© 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

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 Tables ...... 70 5.7 References ...... 83

Chapter 6: CONCLUSIONS ...... 86

Chapter 7: INTEGRATION AND FUTURE WORK ...... 87

APPENDIX A. Abbreviations ...... 90 APPENDIX B. Additional Figures ...... 91

v CHAPTER 1: INTRODUCTION

1.1 Overview

Autoimmune diseases are those in which the body's immune system attacks healthy cells.1 23.5(NIH)2—50(AARD)3 million Americans currently suffer from autoimmune diseases.

The body is perceived as a foreign object in people with autoimmune diseases and the T-cells target healthy cells and tissues as if they are and subsequently, inflammation is characteristic of most diseases. There are no known prevention methods for most autoimmune disorders currently.4,5

Celiac Disease is an inherited autoimmune disorder and is one of the top 10 leading causes of death in female children and women in all age groups up to 64 years of age3 with over

3 million suffer from celiac disease in the U.S.,6 still its prevalence is spread worldwide (Figure

1).7 Celiac disease is activated by gluten, a protein found in wheat, rye, and barley and targets the small intestine. It inhibits nutrients absorption by drastically reducing the microvilli of the small intestine (Figure 2). There are a wide range of symptoms with the most common including recurring abdominal bloating and pain, weight loss, iron-deficiency, anemia that does not respond to iron therapy, fatigue, chronic diarrhea or constipation, and vomiting.6 The current treatment of celiac disease is a lifelong gluten free diet, which is difficult to follow due to hidden gluten in unexpected foods and added expense.8

The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling pathway transfers information from the extracellular chemical signals into the nucleus of the cell (Figure 3).10 The result is DNA transcription and expression of genes involved in a variety of cellular functions such as: immunity, proliferation, differentiation, apoptosis, and oncogenesis.

The JAK/STAT signaling cascade is made up of three main components: a cell surface receptor, a

Janus kinase and two Signal Transducer and Activator of Transcription proteins. JAK/STAT functionality is directly linked to the immune response.9 Janus kinase is part of a family of

Tyrosine kinases: JAK1, JAK2, and TYK2 (tyrosine kinase-2), and JAK3. Inhibition of the

JAK/STAT pathway and antibodies blocks the recruitment of immune cells to the site of inflammation.10 JAK/STAT regulation is mostly facilitated through phosphorylation and dephosphorylation.11 Small intestinal inflammation is caused by these pro-inflammatory cytokines along with immune cell infiltration and inflamed tissue.10 Anthocyanins (ANCs) have been linked with the inhibition of JAK.12,13

ANCs are water-soluble phenolic compounds in the flavonoid family. The six main ANCs found in the diet are shown in Figure 4. Their color profile varies from orange to bright red and deep blue to violet and are found in flowers, vegetables, fruits, and grains.14 Health benefits of

ANCs include their potent antioxidant activity,15 protection from cardiovascular disease,16 potential to decrease weight gain and body fat accumulation,17 protection against diabetes,18 reduction in inflammation,19 inhibition of cancer growth,20 and neurological protection.21 They are becoming more popular as alternatives to food colorants. In fact, the following companies have pledged to make the switch to all natural colorants by 2018: Kraft, Taco Bell, Pizza Hut,

Subway, General Mills (Trix Cereal- lost blue and green), and Noodles & Company.22 Natural colorants are less stable than artificial dyes and though ANC use is increasing especially in red and purple beverages, they are sensitive to light, temperature, and pH.23,24

The first aim of this study was to compare the color stability of 9 unique colored corn varieties (Figure 5).25 Nine corn varieties went through kernel pericarp water extraction and

2 were freeze-dried (Figure 6). Kool-Aid Invisible,® Kool-Aid without color from Red 40, was mixed according to packaging instructions using distilled water. Extract concentration for each variety was initially adjusted to match the color of Kool-Aid Cherry. Corn varieties V1, V2, V3,

V4, V5, V6, V7, V8, and V9 had concentrations of 0.9, 0.7, 0.7, 2.2, 0.6, 1.4, 1.1, 0.6, and 0.5 mg freeze dried extract/mL Kool-Aid Invisible, respectively, depending on the spectrometric absorbance values at 520 nm to mimic Kool-Aid Cherry red color. Solutions were aliquoted in triplicate for each sample for each week tested and were stored without light at 4 °C, 22 °C, or

32 °C for a duration of 12 weeks. Color, spectra of absorption (λmax and absorbance at λmax), total anthocyanin content (TA), total phenolics (TP), condensed tannins, pH, and pigment composition changes were all measured once a month.

The second aim of this study was to determine the protective potential of ANC-rich plant extracts. Extracts from fruits (red and purple grape), vegetables (purple sweet potato and purple carrot), grains (colored corn varieties and colored corn with different extraction methods, black rice, sorghum, and blue wheat), and legumes (black bean, black lentil, black peanut, and purple bean) (Figure 7) were tested to evaluate their protective and anti- inflammatory effects on cells treated with a known celiac toxic peptide using a cell proliferation assay, human inflammation array, kinase binding assay, western blotting, and computational modeling (Figure 8).

1.2 Figures

Figure 1. Prevalence of a) celiac disease compared to b) wheat consumption across the world. 7

Figure 2. Microvilli of a normal small intestine compared to one damaged by celiac disease.

Figure 3. JAK/STAT signalling pathway extracellular to nucleus.10

Figure 4: The six main anthocyanins found in the human diet.

Figure 5. White, yellow, red, and purple colored corn and the counties that yield either %50 (yellow), %75 (light green), or %90 (dark green) of total corn produced in the U.S.25

Figure 6. Anthocyanin extraction process of the nine corn varieties (same as PW).

Figure 7. Plant extract material sources used for phase II.

Figure 8. Study design of phase II.

1.3 References

1. Y. Shoenfeld and A. D. Isenberg. The mosaic of autoimmunity. Immunol. Today. 1989, 10, 123-126. 2. U. S. Department of Health and Human Services, National Institutes of Health, National Institute of Health (NIH). Understanding Autoimmune Diseases. National Institute of Arthritis and Musculoskeletal and Skin Disease. 2016, 16-7582, 1-3. Retrieved from https://www.niams.nih.gov/%5C/Health_Info/Autoimmune/understanding_autoimmune.pdf 3. American Autoimmune Related Diseases Association, Inc. Autoimmune Statistics: Autoimmune Disease Fact Sheet. 2014-2017. Retrieved from https://www.aarda.org/autoimmune-information/autoimmune- statistics/ 4. Bylund DJ, Nakamura RM. Organ-specific autoimmune diseases. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 2011, 22, 1014-1020. 5. D. H. Kono, Theofilopoulos AN. Autoimmunity. Kelley’s Textbook of Rheumatology. 2013, 9, 281-299. 6. M. T. Minaya, DDS, Clinical Research Coordinator Celiac Disease Center at Columbia University; Milka Monegro, Clinical Research Coordinator Celiac Disease Center at Columbia University. https://celiacdiseasecenter.columbia.edu 7. V. Abadie, L.M. Sollid, L.B. Barreiro, and B. Jabri. Integration of genetic and immunological insights into a model of celiac disease pathogenesis. Annu. Rev. Immunol. 2011, 29, 493-525. 8. P. J. Ciclitira, M.W. Johnson, D. H. Dewar, and H. J. Ellis. The pathogenesis of coeliac disease. Mol. Aspects. Med. 2005, 26, 421-458. 9. D. S. Aaronson and C. M. Horvath. A road map for those who don’t know JAK-STAT. Science. 2002, 296, 1653-1655. 10. J. Pedersen, M. Coskun, C. Soendergaard, M. Salem, and O. H. Nielsen. Inflammatory pathways of importance for management of inflammatory bowel disease. World J. Gastroenterol. 2014, 20, 564-77. 11. K. M. Henkels, K. Frondorf, M. E. Gonzalez-Mejia, A. L. Doseff, and J. Gomez-Cambronero. IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3). FEBS Lett. 2011, 585, 159-166. 12. I. T. Nizamutdinova, Y. M. Kim, J. I. Chung, S. C. Shin, Y. K. Jeong, H. G. Seo, J. H. Lee, K.C. Chang, and H. J. Kim. Anthocyanins from black soybean seed coats stimulate wound healing in fibroblasts and keratinocytes and prevent inflammation in endothelial cells. Food Chem. Toxicol. 2009, 47, 2806-2812. 13. K. M. Henkels, H. J. Peng, K. Frondorf, and J. Gomez-Cambronero. A comprehensive model that explains the regulation of phospholipase D2 activity by phosphorylation-dephosphorylation. Mol. Cell Biol. 2010, 30, 2251-2263. 14. V. P. Dia, Z. Wang, M. West, V. Singh, L. West, and E. Gonzalez de Mejia. Processing method and corn cultivar affected anthocyanin concentration from dried distilllers grain with solubles. J. Agric. Food Chem. 2015, 63, 3205-3218. 15. M. Mikulic-Petkovsek, B. Krska, B. Kiprovski, and R. Veberic. Bioactive Components and Antioxidant Capacity of Fruits from Nine Sorbus Genotypes. J. Food Sci. 2017, 82, 647-658. 16. D. Fratantonio, F. Cimino, M. Molonia, D. Ferrari, A. Saija, F. Virgili, and A. Speciale. Cyanidin-3-O- glucoside ameliorates palmitate-induced insulin resistance by modulating IRS-1 phosphorylation and release of endothelial derived vasoactive factors. (BBA)- Mol. Cell Biol. Lipids. 2017, 1862, 351-357. 17. M. L. Bertoia, E. B. Rimm, K. J. Mukamal, F. C. Hu, W. C. Willett, and A. Cassidy. Dietary flavonoid intake and weight maintenance: three prospective cohorts of 124,086 US men and women followed for up to 24. BMJ. 2016, 352, 1-7. 18. C. Chaiyasut, B. S. Sivamuaruthi, N. Pengkunsri, W. Keapai, P. Kesika, M. Saelee, P. Tojing, S. Sirilum, K. Chaiyasut, S. Peerajan, and N. Lailerd. Germinated Thai Black Rice Extracts Protects Experimental Diabetic Rats from Oxidative Stress and Other Diabetes-Related Consequences. Pharmaceuticals. 2017, 10, 1-16. 19. C. A. Morais, V. V. Rosso, D. Estadella, and L. P. Pisani. Anthocyanins as inflammatory modulators and the role of the gut microbiota. The J. Nutr. Biochem. 2016, 33, 1-7. 20. P. Sharma, S. F. McClees, and F. Afaq. Pomegranate for Prevention and Treatment of Cancer: An Update. Molecules. 2017, 22, 1-18. 21. N. Winter, E. K. Ross, S. Khatter, K. Miller, and D. A. Linseman. Chemical basis for the disparate neuroprotective effects of the anthocyanins, callistephin, and kuromanin, against nitrosative stress. Free Radical Bio. Med. 2017, 103, 23-34.

22. K. Bratskeir. 11 Food Companies Removing Artificial Colors and Flavors by 2018. Huffington Post. 2015. http://www.huffingtonpost.com/entry/11-companies-that-plan-to-remove-artificial-flavors-before- 2019_us_55b6a777e4b0074ba5a5d327 23. E. West and L. J. Mauer. Color and chemical stability of a variety of anthocyanins and ascorbic acid in solution and powder forms. J. Agric. Food Chem. 2013, 61, 4169-4179. 24. X. L. He, X. L. Li, Y. P. Lv, and Q. He. Composition and color stability of anthocyanin-based extract from purple sweet potato. Food Sci. Technol. (Campinas). 2015, 35, 468-473. 25. G. Schnitkey. Concentration of Corn and Soybean Production in the U.S. FarmDocDaily. 2013 http://farmdocdaily.illinois.edu/2013/07/concentration-corn-soybean-production.html

CHAPTER 2: REVIEW OF LITERATURE

2.1 Celiac Disease

Celiac disease is characterized as a sensitivity to gluten where the consumption of grains, like wheat, barley and rye (Table 1),1 activates an immune response in the small intestine. Celiac disease occurs when the T-cells are reacting to an immune response (Figure

9)2 and the B lymphocytes respond by attacking its own cell tissue by producing autoantibodies again the gliadin, endomysium, or transglutaminase tissues.3 This type of autoimmune response occurs in people that have a greater amount of heterodimer human leukocyte antigen

(HLA)-DQ2 and HLA-DQ8 in their genes, however approximately 40% of the population carry these alleles and the majority never develop Celiac disease.3,4

Today about 1% of the population has been diagnosed with celiac disease, the majority living in Western Europe.3 The only treatment available for patients with celiac disease is avoiding consumption of products that contain gluten.5 There are still no current treatments to help people that are impacted with celiac diseases besides prevention. However, there are lots of studies that are looking at how it develops on a genetic level to determine the cause.

Preventative measures for celiac disease by introducing gluten into the diet of breast fed babies to help prevent gluten intolerance in the future to reduce risk for other autoimmune disorders have been attempted; this study is inconclusive because human test subjects were used and the long term results have not yet been collected.6 Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease; genetic mapping is being used to understand what genetic traits contribute to celiac disease. After

14 examining thousands of patients they were able to determine the genetic structure of risk regions and minimize the number of genetic signals that may have an impact on celiac disease.7

2.2 JAK/STAT Pathway

The role of the Janus kinase (JAK)/ signal transducers and activators of transcription

(STAT) pathway is to drive biological processes such as cell growth, differentiation, proliferation, and regulatory immune functions in the cytokines.8,9 The JAK/STAT pathway takes signals from various extracellular cytokine stimuli to the nucleus so that the appropriate cellular response occurs.9 JAK stimulates the proliferation, differentiation, migration, and apoptosis of cells, all of which are important for the hematopoiesis, immune development, mammary gland development, lactation and many more processes.10

Phosphorylation is the addition of the phosphate group to a molecule which can activate or deactivate regulatory functions. Trans or auto phosphorylation takes place when JAK proteins are gathered, then intracellular cytokine receptor residues are phosphorylated which create phospho-tyrosines on the cytokine receptor tail that can be used as a binding site for

STAT proteins.8 STAT are latent transcription factors that are in the cytoplasm until they are activated.10 When the JAK/STAT pathway is activated or JAK signaling is poorly regulated, issues such as inflammatory diseases, erythrocytosis, and various leukemia are developed.

One particular inflammatory disease JAK/STAT signaling has played an important role in is called the inflammatory bowel disease, where JAK is used as inhibitor to treat ulcerative colitis by weakening the inflammation process.9 Ulcerative colitis is an inflammatory bowel diseases that impacts the inner lining of the large intestine and rectum. Over time is can cause long lasting inflammation, as well as, ulcers in the digestive tract (“Ulcerative colitis”). Irritable

15 bowel syndrome is another disorder that impacts the large intestine (“Irritable bowel syndrome”). Similar to ulcerative colitis, some of the symptoms include diarrhea, constipation, cramping, abdominal pain, bloating, and gas. However, unlike ulcerative colitis, it is not an inflammatory bowel disease and it cannot change the bowel tissues. Studies have found that the JAK/STAT signaling pathway plays and essential role in the mechanism of irritable bowel disease mainly through the influence of JAK because of its influence on cytokine signaling.9

2.3 Anthocyanins

ANCs, Greek for blue flower, are water soluble,11 phenolic compounds found in various fruits, vegetables, and flowers that range in color from red and orange to blue and violet

(Figure 10). As members of the flavonoid group, there are over 700 different type of ANC12 that have four main structures; flavylium cation, quinoidal base, carbonal pseudobase, and chalcone, all of which vary depending on the pH of the ANC structure.13

Over the past few years, various health benefits regarding ANC have been discovered, these include cardiovascular protection, vision improvement, anti-diabetic and anti-obesity properties, chemoprevention and cancer protection, and anti-inflammatory effects.12 There are currently no dietary recommendations regarding the intake of ANC in the US, Canada, or the

European Union. However, in China there is a recommendation of 50 mg/day because of the positive associations between ANC intake and diet quality.12

2.4 Color Stability

Phenolics are secondary metabolites found in all plant tissues and fruit that have an aromatic ring and have one or more hydroxyl substituent.14 They are an important part of fruits and vegetables due to their antioxidant, anti-inflammatory, and anti-carcinogenic biological

16 properties, among many other, which help in disease prevention.15 Some phenols are polymerized into larger molecules like proanthocyanidins or condensed tannins, or they may be present in plants or fruits as esters or glycosides that are conjugated with compounds like flavonoids, alcohols, or sterols.15

Interest in natural colorants to be used in food products has been growing over the years. ANCs are a desirable colorant due to its ease in incorporation because it is water-soluble and the numerous amount of bright colors that can be produced.11 Unfortunately, there have been numerous studies that have found that ANCs are less stable than synthetic food colorants under certain pHs and temperatures, and after extensive exposure to light.

