IDENTIFICATION AND MECHANISM OF ANTHOCYANINS FROM FOODS [INCLUDING COLORED CORN] WITH ANTIDIABETIC POTENTIAL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1014457
• Project No.: ILLU-698-326
• Project Start Date: Jan 8, 2018
• Project End Date: Sep 30, 2022

Project Director
de Mejia, EL.

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
Food Science & Human Nutrition

Non Technical Summary
Corn (Zea mays L.) is the most widely produced grain in the United States. Anthocyanins (ANC) present in colored cornare natural pigments with food colorant potential that exert different health benefits. However there is a gap in knowledge related to the effect of individual ANC in type-2 diabetes (T2DM), a disease that costs more than $245 billion to the U.S. There is also a need forinformation about the relationship of the ANC composition in colored corn and their antidiabetic potential. Furthermore, there is a need to confirm these effects in a more complex biological system, such as an animal model. The first aim of this study looks for the determination of the antidiabetic potential of individual ANC from colored corn in vitro using different dual-cell culture systems. The second aim will establish the relationship between ANC chemical composition of different varieties of colored corn with their antidiabetic potential. A third aim focuses on the confirmation of the antidiabetic potential of ANC from colored corn and the molecular mechanisms involved in an animal model. This research is relevant since it focuses on the use of dietary bioactive components to decrease T2DM, a disease that causes more than 70,000 deaths per year, affecting approximately 13% of the American population.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	1510	1060	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1510 - Corn;

Field Of Science
1060 - Biology (whole systems);

Keywords

anthocyanins

antidiabetic potential

colored corn

type two diabetes

Goals / Objectives
Specific goal #1: Determine the antidiabetic potential of individual anthocyanins from colored corn in vitro. Our working hypothesis is that ANC from colored corn will interact with the novel molecular targets FFAR1 (free fatty acid receptor 1) and GK (glucokinase) in an in vitro model to promote insulin secretion and glucose utilization. Specific goal #2: Establish the relationship between chemical composition of anthocyanin-rich extracts of different varieties of colored corn with their antidiabetic potential. Our working hypothesis is that the antidiabetic molecular mechanisms of various ANC-rich extracts from colored corn varieties will depend on their different ANC composition. Specific goal #3: Confirm the antidiabetic potential of anthocyanins from colored corn and the molecular mechanisms involved in an animal model. Our working hypothesis is that the ANC from colored corn will reduce T2DM in a rat model through the increase of insulin secretion and insulin sensitization to improve glucose utilization. The animal use portion of this objective will not be conducted at the University of Illinois.This part of the research will be performed as a collaboration with the Institute of Neurobiology of the National University of Mexico (UNAM, Campus Juriquilla), with register SAGARPA-SENESICA No. AUT-B-C-0815-024 and the study protocol approved by the Bioethics Committee of University of Queretaro, Queretaro, Mexico.

