Research Project: Elucidating Phytonutrient Bioavailability, Health Promoting Effects and Mechanisms of Existing/Emerging Foods and Beverages

Location: Diet, Genomics and Immunology Laboratory

2022 Annual Report

Objectives
Objective 1: Determine the accumulation and variability of important bioactive compounds in commonly consumed food crops as affected by factors such as stage of maturity (leafy greens harvested at various stages of maturity) and variety/processing (coffee products derived from different sources). [NP107, C1, PS1A] Objective 2: Determine bioavailability and cellular uptake of potential compounds in coffee products; investigate their effects on subclinical inflammation and its associated events related to chronic metabolic diseases; elucidate mechanisms of action on subclinical inflammation and related events. [NP107, C3, PS3B] Objective 3: Determine effects of brassica vegetables harvested at different stages of maturity (e.g., sprout, microgreen, baby green, mature plant) on high fat diet-induced inflammation, and adipose uncoupling protein 1 (UCP1) as mechanisms for their attenuation of high fat diet-induced weight gain. Elucidate the role of the microbiome in mediating changes in inflammation and liver lipid metabolism. [NP107, C3, PS3B]

Approach
Objective 1: Amounts of bioactive compounds in selected model plant products (e.g., coffee beans grown in different regions/conditions, coffee products, brassica vegetables harvested at different stages of maturity) will be determined using established HPLC, MS/MS and NMR methods. Objective 2: Both in-vivo and in-vitro models will be used for this objective. (1) We will determine bioavailability and cellular uptake of coffee compounds using cell culture (e.g., Caco-2, HepG2, monocytic THP-1) models. HPLC, metabolomic and lipidomic analytical technologies will be used to measure the compounds and associated metabolites. (2) We will determine potential effects of coffee/coffee chemicals (javamide-I/-II) on subclinical inflammation markers using a rodent model. Obesity will be induced in animals (e.g., rats) fed a high-fat diet, and the potential effects of coffee and coffee compounds (e.g., javamide-I/-II) on obesity-associated subclinical inflammation and biological changes will be determined to elucidate the effects. (3) We will determine the cellular/molecular mechanisms responsible for the biological effects using cell culture models. Cell will be treated with the compounds and effects of compounds on cellular pathways related to signal transduction pathways, inflammatory cytokines, adhesion molecular, transcriptional factors will be assessed at protein and message levels using western blots, ELISA and RT-PCR. Objective 3: We will determine effects of brassica vegetables harvested at different stages of maturity (e.g., sprout, microgreen, baby green, mature plant) on high fat diet-induced rodent model. Inflammatory marker and adipose uncoupling protein 1 (UCP1) will be assessed using ELISA at protein level and RT-PCR at message level as mechanisms for their attenuation of high fat diet-induced weight gain. Biochemical and marker genes analysis will be performed in liver and adipose tissues to assess the effects on lipid and energy metabolism. Metagenomic analysis using next generation sequencing technology will be performed to elucidate the role of the microbiome in mediating changes in inflammation and liver lipid metabolism.

