocoa-enriched diet in healthy rats was able to modify the immune system’s functionality. This fact could be observed in the composition and functionality of lymphoid tissues, such as the thymus, spleen, and lymph nodes. Consequently, immune effector mechanisms, such as antibody synthesis, were modified. A cocoa-enriched diet in young rats was able to attenuate the serum levels of immunoglobulin (Ig) G, IgM, and IgA and also the intestinal IgM and IgA secretion. Moreover, in immunized rats, the intake of cocoa decreased specific IgG1, IgG2a, IgG2c, and IgM concentrations in serum. This immune-regulator potential was then tested in disease models in which antibodies play a pathogenic role. A cocoa-enriched diet was able to partially prevent the synthesis of autoantibodies in a model of autoimmune arthritis in rats and was also able to protect against IgE and T helper 2-related antibody synthesis in two rat models of allergy. Likewise, a cocoa-enriched diet prevented an oral sensitization process in young rats. In this review, we will focus on the influence of cocoa on the acquired branch of the immune function. Therefore, we will focus on how a cocoa diet influences lymphocyte function both in the systemic and intestinal immune system. Likewise, its potential role in preventing some antibody-induced immune diseases is also included. Although further studies must characterize the particular cocoa components responsible for such effects and nutritional studies in humans need to be carried out, cocoa has potential as a nutraceutical agent in some hypersensitivity status.

Gut Microbiota in a Rat Oral Sensitization Model: Effect of a Cocoa-Enriched Diet.

Increasing evidence is emerging suggesting a relation between dietary compounds, microbiota, and the susceptibility to allergic diseases, particularly food allergy. Cocoa, a source of antioxidant polyphenols, has shown effects on gut microbiota and the ability to promote tolerance in an oral sensitization model. Taking these facts into consideration, the aim of the present study was to establish the influence of an oral sensitization model, both alone and together with a cocoa-enriched diet, on gut microbiota. Lewis rats were orally sensitized and fed with either a standard or 10% cocoa diet. Faecal microbiota was analysed through metagenomics study. Intestinal IgA concentration was also determined. Oral sensitization produced few changes in intestinal microbiota, but in those rats fed a cocoa diet significant modifications appeared. Decreased bacteria from the Firmicutes and Proteobacteria phyla and a higher percentage of bacteria belonging to the Tenericutes and Cyanobacteria phyla were observed. In conclusion, a cocoa diet is able to modify the microbiota bacterial pattern in orally sensitized animals. As cocoa inhibits the synthesis of specific antibodies and also intestinal IgA, those changes in microbiota pattern, particularly those of the Proteobacteria phylum, might be partially responsible for the tolerogenic effect of cocoa.

Effect of a cocoa-enriched diet on immune response and anaphylaxis in a food allergy model in Brown Norway rats

Previous studies have demonstrated that cocoa intake decreased Th2 immune-related antibodies in rats. In consequence, we aimed to study in depth this cocoa action, particularly assessing its effect on a rat model of food allergy (FA) and also on an anaphylactic response. The involvement of the intestinal immune system was analyzed to allow the action mechanisms to be investigated. The role of cocoa flavonoids in the antiallergic properties of cocoa was also established. Brown Norway rats were fed either a reference diet or diets containing conventional cocoa (CC) or nonfermented cocoa (NFC). FA to ovalbumin (OVA) was induced and, later, an anaphylactic response was provoked. As expected, the synthesis of anti-OVA IgE and other Th2-related antibodies was inhibited by CC diet. In addition, the release of mast cell protease II after anaphylaxis was partially prevented by CC, although other variables were not modified. The CC diet also attenuated the increase of some Th2-related cytokines released from mesenteric lymph node and spleen cells, and modulated the intestinal gene expression of molecules involved in allergic response. These results demonstrated the local and systemic influence of CC diet. The effects of the NFC diet were weaker than those of CC, suggesting that cocoa components other than flavonoids play a role in cocoa's action. In conclusion, by acting on intestinal and systemic immune functions, a cocoa-enriched diet in rats exhibited a protective effect against FA and partially against anaphylaxis, making this a food of high interest to the fields of health and immunonutrition.