Project Methods
The project will be divided into three different phases according to our specific goals. In phase I we will assess the efficacy of individual bioactive compounds, including anthocyanins, from foods (including colored corn)to modulate markers related to chronic diseases using innovative in vitro experiments with intestinal, pancreatic, and hepatic dual-cell culture systems. After selecting the most promising compounds based on their bioactivities, we will move to phase II, where the relation of the chemical composition of bioactive-extracts from unique varieties of colored corn with their antidiabetic mechanism of action will be established. Furthermore, in phase III the antidiabetic potential of bioactive compounds from foods, including colored corn, will be confirmed in an animal model of rats, and the mechanisms of action will be established. It is important to clarify that the animal model work will not be performed at the University of Illinois.The proposed research is innovative because it will systematically investigate at the molecular level the role of individual bioactive compounds from foods, including colored corn, in health and wellness as it relates specifically to type-2 diabetes as well as atherosclerosis attenuation or prevention. This proposal offers transformative solutions as it is seeking to understand the role of dietary compounds extracted from foods on chronic diseases using an in vitro dual-cell culture system, an animal model, confocal immunofluorescence microscopy, and molecular docking experiments. These approaches will allow us to better understand the anti-diabetic mechanisms of dietary foods and will determine the long-term potential to ameliorate or prevent their risk. With the knowledge gained from this research, the role of dietary bioactive compounds from foods, including corn, to reduce chronic diseases will be expanded, thus contributing to the field of phytochemicals on human health.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Members of the target audience included producers, consumers, and the food industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students, one graduate student, and one postdoc were trained as a part of this project. How have the results been disseminated to communities of interest?Institute of Food Technologists, IFT20 in Chicago, IL, USA from July 12-15, 2020. Online presentation. American Society for Nutrition. Current Developments in Nutrition. June 1-4, 2020. Online presentation. What do you plan to do during the next reporting period to accomplish the goals?We plan to publish: Rebollo-Hernanz, M., Aguilera, Y., Martín-Cabrejas, M. andGonzalez de Mejia, E. 2021. Role of anthocyanins in oxidative stress and the prevention of cancer in the digestive system. Chapter 24. In: Cancer: Oxidative Stress and Dietary Antioxidants, Kings College London, Ed. Preedy & Patel -Academic Press. ISBN: 9780128195475. Grancieri, M., Stampini, H. andGonzalez de Mejia, E. 2020. Protein digests and pure peptides from chia seed prevented adipogenesis and inflammation by inhibiting PPARγ and NF-κB pathways. Nutrients nutrients-1003903 Received: 31 October 2020.

