Source: PENNSYLVANIA STATE UNIVERSITY submitted to
IMPORTANCE OF POORLY ABSORBED POLYPHENOLS FOR THE MITIGATION OF GASTROINTESTINAL INFLAMMATION BY COCOA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1018580
Grant No.
2019-67017-29251
Project No.
PENW-2018-07954
Proposal No.
2018-07954
Multistate No.
(N/A)
Program Code
A1341
Project Start Date
Mar 1, 2019
Project End Date
Feb 28, 2023
Grant Year
2019
Project Director
Lambert, J. D.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Food Science
Non Technical Summary
The US cocoa and chocolate industries are economically important. Cocoa contains large amounts of monomeric polyphenols and polymeric proanthocyanidins (PaCs). These compounds exist in both a free (ExPP) form and bound to cell material (NonExPP) and have varying bioavailability. Whereas monomeric ExPP are absorbed from the small intestine, PaCs with higher degree of polymerization (HmwPaCs) and NonExPP are poorly absorbed. These compounds reach the lower gastrointestinal (GI) tract and interact with GI tissues and/or are metabolized by microflora to systemically-available metabolites. Cocoa polyphenols can mitigate GI inflammation in vivo, however, whether these GI-related health effects are due to the systemically-available polyphenols, the interaction between unabsorbed polyphenols and GI tissues, the microbial metabolites, or a combination of these remains unclear. We hypothesize that HmwPaCs and NonExPPs, which have low bioavailability, are primarily responsible for the anti-inflammatory effects of cocoa in the GI tract. We will (1) assess the bioaccessibility of cocoa polyphenol fractions and their microbial metabolites in vitro, (2) determine the efficacy of these fractions in mouse models of GI inflammation, and (3) determine the importance of microbe-derived metabolites for modulation of GI inflammation. This research will provide insight into which polyphenolic fractions are critical for the anti-inflammatory effects of cocoa and probe the underlying mechanisms of action. This work will aid in identifying compounds that may be used as health-related quality control markers to aid processors in maximizing the health-benefits of their products.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70222331010100%
Goals / Objectives
Cocoa contains large amounts of polyphenols (PPs). Monomeric catechins and polymeric proanthocyanidins (PaCs) ranging from degree of polymerization (DP) = 2 to 10 are the most abundant cocoa PPs. These compounds can exist as both a free/solvent extractable (ExPP) form as well as bound to cell material (NonExPP) in the cocoa nib and, as such, have varying digestibility and bioavailability. For example, some small molecular weight ExPP are comparatively well-absorbed from the small intestine and are thus systemically available in humans. By contrast, higher DP PaCs (HmwPaCs) and NonExPP in the cocoa powder are poorly absorbed from the small intestine, but are able to directly interaction with the gut epithelium. NonExPP and HmwPaCs reach the distal gastrointestinal (GI) tract where they are also available for metabolism by GI microbiota resulting in the production of small molecular weight, bioavailable metabolites (microbial PP metabolites [mPPM]). Fermentation of cell wall material in the colon also releases the NonExPP, facilitating specific targeting of the lower GI tract. The poor bioavailability of many cocoa PPs means that the gut is likely the primary target tissue. While promising, the structural complexity, variable bioavailability, and GI biotransformation of cocoa PPs make identification of the active compounds and understanding their health effects challenging. Previous studies have demonstrated that cocoa and cocoa PPs can mitigate GI inflammation in model systems. It is, however, unclear whether the gut-related health effects of cocoa PPs are due to effects of systemically-available lower-molecular weight polyphenols, the direct interaction between HmwPaCs and/or NonExPP and GI tissues, the effects of systemically-available mPPM, or a combination of the three. Based on our preliminary data, we hypothesize that HmwPaCs and NonExPPs, which have low systemic bioavailability, are primarily responsible for the anti-inflammatory effects of cocoa in the context of inflammatory bowel disease. We propose:obj. 1. To assess the bioaccessibility, transport/metabolism, and GI protective effects of cocoa PPs and their microbial metabolites in in vitromodels of the human GI tract.obj. 2. To determine the anti-inflammatory efficacy and biotransformation of cocoa PP fractions in mouse models of inflammatory bowel disease.obj. 3. To determine the importance of microbial metabolism of cocoa PPs for the in vivo modulation of colonic inflammation.
Project Methods
We will use the following methods to accomplish our objectives:1. In vitro models of human digestion including the gastric, small intestinal, and colonic phases.2. In vitro models of gastrointestinal inflammation and gut barrier function including Caco-2 and HT-29 human colon cell lines.3. Two mouse models of gastrointestinal inflammation: dextran-sulfate sodium treated C57BL/6J mice and IL10 deficient mice. Both develop gastrointestinal inflammation that is relevant to humans.4. Germ-free and conventional mice. This will allow us to determine the role of the gut microbiome in the mitigation of gastrointestinal inflammation by cocoa.5. Molecular biological, biochemical, metabolomics, and microbiome analytical approaches will be used to determine the underlying mechanisms of action for the mitigation of gastrointestinal inflammation by cocoa.