There have been multiple studies looking at the impact of pH on ANC in blueberry extracts,16 black carrots,17 and in C. hirsutus, which is a shrub with a pea sized fruit commonly found and consumed throughout India.18 The general consensus in each study was that ANC is more stable at lower pHs, preforming best below pH 5, meaning that ANC may work best in acidic food products. During a study of the stability of ANC as the temperature increased from

5 °C to 35 °C the general trend was that the greater the temperature, the higher the percent of

ANC was destroyed.13 Another study looking at the stability of ANC due to temperature also found that high temperatures, above 100 °C, should be avoided and it concluded that high temperatures and long heating should be avoided for ANC from C. hirsutus in processing and storage.18

An older study looking at the stability of anthocyanin in grape juice showed that light causes the stability of ANC in grape juice to quickly decrease – grape juice stored in the dark for

135 at 20 °C at pH 2 had a 20% of total pigment destroyed but in the grape juice stored in the

17 light for 135 days at 20 °C at pH 2 had 50% of total pigment destroyed.19 A more recent study looking at ANC extracted from C. hirsutus were tested at various temperatures in the dark and in the light to test the effects of light on the stability of the ANC. The results showed that when exposed to light the stability of the ANC was much lower over time.18

2.5 Figures and Tables Figure 9. Celiac disease immune response in the small intestine.2

Figure 10. Freeze dried colored corn extracts with the structures of the three main anthocyanins found in colored corn.

Table 1. Wheat protein, alpha-gliadin, H-Tyr-Pro-Gln-Pro-Gln-Pro-Phe-OH (YPQPQPF).1

Note: Highlighted sequences represent Celiac toxic peptide sequence. YPQPQPF is the sequence used in this study and it is an alpha-gliadin found in wheat protein.

2.6 References

1. A. Cavazos, and E. Gonzalez de Mejia. Identification of Bioactive Peptides from Cereal Storage Proteins and Their Potential Role in Prevention of Chronic Diseases. Comprehensive Reviews in Food Science and Food Safety. 2013, 12, 364-380. 2. R. W. DePaolo, V. Abadie, F. Tang, H. Fehlner-Peach, J. A. Hall, W. Wang, E. V. Marietta, D. D. Kasarda, T. A. Waldmann, J. A. Murray, C. Semrad, S. S. Kupfer, Y. Belkaid, S. Guandalini, and B. Jabri. Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens. Nature. 2011, 471, 220-224 3. S. Pudasaini. Celiac disease and its histopathology. J. Pathol. Nepal. 2017, 7.1, 118-1123. 4. J. Romanos, A. Rosén, V. Kumar, G. Trynka, L. Franke, A. Szperl, J. Gutierrez-Achury, C.C. van Diemen, R. Kanninga, S.A. Jankipersadsing, A. Steck, G. Eisenbarth, D.A. van Heel, B. Cukrowska, V. Bruno, M.C. Mazzilli, C. Núñez, J.R. Bilbao, M.L. Mearin, D. Barisani, M. Rewers, J.M. Norris, A. Ivarsson, H.M. Boezen, E. Liu, C. Wijmenga, and PreventCD Group. Improving coeliac disease risk prediction by testing non-HLA variants additional to HLA variants. Gut. 2014, 63, 415-42. 5. Fasano. Celiac Disease, Gut-Brain Axis, and Behavior: Cause, Consequence, or Merely Epiphenomenon? Pediatrics. 2017, 139, 3. 6. C. E. H. Esch, A. Rosen, R. Auricchio, J. Romanos, A. Chmielewska, H. Putter, A. Ivarsson, H. Szajewska, F. Koning, C. Wijmenga, R. Troncone, M. L. Mearin, and PreventCD Study Group. The PreventCD Study design. Eur. J. Gastoen. Hepat. 2010, 22, 1424-30. 7. G. Trynka, K. A. Hunt, N. A. Bockett, J. Romanos, V. Mistry, A. Szperl, S. F. Bakker, M. T. Bardella, L. Bhaw- Rosun, G. Castillejo, E. G. de la Concha, R. C. de Almeida, K. R. Dias, C. C. van Diemen, P. C. Dubois. R. H. Duerr, S. Edkins, L. Franke, K. Franksen, J. Gutierrez, G. A. Heap, B. Hrdlickova, S. Hunt, L. Plaza Izurieta, V. Izzo, L. A. Joosten, C. Landqford, M. C. Mazzilli, C. A. Mein, V. Midah, M. Mitrovic, B. Mora, M. Morelli, S. Nutland, C. Nuñez, S. Onenqut-Gumuscu, K. Pearce, M. Platteel, I. Polanco, S. Potter, C. Ribes-Koninckx, I. Ricaño-Ponce, S. S. Rich, A. Rybak, J. L. Santiago, S. Senapati, A. Sood, H. Szajewska, R. Troncone, J. Varadé, C. Wallace, V. M. Wolters, A. Zhernakova, Spanish Consortium on the Genetics of Celiac Disease, PreventCD Study Group, Wellcome Trust Case Control Consortium, B. K. Thelma, B. Cukrowska, E. Urcelay, J. R. Bilbao, M. L. Mearin, D. Barisani, J. C. Barrett, V. Plagnol, P. Deloukas, C. Wijmenga, and D. A. van Heel. Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease. Nat. Gen. 2011, 43, 1193–1201. 8. K. Imada and W.J. Leonard. The JAK-STAT pathway. Mol. Immunol. 2000, 37, 1-11. 9. M. Coskun, M. Salem, J. Pedersen, and O.H. Nielsen. Involvement of JAK/STAT signaling in the pathogenesis of inflammatory bowel disease. Pharmacol. Res. 2013, 76, 1-8. 10. J. S. Rawlings, K. M. Rosler, and D. A. Harrison. The JAK/ STAT signaling pathway. J. Cell Sci. 2004, 117, 1281-1283. 11. P. Markakis, Stability of Anthocyanins in Food. Anthocyanins as Food Colors. 2012, 9, 167-180. 12. T.C. Wallace, and M.M. Giusti. Anthocyanins. Adv. Nutr. 2015, 6, 620-622. 13. G.H. Laleh, H. Frydoonfar, R. Heidary, R. Jameei, and S. Zare. The effect of light, temperature, pH and species on stability of anthocyanin pigments in four Berberis species. Pakistan J. Nutr. 2006, 5, 90-92. 14. J. Macheix and A. Fleuriet. Fruit Phenolics. CRC Press. 1990, 392. 15. J. N. Kabera, E. Semana, A. R. Mussa, and X. He. Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties. J. Pharmacy Pharmaco. 2014, 2, 377-392. 16. B. C. Wang, R. He, and Z. M. Li. The Stability and Antioxidant Activity of Anthocyanins from Blueberry. Food Technol. Biotechnol. 2010, 48, 42–49. 17. A. Kirca, M. Ozkan, and B. Cemeroglu. Effect of temperature, solid content and pH on the stability of black carrot anthocyanins. Food Chem. 2007, 101, 212-218. 18. R. Rakkimuthu, S. Palmurugan, and A. Shanmugaprita. Effect of temperature, light, pH on the stability of anthocyanin pigments in Cocculus hirsutus fruit. IJMRME. 2016, 2, 91-95. 19. N. Palamidis, T. Markakis. Structure of anthocyanin. J. Am. Sci. 1975, 111, 742-746.

CHAPTER 3: HYPOTHESIS AND OBJECTIVES

3.1 Hypothesis

The stability of color and phenolic composition of ANC-rich extracts from colored corn varieties in a beverage model will depend on their chemical composition. Colored corn extracts rich in

ANC will selectively inhibit the production of inflammatory biomarkers in a cell model of celiac disease.

3.2 Objectives

To evaluate the effect of anthocyanin profiles characteristics of purple corn varieties on color stability. Furthermore, to evaluate the anti-inflammatory and protective effect of purple and red corn pericarp and other plant extracts on an in vitro model of Celiac disease.

3.3 Evaluations

• Evaluate the effect of temperature on color, of a beverage model, from colored corn

pericarp extracts of different corn varieties.

• Evaluate the effect of corn varieties and other plant extracts with different anthocyanin

profiles on Caco-2 cells viability.

• Evaluate the interaction of main colored corn anthocyanins with Janus kinase 3 (JAK3)

compared to a known inhibitor of JAK3.

• Evaluate the effect of corn varieties with different anthocyanin profile on JAK/STAT

phosphorylation in Caco-2 cells treated with celiac toxic peptide.

• Evaluate the effect of corn varieties with different anthocyanin profile on Caco-2 cell

protection and inflammation caused by a celiac toxic peptide.

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 4.1 Abstract

The objective of this study was to compare the color and chemical stability of nine unique anthocyanin-rich colored corn variety extracts added to a beverage model and stored at

4 ° C, 22 °C, or 32 °C for 12 weeks. After 12 weeks at 32 °C, variety V6 (high condensed form, high cyanidin-3-O-glucoside (C3G), low pelargonidin (Pg), low peonidin (Pn), low in acylated forms) had the longest anthocyanin half-life, based on the quantification by HPLC. 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 some of the smallest changes in color over time. These findings suggest that an abundance of condensed forms with C3G in corn extracts could contribute to the improved stability. However, corn extracts containing more peonidin-derived anthocyanins had reduced stability in the beverage model. Beverage storage parameters also influenced color parameters; low temperatures and low pH enhanced color and anthocyanin stability. The most promising corn varieties for future experiments are V3, V5, and V6 based on color retention.

This chapter is based on the manuscript: Haggard, S., Diego, L., West, L., Juvik, J.A., Chatham, L., Paulsmeyer, M., Gonzalez de Mejia, E. 2017. Comparison of Color Stability and Phenolic Composition from Pericarp Water Extracts of Different Colored Corn (Zea mays L.) Varieties in a Beverage Model. To be submitted to JAFC.

4.2 Introduction

According to the Food and Drug Administration (FDA), artificial food colorants (AFCs) are

“any dye, pigment, or other substance(s) that can impart color to a food, drug, or cosmetic...”1

Despite some consumer concerns regarding use of AFCs,2-4 the FDA’s acceptable amount of added colorants in food products has increased more than 5-fold from 1950 (12 mg/capita/day) to 2012 (68 mg/capita/day).

Anthocyanins (ANCs) are a possible alternative to artificial colorants. They are water- soluble phenolic compounds that are responsible for the red, purple, and blue colors found in fruits and vegetables.5 Structurally based on the flavylium ion or 2-phenylbenzopyrillium, the aglycone form of ANCs are anthocyanidins; they contain hydroxyl and methoxy groups that can influence color depending on their position. Pelargonidin, cyanidin, delphinidin, petunidin, peonidin, and malvidin are the six major anthocyanidins from which the majority of ANCs found in food are derived.6 Once glycosylated, ANCs can be further modified by the addition of acyl groups or by condensation via covalent bonding to flavan-3-ols. ANCs can be found in a variety of plants such as dried purple waxy corn cobs,7 wild blueberries,8 Chinese bayberries,9 among others, and all have potential health benefits. ANC consumption has been linked with a lower risk of type 2 diabetes, Parkinson’s disease, myocardial infarction, cancer, and inflammation.10

Even in small concentrations, ANC-rich polyphenolic purple corn extracts lead to a decrease in glomerulosclerosis which is associated with diabetes.11

There are many species of corn that are produced around the world and grow in a variety of colors such as red, yellow, purple, green, blue, brown, and white. Some of the different types of ANCs include cyanidin 3-O-β-D-glucoside, pelargonidin (Pg) 3-O-β-D-glucoside, cyanidin 3-O-

β-D-(6-malonyl-glucoside), pelargonidin 3-O-β-D-(6-malonyl-glucoside), and peonidin (Pn) 3-O-

β-D-(6-malonyl-glucoside).12 Many of which were isolated from Peruvian purple corn grains

(Zea mays L.) with cyanidin 3-O-β-D-glucoside having the largest concentration.12 Even though there are several physio-chemical methods to improve natural pigments stability,13 it is also relevant to generate plant varieties with more stable compounds.14

There is a need to meet the consumer desire to find foods with natural colorants that will be functional for the food industry. The purpose of this study was to test and compare the stability of color and phenolic composition of nine ANC rich colored corn variety extracts in a beverage model at 4 ˚C, 22 ˚C, and 32 ˚C over a 12-week period.

4.3 Materials and Methods

4.3.1 Chemical and Reagents

Varieties of purple corn each distinguished by their unique pigment composition were generated at the University of Illinois at Urbana-Champaign (UIUC) from materials collected from the North Central Regional Plant Introduction Station (PI; Ames, IA, USA), the Maize

Genetic Cooperation Stock Center (MGCSC; Urbana, IL, USA) or Siskiyou Seeds (Williams, OR,

USA). Pericarp was removed from each variety as previously described.15 Some of the key characteristics of each variety tested can be found in Table 2. Kraft Kool-Aid Invisible Cherry

Sugar-Sweetened Soft Drink Mix and Kraft Kool-Aid Jammers Cherry made with Red 40 were purchased online. The Kraft Kool-Aid Jammers Cherry colored with synthetic Red 40 served as a comparative control. All chemicals used in this study were purchased from Sigma-Aldrich (St.

Louis, MO, USA) unless otherwise stated.

4.3.2 Experimental Design

Nine corn varieties described in Table 2 went through kernel pericarp water extraction and were freeze-dried. Kool-Aid Invisible® was mixed according to package instructions using distilled water. Extract concentration for each variety was initially adjusted to match the color of Kool-Aid Cherry. Corn varieties V1, V2, V3, V4, V5, V6, V7, V8, and V9 had concentrations of

0.9, 0.7, 0.7, 2.2, 0.6, 1.4, 1.1, 0.6, and 0.5 mg freeze dried extract/mL Kool-Aid Invisible, respectively, depending on the spectrometric absorbance values at 520 nm to mimic the Kool-

Aid Cherry red color. Solutions were aliquoted in triplicate for each sample for each week tested and were stored without light at 4 °C, 22 °C, or 32 °C for a duration of 12 weeks. Color, spectra of absorption (λmax and absorbance at λmax), total anthocyanin concentration (TA), total phenolics (TP), condensed tannins, pH, and pigment composition changes were all measured once a month.

4.3.3 Corn pericarp pigment extraction

ASE 300 Accelerated Solvent Extraction System was used for extraction (Thermo Scientific,

Waltham, Massachusetts, USA). Corn pericarp (5-10 g) was added to stainless steel 34 mL or 66 mL cells with cellulose filters. Only water was used as solvent for extraction. The cell was preheated for 5 min and heated to 50 °C for extraction. Static solvent extraction time was 5 min. Pressure in the cell during extraction was maintained at 1500 psi. Amount of solvent to flush through the cell following the static heating step was 100% of the cell volume. Extraction cycles to perform the static heating and flushing steps were repeated 5 times. Cells were purged with nitrogen for 60 sec at the end of the extraction. Extracts were passed through 0.45 micron Nylon membrane filters (Sigma Aldrich, MO, USA), freeze-dried (VirTis Sentry 5 L freeze

27 drier, Gardiner, NY, USA), and stored at -20 °C until analysis. Time between freeze-drying and sample analysis was less than one month.

4.3.4 Kool-Aid mixture and color matching

A Synergy 2 multiwell plate reader (BioTek, Winooski, VT, USA) was used to measure spectra of all samples at a range of 0.2-1.0 mg dry powder/mL Kool-Aid Invisible.

Concentrations were calculated for each sample to match absorption of Kool-Aid Cherry at λmax equal to 0.853. Pericarp water extracts were mixed with Kool-Aid Invisible to reach the desired color then solutions were filtered using 0.45 micron Nalgene filters (Thermo Scientific,

Waltham, Massachusetts, USA) and aliquoted in triplicate to be stored with no light at 4 °C, 22

°C, and 32 °C for weeks 0, 4, 8, and 12.

4.3.5 Tristimulus colorimetric measurement

Color was measured using a Lovibond Tintometer PFX990 (The Tintometer Ltd, Amesbury,

England, UK) instrument. The following color scale parameters were used: CIELAB values L*, a*, and b*; observer/illuminant 10° and D65, and path length of 1 cm. Briefly, 2-3 mL extracts acclimated at room temperature were placed in a disposable UV-Vis cuvette and the color parameters L*, a* and b* were measured and recorded. Color squares were generated by converting L*, a* and b* values to RGB values using http://colormine.org/convert/rgb-to-lab and Microsoft power point software. The color difference (ΔE) was calculated according to CIE

1976 guideline16 as follows:

! � ! ΔE = ( L∗ − L!) + (a∗ − a!) + (b∗ − b!)

The hue angle and chroma were calculated17 as follows:

b∗ Hue angle h = tan!! !" a∗

Chroma C∗ = a∗ ! + b∗ !

In the equations, L, a, and b correspond to lightness, redness/greenness, and blueness/yellowness, respectively, at the final time either 4, 8, or 12 weeks (*) and initial time week 0 (0) measurement.