Impacts
What was accomplished under these goals? The aim was to compare the anti-obesity efficacy of different concentrations of phenolic-rich water extract from purple maize pericarp (PPE) in a murine model of obesity for twelveweeks. Forty C57BL/sixmice (n= 10/group) were randomized: standard diet (SD), high-fat diet (HFD), HFD and HFD + 500 mg PPE/kg (500 PPE). PPE contained mainly ferulic acid, anthocyanins, and other phenolics (total phenolics: 448.5 µg/mg dry weight, DW). Body weight (-27.9%), blood glucose (-26.5%), and blood triglycerides (-22.1%) were most attenuated (p < 0.05) in 500 PPE group compared to HFD group. Also, 500 PPE group had reduced (p < 0.05) plasma levels of TNF-α, MCP-1, resistin, and leptin compared to HFD group. Fatty liver disease scores were highest for HFD (8.4), followed by 200 PPE (6.1), 500 PPE (2.7), and SD (0.4) groups. Relative adipose tissue was lower (p < 0.05) in 200 PPE (7.6%), 500 PPE (8.0%), and SD (0.8%), compared to HFD (12.1%) group. In 500 PPE, compared to HFD group, important genes were modulated related to adipogenesis (Mmp3, fold-change [FC] = 7.4), inflammation (Nfkb1, FC = -1.8), and glucose metabolism (Slc2a4, FC = 23.6) in adipose tissue. In liver, 500 PPE group showed modulation of genes related to gluconeogenesis (Pck1, FC = -2.9), lipogenesis (Fasn, FC = -2.4), and β-oxidation (Cpt1b, FC = 3.1). Maize rich in ferulic acid and anthocyanins prevented obesity through the modulation of TLR and AMPK signaling pathways reducing adipogenesis and adipose inflammation, and promoting energy expenditure.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhang, Q. and Gonzalez de Mejia, E. 2020. Protocatechuic Acid Attenuates Adipogenesis-Induced Inflammation and Mitochondrial Dysfunction in 3T3-L1 Adipocytes via Regulation of AMPK Pathway. Submission ID: 835710. American Society for Nutrition. Current Developments in Nutrition 4, Issue Supplement_2, 495-495.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Gonzalez de Mejia, E. and Mazewski, C. 2020. Black Lentil Water Extract Inhibited Inflammatory Cytokines in a Colitis-Associated Colon Carcinogenesis Model. Submission ID: 837347. American Society for Nutrition. Current Developments in Nutrition 4, Issue Supplement_2, 317-317.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, Q. and, Gonzalez de Mejia, E. 2020. Protocatechuic Acid Attenuates Adipogenesis-Induced Inflammation and Mitochondrial Dysfunction in 3T3-L1 Adipocytes by Regulation of AMPK Pathway. Journal of Functional Foods 69, 103972.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Luna-Vital, D., Luzardo-Ocampo, I., Cuellar-Nu�ez, L., Loarca-Pina, G. and Gonzalez de Mejia, E. 2020. Maize Extract Rich in Ferulic Acid and Anthocyanins Prevented High-Fat Induced Obesity in Mice by Modulating SIRT1, AMPK, and IL-6 Associated Metabolic and Inflammatory Pathways. The Journal of Nutritional Biochemistry 79, 108343.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Luna-Vital, D.A. and de Mejia, E.G. 2020. Ferulic Acid and Anthocyanin from Colored Maize Correlated with Prevention of High-Fat Induced Obesity in Mice by Modulating Lipid, Glucose and Inflammatory Pathways. Submission ID: 830250. American Society for Nutrition. Current Developments in Nutrition 4, Issue Supplement_2, 433-433.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kowalski, R. and Gonzalez de Mejia, E. 2020. Blackcurrant (Ribes nigrum) Characterization and Fermented Product Development. IFT20 in Chicago, IL, USA from July 12-15, 2020. Online presentation.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kowalski, R., Gustafson, E., Carroll, M. and Gonzalez de Mejia, E. 2020. Enhancement of Biological Properties of Blackcurrants by Lactic Acid Fermentation and Incorporation into Yogurt: A Review. Antioxidants 9(12), 1194.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Luzardo Ocampo, I., Loarca-Pina and Gonzalez de Mejia, E. 2020. Gallic and Butyric Acids Modulated NLRP3 Inflammasome Markers in a Co-Culture Model of Intestinal Inflammation. Food and Chemical Toxicology 146, 111835https://doi.org/10.1016/j.fct.2020.111835.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Mazewski, C., Luna-Vital, D., Berhow, M. and Gonzalez de Mejia, E. 2020. Reduction of Colitis-Associated Colon Carcinogenesis by a Black Lentil Water Extract Through Inhibition of Inflammatory and Immunomodulatory Cytokines. Carcinogenesis 41 (6), 790803.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Members of the target audience included producers, consumers, and the food industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students, one graduate student, and one postdoc were trained as a part of this project. How have the results been disseminated to communities of interest? American Society for Nutrition (June 8-11, 2019, Baltimore, MD). Gonzalez de Mejia, E. 2018. Biological activity of anthocyanins.In: The Future of Food Color. International Association of Color Manufacturers (IACM). Global Color Conference, held at The Ritz-Carlton, Pentagon City in Arlington, VA on November 6-7, 2018. Gonzalez de Mejia, E. andMazewski, C., 2019. Black lentil aqueous extract attenuates colitis-associated colon carcinogenesis in mice.American Society for Nutrition (June 8-11, 2019, Baltimore, MD).Current Developments in Nutrition, 3, Issue Supplement_1, June 2019, nzz030.OR04-06-19. Mazewski, C. andGonzalez de Mejia, E. 2018. Impact of anthocyanins on colorectal cancer. In: American Chemical Society Books. Book: "Chemistry and Biological Activities of Phenolic Compounds from Fruits and Vegetables". Chapter 19, pp 339-370. DOI: 10.1021/bk-2018-1286.ch019. ISBN13: 9780841232969; eISBN: 9780841232952. What do you plan to do during the next reporting period to accomplish the goals?We intend to publish the following manuscript: Luna-Vital, D., Luzardo-Ocampo, I., Cuellar-Nuñez, L., Loarca-Pina, G. andGonzalez de Mejia, E. 2020. Maize extract rich in ferulic acid and anthocyanins prevented high-fat induced obesity in mice by modulating SIRT1, AMPK, and IL-6 associated metabolic and inflammatory pathways. The Journal of Nutritional Biochemistry.