Cocoa and cocoa flavanol epicatechin improve hepatic lipid metabolism in in vivo and in vitro models. Role of PKCζ

Diabetes alters lipid metabolism that could lead to hepatic steatosis. The present study aimed to investigate the effect of a cocoa-enriched diet in type 2 diabetic Zucker diabetic fatty (ZDF) rats and that of cocoa-flavanol epicatechin (EC) in high-glucose-exposed HepG2 cells on hepatic lipid metabolism. Male ZDF cocoa-fed-rats had decreased body weight gain and improved circulating and hepatic lipid levels. Similarly, EC alleviated the altered lipid values induced in high-glucose-challenged HepG2 cells. The lipid-lowering effect was related to diminished fatty acid synthesis (sterol-regulatory-element-binding protein-1-c and fatty-acid synthase down-regulation), and increased fatty-acid oxidation (proliferator-activated-receptor α up-regulation). These effects depended on 5′-AMP-activated-protein-kinase (AMPK), protein kinase B (AKT) and protein kinase C (PKC)-ζ, which phosphorylated levels returned to control values upon cocoa or EC administration. Moreover, PKCζ played a role on AKT and AMPK regulation. These findings suggest that cocoa and EC protect hepatocytes in vivo and in vitro by improving lipid metabolism through multiple-signalling pathways.

Cocoa intake ameliorates hepatic oxidative stress in young Zucker diabetic fatty rats

Chronic hyperglycemia in diabetes is associated with oxidative stress-mediated tissue damage. The present study is aimed to explore the role of a cocoa-enriched diet in ameliorating the oxidative stress-induced damage in the liver of young type 2 diabetic Zucker diabetic fatty (ZDF) rats. Male ZDF rats were fed a control or cocoa-rich diet (10%), and Zucker Lean (ZL) animals received the control diet. ZDF rats fed with cocoa (ZDF-Ca) decreased body weight gain, glucose and insulin levels, and improved glucose tolerance and insulin resistance. Cocoa diet further reduced reactive oxygen species (ROS) levels and carbonyl content in the liver of ZDF animals. The diminished activity of superoxide dismutase (SOD) and the enhanced activity of heme oxygenase (HO-1) in ZDF-C were returned to ZL values upon cocoa administration. Cocoa did not restore the decreased glutathione-S-transferase (GST) activity in both ZDF groups in comparison to ZL rats. Glutathione (GSH) content and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) remained unaltered among all animal groups. Moreover, cocoa-rich diet suppressed total and phosphorylated nuclear factor erythroid-derived 2-like 2 (Nrf2), as well as p65-nuclear factor-kappaB (NF-ĸB) enhanced levels observed in ZDF rats. The results indicate that cocoa protects the hepatocytes by improving the antioxidant competence in the liver of young type 2 diabetic ZDF rats.

Inclusion of cocoa as a dietary supplement represses expression of inflammatory proteins in spinal trigeminal nucleus in response to chronic trigeminal nerve stimulation.

Central sensitization is implicated in the pathology of temporomandibular joint disorder and other types of orofacial pain. We investigated the effects of dietary cocoa on expression of proteins involved in the development of central sensitization in the spinal trigeminal nucleus (STN) in response to inflammatory stimulation of trigeminal nerves. Male Sprague-Dawley rats were fed either a control diet or an isocaloric diet consisting of 10% cocoa powder 14 days prior to bilateral injection of complete Freund's adjuvant (CFA) into the temporomandibular joint to promote prolonged activation of trigeminal ganglion neurons and glia. While dietary cocoa stimulated basal expression of glutamate-aspartate transporter and mitogen-activated protein kinase phosphatase-1 when compared to animals on a normal diet, cocoa suppressed basal calcitonin gene-related peptide levels in the STN. CFA-stimulated levels of protein kinase A, P2X3 , P-p38, glial fibrillary-associated protein, and OX-42, whose elevated levels in the STN are implicated in central sensitization, were repressed to near control levels in animals on a cocoa-enriched diet. Similarly, dietary cocoa repressed CFA-stimulated inflammatory cytokine expression. Based on our findings, we speculate that cocoa-enriched diets could be beneficial as a natural therapeutic option for temporomandibular joint disorder and other chronic orofacial pain conditions.

The effects of cocoa on the immune system.