4.3.6 Chemical Analysis

4.3.6.1 Measurement of total polyphenols concentration

All samples were analyzed for total phenolics using methods previously reported.5,18 A standard curve was created using gallic acid; 0, 40, 50, 75, 100, 150, 175, and 200 µg/mL. Fifty

µL standard and 1:2 diluted samples were added to a 96-well plate in triplicate. Fifty µL 1 N

Folin-Ciocalteu reagent was added to each well, mixed, and let stand for 2-5 minutes, then 100

µL 20% Na2CO3 (sodium carbonate) was added to each well, mixed, and let stand for 10 min.

Absorbance was read using a Synergy 2 multiwell plate reader (BioTek, Winooski, VT, USA) at

690 nm and results were expressed as mg gallic acid per gram of dry extract.

4.3.6.2 Measurement of condensed tannin concentration

All samples were analyzed for condensed tannins using methods previously reported.19,20 A standard curve was constructed using a serial diluted catechin/methanol solution (0.1, 0.2, 0.4,

0.6, and 0.8 mg/mL). Samples were diluted to a factor of 1:10 or 1:20 with methanol. Vanillin reagent was prepared fresh by mixing equal volumes of 1% vanillin in methanol and 8% concentrated HCl in methanol. Five mL of 1:1 8% HCl:methanol with 1% vanillin at was added at 1-min intervals to 1-mL aliquots of the samples. Five milliliters of 4% concentrated HCl in methanol was added to a second 1-mL aliquot (the blank) also at 1-min intervals. Absorbance at 500 nm was read after 20 min and the absorbance of the blank was subtracted from the

29 absorbance with vanillin. The standard curve was always run at the same temperature, and temperatures should be reported for all assays. Measurement of each sample was repeated three times. The total tannin content is expressed as milligram catechin per milliliter of aqueous extract based on the standard curve. Solutions were protected from light during preparation, and wells were covered with aluminum foil to avoid methanol evaporation.

4.3.6.3 HPLC Analysis

HPLC analysis of samples for ANC profile was performed in triplicate using a Hitachi HPLC

System (Hitachi High Technologies America, Inc., Schaumburg, IL, USA) equipped with a multi- wavelength detector, L-7100 pump following previously reported protocol22 with some modifications. The injection volume was 20 µL. The flow rate was 1 mL/min and the gradient used was from phase A 2% formic acid in water and phase B 0% acetonitrile to 40% acetonitrile in a linear fashion using a Grace Prevail C18 (5 µm, 250 × 4.6 mm, Columbia, MD, USA) for 30 min. Peaks were identified based on the same retention time as available standards or literature.21 The concentration of each ANC was determined using a calibration curve with 5 points in the range of 50–2000 µg/mL of C3G standard obtained by plotting the area of C3G peak versus its concentration with a correlation coefficient (R2) of 0.99. Results were expressed as ANC peak concentration µg/mL.

4.3.6.4 First-order reaction kinetics

Storage stability data for the concentration of ANC obtained from the HPLC quantifications were plotted using the first-order reaction rate kinetics22 by the following equation:

ln�! = ln�! − ��

Where A0 is the initial value, and At is the final value of the different parameters at an initial and final time.

The first-order reaction rate constant (k) and half-life (t1/2) (the time needed for 50% degradation) were calculated using the following equation:

!! t!/! = −ln0.5 ∙ k

4.3.6.5 Statistical analysis

The results were expressed as the mean of three independent experiments. An analysis of variance was performed, and multiple mean comparisons were assessed with a Tukey test using

JMP version 7 (SAS Institute Inc., Cary, NC, USA). To explain the variability of the results, a principal component (PC) analysis was performed using SPSS version 21 (IBM Corporation,

Armonk, NY, USA).

4.4 Results and Discussion

4.4.1 Tristimulus colorimetric measurements

Color squares of the nine corn varieties presented in Figure 11 show the degradation of color over time for each temperature. Colors from ANC-rich extracts were acceptable for all varieties at week 0 color squares based on industry standards. Corn varieties V3 and V5 retained their color best of the nine varieties. V3 contained high Pg, low condensed forms, low

Pn, high C3-mal, high Pg3-mal, and high Pn3-mal; and V5 had low levels of Pn, high condensed forms, high C3G, low Pg, high C3-mal, and low acylated forms. A recent study suggests that condensed forms alone do not stabilize color,23 but can contribute to stability in the presence of higher proportions of C3G. For all samples, pH remained constant at all temperatures over all

12 weeks.

4.4.2 Hue angle and Lightness

Figure 12 shows the hue angles of each corn variety over time at the three different temperatures and the values are shown in Table 3. In general, V3 and V5 corn varieties had the lowest change of Hue angle throughout time. Corn varieties V4 and V9 showed the largest changes in Hue. In a study on purple sweet potato extract, a similar loss of color was observed and in some cases developed browning at much higher temperatures.24 In a study using malvidin 3-O-β-D-glucoside as pigment and comparing epicatechin, catechin, procyanidin B2 or isolated compounds as copigments; proanthocyanidins B3 and C2 in barley, procyanidin B2 in apple, and trimeric and tetrameric proanthocyanidins in brown rice were all stronger copigments than flavanols.25 Even though catechin and dimers B2 and B3 acted as weak copigments, only slightly enhancing the red color in solutions, the samples rapidly turned yellow upon heating.25 Under the same conditions, trimeric and tetrameric proanthocyanidins more efficiently protected the red color.25 Figure 13 shows the lightness of samples over time and the values are also given in Table 3. V1 and V4 had the most unfavorable values after 12 weeks at 32 °C and V5 and V7 behave most favorably.

4.4.3 Chroma and ΔE

Figure 14 shows the chroma and Table 4 gives the values of chroma and ΔE of the varieties over time. At 4 °C there was not much difference among varieties over time but V4 had the largest change in chroma at 4, 22, and 32 °C. After 12 weeks, at 22 °C V5 had the smallest change in chroma and at 32 °C V6 had the smallest change in chroma. In a study looking at color from black beans, for pH 3.5 and 4.3 there was an increasing tendency on the chroma

32 value with time.26 In an ideal system, chroma and ΔE would not change among temperature conditions. Varieties had similar behavior patterns in respect to change in temperature.

4.4.4 Measurement of total polyphenols and condensed tannin concentration

Table 5 shows the total polyphenols over time at all temperatures. All corn varieties showed an increase in polyphenols after 4 weeks at 4 °C and 22 °C, probably due to the formation of degradation products of ANCs. At 22 °C and 32 °C there was a modest change in the concentration of polyphenols, and some samples did not have significant difference amongst samples at 12 weeks. V5 also had the highest starting concentration of TP. In a study comparing black currant juices with differing TP, the sample with the lowest concentration of

TP had the least stability at a storage time of 12 weeks.27 In a study comparing extraction methods of grape pomace a higher level of polyphenols was linked to increased color stability.28

Temperate (pasteurization or freezing) had a significant effect on total polyphenols of strawberry pulp while not affecting total ANCs content even after 4 months storage while frozen.29 Table 5 shows the condensed tannins of the varieties. After 12 weeks V2 and V3 had the smallest percent decrease in tannins at 4 °C. After 12 weeks V4 had the highest percent decrease in tannins at all three temperatures. V7 was the most stable over time at 4 °C. V9 was the most stable up to week 8 at 22 °C. At 32 °C V7 was most stable after 12 weeks. In wines treated with tannins, color was more stable.30

4.4.5 HPLC Analysis

Table 6 shows the concentration of condensed forms, C3G, Pg3G, and Pn3G and the Table 7 shows the concentration of acylated forms present in the purple corn varieties. In agreement with our previous study,22 acylated forms of ANCs from purple corn were mostly degraded. In a

33 recent study looking at the effects of growth and post-harvest temperature conditions on red cabbage, they found that even at 4 °C, acylated forms were not stable.31 Over time at each temperature condensed forms were most stable in V5, C3G was most stable in V9, and both

Pg3G and Pn3G were most in V3. Lingonberry, strawberry, raspberry, and cranberry juices all showed similar ANC profiles when kept at room temperature for an extended time period.32

Correlations between parameters are shown in Table 8. The strongest positive correlations were Chroma to TA, C3G, Pn3G, and acylated forms, TA to C3G, Pn3G, and acylated forms, condensed forms to C3G, C3G to Pn3G and C3-mal, Pn3G to C3-mal and Pn3-mal, and finally C3- mal to Pn3mal. The strongest negative correlations were time to chroma, TA, Pn3G, C3-mal, and Pn3-mal and chroma to hue.

4.4.6 First-order reaction kinetics

Table 6 shows the total ANCs first-order reaction kinetics based on the results of the quantification of each ANC by HPLC. V6 (high condensed form, high C3G, low Pg, low Pn, low

C3-mal, low other acylated forms) and V7 (no condensed form, low C3G, low Pg, high Pn, high

C3-mal, low Pg3-mal, high Pn3-mal) had the longest half-lives and lowest reaction rates at all temperatures. V9 had the shortest half-life and highest reaction rates at 4 °C but at 22 °C, V8 had the shortest half-life and at 32 °C it was V4. The half-life of V6 ANCs was roughly 5 times longer than V9. V6, one of the varieties with longest TA half-life has a high concentration of condensed forms. According to the chroma kinetics, V5 and V3 had the longest half-live if stored at 4 °C (Table 9). Although chroma parameter in anthocyanin studies follows a first order reaction, Hue cannot be fitted in the model due to its angle nature. In a study comparing the light and temperature stability of black carrot and red daisy, black carrot had more color

34 stability under temperature stress and this was attributed to the increased presence of acylated anthocyanins, which were missing from red daisy.33 In previous reports it has been stated that copigmentation among ANCs improves their chemical and color stability.34 When comparing half-lives of individual anthocyanins from black rice, C3G had the longest half-life compared to total ANCs, pelargonidin, and delphinidin.35 Due to the unique ANC composition of the purple corn varieties tested, stability among them was attributed to their distinct pigment composition and interactions.

4.4.7 Principal components statistical analysis

A principal component (PC) analysis is shown in Figure 15 of the nine varieties at different temperatures for 4, 8, and 12 weeks. True values of chroma, hue, TA, condensed forms (CF),

C3G, Pg3G, Pn3G, C3-mal, Pg3-mal, and Pn3-mal for each variety at each temperature at 4, 8, and 12 weeks were analyzed. Most of the treatments were grouped together from the 22 °C and 32 °C treatment groups, indicating that there is no extreme changes in the behavior of these members. The low temperature treatment conferred protection of color stability to the varieties shown in the upper portion of the figure, which was comprised of samples stored at 4

°C. This protection at lower temperatures has been described in other studies.36,37 At each time point V5 at 4 °C can be seen in the upper left separated from the other 4 °C treatment which suggests it exhibited a unique behavior relating to color stability.

4.5 Conclusions

In summary, nine unique anthocyanin-rich colored corn extracts were added to a beverage model and stored at different temperatures for 12 weeks. It was found that the high presence of condensed forms with C3G in the pigments from corn pericarp could potentially contribute

35 to the color parameters and stability. Corn samples with high peonidin had less stable pigments. Low temperatures and low pH promoted color and anthocyanin stability. The ideal pigment composition for purple corn varieties with stable pigments are those similar to V3

(high Pg, high C3-mal, Pn3-mal), V5 (high condensed, high C3G, C3-mal), and V6 (high condensed, high C3G, low acylated). There is not enough evidence to support whether condensed forms improved the stability or not as a general conclusion, the clustering and other analysis with different tools did not allow a clear trend since the samples are heterogeneous mixtures of anthocyanins.

4.6 Figures and Tables Figure 11. Color squares of nine corn varieties dissolved in Kool-Aid Invisible® stored at 4 °C, 22 °C, and 32 °C for 12 weeks in comparison to Kool-Aid Jammers Cherry.

Figure 12. Hue angles of nine corn varieties dissolved in Kool-Aid Invisible stored at 4 °C, 22 °C, and 32 °C for 12 weeks.

4 °C

38 V1 36 34 V2 32 V3 30 V4 28 V5 Hue (deg) Hue(deg) 26 24 V6 22 V7 20 0 2 4 6 8 10 12 V8 Time (weeks) V9

22 °C

60 55 V1 50 V2 45 V3 40 V4 35 Hue (deg) Hue(deg) 30 V5 25 V6 20 V7 0 2 4 6 8 10 12 Time (weeks) V8

32 °C

70 65 V1 60 V2 55 50 V3 45 V4

Hue (deg) Hue(deg) 40 V5 35 30 V6 25 V7 0 2 4 6 8 10 12 Time (weeks) V8

Figure 13. Lightness (L*) of nine corn varieties dissolved in Kool-Aid Invisible stored at 4 °C, 22 °C, and 32 °C for 12 weeks.

4 °C

75 V1

70 V2 V3 65 V4 60 V5

Lightness (L*) Lightness 55 V6

50 V7 0 2 4 6 8 10 12 V8

Time (weeks) V9

22 °C

80 V1

75 V2

70 V3

65 V4 60 V5

Lightness (L*) Lightness 55 V6 50 V7 0 2 4 6 8 10 12 V8

Time (weeks) V9

32 °C

85 V1 80 V2 75 V3 70 V4 65 V5 60 Lightness (L*) Lightness V6 55 V7 50 0 2 4 6 8 10 12 V8 Time (weeks) V9

Figure 14. Chroma of nine corn varieties dissolved in Kool-Aid Invisible stored at 4 °C, 22 °C, and 32 °C for 12 weeks.

4 °C

75 V1 V2 65 V3 55 V4

Chroma 45 V5

35 V6 V7 25 0 2 4 6 8 10 12 V8 Time (weeks) V9

22 °C

80 V1

70 V2

60 V3

50 V4

Chroma 40 V5 30 V6 20 V7 0 2 4 6 8 10 12 V8

Time (weeks) V9

32 °C

75 V1

65 V2

55 V3 V4 45

Chroma V5 35 V6 25 V7 15 0 2 4 6 8 10 12 V8 Time (weeks) V9

Figure 15. Principal component analysis of colored corn pericarp water extracts added to Kool-Aid Invisible® stored at 4 °C, 22 °C, and 32 °C for A) 4, B) 8, and C) 12 weeks.

A B

Table 2. Accession details and ANC general composition of the nine purple corn varieties.

Corn Accessions Used ANC Composition Varieties High condensed form, presence of C3G, Pg3G, V1 Apache Red x PI514747 Pn3G, C3-mal, Pn3-mal Very low condensed form, C3G, no Pg, high V2 PI550558 x PI571427 Pn3G, high C3-mal, high Pn3-mal Very low condensed form, high Pg, low Pn, high V3 MGCSC 506B x PI571427 C3-mal, high Pg3-mal, high Pn3-mal No condensed form, low Pg, high Pn, high Pn3- V4 MGCSC X131 mal, low other acylated forms MGCSC X131 x PI514862, High condensed form, high C3G, low Pg, low Pn, V5 MGCSC X131 x PI571427, high C3-mal, low other acylated forms PI213730 x PI571427 High condensed form, high C3G, low Pg, low Pn, V6 PI213730 x PI571427 low C3-mal, low other acylated forms No condensed form, low C3G, low Pg, high Pn, V7 PI550473 x Ames 8488 high C3-mal, low Pg3-mal, high Pn3-mal MGCSC X131 x PI571427, MGCSC X131 x PI514862, No condensed form, high C3G, low Pg, high Pn, V8 Apache Red x PI514862, high C3-mal, low Pg3-mal, high Pn3-mal PI550473 x PI571427 MGCSC X131 x Ames 8488, Very low condensed form, C3G, no Pg, high Pn, V9 PI213730 x Ames 8488, very high C3-mal, low Pg3-mal, high Pn3-mal PI550473 x Ames 8488

Table 3. Hue angle (degree) and Lightness (L*) of nine corn varieties dissolved in Kool-Aid Invisible® stored at three different temperatures (4, 22, and 32 °C) during 4, 8, and 12 weeks.

