ISOLATION OF ANTHOCYANIN MIXTURES FROM FRUITS AND VEGETABLES AND EVALUATION OF THEIR STABILITY, AVAILABILITY AND BIOTRANSFORMATION IN THE GASTROINTESTINAL TRACT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jian He, M.S. * * * * * The Ohio State University 2008 Dissertation Committee: Approved by Assistant Professor M. Mónica Giusti, Adviser Professor Steven J. Schwartz ______________________________ Professor Mark L. Failla Adviser Professor W. James Harper Food Science and Nutrition Graduate Program Associate Professor Timothy A. Schroeder © Copyright by JIAN HE 2008 ABSTRACT Anthocyanins are among the most abundant polyphenols in fruits and vegetables and exhibit potent antioxidant activity. Our previous studies suggested that anthocyanins present in the gastrointestinal tract (GIT) of rats exert chemopreventive effect on colon cancer. Since such effect was dose dependent, it is important to examine the stability and transformation of anthocyanins in the GIT. Based on some in vitro studies and limited in vivo evidence, we hypothesized that anthocyanins were moderately stable under the influence of digestive enzymes and physiological conditions in the GIT, and their chemical structures determine the stability, accessibility, biotransformation, and health- promoting effects. To validate our hypothesis a series of studies were conducted. We first examined the transit, stability, transformation, and uptake of black raspberry anthocyanins in rat GIT. Our results suggest that ingested anthocyanin glycosides remained relatively stable in the GIT lumen and were efficiently taken up into the GIT tissues with limited transfer to the plasma compartment. We also observed selective degradation of cyanidin-3- glucoside in the small intestine lumen, which was tentatively attributed to β-glucosidase activity in the small intestine. ii In order to eliminate interferencing non-anthocyanin compounds in bioassays, a novel mixed mode cation-exchange/reversed-phase (MCX) solid-phase extraction (SPE) technique was developed for high purity isolation of anthocyanins. The new methodology drastically increased purity and efficiency as compared to three widely used SPE techniques – C18, HLB, and LH-20 columns. For the majority of plant materials evaluated, the new technique achieved above 99% anthocyanin purity while maintaining excellent yield (93.6 ± 0.55%) and low cost. The resulted high purity anthocyanins were later used in the enzymatic assays. To elucidate the effect of intestinal enzymes on anthocyanin digestion, an in vitro model was employed. Cell-free extract of pig small intestinal mucosa and crude extract of lactase containing foods/supplement were examined with respect to their β-glycosidase activities on highly purified anthocyanins. Selective degradation of anthocyanin glucosides and galactosides was observed in the presence of small intestinal mucosa cell- free extract and lactase supplement extract, respectively. The type of aglycone also substantially affected the resistance of anthocyanins to enzymatic degradation. The outcome of this dissertation demonstrates that chemical structure of anthocyanins greatly affects their stability in the GIT. Such information will contribute to the exploration of anthocyanin’s health benefits in vivo, and may eventually facilitate the development of value added foods/nutraceuticals that promote the healthiness of people. iii The dissertation is dedicated to my mother Zonghui Hu, my father Shide He, and my dear wife Weishu Xue for their love and ultimate support to me. iv ACKNOWLEDGMENTS I would like to thank my advisor Dr M. Mónica Giusti for her intelligent advices, warm encouragement, and ultimate patience throughout my graduate study. My success in academia is not possible without her guidance. I wish to thank Dr Mark L. Failla for sharing his incredible knowledge in nutrition to help me designing my experiments and fulfilling my academic goals. I also thank the other members of my advisory committee, Dr Steve J. Schwartz and Dr W. James Harper for their invaluable opinions to my research. I acknowledge Dr Luis Rodriguez-Saona for advising me of the FT-IR work. I appreciate the chance of collaborating with Kristin E. Keatley who successfully graduated from the Department of Human Nutrition last year. Great thanks to my colleagues and friends, especially all my lab mates Fer Pólit, Nuryati Pangestu, Fabiola Gutierrez, Taylor Wallace, Dante Vargas, Christian Sweeney, Jennifer Willig, Jodee Johnson, Pu Jing, and Matthew Bunce. All of these lovely people helped me as much as they could and I really enjoyed the happy days spent with them. I am grateful to USDA for funding my doctoral study. My doctoral research was supported