Progress Report
This report is for a NP107 project entitled "Elucidating Phytonutrient Bioavailability, Health Promoting Effects and Mechanisms of Existing/Emerging Foods and Beverages." We focus on two foods, coffee and kale, at different growth/maturation states. The following describes the current progress of the project. For Objective 1, we investigated the amounts of javamide-I/-II in Arabica and Robusta coffee beans and coffee products (instant and ground coffees) in the market using HPLC and MS/MS methods. Also, principal component analysis (PCA) was performed using the variables of bio-active compounds (javamide-I/-II, chlorogenic acids, caffeine) in the products. The data showed that there is a significant disparity in the amounts of javamide-I/-II in the coffee products in the market, suggesting that coffee products in the market may not provide the same purported health effects. These data suggest that the amounts of javamide-I/-II may be an important factor in the potential for different coffees to improve health and should be investigated further. Food bioactive components, such as flavonoids and glucosinolates, contribute to health effects of a diet and are subject to regulation by the environment. However, their biological activity is content and amount dependent. Therefore, it is important to identify and document the content and amounts of bioactive components in foods. Cruciferous vegetable microgreens are new foods available to the U.S. consumer and purportedly rich in health-promoting bioactive components. Quantitative measure of the bioactive components remains largely unknown. ARS scientists in Beltsville analyzed cruciferous vegetable microgreens grown in different conditions to assess the composition of flavonoids and glucosinolates. Kale and broccoli microgreens grown under commercial- and in-home conditions were found to have different secondary metabolite composition. The results suggested that environmental factors could strongly affect the chemical composition of cruciferous vegetables such as kale and broccoli microgreens that are related to human health promotion. For Objective 2, we investigated potential effects of coffee containing javamide-I/-II (CCJ12) on obesity, COX-I/-II enzymes, and metabolic/inflammatory factors. We also performed biological analyses of metabolic factors (bodyweight, cholesterol, HDL/LDL, lipids), inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, MCP-1), and adipokines (adiponectin) in the blood samples from the animal study. Also, investigated inflammatory cytokine-related signal transduction pathways (e.g., MAP Kinase, NF-KB). Our study suggests that CCJ12 may have no negative effects on bodyweight, LDL, HDL, total cholesterol, adiponectin, leptin, C-reactive protein, sE-selectin, rather some positive effects on inflammatory factors (e.g., TNF-alpha and MCP-1) in the rats fed a normal diet. The outcomes of this study suggest that the amounts of javamide-I/-II in coffee products may be an important factor in understanding the potential health effects of different types of coffees, which are closely associated with bioactive compounds in coffee. For Objective 3 experiment 1, we focused on performing proteomics and metabolomics analysis of tissue samples. Liver, plasma and fecal samples were harvested from mice fed with following diet groups: 1) Low fat diet (LF), 2) High fat diet (HF), 3) Low fat diet + kale microgreen (LFMG), 4) High fat diet + kale microgreen (HFMG), 5) Low fat diet + mature Kale (LFMK), 6) High fat diet + mature kale (HFMK). Livers from all diet groups were analyzed using proteomics technique of mass spec-based data independent analysis (DIA). A total of 2,052 proteins were identified in the liver samples using the DIA method. Paired comparison, using p<0.05 and 1.5-fold cut off, identified 144 proteins that were differentially expressed between the LF diet vs. the HF diet. Compared to the HF diet, supplementation of microgreen (MG) and mature kale (MK) lead to 52 and 69 differentially expressed proteins, respectively. Importantly, DIA analysis identified fatty acid binding protein 5 (FABP5) as a robust marker that allows for differentiation of mice consuming a LF diet vs. HF diet. HF diet animal’s liver FABP5 protein level was ~10x lower than that of LF diet control. Moreover, FABP5 also appeared to be differentially affected by supplementation of kale microgreen and mature kale in the diet. Supplementation of kale microgreen in the diet lead to ~8-fold lower FABP5 level then animals fed HF diet. In contrast, there were no difference in liver FABP5 levels between HF diet fed animals and mature kale supplemented animals. These data support liver FABP5 may be a candidate biomarker for consumption of HF diet. Also, vegetables with different growing stage may act mechanistically different at tissue level. Using the ingenuity pathway analysis (IPA) platform, we further identified sets of canonical pathways affected by MG or MK are different. Animals fed an MG diet appeared to exert effects on similar canonical pathway to those fed an LF diet. Additionally, liver plasma and fecal samples were analyzed using mass spec-based metabolomic techniques for lipid (plasma) and secondary metabolites (liver, plasma, feces). Bio-informatics analysis of the metabolomics results are currently in progress. For Objective 3 experiment 2, fecal samples for mice fed with following diet groups: 1) Low fat diet (LF), 2) High fat diet (HF), 3) Low fat diet + kale microgreen (LFMG), 4) High fat diet + kale microgreen (HFMG), 5) Low fat diet + mature Kale (LFMK), 6) High fat diet + mature kale (HFMK) were harvested. The samples were processed for DNA purification, library construction and currently in the que for 16S metagenomics analysis using the next generation sequencing (NGS) platform. Bio-informatic analysis of NGS data will follow to compare and elucidate the effects of various diet treatments on the gut microbiome. Rice is one of the major commodities consumed worldwide. Due to its easily digestible starch content, rice consumption may contribute to development of chronic diseases, such as obesity and diabetes. Development and identification of rice varieties containing higher amount of non-digestible starch, such as resistant starch, may help to overcome such a problem. ARS scientists in Beltsville, Maryland, in collaboration with ARS scientists from the Dale Bumper International Rice Center and Southern Regional Research Center, fed cooked rice with different resistant starch levels to diet-induced-obesity model rodents. Cooked rice with resistant starch higher than 0.44% modified the gut microbiome, and this change correlated with attenuation of obesity-related risks factors such as body weight gain and lipid profile changes. This study demonstrated potential use of high resistant starch rice varietal, as part of a healthy diet, to prevent health risks associated with diet-induced chronic diseases.