4 °C 22 °C 32 °C

Time 0 week 4 week 8 week 12 week 4 week 8 week 12 week 4 week 8 week 12

V1 28.5eD 25.1fBCD 25.4fCD 24.2fCD 31.1dC 32.8dD 36.6cD 37.3cC 43.1bD 48.4aBC

V2 27.2gE 22.0hD 22.5hE 22.5hD 28.6fD 29.6eE 35.0cE 33.0dCD 39.7bE 48.4aBC

V3 37.2bcA 30.4eA 32.9dA 31.6deA 36.1cB 32.8dD 35.5cE 39.3bC 38.8bF 43.8aDE

V4 29.1iC 27.9iAB 31.6hB 34.4gA 48.9fA 51.9eA 56.0dA 58.2cA 65.8bA 68.5aA 24.3efC V5 26.9cdE 25.7deBC 24.7efD D 23.7fE 25.4eG 27.8cG 28.1cD 37.2bG 43.1bDE

Hue (angle) V6 27.4dE 25.1dBC 26.4dC 27.8dB 30.5cdC 35.4bcC 38.3bC 35.3bcC 46.3aC 50.0aB 25.2fBC V7 29.4cBC 27.7dAB 26.0efC D 24.4gE 23.5hH 25.1fH 27.3dD 37.8bG 46.4aCD

V8 29.9cdB 25.9efBC 24.7efD 23.6fCD 24.1efE 26.6defF 30.4cF 27.4cdeD 34.1bH 42.2aE

V9 25.5cF 23.3dCD 22.3eE 26.7bBC 48.9aA 48.9aB 48.9aB 48.9aB 48.9aB 48.9aBC 69.0abA V1 57.1dF 59.6cdCD 61.2bcdD B 67.8abcC 68.9abE 70.1aD 70.4aD 70.0aDE 74.3aAB

V2 62.1hBC 64.4gAB 66.7fB 69.1eAB 73.8dA 76.5cAB 79.0bA 75.2cdB 79.9bAB 82.1aA 66.1cdA 72.8abC V3 61.8eCD 64.7deAB 64.6deBC B 70.3bcB 72.2bCD 74.6abC 71.6bCD D 76.9aAB 77.0abA 75.9bcA V4 62.5fAB 65.8efA 69.3deA 72.5cdA 74.9bcA B 76.1bBC 78.4abA 80.4aAB B

V5 58.8gE 58.2gD 61.3fD 62.1fAB 64.4eD 68.3dE 71.7bcD 70.0cdDE 73.7bCD 76.9aAB

Lightness (L*) V6 55.3cG 57.6bcD 61.0abcD 56.3cB 63.5abcD 65.5abcF 66.9abE 67.7abE 68.7aE 69.7aB

V7 61.6ghD 61.4hBCD 63.8fgC 64.7fAB 67.1eC 71.9dD 74.7cC 74.1cdBC 78.4bAB 81.7aA 66.4cdA 74.5abB 73.7abcB 74.8abA V8 61.6dD 62.5dABC 64.9dBC B 69.4bcdB C 77.7aAB C 78.6aAB B 66.9cdA V9 62.9fA 63.8efABC 65.8deBC B 67.9cC 70.8bDE 71.7bD 72.4bCD 76.4aBC 77.8aAB One-way ANOVA was conducted across the same row. Absence of the same small letter represents a significant difference (p < 0.05) in the same row. One-way ANOVA was also conduced across the same column. Absence of the same capitalized letter represents a significant difference (p < 0.05) in the same column. Time 0 gives the starting values for each variety.

Table 4. Chroma and ΔE of nine corn varieties dissolved in Kool-Aid Invisible® stored at three different temperatures (4, 22, and 32 °C) during 4, 8, and 12 weeks.

4 °C 22 °C 32 °C

Time 0 week 4 week 8 week 12 week 4 week 8 week 12 week 4 week 8 week 12 V1 55.6abA 52.3abcB 37.2cdAB 39.0bcdA 65.5aC B C 33.2dBC 46.2bcdAB C 34.8cdC B 34.4cdB 34.1cdB V2 44.3bcAB 62.6aF 44.3bcB 49.7bC C 38.9cB 30.6deCD 25.2efE 31.8dBC 24.4efEF 20.4fF V3 42.9bcd 52.3bcAB 32.9deB 68.3aA B 54.7bABC C 49.3bcdA 40.3cdeA 34.3deC 42.7bcdA C 29.0eC V4

60.2aH 43.6bB 40.7bD 28.5cC 29.2cC 28.1cD 26.0cE 25.9cC 22.7cF 21.4cEF V5 63.8aD 59.4aAB 58.2aAB 55.6abAB 47.2bcA 39.3cdAB 36.9cdB 36.1dAB 29.1dCD 29.8dC Chroma V6 52.4abA 63.0aE B 50.9abcC 39.1cABC 46.4bcAB 40.1bcA 41.8bcA 41.3bcAB 41.9bcA 41.3bcA V7 65.8aBC 63.1bA 59.0cA 57.2cA 53.0dA 40.8eA 34.1gC 36.7fAB 21.9hF 19.0iG V8 56.4abAB 32.8deBC 66.0aB 55.6bAB C 52.7bAB 47.4bcA D 28.5efD 38.9cdAB 27.5efDE 22.4fE V9 29.8eBC 61.0aG 55.9bAB 49.8cC 51.1cABC 49.8cA 41.0dA 38.5dB 39.6dAB D 26.4eD V1 - 10.9dB 14.3dBC 34.6aA 22.2cBC 31.0bBC 34.1aC 30.6bBC 35.8bCD 39.3aEF V2 - 19.1eAB 14.4eB 20.1aAB 26.5dB 35.1cAB 41.4aA 33.8cB 42.9bB 48.5aB V3 26.4bcd 27.5bcdB - AB 14.9dAB 17.7aAB 20.9cdBCD 30.2bcCB 36.4aB C 37.1bCD 42.5aD V4 - 17.0gB 20.8fA 33.6aB 36.3eA 38.8dA 41.1aA 42.7cA 47.6bA 47.7aBC V5 ΔE - 4.6fAB 6.6fD 9.2efAB 17.8deCDE 26.3cdCD 29.8bcD 30.0bcBC 38.5abC 40.4aE V6 25.5abcA 18.8bcdCD 26.1abcC 26.1abc - 11.2dAB 13.5cdBC B E D E 26.7abBC 30.3abE 32.9aG V7 - 3.3iA 8.1hCD 10.2gAB 14.8fDE 27.5eC 34.5cC 31.7dBC 47.4bA 52.0aA V8 - 11.5eAB 11.6eBCD 15.6efAB 21.0deBCD 35.8bcAB 40.8abA 29.8cdBC 42.2abB 46.8aC V9 - 5.7dAB 12.1cBCD 11.1cAB 12.6cE 21.7bD 24.2bF 23.5bC 34.1aD 38.1aF One-way ANOVA was conducted across the same row. Absence of the same small letter represents a significant difference (p < 0.05) in the same row. One-way ANOVA was also conduced across the same column. Absence of the same capitalized letter represents a significant difference (p < 0.05) in the same column. Most stable samples bolded at each temperature and time.

Table 5. Total polyphenols (mg gallic acid eq/g dry extract) and condensed tannins (mg catechin eq/g dry extract) concentrations of nine corn varieties dissolved in Kool-Aid Invisible® stored at three different temperatures (4, 22, and 32 °C) during 4, 8, and 12 weeks.

4 °C 22 °C 32 °C Time 0 week 4 week 8 week 12 week 4 week 8 week 12 week 4 week 8 week 12 V1 147.6bcd 142.1bcd 150.1abc 156.7abB 140.0cd 152.4abc 109.2eC 133.3dC BC C B C C A D 152.9abcC 164.6aAB V2 108.3de 117.5bcd 121.4bc 126.7abD 116.0cd 124.6bcB D 120.2bcD D C E D C 101.2eD 121.5bcD 136.8aBC V3 105.0bc 123.3abC 121.6ab 133.9aCD 118.5ab 120.0abB 122.6abD

D D 116.1abD C E D C 89.5cDE 119.5abD C V4 44.4bcF 47.6bF 43.8bcF 48.8bF 46.7bG 46.3bcF 47.8bE 36.0cF 96.7aE 48.5bE V5 179.4ab 183.8ab 195.9ab 175.6ab A 187.4abA 196.6abA A 189.6abA A 168.8bA A 213.2aA 189.8abA V6 105.4abc 110.0abc 113.3ab 108.5abc 108.8abc

Total Phenolics D 110.5abD D 92.3cD 110.1abcE D 104.7bcC CD 122.8aD DC V7 73.4bcE 76.0bcE 82.2abcE 65.2cEF 78.5bcF 87.7abE 70.3bcD 74.7bcE 86.0abE 101.0aD V8 133.3abB 141.8abC 150.7ab 128.8abB 133.5ab 134.1abB 125.6bC CD 142.8abC 77.1cDE D C C BC 153.2aC C V9 152.9bc 138.3cB 174.6abA 176.9ab 162.2abc 159.0abc B 154.7bcB 175.7abB C B B A AB 191.1aB 184.3abA V1 1.7aBC 1.3bD 1.2bD 1.0cB 0.6deE 0.7dD 0.6deCD 0.5eF 0.5deD 0.5eCD V2 1.8aB 1.5bC 1.3cC 1.1dAB 0.4fgF 0.6eD 0.5efD 0.4fgF 0.5fgDE 0.3gDE V3 2.3aA 1.9bB 1.9bB 1.6cA 0.8eD 1.1dB 0.8eAB 0.7fD 0.8eAB 0.6gC V4 0.6aE 0.4bF 0.4bF 0.3cC 0.3cG 0.2dF 0.1eE 0.2dG 0.1eF 0.1eE V5 2.4aA 2.2abA 2.0bcAB 1.3efAB 1.6deB 1.1fgB 0.8ghAB 1.0fghC 0.8hBC 1.8cdB

Tannins V6 1.1bD 0.8cE 0.7cdE 1.2bAB 0.6deE 0.5deE 0.9cA 0.4eF 0.4eE 2.0aA V7 1.4abCD 1.3bD 1.2bcD 1.4abAB 1.0cdC 0.7eD 0.8deAB 0.6eE 0.3fE 1.6aB Total Condensed V8 2.6aA 2.3bA 2.0cAB 1.2eAB 1.5dB 0.9fC 0.7gBC 1.1efB 0.7gC 1.5dB V9 2.4aA 2.2abA 2.0bcA 1.4deAB 1.8bcdA 1.4deA 0.9fA 1.3eA 0.9fA 1.7cdeB One-way ANOVA was conducted across the same row. Absence of the same small letter represents a significant difference (p<0.05) in the same row. One-way ANOVA was also conduced across the same column. Absence of the same capitalized letter represents a significant difference (p<0.05) in the same column. Most stable samples bolded at each temperature and time.

Table 6. Condensed forms, C3G, Pg3G, Pn3G, and first-order reaction kinetics for anthocyanin stability of nine corn varieties dissolved in Kool-Aid Invisible® stored at three different temperatures (4, 22, and 32 °C) during 4, 8, and 12 weeks based on HPLC concentrations (µg/mL).

4 °C 22 °C 32 °C Time 0 week 4 week 8 week 12 week 4 week 8 week 12 week 4 week 8 week 12 V1 3.4aB 3.2aB 2.7bB 2.6bB 2.3bB 1.8cB 1.6cB 1.6cB 1.0dB 0.5dAB

V2 0.5abE 0.6aD 0.4abcD 0.4abcC 0.5abD 0.3bcD 0.2cC 0.5abD 0.2cD <0.01 V3 0.5abE 0.5aD 0.5abCD 0.5abC 0.5abD 0.4abD 0.3bcC 0.5abD 0.2cdD 0.1dB V4 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 V5 6.9aA 6.1bA 5.8bcA 5.5cA 4.8dA 3.3eA 2.6fA 3.0efA 1.5gA 1.0hA V6 3.0aC 2.4abC 1.5bcdBC 0.3efC 1.9abC 1.3bcdeC 0.1fC 1.1defC 0.5defC 0.3efB V7 0.1aEF 0.4aD 0.1aD 0.2aC 0.1abE <0.01 <0.01 <0.01 <0.01 0.3aB Condensed forms V8 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 V9 0.9aD 0.5bD 0.3bCD 0.4bC 0.5abD 0.4bD <0.01 0.4bD 0.2bD <0.01 V1 13.9aC 9.8bC 7.1cD 5.4dE 2.7eE 1.1dG 0.3gF 2.9eF 0.8dG 0.2gF V2 9.7aE 8.7bC 7.1dD 5.1eE 4.0fDE 2.2hF 1.5iE 7.6cC 2.8gD 1.2iD V3 8.9bF 9.4abC 10.2aC 9.6abC 9.9abB 6.6cB 4.9dB 10.4aB 4.8dC 2.5eC V4 3.0aH 0.8bE 0.4cF 0.3dG 0.2efF 0.1fH <0.01 0.2deG <0.01 <0.01 glucoside - V5 22.2aA 19.7bA 18.4cA 17.2cA 12.9dA 6.0eC 4.0fC 6.4eD 2.0gE 0.9gDE V6 8.8aF 5.0bD 3.0cE 2.0dF 0.3eF <0.01 <0.01 <0.01 <0.01 <0.01 V7 4.8bcdG 5.6abcD 6.0abD 6.2aD 5.3abcDE 4.8bcdD 3.5dC 4.6cdE 1.6eF 0.5eEF 15.5aB 12.8bB 11.1cBC 9.4dC 7.1eC 4.1gE 2.9hD 10.8cB 5.7fB 3.3ghB Cyanidin 3 V8 V9 11.3cdeD 11.5cdB 11.5cdBC 11.9bcB 12.7abA 11.1deA 10.5eA 12.9aA 7.3fA 4.4gA

V1 1.9aC 1.4bCDE 0.3cdeD 0.5cdD 0.5cCD 0.2deB 0.1eC 0.4cdeCDE <0.01 <0.01 V2 1.8abCD 2.2aB 2.0aB 1.8abC 1.6abB 0.5cdB 0.1dC 1.2bcABC 0.2dB <0.01 V3 7.5aA 6.4abA 5.2bA 4.2cA 5.5bA 3.0dA 1.9eB <0.01 4.2cA 2.1eA glucoside

- V4 1.0aF 0.8bF 0.6bCD 0.4cD 0.3cCD 0.1dB <0.01 0.1dE <0.01 <0.01 V5 1.4bE 0.8cEF 0.5cdD 2.5aB 0.4cdCD 0.2dB 0.4dC 0.2dDE <0.01 <0.01 V6 1.2abEF 0.2bcG 1.3aBCD 0.4abcD 0.1cD <0.01 <0.01 <0.01 <0.01 <0.01 V7 1.3bEF 1.3bDE 1.2bBCD <0.01 1.1bBC 0.4cB 2.0aA 1.0bBCD 0.1cB <0.01 V8 3.1aB 1.8bBC 1.6bcBC <0.01 1.4bcB 0.3dB 2.0bA 1.5bcAB 0.2dB 1.0cdB Pelargondin 3 V9 1.5bDE 1.5bCD 1.0bcBCD <0.01 1.7bB 0.5cB 0.4cC 1.9bA 0.3cB 0.6cC V1 2.0aF 3.4aD 2.9aD 0.8aE 1.3aBC 0.4aE <0.01 0.4aBC <0.01 <0.01

V2 11.3aBC 5.6bcBCD 9.0abA 1.7dDE 4.3cdAB 3.1cdB 1.6dB 2.0cdB <0.01 <0.01 V3 5.3abE 7.5aB 4.6abC 5.9aA 0.7cC 2.4bcC 1.1cB 1.0cdBC 0.3cA 0.1c V4 3.2aEF 0.1eE 2.1bE 1.5cDE 0.8dBC <0.01 <0.01 0.1eC <0.01 <0.01 glucoside - V5 15.6aA 14.9aA 3.5cD 2.7cdCD 7.3bA 2.5dC 1.3eB 1.9deBC <0.01 <0.01 V6 0.1aF 0.2aE <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 V7 5.6aDE 4.2bCD 4.3bC 3.9bBC 1.4cBC 1.3cD 0.8cdBC 0.8cdBC 0.2dA <0.01

Peonidin 3 V8 12.1aB 6.5bcBC 5.8cB 4.9dAB 6.9bA 4.3deA 2.7fA 3.9eA 0.3gA <0.01 V9 8.8aCD 4.9bBCD 4.9bC 2.5dCD 4.1cABC 1.3eD 0.2fC 1.1eBC 0.3fA <0.01 First-order reaction kinetics of ANC based on HPLC concentration Temp V1 V2 V3 V4 V5 V6 V7 V8 V9 (°C) t , 4 13.2 10.7 13.1 22.2 12.6 49.1 40.7 10.9 9.8 1/2 weeks 22 8.1 7.3 10.1 6.5 7.1 14.4 13.5 5.6 7.1 32 3.6 3.0 3.9 2.2 5.1 7.3 7.3 4.2 5.4 Reaction 4 0.053 0.065 0.053 0.031 0.055 0.014 0.017 0.064 0.070 rate k, 22 0.086 0.095 0.069 0.107 0.097 0.048 0.051 0.125 0.097 -1 week 32 0.192 0.231 0.178 0.319 0.136 0.095 0.095 0.166 0.129 EA (kJ/mol) 3.3 2.9 2.6 6.7 2.7 5.7 5.2 2.9 1.7 2 0.90 0.84 0.77 0.98 1.00 1.00 1.00 1.00 0.98 R One-way ANOVA was conducted across the same row. Absence of the same small letter represents a significant difference (p<0.05) in the same row. One-way ANOVA was also conduced across the same column. Absence of the same capitalized letter represents a significant difference (p<0.05) in the same column. Most stable samples bolded at each temperature and time. n/a stands for not available. Total anthocyanin concentration was the sum of the quantification of each peak of the HPLC for every corn variety.

