Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2005 Structure - degradation relationships of flavonoids and their correlation to human bioavailability Andrean Llewela Simons Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Medical Toxicology Commons, Microbiology Commons, Nutrition Commons, and the Toxicology Commons Recommended Citation Simons, Andrean Llewela, "Structure - degradation relationships of flavonoids and their correlation to human bioavailability " (2005). Retrospective Theses and Dissertations. 1813. https://lib.dr.iastate.edu/rtd/1813 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. NOTE TO USERS This reproduction is the best copy available. ® UMI Structure - degradation relationships of flavonoids and their correlation to human bioavailability by Andrean Llewela Simons A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY Co-majors: Food Science and Technology; Toxicology Program of Study Committee: Patricia A. Murphy, Co-major Professor Suzanne Hendiich, Co-major Professor Diane Birt Aubrey Mendonca Mark Rasmussen Iowa State University Ames, Iowa 2005 Copyright © Andrean Llewela Simons, 2005. All rights reserved. UMI Number: 3172244 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3172244 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 11 Graduate College Iowa State University This is to certify that the doctoral dissertation of Andrean Llewela Simons has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Committee Member Signature was redacted for privacy. Committee Member Signature was redacted for privacy. Corpmittee Member Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. Co-major Professaissor Signature was redacted for privacy. %!\th Co }or Program Signature was redacted for privacy. iii TABLE OF CONTENTS ABSTRACT 1 GENERAL INTRODUCTION 3 Introduction 3 Dissertation Organization 5 LITERATURE REVIEW 6 A. Chemistry and Sources of Flavonoids 6 B. Function of Flavonoids 11 C. Biosynthesis of Flavonoids 13 D. Dietary Intake 17 E. Biological and Health Effects 18 a. Estrogenic Activity 18 b. Antioxidant Effects 25 c. Flavonoids and Steroid Hormone Dependent Cancers 29 d. Flavonoids and Atherosclerosis 34 F. Metabolism and Bioavailability 39 a. Hydrolysis and Absorption 40 b. Hepatic Metabolism 43 c. Gut Microbial Metabolism 45 d. Bioavailability 54 G. Toxicity 57 IV H. References 62 METABOLISM OF GLYCITEIN (7,4'-DIHYDROXY-6-METHOXYISOFLAVONE) BY HUMAN GUT MICROFLORA 84 Abstract 84 Introduction 84 Materials and Methods 87 Results and Discussion 90 Acknowledgements 97 Literature Cited 97 HUMAN GUT MICROBIAL DEGRADATION OF FLAVONOIDS: STRUCTURE - FUNCTION RELATIONSHIPS 113 Abstract 113 Introduction 113 Materials and Methods 115 Results and Discussion 118 Acknowledgements 124 Literature Cited 125 GUT MICROBIAL DEGRADATION OF FLAVONOIDS AND CORRELATION TO HUMAN BIOAVAILABILITY 135 Abstract 135 Introduction 136 Materials and Methods 138 Results 143 Discussion 146 Literature Cited GENERAL CONCLUSIONS 1 ABSTRACT The chemical structure of flavonoids greatly influences the rate of degradation by human gut microflora and their overall bioavailability. To investigate which soy isoflavone structures were needed for rapid gut microbial degradation, 100 p,mol of daidzein, genistein and glycitein were incubated in fecal fermentation mixtures consisting of brain heart infusion media (BHI) and fresh feces m Wfro from 12 human subjects for 24 hours. Genistein, which possesses a hydroxyl group in the 5 position, degraded most rapidly in all subjects with an average k = 0.43 ± 0.44 h~i (p = 0.018). Glycitein and daidzein degradation rates were not different from each other with an average k = 0.30 ± 0.21 h"' and k = 0.16 ± 0.17 h~i (p = 0.074). Glycitein, the metabolism of which has been less characterized, was metabolized to dihydroglycitein, dihydro-6,7,4' -trihydroxyflavone, 5'-OMe-O-desmethylangolensin, 6- OMe-equol and daidzein. To further determine the relationship between other flavonoids with hydroxyl groups in the 5 position and gut microbial degradation, 80 |iM of 14 flavonoids, flavone, apigenin, chrysin, naringenin, kaempferol, genistein, daidzein, daidzin, puerarin, 