Progress 03/01/19 to 02/28/23

Outputs
Target Audience:The target audience for this project is the cocoa processors and chocolate manufacturers; research scientists in food, nutrition, and biomedical research; public health policy makers; and the general public. Changes/Problems:The COVID-19 pandemic adversely affected progress on this project due to a 6 month closure of all research activities at Penn State followed by reduced access to laboratory facilities for an additional 6 months. Once lab operations were allowed to resume, residual delays occurred do to the need to retrain lab personnel and restart equipment. We used a no-cost extension to try to overcome the effects of COVID-19. Contrary to our expectations, we found the cocoa did not improve, and may have exacerbated DSS-induced colitis. While we pursued additional studies with this model to finalize manuscripts, we switched the majority of our efforts to the high fat-fed mouse model of obesity to study obesity-related inflammation. What opportunities for training and professional development has the project provided?This project provided research training opportunities for one PhD student, one MS student, one post-doctoral fellow, and several undergraduate researchers. The support provided by the project allowed us to purchase consumables and/or pay salary support to enable these trainees to learn techniques in food science and health-related research. The results of these research efforts provided preliminary data that allowed the PhD student to successfully compete for a USDA NIFA predoctoral fellowship. How have the results been disseminated to communities of interest?The results of this project have been disseminated by posters and presentations at national scientific meetings (Experimental Biology, Annual Meeting of the American Chemical Society, and Nutrition 2022 and 2023) and peer-reviewed scientific papers. The results were also shared with industry stakeholders through talks given at the Penn State Chocolate Short Course which was offered in June of 2022 and 2023. Approximately 25 industry members attended each offering. Finally, the results were disseminated through press releases and interviews arranged by the Penn State College of Agricultural Sciences and Penn State University public relations offices. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? For previously reported periods Lack of effect against or enhancement of dextran sulfate sodium-induced colonic inflammationWe found that cocoa and polyphenol-rich cocoa fractions failed to improve colonic inflammation in dextran sulfate sodium (DSS)-treated mice. In fact, treatment with cocoa powder and cocoa fractions enhanced DSS-induced weight loss. Cocoa powder dose-dependently enhanced the plasma levels pro-inflammatory tumor necrosis factor (TNF)a, interleukin [IL]1b, and IL4. Improvements in obesity-related inflammation We found that cocoa improved gut barrier function, gastrointestinal and hepatic inflammation, and systemic inflammation in high fat (HF)-fed obese mice. Compared to HF-fed control mice, cocoa treated mice hadreduced gastrointestinal permeability, reduced plasma endotoxin levels, and reduced expression of inflammation-related genes in the intestine. Cocoa treated mice had decreased levels of inflammation and oxidative stress related markers in the liver compared to HF-fed controls and greater expression of genes related to mitochondrial biogenesis. Although cocoa exerted anti-inflammatory effects in both male and female mice, there were sex-specific differences in the specific markers that were examined. For the final reporting period Sex-specific differences in the anti-inflammatory effect of cocoa in high fat-fed miceDietary cocoa supplementation exhibited sex-specific effects on hepatic markers of antioxidant response and endoplasmic reticulum stress in high fat (HF)-fed obese C57BL/6J mice. Obesity and hepatic lipidosis can lead to the development of ER stress. Protein disulfide isomerase (PDI, mRNA: Pdi) and endoplasmic reticulum oxidoreductin-1a (ERO1a, mRNA: Ero1a) work in tandem to reorganize inappropriate disulfide bridges in misfolded proteins as part of the unfolded protein response. As part of this reaction ERO1a generates hydrogen peroxide. We found that cocoa-treated female mice had higher hepatic mRNA expression of Pdi and reduced hepatic Ero1a expression compared to HF-fed controls. In male mice, hepatic expression of Pdi was not different between cocoa-treated mice and HF-fed control mice. Ero1a expression was higher in cocoa-treated male mice compared to HF-fed controls. Glutathione peroxidase 7 (GPX7, mRNA: Gpx7) catalyzes the detoxification of hydrogen peroxide, including that produced by ERO1a. Hepatic Gpx7 expression was higher in cocoa-treated male mice compared to HF-fed controls. Manganese-dependent superoxide dismutase (SOD2, mRNA: Sod2) detoxifies superoxide produced by the mitochondria. ER stress has been shown to enhance mitochondrial dysfunction and enhance production of superoxide. In male mice treated with cocoa, Sod2 expression was higher than in HF-fed control mice. By contrast, in female mice cocoa-treated mice had lower levels of both Sod2 and Gpx7 than HF-fed controls. Apolipoprotein B (APOB, mRNA: Apob) is an essential component of very low-density lipoprotein (VLDL) particles and plays a key role in exporting lipids from the liver and resolving fatty liver disease. ER stress has been shown to reduce hepatic APOB expression. Cocoa-treated female mice had higher Apob mRNA expression compared to HF-fed controls, whereas in female mice there was no difference in Apob levels between cocoa-treated and HF-fed control mice. Effects of cocoa fractions on obesity-related inflammation We examined the effect of dietary supplementation of HF-fed obese mice with cocoa powder (CP), extractable cocoa polyphenols (ExPP), or non-extractable cocoa polyphenols (NonExPP) on markers of colonic inflammation and fecal short chain fatty acids (SCFAs). Colonic mRNA expression of tumor necrosis factor (TNF)a, NLR family pyrin domain containing (NLRP)3, and caspase 1 (caspase 1) were examined as markers of inflammasome-mediated inflammation. Expression of these markers were not significantly different in cocoa or cocoa fraction treated mice compared to HF-fed control mice.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Weikart DK, Indukuri VV, Racine KC, Coleman KM, Kovac J, Cockburn DW, Hopfer H, Neilson AP, Lambert JD (2022) Effect of processing on the anti-inflammatory efficacy of cocoa in a high fat diet-induced mouse model of obesity. J Nutr Biochem. 109: 109117.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2023 Citation: Navare S, Lam BH, Kwasniewski MT, Anantheswaran R, Lambert JD (2023) Structural characterization of cocoa procyanidins (PACs) using LCMS-based methods. Amer. Chem. Soc. Indianapolis, IN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Weikart DK, Lambert JD (2022) Cocoa and cocoa polyphenol fractions fail to improve colitis in dextran sulfate sodium-treated mice. FASEB J. doi.org/10.1096/fasebj.2022.36.S1.R2663.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Weikart DK, Racine KC, Neilson AP, Lambert JD (2023) Sexual Dimorphism in Inflammation and Fatty Acid Metabolism in High-Fat Diet- Induced Obese Mice. FASEB J. accepted