Table 7. Acylated forms, C3G-Mal, Pg3G-Mal, and Pn3G-Mal present in 9 corn varieties dissolved in Kool-Aid Invisible® stored at three different temperatures (4, 22, and 32 °C) during 4, 8, and 12 weeks, based on HPLC concentration (µg/mL).

4 °C 22 °C 32 °C Time 0 week 4 week 8 week 12 week 4 week 8 week 12 week 4 week 8 week 12 V1 7.7aF 0.3cD 1.5bDE 1.3bB 0.2cD <0.01 <0.01 <0.01 <0.01 <0.01 V2 12.6aE 1.8bABCD 2.3bCD 0.5cC 0.4cD 0.2cA <0.01 0.2cB <0.01 <0.01 V3 14.7aCD 2.9cABCD 6.1bB 3.0cA 2.9cC 0.7cdA 0.2dA 1.1cdA <0.01 <0.01

V4 0.3bG 0.5aCD 0.1cE 0.1cC 0.1cD <0.01 <0.01 <0.01 <0.01 <0.01 Mal - V5 15.4aCD 1.1cBCD 9.5bA 0.5cC 0.4cD <0.01 <0.01 <0.01 <0.01 <0.01

C3G V6 1.3aG 0.1bD <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 V7 13.6aDE 3.2cAB 2.9cCD 2.8cA 5.9bB 0.2dA 0.2dA 0.3dB <0.01 <0.01 V8 18.3aB 3.8cA 3.6cCD 3.0cA 8.5bA 0.5dA 0.6dA 0.5dAB <0.01 <0.01 V9 22.2aA 2.9bABC 2.5bCD 2.7bA 1.0bD 2.2bA 0.8bA 0.7bAB <0.01 <0.01 V1 1.6bC 3.0aCD 2.2abE <0.01 0.4cC <0.01 <0.01 <0.01 <0.01 <0.01 V2 2.0abC 7.3aABCD 0.5abF 5.6abD 1.4abBC 0.1bA <0.01 <0.01 <0.01 <0.01

V3 13.8aA 5.9bBCD 5.2bD 4.2bE 1.4bBC 0.2bA <0.01 0.7bA <0.01 <0.01 V4 0.3bD 0.1bD 0.1bF 0.6aG 0.2bC <0.01 <0.01 <0.01 <0.01 <0.01 Mal - V5 1.9bC 3.3aCD 0.7dF 2.3bF 1.3cBC <0.01 <0.01 <0.01 <0.01 <0.01 V6 0.1D <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Pg3G V7 5.3cB 8.9aABC 8.3aC 7.0bC 2.0dBC 1.3deA 0.8eA 0.8eA <0.01 <0.01 V8 6.1dB 15.0aA 12.6bA 10.2cA 2.5eB <0.01 <0.01 <0.01 <0.01 <0.01 V9 5.5cB 12.3aAB 9.9bB 8.8bB 5.8cA 1.2dA <0.01 <0.01 <0.01 <0.01 V1 5.6aF 0.3bA 0.1bF <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 V2 16.7aC 4.0bA 7.5bA 0.5bB <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

V3 10.4aE 4.0abA 5.4abB 2.7bA 1.8bC 0.1b <0.01 <0.01 <0.01 <0.01 V4 4.3aG 2.2bA 1.3cCD 0.0dC <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Mal - V5 4.3aG 0.2cA 1.9bC <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 V6 0.6H <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Pn3G V7 12.2aD 0.4cA 0.5cDEF <0.01 3.9bB <0.01 <0.01 <0.01 <0.01 <0.01 V8 18.7aA 1.4cA 0.9cDE <0.01 5.8bA <0.01 <0.01 <0.01 <0.01 <0.01 V9 17.9aB 0.9bA 0.3bcEF <0.01 0.2cD <0.01 <0.01 <0.01 <0.01 <0.01 One-way ANOVA was conducted across the same row. Absence of the same small letter represents a significant difference (p<0.05) in the same row. One-way ANOVA was also conduced across the same column. Absence of the same capitalized letter represents a significant difference (p<0.05) in the same column. <0.01 designates non-detectable peak concentrations.

Table 8. Correlation matrix of various parameters evaluated in the nine corn varieties (p < 0.05).

Correlation Matrix Tempera Time Chrom Hue TA CF C3G Pg3G Pn3G C3-mal Pg3- Pn3- ture a mal mal

Tempera 1.000 ture Time 0.000 1.000

Chroma -.418 -.718 1.000

Hue .481 .333 -.555 1.000

TA -.330 -.587 .788 -.477 1.000

CF -.150 -.246 .339 -.329 .317 1.000

C3G -.291 -.482 .658 -.406 .815 .580 1.000 Correlation Pg3G -.100 -.364 .389 -.092 .407 -.105 .367 1.000

Pn3G -.255 -.548 .620 -.425 .809 .339 .732 .283 1.000

C3-mal -.106 -.630 .689 -.285 .850 .161 .590 .386 .735 1.000

Pg3-mal -.421 -.269 .556 -.262 .609 -.139 .422 .473 .424 .479 1.000

Pn3-mal -.077 -.631 .630 -.267 .747 -.079 .400 .370 .666 .905 .384 1.000

Largest positive and negative correlations bolded TA-Total anthocyanins CF-Condensed forms

Table 9. Half-life values of Chroma of nine corn varieties dissolved in Kool-Aid Invisible® stored at three different temperatures (4, 22, and 32 °C) during 12 weeks

Half-life values of Chroma Temp V1 V2 V3 V4 V5 V6 V7 V8 V9 (°C) t , 4 13.2 30.0 49.4 12.0 64.1 19.0 56.9 41.9 43.1 1/2 weeks 22 13.1 9.3 12.2 10.9 15.2 20.1 12.4 9.6 17.7 32 13.3 7.7 9.8 8.6 11.1 22.1 6.5 7.7 9.9

4.7 References

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21. West, M.E.; Mauer, L.J. Color and chemical stability of a variety of anthocyanins and ascorbic acid in solution and powder forms. J. Agric. Food Chem. 2013, 61(17), 4169-4179. 22. Gonzalez de Mejia, E.; Dia, V. P.; West, L.; West, M.; Singh, V.; Wang, Z.; Allen, C. Temperature dependency of shelf and thermal stabilities of anthocyanins from corn distillers’ dried grains with solubles in different ethanol extracts and a commercially available beverage. J. Agric. Food Chem. 2015, 63(45), 10032-10041 DOI: 10.1021/acs.jafc.5b03888 23. Luna-Vital, D.; Li, Q.; West, L.; West, M.; González de Mejia, E. Anthocyanin condensed forms do not affect color or chemical stability of purple corn pericarp extracts stored under different pHs. Food Chem. 2017, In Press. 24. He, X.L.; Li, X.L.; Lv, Y.P.; He, Q. Composition and color stability of anthocyanin-based extract from purple sweet potato. Food Sci. Technol, Campinas. 2015, 35(3), 468-473 25. Malien-Aubert, C.; Dangles, O.; Amiot, M. J. Influence of Procyanidins on Color Stability of Oenin Solutions. J. Agric. Food Chem. 2002, 50, 3299-3305. 26. Mojica, L.; Berhow, M.; Gonzalez de Mejia, E. Black bean anthocyanin-rich extracts as food colorants: physicochemical stability and antidiabetes potential. Food Chem. 2017, 229, 628–639. 27. Mäkilä, L.; Laaksonen, O.; Kallio, H.; Yang, B. Effect of processing technologies and storage conditions on stability of black currant juices with special focus on phenolic compounds and sensory properties. Food Chem. 2017, 221, 422–430. 28. Cardona, J. A.; Lee, J.H.; Talcott, S. T. Color and polyphenolic stability in extracts produced from muscadine grape (Vitis rotundifolia) pomace. J. Agric. Food Chem. 2009, 57, 8421-8425 29. Gonçalves, G.A.; Resende, N.S.; Carvalho, E.E.; Resende, J.V.; Vilas Boas E.V. Effect pasteurization and freezing method on bioactive compounds and antioxidant activity of strawberry pulp. Int. J. Food Sci. Nutr. 2017, 1-16. doi: 10.1080/09637486.2017.1283681 30. Alcalde-Eon, C.; García-Estevez, I.; Puente, V.; Rivas-Gonzalo, J. C.; Escribano-Bailon, M. T. Color stabilization of red wines. A chemical and colloidal approach. J. Agric. Food Chem. 2014, 62, 6984−6994. dx.doi.org/10.1021/jf4055825 31. Socquet-Juglard, D.; Bennet, A. A.; Manns, D. C.; Mansfield, A. K.; Robbins, R. J.; Collins, T. M.; Griggiths, P. D. Effects of growth temperature and postharvest cooling on anthocyanin profiles in juvenile and mature brassica oleracea. J. Agric. Food Chem. 2016, 64, 1484-1493. 32. Rein, M. J.; Heinonen, M. Stability and enhancement of berry juice color. J. Agric. Food Chem. 2004, 52(10), 3106-114. 33. Osmani, S.A.; Halkjær Hansen, E.; Malien-Aubert, C.; Olsen, C.E.; Bak, S.; Lindberg Møller, B. Effect of glucuronosylation on anthocyanin color stability. v. 2009, 57(8), 3149-155. 34. Boulton, R. The copigmentation of anthocyanins and its role in the color of red wine: a critical review. Am. J. Enol. Vitic. 2001, 52(2), 67-87. 35. Loypimai, P.; Moongngarm, A.; Chottanom, P. Thermal and pH degradation kinetics of anthocyanins in natural food colorant prepared from black rice bran. J. Food Sci. Tech. 2016, 53(1), 461-470. doi:http://dx.doi.org/10.1007/s13197-015-2002-1 36. Reque, P.M.; Rosana, S.; Steffens, R. S.; Jablonski, A.; Flores, H. S.; Rios, A. O.; Jong, E. V. Cold storage of blueberry (Vaccinium spp.) fruits and juice: Anthocyanin stability and antioxidant activity. J. Food Compos. Anal. 2014, 33, 111–116. 37. Kamiloglu, S.; Pasli, A.A.; Ozcelik, B.; Camp, J. V.; Capanoglu, E. Influence of different processing and storage conditions on in vitro bioaccessibility of polyphenols in black carrot jams and marmalades. Food Chem. 2015, 186, 74-82, ISSN 0308-8146

CHAPTER 5: PROTECTIVE POTENTIAL OF ANTHOCYANIN-RICH PLANT EXTRACTS ON AN IN VITRO MODEL OF CELIAC DISEASE 5.1 Abstract

The objective was to evaluate the potential of anthocyanin-rich plant extracts to protect human epithelial cells against a celiac toxic peptide (CTP). Healthy differentiated Caco-2 cells were used to represent the intestine and treated with a known CTP from wheat protein, alpha- gliadin, H-Tyr-Pro-Gln-Pro-Gln-Pro-Phe-OH (YPQPQPF). Extracts from fruits (red and purple grape), vegetables (purple sweet potato and purple carrot), grains (colored corn kernels, black rice, sorghum, and blue wheat), and legumes (black bean, black lentil, black peanut, and purple bean) were tested to evaluate their protective and anti-inflammatory effects on cells treated with CTP using a cell proliferation assay and human inflammation array. Corn kernel extracts and black peanut increased protection (p<0.05). Purple corn extract (PAWE) exhibited a 3.6 fold decrease (FD) on granulocyte macrophage-colony stimulating factor (GM-CSF), both PAWE and red corn extract (RAW) exhibited a modest fold increase (FI) on interleukin (IL) 6, RAW had a 4.3 FI on IL-11, and PAWE exhibited a slight FI on metallopeptidase inhibitor 2 (TIMP-2)

(p<0.05). Interleukin 11 and TIMP-2 are anti-inflammatory while GM-CSF is pro-inflammatory, and IL-6 can be either pro- or anti-inflammatory. The ability of plant extracts to inhibit CTP has positive implication for human health; these extracts could potentially decrease the occurrence of inflammation for gluten sensitive individuals.

This chapter is based on the manuscript: Haggard, S., Johnson, J., Li, S., Gonzalez de Mejia, E. 2017. Protective Potential of Anthocyanin-Rich Plant Extracts on an in vitro Model of Celiac Disease. To be submitted to Food and Function.

5.2 Introduction Autoimmune disorders occur when the immune system mistakenly targets and destroys healthy body cells and tissues.1 There are more than 80 types of autoimmune disorders that can occur in almost any part of the body.1 Celiac disease is an inherited autoimmune disorder that affects the digestive process of the small intestine. When a person with celiac disease consumes gluten, a protein found in wheat, rye, and barley, their immune system responds by targeting the small intestine causing inflammation and disrupting nutrient absorption.2

Undiagnosed and untreated, celiac disease can lead to the development of other autoimmune disorders, as well as osteoporosis, infertility, and neurological conditions.3 T-cells recognize and kill infected virus cells in the body. In celiac disease, there are gluten-specific T-cells that are directly involved; they target peptides derived from gluten.4

The Janus-kinase pathway is a causative agent in many diseases and inflammation. The

Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway is a signal transducer that is used by cytokine stimulating factors and hormones. It is found in the cells and transports signals from the transmembrane receptors to the nucleus.5

Anthocyanins (ANCs), water-soluble phenolic compounds, can be found in many fruits and vegetables and are responsible for their red, purple, and blue colors.6 Structurally based on the flavylium ion or 2-phenylbenzopyrillium, the aglycon form of ANCs is called anthocyanidin; it presents hydroxyl and methoxyl groups depending on positioning. Pelargonidin, cyanidin, delphinidin, petunidin, peonidin, and malvidin are the six most commonly found ANCs in food.7

ANCs are found in a variety of plants8-10 as well as in varieties of corn (Zea mays L.) produced around the world; kernels have a range of colors such as: red, yellow, purple, green, blue,

53 brown, and white. Zea mays L. is a corn traditionally cultivated by Native Americans in North and South America. Through HPLC testing, it was found that purple corn kernels contain cyanidin 3-O-β-D-glucoside, pelargonidin 3-O-β-D-glucoside, cyanidin 3-O-β-D-(6-malonyl- glucoside), pelargonidin 3-O-β-D-(6-malonyl-glucoside), and peonidin 3-O-β-D-(6-malonyl- glucoside).11 ANCs are strong antioxidants12 and their consumption protects from cardiovascular disease,13 potentially decreases weight gain and body fat accumulation,14 protects against diabetes,15 reduces inflammation,16 inhibits cancer growth,17 and acts as a neuroprotective agent.18 ANCs are potentially inhibiting the JAK/STAT pathway by keeping the signal transducer and activator of transcription from becoming phosphorylated, stopping the reaction from passing from the cytoplasm to the nucleus.19

The hypothesis of this research was that ANC-rich plant extracts selectively inhibit the production of inflammatory biomarkers in a cell model of celiac disease. The aim of this study was to test the effects of ANC-rich extracts from an assortment of plant sources, enriched ANC extracts from colored corn, and extracts from different colored corn selected varieties (A-F) from purple corn with unique ANC profiles on an in vitro model of celiac disease. Cell protection and viability were assessed as well as the anti-inflammatory effects of ANCs on Caco-

2 cells treated under controlled conditions to determine the effect of differing ANCs on interleukin and other inflammatory protein production.

5.3 Materials and methods

A commercially-available purple corn cultivar was purchased from Angelina’s Gourmet

(Swanson, CT). Red corn was obtained from Siskiyou Seeds (3220 East Fork Rd. Williams, OR

97544). Colored corn varieties A-F were produced at the University of Illinois at Urbana-

Champaign (UIUC). The dry water extracts from purple sweet potato, carrot, and both grapes were from San Joaquin Valley Concentrates (Fresno, CA). The other plant materials were obtained from UIUC. Caco-2 cell line was obtained from the American Type Cell Cultures

(Manassas, VA). Modified Eagle’s Medium (MEM1X) was purchased from Sigma-Aldrich (St.

Louis, MO), fetal bovine serum was purchased from Invitrogen (Grand Island, NY), streptomycin antibiotic was purchased from Mediatech Inc. (Manassas, VA). RayBio® C-Series Human

Inflammation Array C1 was purchased from RayBiotech (Narcross, GA). Omnia® Y Peptide 7 Kit and JAK3 Recombinant Human Protein were purchased from ThermoFisher Scientific (Waltham,

MA). Celiac toxic peptide H-6712 Gliadorphin-7 H-Tyr-Pro-Gln-Pro-Gln-Pro-Phe-OH

Trifluoroacetate salt (YPQPQPF, purity > 95%) was purchased from Bachem (Torrance,

California). RayBio® C-Series Human Inflammation Array C1 was purchased from RayBiotech

(Norcross, GA). Primary antibodies anti-rabbit JAK3 (D1H3), Phospho-JAK3 (Tyr980/981)

(D44E3), STAT1 (D1K9Y), and Phospho-STAT1 (Tyr701) (58D6) were purchased from Cell

Signaling Technology (Danvers, MA). Secondary antibody anti-rabbit IgG horseradish peroxidase conjugated was purchased from GE Healthcare (Buckinghamshire, UK). All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

5.3.1 Extraction and semi-purification of ANC

5.3.1.1. Purple corn pericarp pure water extract (PW)

The pericarp of purple corn was extracted in water at 50 °C for 3 h in the dark. The extract was filtered through grade 2 filter paper followed by 0.45 µm nylon membrane filters and freeze dried (VirTis Sentry 5 L freeze drier, Gardiner, NY, USA).