Cocoa is a food relatively rich in polyphenols, which makes it a potent antioxidant. Due to its activity as an antioxidant, as well as through other mechanisms, cocoa consumption has been reported to be beneficial for cardiovascular health, brain functions, and cancer prevention. Furthermore, cocoa influences the immune system, in particular the inflammatory innate response and the systemic and intestinal adaptive immune response. Preclinical studies have demonstrated that a cocoa-enriched diet modifies T cell functions that conduce to a modulation of the synthesis of systemic and gut antibodies. In this regard, it seems that a cocoa diet in rats produces changes in the lymphocyte composition of secondary lymphoid tissues and the cytokines secreted by T cells. These results suggest that it is possible that cocoa could inhibit the function of T helper type 2 cells, and in line with this, the preventive effect of cocoa on IgE synthesis in a rat allergy model has been reported, which opens up new perspectives when considering the beneficial effects of cocoa compounds. On the other hand, cocoa intake modifies the functionality of gut-associated lymphoid tissue by means of modulating IgA secretion and intestinal microbiota. The mechanisms involved in these influences are discussed here. Further research may elucidate the cocoa compounds involved in such an effect and also the possible medical approaches to these repercussions.

Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-α-stimulated Caco-2 cells

Numerous lines of evidence support a relationship between intestinal inflammation and cancer. Therefore, much attention has recently been focused on the identification of natural compounds with anti-inflammatory activities as a strategy to suppress the early stages of colorectal cancer. Because cocoa is a rich source of bioactive compounds, the present study investigated its anti-inflammatory properties in a rat model of azoxymethane (AOM)-induced colon carcinogenesis and in TNF-α-stimulated Caco-2 cells. A total of forty male rats were fed with control or cocoa-enriched diets (12 %) during 8 weeks and injected with saline or AOM (20 mg/kg body weight) during the third and fourth week (n 10 rats/group). At the end of the experiment, colon samples were evaluated for markers of inflammation. The anti-inflammatory activity of a cocoa polyphenolic extract (10 μg/ml) was examined in TNF-α-stimulated Caco-2 cells, an in vitro model of experimentally induced intestinal inflammation. The signalling pathways involved, including NF-κB and the mitogen-activated protein kinase family such as c-Jun NH₂-terminal kinases (JNK), extracellular signal-regulated kinases and p38, were also evaluated. The results show that the cocoa-rich diet decreases the nuclear levels of NF-κB and the expression of pro-inflammatory enzymes such as cyclo-oxygenase-2 and inducible NO synthase induced by AOM in the colon. Additionally, the experiments in Caco-2 cells confirm that cocoa polyphenols effectively down-regulate the levels of inflammatory markers induced by TNF-α by inhibiting NF-κB translocation and JNK phosphorylation. We conclude that cocoa polyphenols suppress inflammation-related colon carcinogenesis and could be promising in the dietary prevention of intestinal inflammation and related cancer development.

Effects of a cocoa diet on an intestinal inflammation model in rats

Cocoa is a rich source of fiber and flavonoids with recognized antioxidant and anti-inflammatory potential. The aim of this study was to evaluate the effects of a cocoa-enriched diet on rats with dextran sulfate sodium (DSS)-induced colitis. Wistar rats were fed with either a 5% cocoa diet or standard diet. Colon inflammation was induced by DSS in the drinking water: 5% for six days and 2% over the following nine days. Colitis was assessed by body weight loss, stool consistency and blood presence in stools. A group of animals fed standard diet was treated with quercitrin (1 mg/kg) after colitis establishment. After two weeks of DSS treatment, the colon oxidative and inflammatory status and lymphocyte composition from blood and mesenteric lymph nodes (MLNs) were assessed. The cocoa-fed group did not exhibit amelioration of clinical colitis but displayed higher antioxidant activity than the colitic reference group by the restoration of colon glutathione content and prevention of lipid peroxidation. The cocoa diet showed anti-inflammatory potential because it down-regulated serum tumor necrosis factor-α, colon inducible nitric oxide synthase activity and decreased colon cell infiltration. The lymphocyte composition in MLNs was not modified by drinking DSS, but there was an increase in the proportion of natural killer and regulatory T-cells in the blood. These changes were not modified by cocoa. In conclusion, cocoa intake may help to inhibit the negative oxidative effects consequent to colitis, although this action is not enough to abrogate the intestinal inflammation significantly.