Progress 03/01/21 to 02/28/22

Outputs
Target Audience:The results of studies conducted over the last year were presented to other food and nutrition scientists through national scientific meetings as well as seminars presented at Penn State University. We also presented our results to the general public through press releases associated with peer-reviewed publications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD student, one MS student, and one post-doctoral fellow have participated in the research supported by this project. These trainees have had the opportunity to present their results at local scientific meetings held virtually at Penn State, and the PhD student has had the chance to present at national scientific meetings (Nutrition 2021, held remotely). The MS student defended her thesis in January 2022. The PhD student wrote and was awarded a USDA NIFA predoctoral fellowship in part because of experience gained working on this project. How have the results been disseminated to communities of interest?Results have been disseminated in the form of peer-reviewed papers (one published, one under review), a paper given at a national scientific meeting, and through related press-releases from the Penn State College of Agricultural Sciences communications office. What do you plan to do during the next reporting period to accomplish the goals?In the coming year, we will complete our studies examining the effect of cocoa treatment on the expression of pro-inflammatory genes in the colon and liver of high fat-fed obese mice. We will also conduct gut microbiome analysis to determine how cocoa and cocoa polyphenols differentially affect the composition of the colonic microflora in high fat-fed obese mice. In collaboration with Dr. Andrew Neilson at North Carolina State University, we will conduct in vitro fermentation experiments with cocoa and cocoa polyphenols fractions using human fecal samples. We will compare the metabolite profile and microbiome among the different treatment groups. We will also evaluate the in vitro antiinflammatory activity of the metabolites. Experiments will be conducted using fecal samples from normal weight and obese subjects.