Impacts
What was accomplished under these goals? This study aimed to investigate the associations between phenolic composition of selected purple maize genotypes and their anti-inflammatory, anti-adipogenic and anti-diabetic properties in vitro. Anthocyanin-rich water extracts (PMWs) from 20 purple maize genotypes were evaluated in RAW 264.7 macrophages and 3T3-L1 adipocytes under different conditions. Cyanidin-3-O-glucoside (C3G), pelargonidin-3-O-glucoside (Pr3G), peonidin-3-O-glucoside (P3G) and corresponding acylated forms were major anthocyanins in PMW, accompanied by ten tentatively identified non-anthocyanin phenolics. Correlation studies showed that C3G, P3G and derivatives, but not Pr3G and its acylated form contributed to the biological properties of PMW. Besides anthocyanins, quercetin, luteolin and rutin were the dominant anti-inflammatory and anti-diabetic components, in terms of down-regulating pro-inflammatory mediator production in inflamed macrophages and adipocytes, modulating diabetes-related key enzymes and improving insulin sensitivity in insulin-resistant adipocytes. Quercetin and phenolic acids, especially vanillic acid and protocatechuic acid, were closely associated with anti-adipogenic properties of PMW via inhibition of the preadipocyte-adipocyte transition.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Luna-Vital, D., Chatham, L., Juvik, J., Singh, V., Somavat, P. and de Mejia, E. 2019. Activating effects of phenolics from Apache Red zea mays L. on free fatty-acid receptor-1 and glucokinase evaluated with a dual culture system with epithelial, pancreatic and liver cells. Journal of Agricultural and Food Chemistry 67, 9148-9159. Special Issue: Health-Promoting Food Ingredients.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Mazewski, C., Kim, S. and Gonzalez de Mejia, E. 2019. Anthocyanins and their metabolites inhibit colorectal cancer cell progression and stimulate an immune response in vitro and in silico. Scientific Reports 9 (1), 11560. https://doi.org/10.1038/s41598-019-47903-0.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, Q., Luna-Vital, D. and Gonzalez de Mejia, E. 2019. Anthocyanins from colored maize ameliorated the inflammatory paracrine interplay between macrophages and adipocytes through regulation of NF-kappaB and JNK-dependent MAPK pathways. Journal of Functional Foods. 54, 175-186.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, Q., Gonzalez de Mejia, E., Luna-Vital, Tao, T., Chandrasekaran, S., Chatham, L., Juvik, J., Singh, V. and Kumar, D. 2019. Relationship of phenolic composition of selected purple maize (Zea mays L.) genotypes with their anti-inflammatory, anti-adipogenic and anti-diabetic potential. Food Chemistry. 289, 739-750.