5.3.1.2 Purple corn resin-purified acidified water extract (PAW)

This was obtained by extracting 1 kg of ground whole corn kernels with 2 L of 2% v/v aqueous formic acid at 4 °C for 20-24 h in the dark. The extract was filtered through grade 2 filter paper followed by 0.45 µm nylon membrane filters. The clarified extract was passed through a column containing Amberlite FPX66 (Dow Chemical, Midland, MI) resin (~1:1 ratio of extract and resin volumes) previously washed with water. The resin column was then washed with water until readings taken with a conductivity meter equipped with a flow cell

(Traceable®, ThermoScientific, Waltham, MA) suggested that no significant amounts of unretained compounds were present. Absolute ethanol (USP grade) was introduced onto the column to remove ANCs as well as other adsorbed phenolic compounds. Collection was started when color elution was observed and terminated when the eluate was colorless. Ethanol and water were removed under water aspirator vacuum using a rotary evaporator at bath temperatures of <40 °C in the dark. The remaining water extract was freeze-dried, and the dried extract was stored at -20 °C.

5.3.1.3 Red corn resin-purified acidified water extract (RAW)

Whole red corn kernels were used for the extraction. The same procedure was followed as described for PAW.

5.3.1.4 Purple corn PAW extract with additional ethyl acetate purification (PAWE)

PAW (3 mg/mL) was dissolved in water and extracted 5 times with an equal volume of ethyl acetate (v:v) to water in a separating funnel. The ethyl acetate layers were discarded and the aqueous layer was placed on a rotary evaporator with water aspirator vacuum and a bath

56 temperature of 40 °C until all traces of residual ethyl acetate were removed. The aqueous layer was then freeze-dried.

5.3.1.5 Plant extracts and six specialty corn varieties

ANC-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), all referred to as “other plant extracts,” and six specialty corn varieties are presented in Table 10. The six specialty corn varieties were extracted using the same procedure as described for PW. The purple sweet potato, purple carrot, and both grapes were received as dry water extracts. Black rice, sorghum, and blue wheat, black bean, black lentil, black peanut, and purple bean were extracted as indicated for PAW and RAW.

5.3.2 Chemical Analysis

5.3.2.1 Measurement of total monomeric ANC concentrations

All the plant extracts were analyzed for total monomeric ANC (TA) concentration by the pH differential method20 as previously reported using a microplate reader method in three independent replicates. Samples were diluted using two buffers (pH 1.0, 0.25 M KCl buffer and pH 4.5, 0.40 M sodium acetate buffer). Two hundred µL of diluted solutions at each pH were transferred to a 96-well plate and the absorbance read at 520 and 700 nm using a Synergy 2 multiwell plate reader (BioTek, Winooski, VT). The total monomeric ANC concentration was calculated as cyanidin-3-glucoside (C3G) equivalents per L as described below:

Total monomeric ANCs (mg/L) = (A * MW * D * 1000)/(ε * PL* 0.45)

Where: A = (A520 – A700) at pH1.0 – (A520 – A700) at pH4.5; MW = 449.2 g/mol for C3G; D = dilution factor; PL = constant path length 1 cm; ε = 26900 L/mol-cm which is the molar extinction coefficient for C3G, 1000 conversion factor from grams to milligrams and 0.45 as the conversion factor from the established method to the plate reader method. Final results were expressed as mg C3G equivalents per gram of dry extract.

5.3.2.2 Measurement of total polyphenols

All the plant extracts were analyzed for total phenolics using methods previously reported.6,21 A standard curve was created using gallic acid; 0, 40, 50, 75, 100, 150, 175, and

200 µg/mL. Fifty µL standard and 1:2 diluted samples were added to a 96-well plate in triplicate. Fifty µL 1 N Folin-Ciocalteu was add to each well, mixed, and let stand for 2-5 min, then 100 µL 20% Na2CO3 (sodium carbonate) was added to each well, mixed, and let stand for

10 min. Absorbance was read using a Synergy 2 multiwell plate reader (BioTek, Winooski, VT) at 690 nm and results were expressed as mg gallic acid per gram of dry extract.

5.3.2.3 Measurement of condensed tannin concentration

All the plant extracts were analyzed for condensed tannins using methods previously reported.22,23 A standard curve was constructed using a serial diluted catechin/methanol solution (0.1, 0.2, 0.4, 0.6, and 0.8 mg/mL). Samples were diluted to a factor of 1:10 or 1:20 with methanol. Vanillin reagent was prepared fresh by mixing equal volumes of 1% vanillin in methanol and 8% concentrated HCL in methanol. Five mL of 1:1 of 8% HCL/methanol with 1% vanillin at was added at 1-min intervals to 1-mL aliquots of the samples. Five milliliters of 4% concentrated HCL in methanol was added to a second 1-mL aliquot (the blank) also at 1-min intervals. Absorbance at 500 nm was read after 20 min and the absorbance of the blank was

58 subtracted from the absorbance with vanillin. The standard curve and samples were always run at the same temperature. Measurement of each sample was repeated three times. The total tannin content was expressed as milligram catechin per milliliter of aqueous extract based on the standard curve. Solutions were protected from light during preparation, and wells were covered with aluminum foil to avoid methanol evaporation.

5.3.3 HPLC Analysis

HPLC analysis of all extracts for ANC profile was performed in triplicate using a Hitachi

HPLC System (Hitachi High Technologies America, Inc., Schaumburg IL) equipped with a multi- wavelength detector, L-7100 pump following previously reported protocol24 with some modifications. The injection volume was 20 µL. The flow rate was 1 mL/min and the gradient used was from 2% formic acid in water and 0% acetonitrile to 40% acetonitrile in a linear fashion using a Grace Prevail C18 (5 µm, 250 × 4.6 mm, Columbia, MD) for 30 min. Peaks were identified based on the same retention time as available standards or the literature.24 The concentration of each ANC was determined using a calibration curve with 5 points in the range of 50–2000 ng/ml of C3G standard. The calibration curve was obtained by plotting the area of cyanidin-3-O-glucoside peak versus its concentration. The calibration curve obtained a correlation coefficient (R2) of 0.99 or better.

5.3.4 Cell Culture and Celiac Disease Model

Caco-2 were cultured at 37 °C under a 5% CO2 atmosphere using Modified Eagle’s

Medium, supplemented with 20% (v/v) fetal bovine serum, and 1% streptomycin antibiotic.

The growth medium of Caco-2 cultures was replaced every 2–3 days. All experiments were performed on healthy differentiated Caco-2 cell monolayers. In order to mimic celiac disease in

59 the human body and the effect of ANC-rich extracts Caco-2 cells were treated with a pure peptide with known celiac toxicity. Celiac toxic peptide YPQPQPF is an alpha-gliadin wheat protein.25 Cell viability was tested using purple and red corn extracts, corn variety extracts, plant extracts and the peptide. Caco-2 cells were treated directly with different concentrations of extracts and the peptide. Cell viability was measured using the cell proliferation assay was performed using the CellTiter® 96 Aqueous One Solution proliferation following the manufacturer’s instructions. Then Caco-2 cells were treated with both extracts and the peptide to determine cell protection.

5.3.5 Inflammation Array

A RayBio® C-Series Human Inflammation Array C1 was performed following the manufacturers instruction using cell culture media. Caco-2 cells were treated with 12 mM CTP alone, or in combination with PAWE, or RAW at a concentration of 1 mg/mL. Membranes were visualized using a Gel Logic 4000 Pro Imaging System (Carestream Health, Inc., Rochester, NY,

USA).

5.3.6 Janus Kinase Assay

Inhibitory potential of corn extracts were determined using the Invitrogen Omnia®

Kinase Assay Kit User Guide instructions Determining Kinase Inhibitor IC50 Values section. A

Synergy 2 multiwell plate reader (BioTek, Winooski, VT) was used to measure fluorescence.

Because ANCs produce their own fluorescence, values were obtained by taking the difference of samples with the enzyme (JAK3) and samples without. Samples inhibited by ANCs were compared with an uninhibited reaction.

5.3.7 Evaluation of phosphorylation by western blotting

To further evaluate the potential of the ANC-rich colored corn extracts on the JAK/STAT pathway in Caco-2 cells, protein expression of JAK 3, p-JAK3, STAT1, and p-STAT1 was assessed by western blotting using the antibodies mentioned in the Materials section. The cells were treated with 12 mM CTP alone, or in combination with PW, PAW, PAWE, or RAW at a concentration of 1 mg/mL. After the treatments, the cells were lysed using RIPA buffer. The cell lysates were standardized quantifying the protein, and 40 μg of protein were loaded in each lane of an SDS-PAGE gel. Separated proteins were transferred to PVDF membranes; the primary antibody was added at the manufacturer’s recommended dilution and incubated at 4

°C overnight. The secondary antibody was added at the manufacturer’s recommended dilution and incubated for up to 3 h at room temperature. Protein expression was detected using 1:1 chemiluminescent reagents of the ECL Prime Western Blotting kit (GE Healthcare,

Buckinghamshire, UK) and visualized using a Gel Logic 4000 Pro Imaging System (Carestream

Health, Inc., Rochester, NY, USA). The intensity of each band was normalized to GAPDH, and the results were expressed as the expression level relative to a control.

5.3.8 Computational Docking

DockingServer was used for molecular modeling of the ANCs with tyrosine kinase

JAK3.26 The MMFF94 force field and Gasteiger partial charges were used in the modeling. Non- polar hydrogen atoms were merged and rotatable bonds defined. Docking calculations were carried out on receptor JAK3 protein structures with ligands of cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-glucoside, and staurosporine, a synthetic JAK3 inhibitor. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of AutoDock tools.27 AutoDock parameter set- and

61 distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Initial position, orientation, and torsions of the ligand molecules were set randomly. Docking results were derived from 100 different runs that were set to terminate after a maximum of 250,000 energy evaluations. The population size was set up to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied.

5.3.9 Statistical Analysis

Statistical analyses were conducted using one-way ANOVA to compare experimental to control values JMP version 8.0. Comparisons between groups were performed using Tukey’s W test, and differences were considered significant at p < 0.05.

5.4 Results and Discussion

5.4.2 Chemical Analysis

Total Anthocyanin, Polyphenol and Condensed Tannins Concentration

Total anthocyanin, total polyphenol, and total tannin concentrations for all extracts are shown in Table 11. Of the four differently extracted colored corn extracts, PAWE contained the highest concentration of ANCs, which was 217.5 mg C3G equivalent/g extract compared to

PAW with 190 mg C3G equivalent/g extract and PW with 92.3 mg C3G equivalent/g extract.

RAW had much lower total ANC (56.2 mg C3G equivalent/g extract) than its counterpart PAW

(190 mg C3G equivalent/g extract). PAWE and PAW had the highest total polyphenols and

PAWE had the highest tannin concentration. Corn variety F had the highest concentration of

ANCs and there was no difference between polyphenols and tannins amongst corn varieties. Of the other plant extracts black rice had the highest concentration of ANCs, polyphenols, and

62 tannins. In a similar study, it was also found that black rice had the highest concentration of

ANCS, polyphenols, and tannins out of chickpea, sorghum, quinoa, lentil, amaranth, brown rice, oat, white rice and soft wheat.28

5.4.3 HPLC Anthocyanin Profiles

HPLC profiles for PW, PAW, PAWE, and RAW are shown in Figure 16. Profiles for six colored corn varieties are shown in Figure 17. Peaks for corn extracts are labeled 1-7; peak 1 represents condensed forms with a retention time (RT) ~10 min, peak 2 represents cyanidin-3-

O-glucoside with RT ~16 min, peak 3 represents pelargonidin-3-O glucoside with RT ~17 min, peak 4 represents peonidin-3-O-glucoside with RT ~18 min, peak 5 represents cyanidin-3-(6’’- malonyl)-glucoside with RT ~19 min, peak 6 represents pelargonidin-3-(6’’-malonyl)-glucoside with RT ~20 min, and peak 7 represents peonidin-3-(6’’-malonyl)-glucoside with RT ~21 min.

Condensed forms, flavanol-anthocyanins, are a characteristic of purple corn chemical composition and some are esterified with malonic acid on the aglycone.29 Profiles for other plant extracts are shown in Figure 18. Previous studies showed30 that black rice has several ANC peaks consisting of cyanidin-3-O-glucoside, cyanidin-3-O-gentiobioside, cyanidin-3-O-rutinoside, and delphinidin-3-O-glucoside. Black bean’s ANC profile consists of delphinidin-3-O-glucoside, petunidin-3-O-glucoside and malvidin-3-O-glucoside.31 Black peanut32 contains cyanidin-3- sambubioside. Black lentil has one identified major ANC peak of delphinidin 3-O-(2-O-β-d- glucopyranosyl-α-l-arabinopyranoside).33 Purple bean has various ANC including cyanidin-3-O- glucoside, delphinidin O-3-glucoside, cyanidin 3-O-sambubioside, pelargonidin-3-O-glucoside, and peonidin-3-O-glucoside.34 Blue wheat35 is also known to contain many ANCs such as delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside, cyanidin-3-rutinoside,

63 petunidin-3-glucoside, petunidin-3-rutinoside, peonidin-3-rutinoside and malvidin-3-rutinoside.

Both sorghum varieties, red and black, have many unidentifiable ANCs but one main ANC was identified as luteolindin.36 Purple sweet potato is abundant in ANCs including: cyanidin 3- sophoroside-5-glucoside, peonidin 3-sophoroside-5-glucoside, cyanidin 3-(6"- caffeolysophoroside)-5-glucoside and another unspecified pelargonidin compound.37,38 Red grape has one main peak identified as peonidin-3,5-O-glucoside.39 Purple grape, as seen in other experiments and studies40 has several peaks of ANCs including malvidin-3-O-glucoside, delphinidin-3,5-diglucoside, cyanidin-3,5-diglucoside, peonidin-3,5-diglucoside and petunidin-

3,5-diglucoside. Purple carrot has various ANCs41 including cyanidin-3-xylosyl (sinapolyglucosyl) galactoside, cyanidin 3-xylosylgalactoside, cyanidin - xylosyl (p-hydroxybenzoylglucosyl) galactoside, cyanidin 3-xylosyl (feruloylglucosyl) galactoside, cyanidin 3-xylosyl

(coumaroylglucosyl) galactoside, pelargonidin 3-xylosylgalactoside, peonidin 3- xylosylgalactoside, peonidin 3xylosyl (sinapoylglucosyl) galactoside and peonidin 3-xylosyl

(feruloylglucosyl) galactoside.

5.4.4 Effect of ANC-rich corn and other plant extracts on a Celiac Disease Model

Cell viability for the four colored corn extracts had an upper limit concentration of 1 mg/mL; cells began to die off at higher concentrations. The six corn varieties along with purple bean, black peanut, black lentil, purple carrot, and purple sweet potato were all able to sustain

100% survival at 1 mg/mL. Black rice, black bean, blue wheat had an upper limit of 0.1 mg/mL while sorghum, red grape, and purple grape had an upper limit of 0.01 mg/mL; therefore, these concentrations were used to ensure cell viability was higher than 80% and not lower due to these extracts. Cell viability for Caco-2 cells treated with celiac toxic peptide ranging from 0-25

64 mM resulted in an IC50 value of 13.7 mM (data not shown). Two concentrations of CTP were used 12 mM (IC35) and 25 mM (IC95) to represent subtle and chronic inflammation from celiac disease. Table 12 shows the percent survival of Caco-2 cells treated with the four corn extracts at 1 mg dry weight extract/mL and 12 mM celiac toxic peptide (CTP). In a mouse model for celiac disease, humanized HLA-DA8 transgenic mice were created to mimic the human B57 polymorphism to study the anti-gluten T cell response and ability to recruit cross-reactive TCR repertoire. It was found that even after exposure to different forms of dietary gluten peptides, mice developed only low-penetrance enteropathy and intestinal inflammation which demonstrated the presence of additional factors to pathogenesis of celiac disease.42 This can be related to how corn ANCs can help regulate the pathogenesis of celiac disease. In a study on celiac disease and diets, it was shown that polyphenols and carotenoids exerted antioxidant and anti-inflammatory properties by being able to suppress the activation of NF-kB. An effect that is potentially mediated by the inhibition of different protein kinases involved in signal transduction pathway.43 Additionally, another study used purple carrot and sweet potato extracts to inhibit pro-inflammatory cytokines. It was observed that IL-8 production, a cytokine that mediates the activation of immune responses, was significantly inhibited in Caco-2 cells treated with both 50 and 100 µg/mL levels of either purple carrot or sweet potato extract.44

When cells were pretreated with plant extracts (1 mg/mL) for 4 h, before being treated with 12 mM CTP for 24 h, there was no increased cell survival. In the present study, cells treated with plant extracts and CTP together for 24 h had a significantly increased survival rate compared to those treated with CTP alone. RAW and PAWE had the highest survival (%) when cotreated. A study observed the effects of geniposide on TNBS-induced experimental colitis in rats. Doses of

25, 50, and 100 µg/ml geniposide significantly down-regulated NF-kB, COX-2, and iNOS protein expression in LPS-infected Caco-2 cells, which helped suppress inflammation.45 Table 13 shows survival (%) increase of cells treated with colored corn and other plant extracts with 25 mM CTP compared to cells treated with CTP alone. Corn varieties A-E had the most increased survival of the corn extracts with an increase of 65-84%. Black peanut had the highest increased cell survival for the other plant extracts.