Impacts
What was accomplished under these goals? Objective 2. We continued analysis of tissue samples derived from male C57BL/6J mice that had been treated with cocoa polyphenol fractions prior to induction of colonic inflammation with dextran sulfate sodium. Treatment with cocoa, extractable cocoa polyphenols, and nonextractable cocoa polyphenols tended to exacerbate DSS-induced body weight loss and failed to reduced DSS-induced colonic shortening. Treatment with cocoa powder increased colonic mRNA levels of interleukin (IL) 1 beta and IL10 compared to DSS-only treatment. Treatment with extractable polyphenols or non-extractable polyphenols had no significant effect on IL1beta or IL10 mRNA levels. Treatment with nonextractable polyphenols tended to increase colonic mRNA levels of IL6. Objective 2. We initiated a new study using a high fat-fed mouse model of obesity-related colonic inflammation. Male C57BL/6J mice were fed diet containing 60%kcal from fat diet for 8 weeks to induce obesity. Mice were then given high fat diet supplemented with cocoa powder or an equivalent dose of extractable or nonextractable polyphenols for 8 additional weeks. Control mice were maintained on high fat diet. Compared to HF-fed mice, mice treated with cocoa powder had significantly lower final body weight. Mice fed extractable or nonextractable polyphenols tended to have lower final body weights, but these treatments were less effective than cocoa powder and did not reach statistical signficance. Cocoa treatment but not polyphenol fraction treatments reduced spleen wight compared to HF-fed control mice. Colon length was not different among the treatment groups. We are currently examining the expression of pro-inflammatory genes in the colons of mice treated with either cocoa powder or the cocoa polyphenol fractions.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Sun M, Gu Y, Glisan SL, Lambert JD (2021) Dietary cocoa ameliorates non-alcoholic fatty liver disease and increases markers of antioxidant response and mitochondrial biogenesis in high fat-fed mice. J Nutr Biochem. 92:108618.
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Weikart DK, Indukuri VV, Racine KC, Coleman KM, Kovac J, Cockburn DW, Hopfer H, Neilson AP, Lambert JD (2021) Effect of processing on the anti-inflammatory efficacy of cocoa in a high fat diet-induced mouse model of obesity. J Nutr Biochem. under review.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Weikart D, Indukuri V, Racine K, Neilson A, Lambert J (2021) Impact of Processing on the Anti-inflammatory and Gastrointestinal Protective Effects of Cocoa in High Fat-Fed Obese Mice. Curr Dev Nutr 5(S2):380


