IMPROVING SULFORAPHANE BIOAVAILABILITY FROM COOKED BROCCOLI BY YANLING WANG DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Science and Human Nutrition with a concentration in Food Science in the Graduate College of the University of Illinois at Urbana-Champaign, 2019 Urbana, Illinois Doctoral Committee: Associate Professor Michael J. Miller, Chair Professor Emeritus Elizabeth H. Jeffery, Director of Research Professor John A. Juvik Assistant Professor Zeynep Madak-Erdogan Adjunct Professor Mark A. Berhow ABSTRACT Broccoli is a nutritious food containing many macro- and micro-nutrients. Most consumers prefer cooked broccoli (CB); unfortunately, the enzyme myrosinase is inactivated by heat, resulting in the failure of glucoraphanin (GRP) hydrolysis to sulforaphane (SF), the potent anti- cancer compound in broccoli. Certain unknown bacteria in the gut microbiota also have myrosinase-like activities that can hydrolyze GRP and release bioactive SF. However, the activity from bacteria is low compared to that from plant. The overall objective of this study is to identify ways to improve SF formation and absorption (bioavailability) from CB, through better understanding the mechanism. Our first hypothesis was that frequent intake of CB would enrich the bacteria that contain myrosinase-like activity, therefore enhancing SF bioavailability and bioactivity. Our data show that no less than 4 days of CB diet greatly alters the cecal microbiota composition in rats, and increases the myrosinase-like activity of cecal microbiota ex vivo. Increased colonocyte NAD(P)H: quinone oxidoreductase 1 activity (one marker of SF bioactivity) in rats fed with no less than 7 days of a CB diet also indicates enhanced SF bioactivity by frequent intake of CB. The myrosinase-like activity of cecal microbiota is lost when the 4 day CB diet was switched back to a control diet for an additional 3 days, suggesting that the increase in SF bioavailability requires intake of CB on a daily basis. Whether the myrosinase-like activity or the abundance of the myrosinase-containing bacteria is induced by a CB diet remains unknown and needs to be further investigated. In the next study, we compared the effect of frequent intake of CB and raw broccoli (RB) in prevention of dextran sulfate sodium (DSS)-induced colitis in mice. Our data show that frequent intake of a CB diet is as effective as a RB diet in lessening damage by DSS, evidenced by a decreased disease activity index, attenuated colon length shrinkage, less endotoxin (lipopolysaccharide) leakage into blood, and less severe colon lesions as assessed by ii histopathology. Intriguingly, mRNA expression of pro-inflammatory cytokines indicated that broccoli anti-inflammatory action may be through inhibition of the IL-6 trans-signaling pathway, as evidenced by reversal of the DSS-increased expression of IL-6, CCR2 and vascular cell adhesion molecule 1 (VCAM-1). Our next hypothesis was that the quercetin-3-O-sophoroside (QS), the major flavonoid in broccoli, could improve the bioavailability of SF from CB, by modifying the gut microbiota composition. Because no literature existed on health-related properties of QS, we considered that it was essential to first understand the disposition of QS per se. Our data show that QS is absorbed intact in the small intestine and partly methylated to isorhamnetin sophoroside (methylated QS). No quercetin aglycone or its typical phase II metabolites were observed. With this important piece of information about where and how QS is absorbed and metabolized, future studies will be able to study whether the intact QS improves SF bioavailability from CB and whether this effect, if present, is dependent on gut microbiota. iii TABLE OF CONTENTS CHAPTER 1: Introduction ..................................................................................................1 1.1 Introduction ....................................................................................................................1 1.2 References ......................................................................................................................3 CHAPTER 2: Literature Review .........................................................................................4 2.1 Broccoli Composition ....................................................................................................4 2.2 Broccoli and Health .......................................................................................................5 2.3 Glucoraphanin and Sulforaphane ...................................................................................8 2.4 Quercetin Glycosides and Quercetin ...........................................................................12 2.5 Tables and Figures .......................................................................................................26 2.6 References ....................................................................................................................31 CHAPTER 3: Dietary Broccoli Alters Rat Cecal Microbiota to Improve Glucoraphanin Hydrolysis to Bioactive Isothiocyanates ............................................................................40 3.1 Introduction ..................................................................................................................40 3.2 Materials and Methods .................................................................................................42 3.3 Results ..........................................................................................................................46 3.4 Discussion ....................................................................................................................49 3.5 Conclusions ..................................................................................................................52 3.6 Tables and Figures .......................................................................................................53 3.7 References ....................................................................................................................59 CHAPTER 4: Lightly Cooked Broccoli Is as Effective as Raw Broccoli in Mitigating Dextran Sulfate Sodium-Induced Colitis in Mice ..............................................................64 4.1 Introduction ..................................................................................................................64 iv 4.2 Materials and Methods .................................................................................................66 4.3 Results ..........................................................................................................................73 4.4 Discussion ....................................................................................................................78 4.5 Conclusions ..................................................................................................................85 4.6 Tables and Figures .......................................................................................................86 4.7 References ....................................................................................................................93 CHAPTER 5: Purification, Absorption and Metabolism of Quercetin-3-O-Sophoroside, the Major Quercetin in Brassica ........................................................................................97 5.1 Introduction ..................................................................................................................97 5.2 Methods........................................................................................................................98 5.3 Results ........................................................................................................................103 5.4 Discussion ..................................................................................................................107 5.5 Conclusions ................................................................................................................111 5.6 Table and Figures .......................................................................................................112 5.7 References ..................................................................................................................119 CHAPTER 6: Conclusions and Future Directions...........................................................123 6.1 Conclusions and Future Directions ............................................................................123 6.2 References ..................................................................................................................128 APPENDIX A: Dietary Broccoli Alters Rat Cecal Microbiota to Improve Glucoraphanin Hydrolysis to Bioactive Isothiocyanates ..........................................................................129 APPENDIX B: Lightly Cooked Broccoli Is as Effective as Raw Broccoli in Mitigating Dextran Sulfate Sodium-Induced Colitis in Mice ............................................................142 v CHAPTER 1: Introduction 1.1 Introduction Broccoli and other crucifers are well appreciated as important anti-cancer and anti- inflammation vegetables. Whereas most consumers prefer cooked broccoli (CB), the enzyme myrosinase is inactivated by heat, resulting in the failure of glucoraphanin (GRP) hydrolysis to sulforaphane (SF), the potent anti-cancer compound in broccoli. Therefore,