Cocoa extract exerts sex-specific anti-diabetic effects in an aggressive type-2 diabetes model: A pilot study.

Type 2 diabetes (T2D) is characterized by hyperglycemia and insulin resistance. Cocoa may slow T2D development and progression. This study employed male and female BTBR.Cg-Lepob/ob/WiscJ (ob/ob) and wild type (WT) controls to assess the potential for cocoa to ameliorate progressive T2D and compare responses between sexes. Mice received diet without (WT, ob/ob) or with cocoa extract (ob/ob + c) for 10 weeks. Acute cocoa reduced fasting hyperglycemia in females, but not males, after 2 weeks. Chronic cocoa supplementation (6-10 weeks) ameliorated hyperinsulinemia in males and worsened hyperlipidemia and hyperinsulinemia in females, yet also preserved and enhanced beta cell survival in females. The underlying mechanisms of these differences warrant further study. If sex differences are apparent in subsequent preclinical studies, clinical studies will be warranted to establish whether these differences are relevant in humans. Sex differences may need to be considered when designing human dietary interventions for T2D.

Effect of processing on the anti-inflammatory efficacy of cocoa in a high fat diet-induced mouse model of obesity.

Obesity causes inflammation which may lead to development of co-morbidities like cardiovascular diseases. Cocoa is a popular food ingredient that has been shown to mitigate obesity and inflammation in preclinical models. Cocoa typically undergoes fermentation and roasting prior to consumption, which can affect the polyphenol content in cocoa. The aim of this study was to compare the effect of fermentation and roasting protocols on the ability of cocoa to mitigate obesity, gut barrier dysfunction, and chronic inflammation in high fat (HF)-fed, obese C57BL/6J mice. We found that treatment of mice with 80 mg/g dietary cocoa powder for 8 weeks reduced rate of body weight gain in both male and female mice (46-57%), regardless of fermentation and roasting protocol. Colonic length was increased (11-24%) and gut permeability was reduced (48-79%) by cocoa supplementation. Analysis of the cecal microbiome showed that cocoa, regardless of fermentation and roasting protocol, reduced the ratio of Firmicutes to Bacteroidetes. Multivariate statistical analysis of markers of inflammation and body weight data showed sex differences in the effect of both the HF diet as well as cocoa supplementation. Based on this data there was strong protective efficacy from cocoa supplementation especially for the more processed cocoa samples. Overall, this study shows that anti-obesity and anti-inflammatory efficacy of cocoa is resilient to changes in polyphenol content and composition induced by fermentation or roasting. Further, this study shows that although cocoa has beneficial effects in both males and females, there are significant sex differences.

Phenolic-rich beverages reduce bacterial TMA formation in an ex vivo-in vitro colonic fermentation model.

The production of pro-atherogenic trimethylamine N-oxide (TMAO) is dependent on the gut microbiota metabolism of quaternary amines (i.e., choline) into trimethylamine (TMA). Nutritional strategies that target microbial conversion of choline into TMA could reduce cardiovascular disease and atherosclerosis burden by reducing subsequent formation of TMAO. This study aimed to evaluate (1) whether beverages rich in known inhibitors of TMA production (chlorogenic acid, catechin and epicatechin) can reduce TMA formation and (2) the effect of upper gastrointestinal digestion on efficacy. To do this, either raw or digested coffee, tea and cocoa beverages were evaluated for their TMA-d9 production inhibition in our ex vivo-in vitro fermentation model with human fecal slurries and choline-d9 substrate. Results showed that digestion was required to unlock the TMA-d9 production inhibition potential of coffee and cocoa beverages, and that teas did not possess a strong inhibition potential either digested or undigested. By fractionating digested bioactive beverages, we determined that those fractions rich in chlorogenic acid were the most bioactive. Overall, this study suggests that regular cocoa and coffee consumption could be a nutritional strategy able to reduce TMAO levels. In vivo studies should be carried out to confirm the potential of these beverages as strategies to inhibit TMA production.

Development of a High-Throughput Method to Study the Inhibitory Effect of Phytochemicals on Trimethylamine Formation.