Progress 03/01/20 to 02/28/21

Outputs
Target Audience:The target audience for our project are (1) researchers in the area of food and heath; (2) public health decision makers who develop dietary recommendations; (3) stakeholders in the cocoa and chocolate processing industry; and (4) the general public. Changes/Problems:1. Disruption due to COVID-19. Research activities at Penn State were suspended from April 2020 - July 2020. We were not able to do work in our labs during this time. Beginning in July 2020, research was allowed to resume but on a limited basis (no more than 2 people in the lab at one time and strict social distancing protocols in place) and with many core supporting labs working with reduced staff and capacity. These events lead to significant disruptions in our ability to collect data and also compromised graduate student training. As a result, even after research was allowed to resume, it took 3 - 4times longer for a student to master a new technique and 2 - 3 times longer to collect data. We have therefore made significantly less progress than anticipated. Since January 2021, Penn State has begun to reopen and lab occupancy levels have increased. If the number of COVID-19 cases continues to fall, then we expect to return to normal levels of lab access and productivity in 2021/2022. 2. Lack of anti-inflammatory efficacy in the DSS-induced mouse model of colitis.The results of our studies in the DSS-induced mouse model of colitis indicate that cocoa and cocoa polyphenol fractions do not exert significant anti-inflammatory effects in this model. Some of our data (e.g. plasma cytokine levels) suggest that cocoa may exacerbate DSS-induced colitis. These results are in contrast to what we and others have observed in the high fat-fed mouse model of obesity. In that model cocoa can reduce markers of inflammation, metabolic endotoxemia, and gut barrier dysfunction. We hypothesize that this difference in efficacy is due to differences in the severity of gastrointestinal inflammation induced by DSS compared to obesity. We plan to summarize our results and submit them for publication, but we would like to pivot from using the DSS-induced mouse model of colonic inflammation and instead continue our research using the high fat diet-induced mouse model of obesity-related inflammation. Given that thehigh fat diet model is more responsive to the anti-inflammatory effects of cocoa, it will provide a more effective model for accomplishing our proposed goals of (1) identifying the role of non-extractable cocoa polyphenols in the anti-inflammatory effects of cocoa and (2) evaluating the importance of the gut microbiome in mediating these effects. Given our extensive experience with the high fat-fed mouse model, we expect that we will make significant progress towards our original goals even with the switch to the new model. We plan to continue our in vitro studies as written in the original proposal, but will compare the efficacy of whole cocoa powder, extractable polyphenols, and nonextractable cocoa polyphenols in high fat-fed obese mice. What opportunities for training and professional development has the project provided?One PhD student, one MS student, and one post-doctoral fellow have participated in the research supported by this project. These trainees have had the opportunity to present their results at local scientific meetings held virtually at Penn State, but have not had the chance to present at national scientific meetings due to (1) delays in completing experiments and (2) travel restrictions caused by COVID-19. How have the results been disseminated to communities of interest?The results of this project have been disseminated at local scientific meetings held remotely at Penn State. These include Penn State Graduate Exhibition and the Gamma Sigma Delta Research Expo. What do you plan to do during the next reporting period to accomplish the goals?Objective 1.We will continue our efforts to generate microbial metabolites of cocoa polyphenols using an in vitro fecal fermentation system. The resulting metabolites will be purified and used for studies with in vitro models of gastrointestinal inflammation. Objective 2.The results of our studies to date have shown that cocoa and cocoa fractions do not have significant anti-inflammatory activity in the DSS-induced mouse model of colonic inflammation. We speculate that this may be due to the severity of the inflammatory response induced by DSS. We plan to continue our proposed studies in the high fat diet-induced mouse model of obesity. This model exhibits signs of colonic inflammation and loss of gut barrier function, but the pathologies are less severe than the DSS-induced model. Objective 3.We will use the data obtained from additional experiments in objective 2 to modify our plans for Objective 3. Since the cocoa did not produce a robust anti-inflammatory effect in the DSS-induced mouse model, we will refocus our studies for objective 3 on obesity-related colonic inflammation and gut barrier dysfunction.

Impacts
What was accomplished under these goals? Gastrointestinal microbial metabolism of cocoa polyphenols (Objective 1). We have established the in vitro human fecal fermentation model to prepare microbial metabolites of cocoa polyphenols. We have not yet begun the process of identifying and purifying the metabolites present. Dose-dependent anti-inflammatory effects of cocoa in the dextran sulfate sodium (DSS)-induced mouse model of colonic inflammation (Objective 2). We examined the effect of dietary treatment with 0 - 80 mg/g cocoa powder (2 wk pre-treatment plus 1 wk concurrent treatment) in male C57BL/6J mice given DSS as the sole source of drinking fluid. Our hypothesis, based on previous studies in high fat-fed obese C57BL/6J mice was that cocoa would dose-dependently reduce markers of systemic and colonic inflammation. Cocoa treatment lead to a dose-dependent decrease in final body weight compared to mice given DSS only (8.3% lower in mice treated with 80 mg/g cocoa). Plasma levels of tumor necrosis factor (TNF)a and interleukin (IL)1b were increased in a dose-dependent manner compared to those in mice treated with DSS only. Plasma levels of IL4 and interferon (INF)g and IL4 also tended to increase in a dose-dependent manner, but the trend was not statistically significant. No significant trend was observed for IL2, IL6, IL10, and KC/GRO. No significant effect of cocoa treatment on colonic Tnfa and Il1b mRNA levels was observed. These results suggest that, in contrast to our hypothesis, that cocoa treatment does not exert an anti-inflammatory effect in DSS-induced mice and may, in fact, make exacerbate inflammation. Anti-inflammatory effects of cocoa polyphenol fractions in the DSS-induced mouse model of colonic inflammation (Objective 2). To ascertain whether different subfractions might have anti-inflammatory activity in DSS-induced mice even though whole cocoa powder did not have a beneficial effect, we compared the anti-inflammatory efficacy of the acetone/water-extractable polyphenols (ExPP) with that of the non-extractable polyphenols (NonExPP) that were associated with cell wall material. Whole cocoa powder was used as a control. Male C57BL/6J mice were treated with control diet, cocoa powder (80 mg/g diet), or the equivalent dose of ExPP or NonExPP for 2 wks prior to inducing colonic inflammation for 1 wk using DSS. Cocoa treatments continued during the induction period. As observed in our dose-response study, cocoa treatments failed to mitigate DSS-induced decreases in final body weight (7.4% lower than non-DSS treated control). Treatment with cocoa (29% lower), ExPP (32% lower), and NonExPP (20% lower) did significantly reduce relative spleen weight compared to DSS-induced mice, but did not impact colon length or gastrointestinal barrier function as measured by (FITC-dextran permeability). With the exception of positive effects on relative spleen weight, it appears that cocoa, ExPP, and NonExPP do not exert significant anti-inflammatory effects in DSS-induced mice. These results of our studies in the DSS-induced mouse model of colonic inflammation are in stark contrast with previous studies that have examined the impacts of cocoa on gastrointestinal inflammation and barrier function in the high fat diet induced mouse model of obesity. It is possible that this difference in efficacy is due to the severity of inflammation induced in these two models. Whereas DSS induces severe inflammation and leads to ulceration and bleeding in the colon, obesity-related colonic inflammation is comparatively mild and does not manifest as gross pathological changes. It is possible that under these more severe inflammatory conditions, the polyphenols in cocoa act as pro-oxidants and perpetuate damage. We have not initiated studies under objective 3.