7,4'- dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, 5,3'-dihydroxyflavone and 4'-hydroxyflavone, were fermented in fecal fermentation mixtures from 11 human subjects. The degradation rates of 5,7,4'-trihydroxyl-flavonoids, apigenin, genistein, naringenin and kaempferol, were significantly faster with an average k = 0.38 ± 0.11 h~\ compared to flavonoids with other structural motifs with an average k = 0.08 ± 0.02 h"^ (p<0.0001). The bioavailability of flavonoids that are rapidly degraded by the gut microflora may be significantly reduced compared to flavonoids that are slowly degraded. The bioavailability of rapidly degraded flavonoids m v;fro, genistein, naringenin, hesperetin, quercetin, and a 2 slowly degraded flavonoid, daidzein, was analyzed. Five men and five women were fed soymilk containing 28 mg genistein and 16 mg daidzein, grapefruit juice containing 422 mg of naringenin and 8 mg hesperetin, and sautéed onions containing 115 mg quercetin with a 2 week washout period in between feedings. Rapidly degraded flavonoids from m Wfro fecal fermentation systems in 33 subjects, included the 5,7,4'-trihydroxyflavonoids, genistein, apigenin, naringenin, kaempferol, quercetin and luteolin, with an average k = 0.46 ± 0.10 h~\ and the methoxylated flavonoids, glycitein and hesperetin with an average k = 0.24 ± 0.21 h~\ compared to slowly degraded flavonoids with an average k = 0.07 ± 0.02 h~i (p < 0.0001). The bioavailability of the rapidly degraded flavonoids, genistein, naringenin, quercetin and hesperetin with an average k = 5.8 ± 1.9 % were significantly lower than daidzein with an average k = 42.6 ± 15.9 % (p = 0.02) expressed as the amount of flavonoid excreted in urine as a percentage of ingested dose. Subjects with low genistein degrader phenotypes (average k = 0.11 ± 0.07 h~\ n=4) based on average linkage cluster analysis, experienced a higher genistein bioavailability (11.5 ± 8.0 %) compared to the lower genistein bioavailability (3.6 ± 1.9 %, p = 0.007) experienced by subjects with high genistein degrader phenotypes (average k = 1.28 ± 0.45 h~\ n=3). The data from the m Wfro fecal fermentation studies reveal that the chemical structure of flavonoids greatly influences the gut microbial degradation rate. Flavonoids with 5,7,4'- trihydroxyl-flavonoid structures were rapidly degraded by the human intestinal microflora, which in turn, resulted in lesser apparent absorption and excretion in urine over 24 h. Therefore, the chemical structure of flavonoids is a strong determinant of the human bioavailability of flavonoids. 3 GENERAL INTRODUCTION Introduction Flavonoids are polyphenolic compounds that are widely distributed in plants and in the human food supply. Flavonoids are synthesized in plants from the amino acid phenylalanine, and function as pollinator attractors, plant growth regulators, nitrogen fixating bacteria activators and ultraviolet (UV) filters. Fruits, vegetables and herbs are particularly rich sources of flavonoids where these compounds contribute to their color and flavor. Over 5000 flavonoids have been identified to date, and are divided into subclasses including flavones, isoflavones, flavonols and flavonones (Harbome, 1986). The estimated daily intake of flavonoids in humans can reach up to 1 g. Epidemiological studies have suggested possible links between diets rich in fruits and vegetables and reduced risk of cancer and cardiovascular disease. The antioxidant and estrogenic activity of flavonoids may be responsible for these positive correlations. However, some flavonoids have been reported to possess some toxic properties such as pro-oxidant activity and adverse flavonoid-drug interactions, which warrant further investigation. The bioavailability of flavonoids is critical in understanding their biological and health effects in humans. The extent of absorption and gut microbial metabolism may significantly alter the bioavailability and activity of flavonoids at their target site. Therefore, determining how these factors influence human bioavailability is the overall objective of my research. The two major sites of flavonoid