PHENOLIC COMPOUNDS OF SORGHUM, THEIR CHEMOPREVENTIVE PROPERTIES AND ABSORPTION A Dissertation by VICTOR MANUEL TALEON ALBAN Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Lloyd W. Rooney Committee Members, Joseph Awika Susanne Talcott William L. Rooney Head of Department, Jimmy Keeton August 2013 Major Subject: Food Science and Technology Copyright 2013 Victor Manuel Taleon Alban ABSTRACT Sorghum contains many phenolic compounds which have potential antioxidant, anti-inflammatory, and chemopreventive properties as well as natural colorants in foods. Phenolic compounds of stalks, sheaths, leaves, glumes and grains from tan (ARTx631/RTx436), red (Tx2911) and purple (Tx3362) sorghum plants were characterized by UPLC-MS/MS. Antiproliferative properties of selected sorghum extracts were evaluated using HT-29 colon cancer cells and absorption of their polyphenolics was determined by a Caco-2 in vitro model system. Phenolic acids, flavones, 3-deoxyanthocyanidins and chalcones were found in all plant components. Phenolic acids were predominant in the stalks, sheaths and grains of all sorghum types. Flavone glycosides were predominant in leaves, sheaths and stalks while flavone aglycones in glumes and grains. 3-Deoxyanthocyanidins and chalcones were mostly found in sheaths, leaves, glumes and grains of Tx2911 (red) and Tx3362 (purple) genotypes. Sorghum leaves showed high levels of flavone glycosides while glumes had high levels of flavones aglycones. Glume extract of ATx631/RTx436 (tan) and Tx2911 genotypes had the strongest antiproliferation activity (IC50 = 85-178 µg/ml), these extracts had also the highest levels of flavone aglycones (19.6-49.8 mg/g). Absorption of flavones (30.4-42.3 %) was higher than 3-deoxyantocyanidins (1.4-11.3%), while absorption of methoxylated 3- deoxyanthocyanidins (11.3%) was higher than non-methoxylated 3-deoxyanthocyanidins (1.4 – 1.6 %). Flavones had high absorption compared to other sorghum phenolics ii suggesting that sorghum flavone aglycones are more bioavailable than other sorghum phenolic compounds. Consequently, sorghum glumes could be used as used as a source of phytochemicals to increase value of sorghum crop. iii DEDICATION To my family. iv ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Lloyd Rooney, and my committee members, Dr. Awika, Dr. William Rooney, and Dr. Susanne Talcott, for their guidance and support throughout the course of this research. Thanks also go to Linda Dykes, Liyi Yang and Kimberly Krenek for their help with my research, and friends and colleagues from the Cereal Quality Lab for making my time at Texas A&M University a great experience. I also want to extend my gratitude to Dr. Javier Bueso for given me the opportunity to work with him on research and academic areas at Zamorano University and for helping me seeking for graduate studies. Finally, thanks to my mother and father for their encouragement. v TABLE OF CONTENTS Page ABSTRACT .......................................................................................................................ii DEDICATION .................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................... v TABLE OF CONTENTS .................................................................................................. vi LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ............................................................................................................ xi CHAPTER I INTRODUCTION ....................................................................................... 1 CHAPTER II LITERATURE REVIEW ........................................................................... 5 Sorghum Grain Utilization ............................................................................................. 5 Uses of Sorghum Plant Material .................................................................................... 5 Sorghum Color ............................................................................................................... 6 Polyphenolic Compounds of Sorghum Grains ............................................................... 7 Polyphenolic Compounds of Sorghum Leaves, Sheaths, Glumes and Stalks .............. 12 Biosynthesis of Flavonoids .......................................................................................... 12 Importance of Sorghum Polyphenolics ........................................................................ 15 Extraction of Phenolics ................................................................................................ 15 Mass Spectrometry ....................................................................................................... 17 Mass Spectrometry to Identify Phenolic Compounds .................................................. 17 Fragmentation Nomenclature of Polyphenols .............................................................. 19 Structural Characteristics of Polyphenolics with Glycoside Groups ........................... 20 Fragmentation Properties of Phenolic Compounds by MS/MS ................................... 21 Importance of Cancer in the United States................................................................... 22 Neoplasia and Cancer ................................................................................................... 23 Phases and Causes of Cancer ....................................................................................... 23 Chemopreventive Agents ............................................................................................. 24 Relevance of Sorghum Polyphenolics in Cancer Research ......................................... 25 Cancer Cell Proliferation Assays ................................................................................. 26 Proliferation and Mitochondrial Activity ..................................................................... 26 Xenobiotic Transport Mechanisms .............................................................................. 27 Absorption of Flavonoids. ............................................................................................ 27 vi Page In vitro Models to Measure Phenolic Absorption ........................................................ 29 Data Analysis of Caco-2 Transport Model .................................................................. 30 CHAPTER III IDENTIFICATION OF PHENOLICS FROM DIFFERENT SORGHUM MORPHOLOGICAL PARTS ..................................................................... 32 Materials and Methods ................................................................................................. 32 Samples .................................................................................................................... 32 Standards and Reagents ............................................................................................ 32 Sample Preparation ................................................................................................... 33 Extraction for Total Phenols and Antioxidant Activity ............................................ 33 Total Phenol Analysis of Plant Components ............................................................ 34 Antioxidant Activity of Plant Components .............................................................. 34 Extraction of Phenolic Compounds for Mass Spectrometry Analysis ..................... 35 Identification of Phenolic Compounds Using UPLC-DAD and UPLC-TQD- MS/MS ..................................................................................................................... 35 Results and Discussion ................................................................................................. 37 Phenolic Acids of Sorghum ...................................................................................... 37 Flavones of Sorghum ................................................................................................ 45 3-Deoxyanthocyanidins of Sorghum ........................................................................ 53 Chalcones of Sorghum ............................................................................................. 60 Flavanones of Sorghum ............................................................................................ 63 Other Compounds on Sorghum ................................................................................ 64 Total Phenols of Sorghum ........................................................................................ 67 Antioxidant Activity of Sorghum Plant Components .............................................. 67 Correlation Between Total Phenols and Antioxidant Activity of Sorghum. ............ 69 Sorghum Plant Components as Source of Phytochemicals ...................................... 70 Conclusions .................................................................................................................. 72 CHAPTER IV CHEMOPREVENTIVE POTENTIAL OF SORGHUM PHENOLIC EXTRACTS ..................................................................................................................... 73 Materials and Methods ................................................................................................