Progress 01/08/18 to 09/30/18

Outputs
Target Audience:Members of the target audience included producers, consumers, and the foodindustry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Participation and networking in scientific meetings: Americal Chemical Society. Division of Agricultural and Food Chemistry. Oral presentation by invitation. August 19, 2018 - Boston Convention and Exhibition Center. American Chemical Society. Division of Agricultural and Food Chemistry. Natural Alternatives to Artificial Food Additives. Oral presentation. August, 23, 2017 - Walter E. Washington Convention Center. American Society for Nutrition (June 9-12, 2018, Boston). How have the results been disseminated to communities of interest?Presentation in scientific meetings: Luna-Vital, D. A and, de Mejia, E.G. 2018. Current evidence of beneficial effects of anthocyanins from colored corn in in vitro models of obesity and diabetes (AGFD 59). In: Health Promoting Food Ingredients. Division of Agricultural and Food Chemistry. Oral presentation by invitation. August 19, 2018 - Boston Convention and Exhibition Center. de Mejia, E. andLuna-Vital, D. 2017. Stabilization of anthocyanins with food pigment potential and their insulin sensitizing effect in adipocytes under inflammatory status (final paper number: AGFD 219) PAPER ID: 2739944. Division of Agricultural and Food Chemistry. Natural Alternatives to Artificial Food Additives. Oral presentation. August, 23, 2017 - Walter E. Washington Convention Center. Luna-Vital, D. and Gonzalez de Mejia, E. 2018. Activation of novel type-2 diabetes markers free fatty acid receptor-1 and glucokinase by anthocyanins from colored corn in vitro. American Society for Nutrition (June 9-12, 2018, Boston). Abstract ID: 435046. What do you plan to do during the next reporting period to accomplish the goals?Continue with the analyses of the samples from the animal study and submit for publication the following manuscripts: Zhang, Q., Luna-Vital, D. and Gonzalez de Mejia, E. 2018. Anthocyanins from colored maize ameliorated the inflammatory paracrine interplay between macrophages and adipocytes through regulation of NF-κB and JNK-dependent MAPK pathways. Luna-Vital, D., Chatham, L., Juvik, J., Singh, V., Somavat, P. and de Mejia, E. 2019. Water soluble phenolics from an improved variety of Apache red maize activated free fatty acid receptor-1 and glucokinase in vitro. Journal of Agricultural and Food Science. Zhang, Q., Gonzalez de Mejia, E., Luna-Vital, Tao, T., Chandrasekaran, S., Chatham, L., Juvik, J., Singh, V. and Kumar, D. 2019. Anthocyanins from colored maize ameliorated the inflammatory paracrine interplay between macrophages and adipocytes through regulation of NF-kB and JNK-dependent MAPK pathways.

Impacts
What was accomplished under these goals? The objective of this study was to evaluate the ability of anthocyanins (ANC) present in purple corn to enhance insulin secretion and hepatic glucose uptake in pancreatic cells and hepatocytes, through activation of the free fatty acid receptor-1 (FFAR1) and glucokinase (GK), respectively. Using a dual-layer cell culture with Caco-2 cells, INS-1E or HepG2 cells were treated with an anthocyanin-rich extract from the pericarp of purple corn (PCW), as well as pure ANC cyanidin-3-O-glucoside (C3G), peonidin-3-O-glucoside, pelargonidin-3-O-glucoside. Delphinidin-3-O-glucoside (D3G) was used for comparative purposes. Semipurified C3G (C3G-P) and condensed forms (CF-P) isolated from PCW were also used. At 100 μM, the pure ANC enhanced glucose-stimulated insulin secretion (GSIS) in INS-1E cells ranging from 18% to 40% (p<0.05) compared to untreated cells. PCW increased GSIS by 51%. D3G was the most effective anthocyanin activating FFAR1 (EC50: 196.6 μM). PCW had activating potential on FFAR1 (EC50: 77 μg/mL). PCW, as well as C3G and D3G increased the expression of FFAR1, PLC, and phosphorylation of PKD, related to the FFAR1-dependent insulin secretory pathway. The treatment with 100 μM of P3G and C3G increased (p<0.05) glucose uptake in HepG2 cells by 19% and 31%. PCW increased the glucose uptake in HepG2 cells by 48%. It was determined that CF-P was the most effective for activating GK (EC50: 39.9 μM) and the PCW extracts had an efficacy of EC50: 44 μg/mL. The ANC in purple corn also reduced AMPK phosphorylation and PEPCK expression in HepG2 cells, known to be related to reduction in gluconeogenesis. It is demonstrated for the first time that dietary ANC can enhance the activity of novel biomarkers FFAR1 and GK and potentially ameliorate type-2 diabetes comorbidities.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Luna-Vital, D.A. and Gonzalez de Mejia, E. 2018. Anthocyanins from purple corn activate free fatty acid-receptor 1 and glucokinase enhancing in vitro insulin secretion and hepatic glucose uptake. PLoS ONE 13(7): e0200449. https://doi.org/10.1371/journal.pone.0200449.