Accomplishments

Review Publications
Park, J.B. 2021. In vivo effects of coffee containing javamide-I/-II on body weight, LDL, HDL, total cholesterol, triglycerides, leptin, adiponectin, C-reactive protein, sE-selectin, TNF-alpha, and MCP-1. Current Developments in Nutrition. 6(1). https://doi.org/10.1093/cdn/nzab145.
Park, J.B. 2022. Finding a cell-permeable compound to inhibit inflammatory cytokines: Uptake, biotransformation, and anti-cytokine activity of javamide-I/-II esters. Life Sciences. 291:120280. https://doi.org/10.1016/j.lfs.2021.120280.
Muresan, L., Clapa, D., Rusu, T., Wang, T.Y., Park, J.B. 2021. Soybean callus - a potential source of tocopherols. Plants. 10:2571. https://doi.org/10.3390/plants10122571.
Wan, J., Wu, Y., Pham, Q., Li, R.W., Yu, L., Chen, M., Boue, S.M., Yokoyama, W., Li, B., Wang, T.T.Y. 2021. Effects of differences in resistant starch content of rice on intestinal microbial composition. Journal of Agricultural and Food Chemistry. 69(28):8017-8027. https://doi.org/10.1021/acs.jafc.0c07887.
Liu, Z., Shi, J., Wan, J., Pham, Q., Zhang, Z., Sun, J., Yu, L., Luo, Y., Wang, T.T.Y., Chen, P. 2021. Profiling of polyphenols and glucosinolates in kale and broccoli microgreens grown under chamber and windowsill conditions by ultrahigh-performance liquid chromatography high-resolution mass spectrometry. ACS Food Science and Technology. 2(1):101-113. https://doi.org/10.1021/acsfoodscitech.1c00355.
Feng, L., Yasmeen, R., Schoene, N., Lei, K.Y., Wang, T.T. 2019. Resveratrol differentially modulates immune responses in human THP-1 monocytes and macrophages. Nutrition Research. 72:57-69. https://doi.org/10.1016/j.nutres.2019.10.003.
Bian, X., Garber, J.M., Cooper, K.K., Huynh, S., Jones, J., Mills, M., Rafala, D., Nasarin, D., Kotloff, K.L., Parker, C., Tennant, S.M., Miller, W.G., Szymanski, C.M. 2020. Campylobacter abundance in breastfed infants and identification of a new species in the global enterics multicenter study. mSphere. 5(1):e00735-19. https://doi.org/10.1128/mSphere.00735-19.
Choe, U., Sun, J., Bailoni, E., Chen, P., Li, Y., Goa, B., Wang, T.T., Rao, J., Yu, L. 2021. Chemical composition of tomato seed flours, and their radical scavenging, anti-inflammatory and gut microbiota modulating properties. Molecules. 26(5):1478. https://doi.org/10.3390/molecules26051478.
Li, Y., Hao, Y., Gao, B., Geng, P., Huang, H., Yu, L., Choe, U., Liu, J., Sun, J., Chen, P., Wang, T.T.Y., Yu, L. 2019. Chemical profile and in vitro gut microbiota modulatory, anti-inflammatory and free radical scavenging properties of chrysanthemum morifolium cv. Fubaiju. Journal of Functional Foods. 58:114-122. https://doi.org/10.1016/j.jff.2019.04.053.
Mattes, R., Rowe, S., Ohlhorst, S., Brown, A., Brown, A., Hoffman, D., Liska, D., Feskens, E., Dhillon, J., Tucker, K., Epstein, L., Neufeld, L., Kelley, M., Fukagawa, N.K. 2022. Valuing the diversity of research methods to advance nutrition science. Advances in Nutrition. https://doi.org/10.1093/advances/nmac043.