by the USDA NRI Competitive Grants Program 2004-35503-15190. v Very special thanks to my mother Zonghui Hu, my parents in law Zhiguang Xue and Aiping Zheng, and my wife Weishu Xue. Without their unselfish support I could never achieve so much. vi VITA Feb 19, 1980·······································Born – Hefei, China 1998 – 2002········································B.E. Food Science and Engineering, Hefei University of Technology, China 2002 – 2004········································M.S. Nutrition and Food Science University of Maryland, College Park, MD 2004 – 2008········································Graduate Research and Teaching Assistant The Ohio State University, Columbus, OH PUBLICATIONS 1. He, J.; Rodriguez-Saona, L. E.; Giusti, M. M., Mid-infrared spectroscopy for juice authentication - rapid differentiation of commercial juices. J Agric Food Chem 2007, 55, 4443-4452. 2. He, J.; Magnuson, B. A.; Lala, G.; Tian, Q.; Schwartz, S. J.; Giusti, M. M., Intact anthocyanins and metabolites in rat urine and plasma after 3 months of anthocyanin supplementation. Nutr Cancer 2006, 54, 3-12. vii 3. Lala, G.; Malik, M.; Zhao, C.; He, J.; Kwon, Y.; Giusti, M. M.; Magnuson, B. A., Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats. Nutr Cancer 2006, 54, 84-93. 4. He, J.; Magnuson, B. A.; Giusti, M. M., Analysis of anthocyanins in rat intestinal contents – impact of anthocyanin chemical structure on fecal excretion. J Agric Food Chem 2005, 53, 2859-2866. FIELDS OF STUDY Major Field: Food Science and Nutrition viii TABLE OF CONTENTS Page ABSTRACT······················································································································ii ACKNOWLEDGMENTS·································································································v VITA·······························································································································vii TABLE OF CONTENTS ·································································································ix LIST OF TABLES ·········································································································xvi LIST OF FIGURES·······································································································xvii LIST OF ABBREVIATIONS ·························································································xx 1. INTRODUCTION····································································································1 2. LITERATURE REVIEW·························································································3 2.1 ANTHOCYANINS AS NATURAL PIGMENTS ···········································3 2.2 CHEMICAL STRUCTURE OF ANTHOCYANINS ······································4 2.2.1 Chemical structure of flavonoids ·························································4 2.2.2 Anthocyanin aglycones········································································5 2.2.3 Glycosylation and acylation·································································7 ix 2.2.4 The influence of pH on anthocyanin chemical structure ······················8 2.2.5 Pyranoanthocyanins···········································································11 2.3 ANTHOCYANINS IN HUMAN DIET·························································13 2.3.1 Occurrence of anthocyanins in plant materials···································13 2.3.2 Anthocyanins in foods and beverages················································14 2.3.3 Toxicity of anthocyanins····································································15 2.4 STABILITY OF ANTHOCYANINS ····························································16 2.4.1 The effect of pH·················································································16 2.4.2 The effect of temperature···································································17 2.4.3 The effect of light ··············································································18 2.4.4 The effect of enzymes········································································18 2.4.5 The influence of oxygen, peroxide and ascorbic acid·························19 2.4.6 Self-association, acylation and co-pigmentation ································21 2.5 PUTATIVE HEALTH-PROMOTING EFFECTS OF ANTHOCYANINS···22 2.5.1 Relief of oxidative stress····································································23 2.5.2 Prevention of cardiovascular diseases················································24 2.5.3 Anti-inflammatory activity ································································26 x 2.5.4 Anti-carcinogenic