Cocoa intake attenuates oxidative stress associated with rat adjuvant arthritis

Cocoa contains flavonoids with antioxidant properties. The aim of this study was to ascertain the effect of cocoa intake on oxidative stress associated with a model of chronic inflammation such as adjuvant arthritis. Female Wistar rats were fed with a 5% or 10% cocoa-enriched diet or were given p.o. a quercetin suspension every other day for 10days. Arthritis was induced by a heat-killed Mycobacterium butyricum suspension. Reactive oxygen species (ROS) produced by macrophages, and splenic superoxide dismutase (total, cytoplasmic and mitochondrial) and catalase activities were determined. Clinically, joint swelling in arthritic rats was not reduced by antioxidants; however, the 5% cocoa diet and quercetin administration reduced ROS production. Moreover, the 5% cocoa diet normalized the activities of superoxide dismutase and catalase. In conclusion, a cocoa diet reduces the oxidative stress associated with a chronic inflammatory pathology, although it was not enough to attenuate joint swelling.

Cocoa modulatory effect on rat faecal microbiota and colonic crosstalk

Previous studies have reported the effect of a cocoa-enriched diet on the intestinal immune system in rats. Cocoa contains fibre and polyphenols that can directly influence the intestinal ecosystem and its relationship with the immune system. The aim of this study was to evaluate the effects of a cocoa-enriched diet on gut microbiota, toll-like receptor (TLR) expression and immunoglobulin (Ig) A (IgA) intestinal secretion in rats. Four-week-old Wistar rats were fed a standard or cocoa diet for 6weeks. Faecal samples were collected before the beginning of the diet and at the end of the study. After the nutritional intervention, colon samples were obtained to quantify TLR and IgA gene expression and IgA protein. Microbiota composition was characterized by fluorescent in situ hybridization (FISH) coupled to flow cytometry (FCM) analysis using specific probes directed to 16S rRNA of the main bacteria genus present in rat intestine. The cocoa dietary intervention resulted in a differential TLR pattern and a decrease in the intestinal IgA secretion and IgA-coating bacteria. Moreover there was a significant decrease in the proportion of Bacteroides, Clostridium and Staphylococcus genera in the faeces of cocoa-fed animals. In conclusion, cocoa intake affects the growth of certain species of gut microbiota in rats and is associated with changes in the TLR pattern which could be responsible for the changes observed in the intestinal immune system.

A diet enriched with cocoa prevents IgE synthesis in a rat allergy model

Previous studies in young rats reported the impact of cocoa intake on healthy immune status and allow suggesting it may have a role in the prevention of some immune-mediated diseases. The aim of this study was to ascertain the effect of a cocoa diet in a model of allergy in young rats. Three-week-old Brown Norway rats were immunized by i.p. injection of ovalbumin (OVA) with alum as adjuvant and Bordetella pertussis toxin. During the next 4 weeks rats received either a cocoa diet (containing 0.2% polyphenols, w/w) or a standard diet. Animals fed a standard diet showed high concentrations of anti-OVA IgG1, IgG2a, IgG2b and high anti-OVA IgE titres, which is the antibody involved in allergic response. In contrast, animals fed a cocoa diet showed significantly lower concentrations of anti-OVA IgG1 and IgG2a antibodies. Interestingly, the cocoa diet prevented anti-OVA IgE synthesis and decreased total serum IgE concentration. Analysis of cytokine production in lymph node cells at the end of the study revealed that, in this compartment, the cocoa diet decreased the tumor necrosis factor (TNF)-α and the interleukin (IL)-10 secretion but not IL-4 production. In conclusion, a cocoa-enriched diet in young rats produces an immunomodulatory effect that prevents anti-allergen IgE synthesis, suggesting a potential role for cocoa flavonoids in the prevention or treatment of allergic diseases.

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor β, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RARα and RARβ), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL‑6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RARα and RARβ. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RARα and RARβ, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.