5.4.5 Effect of colored corn and other plant extracts on proteins related to inflammation

Figure 19 shows the x-fold increase of the proteins in the human inflammation array that were significantly different among cells treated with either CTP, PAWE, or RAW as compared to untreated cells. PAWE exhibited a 3.6 fold decrease on granulocyte macrophage- colony stimulating factor (GM-CSF). GM-CSF is a pro-inflammatory cytokine. The study that looked at the effects of geniposide on TNBS-induced experimental colitis in rats showed that the geniposide in the treated rats had down-regulation in the increased expression of NF-kB,

COX-2 and iNOS proteins in TNBS treated rats.45 RAW exhibited a 4.3 fold increase on interleukin (IL) 11. IL-11 is an anti-inflammatory pleiotropic cytokine. Both PAWE and RAW exhibited a small fold increase on interleukin 6. IL-6 is an anti-inflammatory myokine and a pro- inflammatory cytokine. In another study, it was observed that IL-6, promptly and transiently was produced in response to infections and tissue injuries. It contributed to the host defense through the stimulation of acute phase responses, hematopoiesis and immune reactions. It also deregulated continual synthesis of IL-6 played a pathological effect on chronic inflammation and autoimmunity.46 In the same purple carrot and sweet potato study previously mentioned, there were no significant effects on IL-6 from the 50 µg/mL purple carrot or potato

66 extract treatment.44 However, in the present study, we were trying to evaluate the more specific effects of corn ANCs on autoimmune antibodies. PAWE exhibited a 0.35 fold increase on metallopeptidase inhibitor 2 (TIMP-2). TIMP-2 is anti-inflammatory. In a mechanistic study, it was revealed that overexpression of TIMP-2 suppresses microglial activation via inhibition of the activity of mitogen-activated protein kinases and NF-kB. This resulted in the enhancement of the activity of anti-inflammatory Nrf2 and cAMP-response element binding protein transcription factors and TIMP-2 also inhibited activity and expression of LPS-induced MMP-3, -

8 and -9 contributing to the reduction in inflammation.47 These results suggest that depending on their composition ANC-rich extracts affect the production of inflammatory biomarkers, the amount of fold change required to see biological changes was not evaluated in this study.

5.4.6 Effect of ANC-rich colored corn and other plant extracts on Janus Kinase 3

Inhibition of JAK3 was performed using different concentrations PW, PAW, PAWE, RAW, and pure C3G in a kinase assay, ex vitro. In a similar study, mice were transplanted with tumor cells and given a JAK3 inhibitor (CP690550 blocker) to evaluate the effects of the inhibitor on the tumor over a period of time. JAK-STAT signaling is one of the most important pathways to control the cell cycle in virtually all cell types in a healthy organism,48 the pathway effects of the tumor cell was similar to the present study and the JAK3 inhibition method used. IC50 values were calculated using percent inhibition at 1 mg/mL dry weight extract and it was compared among extracts and pure C3G (Figure 20). When IC50 values were corrected for C3G equivalents, the concentration for the four corn extracts was much lower compared to pure

C3G. The inhibition curve of JAK3 by PW, PAW, RAW, and pure C3G is shown in Figure 20. In a similar study that studied the effects of the drug tofacitnib on JAK inhibition, it was stated that

67 in a cellular setting, tofacitnib demonstrated preferential inhibition of signaling pathways associated with JAK1 and/or JAK3. Those individuals who took tofacitnib 5 mg twice daily, showed average inhibited cytokines at 50-60%,49 comparable to the effectiveness of ANCs in

JAK3 inhibition. The JAK/STAT pathway is involved in healthy cellular function and only poses an issue when there is an unbalance or dysregulation in the pathway.

5.4.7 Effect of colored corn and other plant extracts on phosphorylation on JAK 3, STAT 1 by western blotting

Figure 21 shows western blots of JAK 3, phosphorylated JAK 3, STAT 1, and phosphorylated STAT 1 in Caco-2 cells and the %phosphorylation of JAK 3 and STAT 1 in untreated Caco-2 cells, those treated with 12 mM CTP, and those cotreated with 12 mM CTP and PW, PAW, PAWE, and RAW (1 mg/mL). There were no significant differences, which suggested that either the concentration of CTP was not appropriate to promote phosphorylation or phosphorylation is not initiated by CTP. The presence of JAK and STAT in

Caco-2 cells suggests they have a functional role within the cells, but the relationship between

JAK/STAT and Celiac disease has not yet been established and further research is needed in this area to determine if there is in fact a link.

5.4.8 Computational Docking

Table 14 shows the results of computational docking for interaction between JAK 3 and the three main ANCs of colored corn and a known JAK 3 inhibitor, staurosporine. Molecular docking results demonstrated that free binding energies between each ANC and JAK3 favored interactions. Each of the three main ANCs had at least one binding site in common with staurosporine. Figures 22 shows the interaction between C3G and JAK 3.

5.5 Conclusions

Pure C3G had the highest IC50 value on the activity of JAK3, in comparison to all the colored corn extracts, suggesting that the ANC in the extracts had an interaction, which inhibited JAK3 more effectively. Colored corn extracts were able to inhibit JAK3 completely at high concentration. The high consumption of ANCs in the diet has not been proven to be toxic.

Differentiated Caco-2 cells survived the celiac toxic peptide if colored corn extracts were exposed simultaneously, not as a pretreatment. RAW and PAWE were able to protect cells the most and to decrease inflammatory markers in cells treated with lower concentrations of CTP.

When cells were treated with higher concentrations of CTP, corn specialty varieties A-E protected cells the best out of the corn extracts and black peanut protected the best for the other plant extracts. ANC-rich extracts protected Caco-2 cells, inhibited JAK3, and reduced inflammatory proteins.

5.6 Figures and Tables

Figure 16. HPLC profiles for four corn extracts at 520 nm. Peaks: 1 Condensed form Retention time (RT) ~10 min. 2 Cyanidin-3-O-Glc RT ~16 min. 3 Pelargonidin-3-O-Glc RT ~17 min. 4 Peonidin-3-O-Glc (RT) ~18 min. 5 Cy3-6''Malonylglc- RT ~19 min. 6 Pg3-6''Malonylglc- RT ~20 min. 7 Pn3-6''Malonylglc- RT ~21 min.

Figure 17. HPLC at 520 nm of six colored corn varieties. Peaks: 1 Condensed form Retention time (RT) ~10 min. 2 Cyanidin-3- O-Glc RT ~16 min. 3 Pelargonidin-3- O-Glc RT ~17 min. 4 Peonidin-3- O-Glc (RT) ~18 min. 5 Cy3-6''Malonylglc- RT ~19 min. 6 Pg3-6''Malonylglc- RT ~20 min. 7 Pn3-6''Malonylglc- RT ~21 min.

Figure 18. HPLC profiles of other ANC-rich plant extracts at 520 nm.

Figure 19. Inflammatory proteins expressed by Caco-2 cells treated with celiac toxic peptide (CTP) 12 mM alone (C+), 1 mg/mL purple corn acidified water extract further purified with ethyl acetate (PAWE), or red corn acidified water extract (RAW) and CTP cotreated compared to untreated cells.

a 6 a 4 a 2 0 -2 -4 X-fold increase -6 b -8 b -10 GM-CSF b IL-11

C+ PAWE RAW

0.4 a 0.35

0.3 ab 0.25 a 0.2

0.15 b b X-fold increase 0.1 0.05 c 0 IL-6 TIMP-2

C+ PAWE RAW

Figure 20. Inhibition of Janus kinase 3 by purple corn water extract (PW), purple corn acidified water extract (PAW), PAW further purified with ethyl acetate (PAWE), red corn acidified water extract (RAW), and pure C3G.

PW PAW 400 350 400 300 350 250 300 200 250 200 RFU 150 RFU 150 100 100 50 50 0 0 -50 0 20 40 60 -50 0 20 40 60 Time (min) Time (min) Uninhibited 0.1mg/mL Uninhibited 0.1mg/mL

1mg/mL 10mg/mL 1mg/mL 10mg/mL

RAW Pure C3G 400 400 350 350 300 300 250 250 200 200 RFU RFU 150 150 100 100 50 50 0 0 -50 0 20 40 60 -50 0 10 20 30 40 50 60 Time (min) Time (min) Uninhibited 0.1mg/mL Uninhibited 0.01mg/mL

1mg/mL 10mg/mL 0.1mg/mL 1mg/mL

Inhibitors IC50 (µg dry weight IC50 (µg C3G % JAK3 activity decrease at extract/mL) equivalent/mL) 1 mg dry weight extract/mL PW 324.5 ± 155.5a 29.9 ± 14.3b 73.7 ± 8.2a RAW 660.0 ± 210.0a 37.1 ± 11.8b 58.2 ± 5.0a PAWE 230.0 ± 30.0a 51.2 ± 6.7b 74.2 ± 4.2a PAW 301.5 ± 78.5a 59.1 ± 15.4b 59.7 ± 6.4a Pure C3G 565.0 ± 45.0a 565.0 ± 45.0a 78.1 ± 6.5a

Figure 21. A) Western blots of Caco-2 cells Lane 1 is C-, 2 is C+, 3 is purple corn water extract (PW), 4 is purple corn acidified water extract (PAW), 5 is PAW further purified with ethyl acetate (PAWE), and 6 is red corn acidified water extract (RAW) of non-phosphorylated Janus kinase 3 (JAK3), phosphorylated Janus kinase 3 (Phospho-JAK3), non-phosphorylated signal transducer and activator of transcription 1 (STAT1), and phosphorylated signal transducer and activator of transcription 1 (Phospho-STAT1). B) Table corresponding to percent phosphorylation of JAK 3 and STAT1 treated with C- media, C+ 12 mM celiac toxic peptide (CTP), plant extracts (1 mg/mL) cotreated with 12 mM CTP (p>0.05). C) Graph corresponding to percent phosphorylation of JAK 3 and STAT1 treated with C- media, C+ 12 mM celiac toxic peptide (CTP), plant extracts (1 mg/mL) cotreated with 12 mM CTP (p>0.05).

B A 1 2 3 4 5 6 5 1 % Phosphorylation % Phosphorylation JAK 3 STAT 1 1 C- 35.9 ± 9.6 46.3 ± 0.6 2 C+ 37.9 ± 6.3 47.2 ± 7.0 3 PW 51.7 ± 11.5 54.1 ± 10.4 4 PAW 48.6 ± 12.9 43.2 ± 2.0 5 PAWE 59.0 ± 11.5 45.9 ± 3.4 6 RAW 51.6 ± 18.9 37.1 ± 9.0

C % Phosphorylaon

80 70 60 50 40 30

% Phosphorylaon 20 10 0 JAK 3 STAT 1

C- C+ PW PAW PAWE RAW

Note: Percent phosphorylation was calculated as the phosphorylated portion divided by the total non-phosphorylated plus phosphorylated portions of either JAK3 or STAT1 after being normalized to GAPDH.

Figure 22. Interactions between A) Cyanidin-3-O-Glucoside and B) Pelargonidin-3-O-Glucoside with Janus Kinase 3.

Figure 22. (cont.) Interactions between C) Peonidin-3-O-Glucoside and D) Staurosporine with Janus Kinase 3.

Table 10. Description of six colored corn specialty varieties

Variety Description Corn varieties

VA Condensed form, high C3G, 50-4 D7-3-6 + 50-4 D8-3-13 Pg3G, Pn3G, high C3-6’’mal, Pg3-6’’mal, high Pn3-6’’mal

VB No condensed form, C3G, 50-5 D6-4-9 + 50-5 D5-4-5 + 50-5 D9-4- Pg3G, high Pn3G, high C3- 12 6’’mal, Pg3-6’’mal, high Pn3- 6’’mal

VC Condensed form, high C3G, 50-4 D5-3-15 + 50-4 D5-3-2 Pg3G, high Pn3G, C3-6’’mal, low Pg3-6’’mal, high Pn3-6’’mal

VD Very low condensed form, very 50-5 D8-4-1 + 50-5 D5-4-7 + 50-2 D9-2- low C3G, Pg3G, low Pn3G, high 12 C3-6’’mal, low Pg3-6’’mal, high Pn3-6’’mal

VE No condensed form, very low 50-1 D5-1-12 + 50-1 D5-1-6 + C3G, low Pg3G, low Pn3G, very 50-9 D10-5-1 + 50-3 D7-1-14 low C3-6’’mal, very low Pg3- 6’’mal, low Pn3-6’’mal

VF Very low condensed form, low 50-2 D7-2-13 + 50-2 D5-2-9 + 50-2 D5-2- C3G, very low Pg3G, low Pn3G, 2 + 50-2 D9-2-6B + 50-2 D9-2-8 high C3-6’’mal, very low Pg3- 6’’mal, very low Pn3-6’’mal

78 Table 11. Total anthocyanins (TA), total phenolics (TP), and condensed tannins concentration of four corn extracts, six corn specialty varieties, and other plant extracts Extracts TA (mg C3G TP (mg gallic acid/g Tannin (mg equivalent/ g dry dry extract) catechin/g dry extract) extract) Purple corn acidified 222.7 ± 5.4a 367.4 ± 9.6a 6.3 ± 0.2a water extract further purified with ethyl acetate (PAWE) Purple corn acidified 196.0 ± 1.9b 382.9 ± 7.5a 5.4 ± 0.1b water extract (PAW) Purple corn water 92.3 ± 1.1c 116.4 ± 3.5b 1.4 ± 0.1d extract (PW) Red corn acidified 56.2 ± 1.3d 141.0 ± 17.2b 2.9 ± 0.2c water extract (RAW) Variety A1 38.8 ± 1.5b 128.9 ± 10.9a 4.2 ± 0.04a Variety B 26.0 ± 2.2bc 114.5 ± 23.6a 2.9 ± 0.4a Variety C 31.0 ± 1.7b 143.7 ± 8.5a 3.5 ± 0.1a Variety D 34.1 ± 7.3b 139.9 ±16.6a 5.2 ± 1.2a Variety E 13.9 ± 0.3c 122.8 ± 20.8a 1.9 ± 0.1a Variety F 55.4 ± 3.1a 191.8 ± 14.6a 9.4 ± 0.2a Black Rice 287.5 ± 21.7a 382.3 ± 13.2a 6.0 ± 0.4a Red Grape 157.5 ± 1.4b 266.2 ± 42.0b 1.5 ± 0.3e Purple Sweet Potato 119.9 ± 5.4c 270.5 ± 14.6b 3.7 ± 0.07b Black Bean 81.5 ± 2.5d 187.03 ± 10.5d 0.2 ± 0.01f Purple Grape 77.8 ± 3.5d 270.9 ± 4.0b 1.7 ± 0.1de Purple Carrot 57.7 ± 0.4d 177.5 ± 1.9d 3.4 ± 0.5c Black Lentil 57.6 ± 3.8d 295.8 ± 13.6b 1.4 ± 0.2de Black Peanut 11.6 ± 1.0e 87. 4 ± 2.9e 1.6 ± 0.04de Purple Bean 7.5 ± 0.3e 167.8 ± 2.3d 1.2 ± 0.02e Blue Wheat 5.0 ± 0.3e 47.4 ± 1.5f 0.2 ± 0.04f Sorghum -0.1 ± 0.3e 225.7 ± 10.0c 0.3 ± 0.05f Different letters indicate significant differences among groups by column, p < 0.05. 1 Specialty corn varieties

79 Table 12. Percent survival of Caco-2 cells treated with 1 mg dry weight ANC-rich corn extract/mL and 12 mM celiac toxic peptide (CTP) individually and combined. Cell Survival (%) ANC- ANC-rich corn ANC-rich corn ANC-rich corn rich corn extract extracts & CTP extracts extracts3 Combined1 Pretreatment (4 h)2 a a b RAW 98.7 ± 1.8 38.3 ± 0.7 111.4 ± 9.7 ab a b PAWE 84.8 ± 0.7 44.1 ± 2.5 141.1 ± 3.7 b a b PAW 69.7 ± 4.6 49.4 ± 3.3 119.7 ± 10.4 b a a PW 79.5 ± 6.1 42.9 ± 4.6 198.0 ± 6.6 4 c a — CTP 37.1 ± 4.7 37.1 ± 4.7 1Combined-Extracts and CTP applied together 2Pretreatment-Extracts applied—Removed—CTP applied 3Extracts-Only extracts applied, no CTP added 4CTP-Positive control, only celiac toxic peptide applied Different letters indicate significant differences among groups by column, p < 0.05.

