COFFEE CONSTITUENTS AND MODULATION OF ANTIOXIDANT STATUS IN CACO-2 CELLS by YAZHENG LIU B. Sc., Shandong University, 2006 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Food Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2010 © Yazheng Liu 2010 ABSTRACT Coffee contains biologically active components which may affect chronic disease risk. These biologically active components include caffeine, cafestol and kahweol, and antioxidants such as chlorogenic acids and Maillard reaction products (MRPs) that are generated during roasting. Although MRPs are regarded as being the most abundant group of antioxidants present in coffee, the mechanism underlying the antioxidant effects of coffee MRPs in both in vitro and in biological systems has yet to be elucidated. In this study, the in vitro antioxidant properties of roasted and non-roasted coffee extracts (Coffea arabica L.) were tested using oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC) and reducing power assays. MRPs were shown to be the prevailing antioxidants in roasted coffee extracts. The mechanisms of the antioxidant action associated with coffee MRPs involve the hydrogen atom transfer (HAT) mechanism and the single electron transfer (SET) mechanism. The biological effects of MRPs derived from coffee extracts on the enzymatic antioxidant defense in human colon adenocarcinoma Caco-2 cells were also investigated. No induction of antioxidant enzyme activities of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase were observed in Caco-2 cells after exposure to coffee MRPs, except for an increased glutathione peroxidase activity after 24 h exposure. In contrast, significantly decreased activities of catalase and glutathione peroxidase, and a reduced glutathione content were observed in Caco-2 cells after treatment with coffee MRPs (p<0.05). ii The antioxidant gene expression profile in Caco-2 cells after coffee treatment was further investigated using a Real-Time Polymerase Chain Reaction (PCR) array technology. Results demonstrated that roasted coffee extracts induced the expression of specific antioxidant response element (ARE)-driven genes in Caco-2 cells, thus enhancing cellular endogenous defense systems. This is the first report of the molecular mechanism underlying the antioxidant effect of coffee in Caco-2 cells. Hydrogen peroxide generated in the cell culture system as a consequence of coffee exposure, may serve as a signaling molecule that is involved in the gene regulatory effect associated with coffee extracts. iii TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii TABLE OF CONTENTS................................................................................................... iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES ............................................................................................................ x LIST OF ABBREVIATIONS..........................................................................................xiii ACKNOWLEDGEMENTS............................................................................................. xvi CHAPTER I OVERVIEW: GENERAL INTRODUCTION, LITERATURE REVIEW, AND RESEARCH HYPOTHESES AND OBJECTIVES........................................................... 1 1.1 GENERAL INTRODUCTION..................................................................................... 2 1.2 LITERATURE REVIEW ............................................................................................. 6 1.2.1 Oxidative stress and antioxidants........................................................................... 6 1.2.1.1 Reactive oxygen species and oxidative stress................................................. 6 1.2.1.2 Antioxidant mechanisms................................................................................. 7 1.2.1.3 Gene regulations by oxidative stress............................................................... 7 1.2.2 Maillard Reaction (MR)....................................................................................... 10 1.2.2.1 Chemistry of Maillard Reaction (MR).......................................................... 10 1.2.2.2 Chemistry of Maillard reaction products (MRPs) ........................................ 11 1.2.2.3 Antioxidant properties of MRPs ................................................................... 14 1.2.2.4 MRPs and chemoprotective enzymes ........................................................... 17 1.2.3 Coffee – a source of MRPs .................................................................................. 18 1.2.3.1 Composition of coffee bioactive components............................................... 19 iv 1.2.3.2 Coffee as a source of dietary antioxidant...................................................... 25 1.2.3.3 Coffee consumption and health .................................................................... 29 1.3 RESEARCH HYPOTHESES AND OBJECTIVES................................................... 32 CHAPTER II CHEMICAL CHARACTERISTERCS AND ANTIOXIDANT PROPERTIES OF COFFEE............................................................................................................................ 35 2.1 Introduction............................................................................................................. 36 2.2 Materials and methods ............................................................................................ 38 2.2.1 Coffee preparation ........................................................................................... 38 2.2.2 Ultrafiltration ................................................................................................... 38 2.2.3 Physical chemical analyses .............................................................................. 39 2.2.4 Chemical based antioxidant assays.................................................................. 40 2.2.5 Cell based assays.............................................................................................. 42 2.2.6 Statistical analysis............................................................................................ 47 2. 3 Results.................................................................................................................... 48 2. 3.1 Yields and recovery of coffee brews and ultrafiltration fractions .................. 48 2.3.2 Chemical characteristics of coffee................................................................... 50 2.3.3 Antioxidant activity of coffee extracts in chemical systems ........................... 55 2.3.4 Biological effects of coffee extracts ................................................................ 60 2.4 Discussion............................................................................................................... 66 2.4.1 Chemical characteristics of coffee................................................................... 66 2.4.2 Antioxidant activity of coffee extracts............................................................. 68 2.4.3 Biological effects of coffee extracts ................................................................ 70 v CHAPTER III COFFEE CONSTITUENTS AND MODULATION OF OXIDATIVE STATUS IN CACO-2 CELLS............................................................................................................... 72 3.1 Introduction............................................................................................................. 73 3.2 Materials and method.............................................................................................. 75 3.2.1 Preparation of coffee and Maillard reaction products (MRPs)........................ 75 3.2.2 Chemical analyses and Antioxidant assays...................................................... 76 3.2.3 Cellular in vitro Assay ..................................................................................... 77 3.2.4 Real-Time Quantitative Reverse Transcription PCR (RQ RT-PCR) Array .... 78 3.2.5 Statistical analysis............................................................................................ 81 3.3 Results..................................................................................................................... 82 3.3.1 Recovery of coffee brews and fractions........................................................... 82 3.3.2 Chemical characteristics of coffee................................................................... 83 3.3.3 Antioxidant activity of roasted and green coffee............................................. 88 3.3.4 Biological effects of coffee bean extracts........................................................ 91 3.3.5 Biological effects of coffee fractions on Caco-2 cells ..................................... 99 3.3.6 The regulatory effects of coffee on the expression of the genes involved in the oxidative stress and antioxidant defense system in Caco-2 cells............................ 105 3.3.7 Chemical characteristics and antioxidant activity of Suc-Ser and Ara-Ser model MRPs............................................................................................................ 109 3.3.8 Chemical characteristics and antioxidant activity Ara-Ser model