PHYTOCHEMISTRY AND HEALTH BENEFITS OF GRAPES AND WINES RELEVANT TO THE STATE OF TEXAS A Dissertation by ARMANDO DEL FOLLO MARTINEZ 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 August 2011 Major Subject: Food Science and Technology Phytochemistry and Health Benefits of Grapes and Wines Relevant to the State of Texas Copyright 2011 Armando Del Follo Martinez PHYTOCHEMISTRY AND HEALTH BENEFITS OF GRAPES AND WINES RELEVANT TO THE STATE OF TEXAS A Dissertation by ARMANDO DEL FOLLO MARTINEZ 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 Approved by: Chair of Committee, Susanne Mertens-Talcott Committee Members, Stephen T. Talcott Alejandro Castillo Weston Porter Intercollegiate Faculty Chair, Alejandro Castillo August 2011 Major Subject: Food Science and Technology iii ABSTRACT Phytochemistry and Health Benefits of Grapes and Wines Relevant to the State of Texas. (August 2011) Armando Del Follo Martinez, B.En., Instituto Tecnologico y de Estudios Superiores de Monterrey, Mexico; M.S., Instituto Tecnologico y de Estudios Superiores de Monterrey, Mexico Chair of Advisory Committee: Dr. Susanne Mertens-Talcott The overall objective of this work was to increase the knowledge regarding American hybrid grapes and wine-making techniques relevant to the State of Texas, specifically to investigate grape chemistry of hybrid grapes, to evaluate the effects of micro-oxygenation on wine chemistry, and to elucidate anti-cancer effects of wine compounds and extract in colon cancer cells in vitro. The methods used include HPLC- PDA-EIS-MSn and molecular bioassays. The American hybrid grapes, Black Spanish (Vitis aestivalis hybrid) and Blanc Du Bois (Vitis aestivalis hybrid), were compared to Cabernet Sauvignon and Merlot (Vitis vinifera) in their phytochemical composition. Total phenolics were similar in red grape varieties, but lower in white grapes. In Black Spanish grapes, anthocyanins and antioxidant capacity (ORAC) exhibited the highest values. Non-anthocyanin polyphenolics did not show qualitative differences in the four grape varieties. The presence of anthocyanins diglucosides was unique to Black Spanish grapes. iv The second experiment involved application of micro-oxygenation with oak inner staves to evaluate the effect of this new vinification technology on the stability of anthocyanins. Overall, anthocyanins exhibited significant decreases over time in the following order: control, wine with oak pieces, oak barrel, and micro-oxygenation. The anti-cancer effect of a combination of wine compounds, resveratrol/quercetin (RQ), and a polyphenolic extract from Black Spanish wine were investigated in colon cancer cells HT-29. RQ reduced the generation of reactive oxygen species (ROS), whereas the ORAC increased. RQ reduced cancer cell viability and proliferation, induced caspase-3-cleavage, and increased PARP-cleavage. Additionally, Sp1, Sp3, Sp4, and survivin were down-regulated at mRNA and protein levels. Furthermore, RQ decreased microRNA-27a (miR-27a) and induced ZBTB10, suggesting that RQ interactions with the miR-27a-ZBTB10-axis play a role in Sp down-regulation. Similar results were obtained for the wine extract. This work will provide valuable information regarding grape varieties, potential health benefits of wine, and wine production techniques to the wine industry in Texas and beyond. v To my family vi ACKNOWLEDGEMENTS I would like to thank Dr. Susanne Talcott, my advisor, for giving me the opportunity to work in her lab after difficult times and for guiding me through the Ph.D. adventure. Also, I wish to thank her for believing in me and making many opportunities and experiences possible throughout my studies. I am also very grateful to Dr. Steve Talcott for helping me to gain a better understanding and broader perspective of food chemistry and food science, and for the hours spent on the mass spectrometer. I appreciate the support and time dedicated to this research by Dr. Weston Porter and Dr. Alejandro Castillo. I also thank my lab mates and friends: Lisbeth, Chris, Jorge, and Thelma for all the good and bad times we spent together, Mike for his help collecting grape and wine samples, and especially Gaby and Giuliana for the chance to tackle the same learning experience together. Also, a special thanks goes to my parents, Dr. Armando Del Follo and Dr. Teresita Martinez, and my sister, Dr. Lourdes Del Follo, for all their support and for inspiring me by their example. Finally, to CONACyT for the economic support to complete my Ph.D. vii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES ................................................................................................... xii LIST OF TABLES .................................................................................................... xvi CHAPTER I INTRODUCTION ................................................................................ 1 II LITERATURE REVIEW ..................................................................... 3 Wine and Texas Wine Industry ...................................................... 3 North American Grapes ................................................................. 5 Vitis labrusca ............................................................................ 6 Vitis rotundifolia ...................................................................... 6 Vitis riparia .............................................................................. 7 Vitis aestivalis .......................................................................... 7 Wine Chemistry .............................................................................. 8 Wine Polyphenolics .................................................................. 8 Non-Flavonoid Phenolic Compounds ................................ 8 Hydroxycinnamic Acids ............................................. 8 Hydroxybenzoic Acids ............................................... 9 Stilbenes ...................................................................... 10 Flavonoid Phenolic Compounds ........................................ 11 Flavonols ..................................................................... 12 Flavan-3-ols, Condensed Tannins and Hydrolyzable Tannins ................................................. 13 Anthocyanins .............................................................. 17 Volatile Compounds ................................................................. 19 viii CHAPTER Page Organic Acids ...................................................................... 19 Alcohols ............................................................................. 20 Phenolics ............................................................................ 20 Nitrogen Compounds ......................................................... 21 Lactones ............................................................................. 22 Ketones ............................................................................... 23 Aldehydes ........................................................................... 23 Esters .................................................................................. 24 New Technologies for Wine Production ........................................ 25 Micro-oxygenation ................................................................... 26 Accelerated Aging .................................................................... 27 Health Benefits of Wine Against Chronic-Degenerative Diseases .......................................................................................... 29 Molecular Aspects of Cancer ......................................................... 31 Colon Cancer ............................................................................ 32 Dietary Risk Factors ................................................................. 33 Non-Dietary Risk Factors ......................................................... 34 Genetic Risk Factors ................................................................ 35 Colorectal Criteria for Stage Classification ............................. 35 Apoptosis .................................................................................. 37 MicroRNAs and Phytochemicals ............................................. 39 III CHARACTERIZATION AND COMPARISON OF AMERICAN HYBRID GRAPES RESISTANT TO PIERCE‟S DISEASE WITH TRADITIONAL VITIS VINIFERA GRAPE VARIETIES GROWN IN TEXAS .................................................... 42 Summary ........................................................................................ 42 Introduction .................................................................................... 43 Materials and Methods ................................................................... 44 Grape Sources .......................................................................... 44 Extraction and Fractionation of Grape Polyphenols