80 Table 13. Increase percent survival of Caco-2 cells treated with anthocyanin-rich plant extracts and celiac toxic peptide (CTP) compared to cells treated only with CTP (25 mM). Corn extracts Other Plant extracts Sample % survival Sample % survival increase increase Concentration, mg/mL 1.0 Concentration 1.0 mg/mL Corn variety C 84.3 ± 0.8a Black Peanut 67.3 ± 0.8a Variety B 81.6 ± 2.5a Purple Carrot 26.8 ± 3.6b Variety A 73.3 ± 2.1a Purple Sweet Potato 19.4 ± 0.3bc Variety D 73.0 ± 0.4a Black Lentil 14.2 ± 2.1cd Variety E 66.9 ± 5.6ab Purple Bean 3.9 ± 1.5d Purple corn acidified Concentration water extract 49.8 ± 5.4bc mg/mL 0.1 Red corn acidified water extract 48.6 ± 2.8bc Black Bean 64.1 ± 2.7a Variety F 45.1 ± 1.7c Black Rice 54.4 ± 1.9a Purple corn water extract 45.9 ± 5.5c Blue Wheat 48.9 ± 5.0a Purple corn acidified water extract further purified with ethyl Concentration acetate 20.9 ± 4.9d mg/mL 0.01 Purple Grape 46.2 ± 10.0a Red Grape 41.4 ± 1.3a Sorghum 24.3 ± 1.4a Different letters indicate significant differences among groups by column, p < 0.05.

81 Table 14. Established Free Energy of Binding, Total Intermolecular Energy, and Ki are given for main corn anthocyanins and staurosporine interacting with Janus kinase 3 as well as the interaction type and site.

Ki Hydrogen Polar Hydrophobic Cation-pi Est. Free Total (µM) bonds Energy of Intermolecular Binding Energy (kcal/mol) (kcal/mol) Anthocyanin

-4.71 -6.57 351.59 HIS1071- GLU1033- MET1037- HIS1071- [3.11] [2.96] [3.38] [3.77] GLN1058- PRO1061- Glucoside - [2.95] [3.43] O -

3 ARG1059- - [3.45] HIS1071-

Cyanidin [3.13]

-4.22 -6.48 808.3 ARG1036- GLU1033- MET1037- GLU1033- [3.05] [2.94] [3.79] [3.64] GLU1033- PRO1061-

Glucoside [1.98] [3.46] - O - ARG1036- 3 - [2.98] GLN1058- [3.29] ARG1059-

Pelargonidin [3.66] -4.76 -6.81 323.16 GLU1033- MET1037- HIS1071- GLU1033- [2.91] [3.72] [3.59] [3.71]

GLU1033- MET1037- [2.09] [3.48] ARG1036- PRO1061- Glucoside - [3.49] [3.83] O -

3 GLN1058- - [3.11] ARG1059-

Peonidin [3.43] HIS1071- [3.51] -6.31 -5.22 23.66 GLN1058- HIS1071- MET1037- HIS1071-

[3.29] [3.68] [3.25] [2.48]

orine PRO1061- Staurosp [3.32]

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85 CHAPTER 6: CONCLUSIONS

• Corn samples with high concentrations of peonidin had less stable pigments.

• Low temperatures and low pH promoted color and ANC stability.

• The most promising corn varieties for future experiments were V3 (high Pg, high C3-

6’’mal, Pn3-6’’mal), V5 (high condensed, high C3G, C3-6’’mal) and V6 (similar to V5).

• Molecular docking results demonstrated that free binding energies between each ANC

and JAK3 favored interactions.

• Pure C3G had the highest IC50 value on the activity of JAK3, in comparison to all the

color corn extracts, suggesting that the ANCs in the extracts had an interaction, which

inhibited JAK3 more effectively.

• Higher concentrations of extracts were able to inhibit JAK3 more but the cells were not

able to survive at these higher concentrations. High consumption of ANCs in the diet

has not been proven to be toxic.

86 CHAPTER 7: INTEGRATION AND FUTURE WORK

Anthocyanins are phytochemicals with bright natural pigments that possess a range of positive health benefits. They have been shown to help fight diabetes and could benefit other autoimmune disorders. Celiac disease is an inherited autoimmune disease that targets the small intestine with gluten, a protein found in wheat, rye, and barley, is consumed. Current treatment for this disease is limited to abstaining from gluten consumption, which can be difficult and expensive because gluten is often present in unexpected products and gluten-free products often cost more.

Because of this problem, our study aimed to determine the protective capability of

ANCs in an in vitro model of celiac disease but before that it was necessary to determine the probability of incorporating ANCs into consumable products. ANCs are known for their bright red, purple, and blue colors and are becoming more popular as natural colorants despite being less stable than synthetic dyes.

Phase I of this study was dedicated to comparing the color stability of nine unique ANC- rich extracts from colored corn varieties. By comparing different profiles, the trend in color stability can be related back to the interactions of anthocyanins and their forms present. Nine corn varieties went through kernel pericarp water extraction and were freeze-dried. Kool-Aid

Invisible® was mixed according to packaging instructions using distilled water. Extract concentration for each variety was initially adjusted to match the color of Kool-Aid Cherry.

Corn varieties V1, V2, V3, V4, V5, V6, V7, V8, and V9 had concentrations of 0.9, 0.7, 0.7, 2.2, 0.6,

1.4, 1.1, 0.6, and 0.5 mg freeze dried extract/mL Kool-Aid Invisible, respectively, depending on the spectrometric absorbance values at 520 nm to mimic Kool-Aid Cherry red color. Solutions

87 were aliquoted in triplicate for each sample for each week tested and were stored without light at 4 °C, 22 °C, or 32 °C for a duration of 12 weeks. Color, spectra of absorption (λmax and absorbance at λmax), total anthocyanin content (TA), total phenolics (TP), condensed tannins, pH, and pigment composition changes were all measured once a month.

There was a difference in color stability and more testing is required to determine exactly which elements contribute the most lasting color over time. Because the extracts were from plant materials, they were not completely pure. It would be beneficial to perform tests on known mixtures of pure ANCs that mimic those present in the plant samples to quantify the color stability and then mimic the profiles through selective breeding.

Phase II of this study aimed to determine the protective potential of ANC-rich plant extracts on a Celiac disease cell model. Caco-2 cells were cultured at 37 °C under a 5% CO2 atmosphere using Modified Eagle’s Medium, supplemented with 20% (v/v) fetal bovine serum, and 1% streptomycin antibiotic. The growth medium of Caco-2 cultures was replaced every 2–3 days. All experiments were performed on healthy differentiated Caco-2 cell monolayers. To mimic Celiac disease in the human body and the effect of ANC rich extracts, Caco-2 cells were treated with a pure peptide with known celiac toxicity. Celiac toxic peptide YPQPQPF is an alpha-gliadin wheat protein. Cell viability was tested using purple and red corn extracts, corn variety extracts, plant extracts and peptide. Caco-2 cells were treated directly with different concentrations of extracts and peptide. Cell viability was measured using the cell proliferation assay was performed using the CellTiter® 96 Aqueous One Solution proliferation (Promega

Corporation, Madison, WI) following the manufacturer’s instructions. Then Caco-2 cells were treated with both extracts and peptide to determine cell protection.

88 It was found that ANC-rich extracts were effective in protecting Caco-2 cells when treated at the same time as CTP. Extracts were also able to inhibit the activity of JAK3 which is involved in an inflammatory pathway as well as reducing inflammatory proteins produced by the cells.

In future studies, it is necessary to identify the actual pathways involved in Celiac disease and to develop a working animal model to research possible treatment methods. It would be beneficial to conduct human intervention studies along with epidemiological studies to assess the role anthocyanins and gluten play for patients with Celiac disease. From this study, it is too soon to conclude any recommendations for patients with Celiac disease, but perhaps with more research we could find recommended intake of ANCs in case of accidental gluten ingestion.

89 APPENDIX A. Abbreviations

AFC Artificial food colorant

ANC Anthocyanins

CF Condensed forms

C3G Cyanidin-3-glucoside

GM-CSF Granulocyte macrophage-colony stimulating factor

IL Interleukin

JAK Janus kinase

Mal Malonyl-glucoside

PAW Purple corn acidified water extract

PAWE Purple corn acidified water extract with additional ethyl acetate purification

PC Principal component

Pg Pelargonidin

Pn Peonidin

PW Purple corn pericarp water extract

RAW Red corn acidified water extract

STAT Signal transducers and activators of transcription

TIMP-2 Metallopeptidase inhibitor 2

V Variety

90 Appendix B. Additional Figures

Figure 23. HPLC at 520 nm of 9 colored corn varieties dissolved in Kool-Aid Invisible® stored at three different temperatures (4, 22, and 32 °C) during 4, 8, and 12 weeks, concentration (µg/mL).

Variety 1 Variety 2 Variety 3 30

25 20 20

20 15 15 15 10 10 10 5 5 5 Intensity (mv) Intensity (mv) Intensity (mv) 0 0 0 -5 Week 0 -5 -5

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 4 °C 15 15

10 10 5

5 5

0 0 Intensity (mv) Intensity (mv) Intensity (mv) -5 -5 -5 Week 4

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 4 6 8 2 4 6

0 2 4 -2 0 2 -4 -2

Intensity (mv) Intensity (mv) Intensity (mv) 0 -4 -6 -2 -6 -8 Week 8 -4

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 4 4 6 2 2

4 0 0 2 -2 -2

0 -4 -4 Intensity (mv) Intensity (mv) Intensity (mv) -6 -6 -2

-8 -8 -4

Week 12 -6

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 22 °C

2 10 2 0

0 5 -2 -2

-4 0 -4 Intensity (mv) Intensity (mv) Intensity (mv) -6 -6 -5 -8 -8 Week 4

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 0 2 -1 2 -2

0 0 -3 -2 -4 -2

-4 -5

Intensity (mv) Intensity (mv) Intensity (mv) -4 -6 -6 -6 -7 Week 8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 0 -2 2 -1 -3

-2 0 -4 -3 -2 -4 -5

-5 -4

Intensity (mv) Intensity (mv) -6 Intensity (mv) -6 -7 -6 -7 Week 12

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min)

91 Appendix B (cont.)

Figure 23. (cont.)

Variety 1 Variety 2 Variety 3

32 °C

0 10 5

-2 5

-4 0

Intensity (mv) -6 Intensity (mv) Intensity (mv) -5

-8 -5 Week 4

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -2 -1 0 -2 -3 -1

-3 -4 -2

-4 -3 -5 -5 -4

Intensity (mv) -6 Intensity (mv) Intensity (mv) -5 -6 -6 -7 -7 -7 Week 8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -2 0 4

-3 2 -2

0 -4 -4 -2

-5 -4 -6 Intensity (mv) Intensity (mv) Intensity (mv) -6 -6 -8 -8 Week 12

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) Variety 4 Variety 5 Variety 6 30 30

25 25 20

20 20 15 15 15 10 10 10

5 5 5 Intensity (mv) Intensity (mv) Intensity (mv) 0 0 0

-5 -5 Week 0 -5

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 4 °C 6

4 15 2 5

10 0

-2 5 0

-4

Intensity (mv) Intensity (mv) 0 Intensity (mv) -6 -5 -5 -8 Week 4

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 15 2 2

10 0 0

-2 5 -2

-4 0 -4

Intensity (mv) -6 Intensity (mv) Intensity (mv) -6 -5 -8

Week 8 -8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -1 15 4

10 -2 2

5 0 -3 -2 0

-4 -4

Intensity (mv) Intensity (mv) -5 Intensity (mv) -6 -5 -10 -8

Week 12 -6 -15

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min)

92 Appendix B (cont.)

Figure 23. (cont.)

Variety 4 Variety 5 Variety 6

22 °C -1 15 4 -2 10 -3 2

-4 5 0

-5 -2 0 -6 -4

Intensity (mv) -7 Intensity (mv) -5 Intensity (mv) -6 -8 -10 -8

Week 4 -9 -15

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -3 0 2 -1 -4 -2

-3 -5 -2 -4

-6 -4 -5 Intensity (mv) Intensity (mv) Intensity (mv) -6 -7 -6 -7 Week 8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 0 4 -2 0 2

-4 -1 0 -6 -2 -2 -8 -3 -4

Intensity (mv) -10 Intensity (mv) Intensity (mv) -6 -4 -12 -8 -5 -14 Week 12 -6

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 32 °C -3 0

2 -1 -4

0 -2

-5 -2 -3

-4 -4 -6 Intensity (mv) Intensity (mv) Intensity (mv) -6 -5 -7 -8 -6 Week 4

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -3 -2 -2

-3 -3 -4

-4 -4 -5

-5 -5

-6

Intensity (mv) Intensity (mv) -6 Intensity (mv) -6

-7 -7 -7 Week 8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -1 -1 -1

-2 -2 -2

-3 -3 -3 -4 -4 -4 -5

Intensity (mv) Intensity (mv) -5 Intensity (mv) -5 -6

-7 -6 -6 Week 12

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min)

93 Appendix B (cont.)

Figure 23. (cont.)

Variety 7 Variety 8 Variety 9

30 20 20 25

15 15 20

15 10 10

10 5 5

Intensity (mv) 5 Intensity (mv) Intensity (mv) 0 0 0 -5 -5 -5 Week 0

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 4 °C 15 15

15 10 10

10 5 5

5 0

Intensity (mv) 0 Intensity (mv) Intensity (mv) -5

-5 -5 -10 Week 4 -15

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 6 10 6

4 4

2 5 2 0 0 -2 0 -2

Intensity (mv) Intensity (mv) Intensity (mv) -4 -4 -6 -5 -6 -8 Week 8 -8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 15 15

10 10

5 5 5

0 0 0

Intensity (mv) -5 Intensity (mv) Intensity (mv) -5 -5 -10 -10

Week 12 -15 -15

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 22 °C

2 5 5

-2 0 0

-4 Intensity (mv) Intensity (mv) Intensity (mv) -5 -6 -5

-8 Week 4

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -1 4 4 -2 2 2

-3 0 0 -4 -2 -2 -5 -4

Intensity (mv) Intensity (mv) Intensity (mv) -4 -6 -6

-8 -7 -6 Week 8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) 4 0 6

4 2 -1 2 -2 0 0 -3 -2 -2 -4

Intensity (mv) Intensity (mv) Intensity (mv) -4 -4 -5 -6

-6 -8 -6 Week 12

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min)

94 Appendix B (cont.)

Figure 23. (cont.)

Variety 7 Variety 8 Variety 9

32 °C

4 8 5 2 6

4 0 2 0

-2 0

Intensity (mv) Intensity (mv) -2 Intensity (mv) -4 -5 -4

Week 4 -6 -6

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -1 2 2 -2

-3 0 0

-4 -2 -2

-5 -4 -4 Intensity (mv) Intensity (mv) Intensity (mv) -6

-6 -6 -7 Week 8

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min) -1 -1 -1

-2 -2 -2

-3 -3 -3 -4 -4 -4 -5

Intensity (mv) Intensity (mv) -5 Intensity (mv) -5 -6

-7 -6 -6 Week 12

10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 10 12 14 16 18 20 22 24 26 28 30 Retention Time (min) Retention Time (min) Retention Time (min)

95 Appendix B (cont.)

Figure 24: Cell viability of Caco-2 cells treated with different concentrations of Celiac toxic peptide. 140

120 y = 116.34ln(x) - 256.46 100 R² = 0.93498

60 Series1 %inhibion Log. (Series1) 40

0 0 5 10 15 20 25 30 mM CTP

Concentration of % Inhibition YPQPQPF (mM) a 25 95.9 ± 0.9 b 12 35.4 ± 0.4 c 10 0.05 ± 1.6 One-way ANOVA was conducted across a column. Absence of the same small letter represents significant difference (p < 0.05).

96 Appendix B (cont.)

Figure 25: Increased protection from plant extracts to Caco-2 cells treated with 25 mM Celiac toxic peptide.

97 Appendix B (cont.)

Figure 26: Human inflammation array membranes treated with A) media, B) 12 mM Celiac toxic peptide (CTP), C) 1 mg/mL purple corn acidified water extract further purified with ethyl acetate and CTP, and D) 1 mg/mL red corn acidified water extract and CTP.

A B

D C

98 Appendix B (cont.)

Figure 27: Nutrition facts and ingredients of A) Kool-Aid Invisible Cherry Sugar Sweetened Soft Drink Mix® and B) Kool-Aid Jammers Cherry® (Kraft-Heinz)