Choline is metabolized by the gut microbiota into trimethylamine (TMA), the precursor of pro-atherosclerotic molecule trimethylamine N-oxide (TMAO). A reduction in TMA formation has shown cardioprotective effects, and some phytochemicals may reduce TMA formation. This study aimed to develop an optimized, high-throughput anaerobic fermentation methodology to study the inhibition of choline microbial metabolism into TMA by phenolic compounds with healthy human fecal starter. Optimal fermentation conditions were: 20% fecal slurry (1:10 in PBS), 100 µM choline, and 12 h fermentation. Additionally, 10 mM of 3,3-dimethyl-1-butanol (DMB) was defined as a positive TMA production inhibitor, achieving a ~50% reduction in TMA production. Gallic acid and chlorogenic acid reported higher TMA inhibitory potential (maximum of 80-90% TMA production inhibition), with IC50 around 5 mM. Neither DMB nor gallic acid or chlorogenic acid reduced TMA production through cytotoxic effects, indicating mechanisms such as altered TMA-lyase activity or expression.

Diet-induced obesity in genetically diverse collaborative cross mouse founder strains reveals diverse phenotype response and amelioration by quercetin treatment in 129S1/SvImJ, PWK/EiJ, CAST/PhJ, and WSB/EiJ mice.

Significant evidence suggests protective effects of flavonoids against obesity in animal models, but these often do not translate to humans. One explanation for this disconnect is use of a few mouse strains (notably C57BL/6 J) in obesity studies. Obesity is a multifactorial disease. The underlying causes are not fully replicated by the high-fat C57BL/6 J model, despite phenotypic similarities. Furthermore, the impact of genetic factors on the activities of flavonoids is unknown. This study was designed to explore how diverse mouse strains respond to diet-induced obesity when fed a representative flavonoid. A subset of Collaborative Cross founder strains (males and females) were placed on dietary treatments (low-fat, high-fat, high-fat with quercetin, high-fat with quercetin and antibiotics) longitudinally. Diverse responses were observed across strains and sexes. Quercetin appeared to moderately blunt weight gain in male C57 and both sexes of 129S1/SvImJ mice, and slightly increased weight gain in female C57 mice. Surprisingly, quercetin dramatically blunted weight gain in male, but not female, PWK/PhJ mice. For female mice, quercetin blunted weight gain (relative to the high-fat phase) in CAST/PhJ, PWK/EiJ and WSB/EiJ mice compared to C57. Antibiotics did not generally result in loss of protective effects of quercetin. This highlights complex interactions between genetic factors, sex, obesity stimuli, and flavonoid intake, and the need to move away from single inbred mouse models to enhance translatability to diverse humans. These data justify use of genetically diverse Collaborative Cross and Diversity Outbred models which are emerging as invaluable tools in the field of personalized nutrition.

Flavanol Polymerization Is a Superior Predictor of α-Glucosidase Inhibitory Activity Compared to Flavanol or Total Polyphenol Concentrations in Cocoas Prepared by Variations in Controlled Fermentation and Roasting of the Same Raw Cocoa Beans.

Raw cocoa beans were processed to produce cocoa powders with different combinations of fermentation (unfermented, cool, or hot) and roasting (not roasted, cool, or hot). Cocoa powder extracts were characterized and assessed for α-glucosidase inhibitory activity in vitro. Cocoa processing (fermentation/roasting) contributed to significant losses of native flavanols. All of the treatments dose-dependently inhibited α-glucosidase activity, with cool fermented/cool roasted powder exhibiting the greatest potency (IC50: 68.09 µg/mL), when compared to acarbose (IC50: 133.22 µg/mL). A strong negative correlation was observed between flavanol mDP and IC50, suggesting flavanol polymerization as a marker of enhanced α-glucosidase inhibition in cocoa. Our data demonstrate that cocoa powders are potent inhibitors of α-glucosidase. Significant reductions in the total polyphenol and flavanol concentrations induced by processing do not necessarily dictate a reduced capacity for α-glucosidase inhibition, but rather these steps can enhance cocoa bioactivity. Non-traditional compositional markers may be better predictors of enzyme inhibitory activity than cocoa native flavanols.

Flavanol supplementation protects against obesity-associated increases in systemic interleukin-6 levels without inhibiting body mass gain in mice fed a high-fat diet.