Publications


    Progress 03/01/19 to 02/29/20

    Outputs
    Target Audience:The objectives of this project and the nature of the on-going activities have been shared with other scientists in the field of food and health researchers at the International Conference on Food Factors in Kobe, Japan. We have also discussed our on-going activities with stakeholders in the cocoa and chocolate industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate student and one graduate student performed the experiments intiated on this project so far. Both individuals learned new laboratory techniques and had the opportunity to practice their presentation skills in lab meetings. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We have the following activities planned and initiated to make progress on accomplishing our research goals in the next reporting period: 1. We have on-going experiments to analyze changes induced by cocoa in markers of colonic inflammation and the gut microbiome in a mouse model of colitis. (Objective 2) 2. We have begun to set up an in vitro fecal fermentation system to (1) study the imapact of cocoa fractions on the gut microbiome and (2) the metabolism of cocoa polyphenols and other components by the gut microbiome. (Objective 1) 3. We will complete the fractionation of cocoa samples begun during the first reporting period and initiate studies in mice to compare the anti-inflammatory activity of the different fractions. (Objective 2 and 3)

    Impacts
    What was accomplished under these goals? Impact: The proposed studies will provide insight into the role of different components in cocoa in the anti-inflammatory effects of the whole product. This information will be useful to cocoa processors who are interested in modifying processing paramaters to maximize the content of the most bioactive components in cocoa powder. The studies will also provide insight into the potential anti-inflammatory efficacy of cocoa powder in the context of inflammatory bowel disease. This information will be useful to public health officials and dieticians in developing dietary recommendations to ameliorate iflammatory bowel disease. Objective 1:We have begun the process of preparing cocoa fractions for in vitro digestion and in vitro bioaccessibility studies. Our work is currently focused on optimizing the yield of each fraction and performing chemical analysis to characterize the components. Preparation of these fractions are on-going and we have no data to report at this time. Objective 2: We have conducted an initial experiment to evaluate the dose-dependent anti-inflammatory activity of cocoa powder in dextran sulfate sodium (DSS)-treated C57BL/6J mice. Male mice (n = 10/dose) were treated with 0, 20, 40, or 80 mg/g diet cocoa powder for 2 wks. Mice were then given 1.5% DSSas the sole source of drinking fluidfor 7 days to induce colonic inflammation. Cocoa treatment continued during DSS treatment. Body weight was collected daily. After DSS treatment, mice were euthanized and blood and tissue samples were collected. Colon length and spleen weight were determined as markers of inflammation. Cocoa-treated mice, at all dose levels, had significantly smaller spleen weights than the DSS-treated control mice (p < 0.05) indicating reduced inflammation. Cocoa treatment dose-dependently increased weight during the DSS-treatment period (p = 0.003 for trend). Objective 3.We have not begun the activities listed under this objective.

    Publications