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© 2017 Sage Willow Haggard © 2017 Sage Willow Haggard COLOR STABILITY AND PHENOLIC COMPOSITION FROM CORN PERICARP EXTRACTS IN A BEVERAGE MODEL AND PROTECTIVE AND ANTI- INFLAMMATORY POTENTIAL OF ANTHOCYANIN-RICH PLANT EXTRACTS ON CELIAC DISEASE IN VITRO BY SAGE WILLOW HAGGARD THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in Food Science and Human Nutrition with a concentration in Food Science in the Graduate College of the University of Illinois at Urbana-Champaign, 2017 Urbana, Illinois Master’s Committee: Professor Elvira Gonzalez de Mejia Professor John A. Juvik Professor Kelly Tappenden ABSTRACT Anthocyanins are increasing in popularity as natural colorants but are less stable than synthetic colorants. Celiac disease is an autoimmune disorder that affects the small intestine when gluten, a protein found in wheat, rye and barley is consumed, the immune system responds producing inflammation and inhibiting the absorption of nutrients into the body. The objective of phase I was to evaluate color stability of nine anthocyanin-rich color corn variety pericarp extracts and the objectives of phase II were to evaluate the potential of anthocyanin- rich plant extracts to protect human epithelial cells against a celiac toxic peptide (CTP) and to compare the effect of different anthocyanin extracts on inflammatory proteins using a human inflammation array. In phase I of this study we compared the color stability of nine anthocyanin-rich extracts from different colored corn varieties pericarp that were added to a beverage model and stored at different temperatures for 12 weeks. After 12 weeks at 32 °C, variety (V)6 (High condensed form, high cyanidin-3-O-glucoside (C3G), low pelargonidin (Pg), low peonidin (Pn), low in acylated forms), had the smallest percent decrease in chroma and longest anthocyanin half-life. V3 (very low condensed form, high Pg, low Pn, high C3-mal, high Pg3-mal, high Pn3-mal) and V5 (high condensed form, high C3G, low Pg, low Pn, high C3-mal, low other acylated forms) had the most favorable hue. V5 and V6 had the smallest change in color over time. These findings suggest that an abundance of condensed forms with C3G in extracts could contribute to the improved stability. However, corn extracts containing more peonidin-derived anthocyanins had reduced stability in the beverage model. The most promising corn varieties for future experiments are V3, 5, and 6 based on color retention. In phase II, healthy differentiated Caco-2 epithelial-like human cells were treated with a known ii CTP from wheat protein, alpha-gliadin H-6712 Gliadorphin-7, H-Tyr-Pro-Gln-Pro-Gln-Pro-Phe- OH (YPQPQPF). Anthocyanin-rich extracts from fruits (red and purple grape), vegetables (purple sweet potato and purple carrot), grains (colored corn, black rice, sorghum, and blue wheat), and legumes (black bean, black lentil, black peanut, and purple bean) were tested to evaluate their protective effects on Caco-2 cells treated with CTP using a cell proliferation assay. Cell protection was evaluated using 0.001-1 mg dry weight extract/mL in the presence of 25 mM CTP (C+) (IC95). A human inflammation array was performed comparing untreated Caco-2 cells (C-) with Caco-2 cells treated with C+, red corn acidified water extract (RAW), and purple corn acidified water extract (PAW) with additional ethyl acetate purification (PAWE) 1 mg/mL extract in the presence of C+. Corn varieties increased protection (60-80%) except for variety F (pelargonidin, low acylated), purple corn pure water extract (PW) and PAW increased cell protection (20-50%), and black peanut increased protection over 60% at low concentrations (p<0.05). PAWE exhibited a 3.6 fold decrease (FD) on granulocyte macrophage-colony stimulating factor (GM-CSF), both RAW and PAWE exhibited a 0.14 and 0.22 fold increase (FI) respectively on interleukin (IL) 6, RAW had a 4.3 FI on IL-11, and PAWE exhibited a 0.35 FI on metallopeptidase inhibitor 2 (TIMP-2) (p<0.05). Interleukin 11 and TIMP-2 are anti- inflammatory while IL-6 can be both anti- or pro-inflammatory and GM-CSF is pro- inflammatory. The ability of anthocyanin-rich plant extracts to inhibit CTP and JAK3 has positive implication for human health; these extracts could potentially decrease the occurrence of inflammation for gluten sensitive individuals. iii ACKNOWLEDGEMENTS Big thanks to my advisor Dr. de Mejia My mentors Natalia Galdino, Luis Mojica, Diego Luna, Grace Li My lab mates Candice Maszeski and Cheng Chen My undergraduate research assistants Kathy Johnson and Sharon Li Our lab undergrads during my time here Helen Bengston, Regina Cortez, Katie Lang, Juliet Lucient, Thomas Novak, Luis Real The Juvik lab UIUC Instructors, faculty, and staff Friends & Family iv TABLE OF CONTENTS Chapter 1: INTRODUCTION .......................................................................................................................................... 1 1.1 Overview ................................................................................................................................................... 1 1.2 Figures ...................................................................................................................................................... 4 1.3 References ............................................................................................................................................... 12 Chapter 2: REVIEW OF LITERATURE ............................................................................................................................ 14 2.1 Celiac Disease .......................................................................................................................................... 14 2.2 JAK/STAT Pathway ................................................................................................................................... 15 2.3 Anthocyanins ........................................................................................................................................... 16 2.4 Color Stability .......................................................................................................................................... 16 2.5 Figures and Tables ................................................................................................................................... 19 2.6 References ............................................................................................................................................... 22 Chapter 3: HYPOTHESIS AND OBJECTIVES ................................................................................................................. 23 3.1 Hypothesis ............................................................................................................................................... 23 3.2 Objectives ................................................................................................................................................ 23 3.3 Evaluations .............................................................................................................................................. 23 Chapter 4: COMPARISON OF COLOR STABILITY AND PHENOLIC COMPOSITION FROM PERICARP WATER EXTRACTS OF DIFFERENT COLORED CORN (ZEA MAYS L.) VARIETIES IN A BEVERAGE MODEL .................................................... 24 4.1 Abstract .................................................................................................................................................. 24 4.2 Introduction ............................................................................................................................................ 25 4.3 Materials and Methods .......................................................................................................................... 26 4.4 Results and Discussion ........................................................................................................................... 31 4.5 Conclusions ............................................................................................................................................. 35 4.6 Figures and Tables ................................................................................................................................... 37 4.7 References ............................................................................................................................................... 50 Chapter 5: PROTECTIVE POTENTIAL OF ANTHOCYANIN-RICH PLANT EXTRACTS ON AN IN VITRO MODEL OF CELIAC DISEASE ........................................................................................................................................................... 52 5.1 Abstract .................................................................................................................................................. 52 5.2 Introduction ............................................................................................................................................ 53 5.3 Materials and Methods .......................................................................................................................... 54 5.4 Results and Discussion ........................................................................................................................... 62 5.5 Conclusions ............................................................................................................................................. 69 5.6 Figures and
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