Effect of a cocoa flavonoid-enriched diet on experimental autoimmune arthritis

Previously we established that a cocoa-enriched diet in young rats reduces specific antibody production and the T helper (Th) lymphocyte proportion in lymphoid tissues. The aim of the present study was to ascertain the modulatory ability of a cocoa flavonoid-enriched diet on collagen-induced arthritis (CIA), which is mediated by anti-collagen autoantibody response and Th lymphocyte activation. Female Louvain (LOU) rats were fed with a cocoa-enriched diet, beginning 2 weeks before CIA induction. Hind-paw swelling and serum cytokine and anti-collagen antibody concentrations were determined. Anti-collagen antibody-secreting cell counts and lymphocyte subset proportions were established in inguinal lymph nodes (ILN). Reactive oxygen species (ROS), nitric oxide (NO) and TNFα produced by peritoneal macrophages were determined. Although arthritic cocoa-fed rats showed a similar hind-paw swelling time course as the arthritic animals fed a standard diet, the cocoa intake was able to decrease specific IgG2a, IgG2b and IgG2c titres. Moreover, cocoa intake in CIA rats reduced ROS production, TNFα and NO release from peritoneal macrophages, and decreased the Th:cytotoxic T cell ratio in ILN. In conclusion, a cocoa flavonoid-enriched diet in LOU rats with CIA produced no effect on hind-paw swelling but was able to modulate the specific antibody response and also the Th lymphocyte proportion, as well as the synthesis of pro-inflammatory mediators from peritoneal macrophages. Therefore, a cocoa-enriched diet could be a good adjuvant therapy in disorders with oxidative stress or autoimmune pathogenesis.

Effect of cocoa-enriched diets on lymphocytes involved in adjuvant arthritis in rats

Cocoa and its flavonoids have potential anti-inflammatory properties in vitro and in acute inflammation models in vivo. The aim of the present study was to ascertain the effects of two cocoa-enriched diets on adjuvant arthritis (AA) in rats, considering not only clinical and biochemical inflammatory indices, but also antibody response and lymphocyte composition. Female Wistar rats were fed with a 5 or 10 % cocoa-enriched diet beginning 2 weeks before arthritis induction and until the end of the study. AA was induced by an intradermal injection of heat-killed Mycobacterium butyricum suspension. The hind-paw swelling (plethysmometry), serum anti-mycobacterial antibody concentration (ELISA), blood and inguinal lymph node lymphocyte subset percentage (flow cytometry), and IL-2, interferon γ and PGE₂ released from splenocytes (ELISA) were assessed. Although the cocoa diets had no significant effect on hind-paw swelling, a tendency to reduce it was observed at the end of the study. Cocoa-enriched diets were able to decrease the serum anti-mycobacterial antibody concentration and the splenocyte PGE2 production, as well as the proportion of T-helper (Th) lymphocytes in blood and regional lymph nodes, which probably includes cells responsible for the arthritic process. The cocoa diets prevented a decrease in the proportion of regulatory T-cells in blood and a disequilibrium between inguinal lymph node natural killer (NK) CD8⁺ and NK CD8⁻ subsets. In conclusion, the cocoa-enriched diets during AA were not able to significantly decrease joint inflammation but modified Th-cell proportions and prevented specific antibody synthesis.

Cocoa-enriched diets enhance expression of phosphatases and decrease expression of inflammatory molecules in trigeminal ganglion neurons

Activation of trigeminal nerves and release of neuropeptides that promote inflammation are implicated in the underlying pathology of migraine and temporomandibular joint (TMJ) disorders. The overall response of trigeminal nerves to peripheral inflammatory stimuli involves a balance between enzymes that promote inflammation, kinases, and those that restore homeostasis, phosphatases. The goal of this study was to determine the effects of a cocoa-enriched diet on the expression of key inflammatory proteins in trigeminal ganglion neurons under basal and inflammatory conditions. Rats were fed a control diet or an isocaloric diet enriched in cocoa for 14 days prior to an injection of noxious stimuli to cause acute or chronic excitation of trigeminal neurons. In animals fed a cocoa-enriched diet, basal levels of the mitogen-activated kinase (MAP) phosphatases MKP-1 and MKP-3 were elevated in neurons. Importantly, the stimulatory effects of acute or chronic peripheral inflammation on neuronal expression of the MAPK p38 and extracellular signal-regulated kinases (ERK) were significantly repressed in response to cocoa. Similarly, dietary cocoa significantly suppressed basal neuronal expression of calcitonin gene-related peptide (CGRP) as well as stimulated levels of the inducible form of nitric oxide synthase (iNOS), proteins implicated in the underlying pathology of migraine and TMJ disorders. To our knowledge, this is the first evidence that a dietary supplement can cause upregulation of MKP, and that cocoa can prevent inflammatory responses in trigeminal ganglion neurons. Furthermore, our data provide evidence that cocoa contains biologically active compounds that would be beneficial in the treatment of migraine and TMJ disorders.