Weight gain and obesity are associated with increased levels of proinflammatory cytokines. Studies have demonstrated the ability of dietary flavanols to reduce the severity of metabolic derangements due to high-fat (HF) feeding. The degree of polymerization of the flavanols appears to play a role in determining the extent of these protective effects. This study evaluated the preventative effects of grape seed and pine bark flavanol supplementation, with significantly different flavanol degree of polymerization, in the context of an HF diet. For 13 weeks, mice were given 35 mg/kg body weight per day grape seed or pine bark as part of an HF diet and compared to mice fed a low-fat diet and control HF diet. All flavanol-supplemented groups and the HF control incurred significantly higher weight gain compared to the lean control, and the grape seed group gained significantly more weight than the HF control. Increased weight gain of treatment groups was likely caused by hyperphagia. Despite lack of improvements to weight gain and glycemic control, it was observed that all flavanol treatment groups were able to significantly reduce interleukin-6 compared to HF control. The grape seed group, which gained the most weight overall, also exhibited the lowest levels of interleukin-6 compared to other groups. Overall, low-dose flavanol extract supplementation, regardless of mean degrees of polymerization, blunted cytokine production despite increased weight gain. This obesity-independent effect suggests flavanols may be used as complementary interventions to ameliorate increased inflammatory tone in the contexts of obesity and diabetes. Furthermore, flavanol-induced hyperphagia may have use for attenuation of cachexia.

Development of a rapid ultra performance hydrophilic interaction liquid chromatography tandem mass spectrometry method for procyanidins with enhanced ionization efficiency.

Cocoa flavanols (catechins and procyanidins) can exist in various polymerization states and are commonly classified by their degree of polymerization (DP). There is increasing evidence that flavanols of distinct DP possess different biological activities, but separation and quantification of the higher DP procyanidins is challenging and has thus created the need for new methodologies that utilize advancements in columns and LC-MS/MS systems. An aqueous normal phase (hydrophilic interaction liquid chromatography, HILIC), UPLC method with post-column ESI adjuvant infusion was developed to reduce the total analysis time, increase peak separation, and increase detection specificity (compared to traditional fluorescence methods) by coupling with mass spectrometry detection. The total elution time was reduced from 70 to 90 min (typically used for normal phase and HILIC HPLC separation of procyanidins) down to 9 min by employing UPLC. Results indicate that by using a post-column 0.04 M ammonium formate infusion (5 µL/min), ionization of procyanidins was significantly enhanced. Lower limits of detection ranged from 3.19 × 10-2 to 4.56 pmol-on-column, and lower limits of quantification ranged from 2.79 × 10-2 to 1.17 × 102 pmol-on-column across compounds DP 1-9. This method builds upon the foundation set by existing analytical methods and employs new technologies to dramatically increase sample throughput and enhance detection limits and specificity, facilitating improved analysis for procyanidins.

Development and Characterization of a Pilot-Scale Model Cocoa Fermentation System Suitable for Studying the Impact of Fermentation on Putative Bioactive Compounds and Bioactivity of Cocoa.

Cocoa is a concentrated source of dietary flavanols-putative bioactive compounds associated with health benefits. It is known that fermentation and roasting reduce levels of native flavonoids in cocoa, and it is generally thought that this loss translates to reduced bioactivity. However, the mechanisms of these losses are poorly understood, and little data exist to support this paradigm that flavonoid loss results in reduced health benefits. To further facilitate large-scale studies of the impact of fermentation on cocoa flavanols, a controlled laboratory fermentation model system was increased in scale to a large (pilot) scale system. Raw cocoa beans (15 kg) were fermented in 16 L of a simulated pulp media in duplicate for 168 h. The temperature of the fermentation was increased from 25⁻55 °C at a rate of 5 °C/24 h. As expected, total polyphenols and flavanol levels decreased as fermentation progressed (a loss of 18.3% total polyphenols and 14.4% loss of total flavanols during fermentation) but some increases were observed in the final timepoints (120⁻168 h). Fermentation substrates, metabolites and putative cocoa bioactive compounds were monitored and found to follow typical trends for on-farm cocoa heap fermentations. For example, sucrose levels in pulp declined from >40 mg/mL to undetectable at 96 h. This model system provides a controlled environment for further investigation into the potential for optimizing fermentation parameters to enhance the flavanol composition and the potential health benefits of the resultant cocoa beans.