Extraction and analysis of antioxidant capacity in rice bran extracts from different Sarawak local rice varieties

II Acknowledgement . IV Declaration by Candidate . VI Conference Presentation . XI Conference Awards . XI List of Figures and Tables . XII Chapter 1: Introduction . 1 1.1. Research Background . 1 1.2. Research Aims and Objectives . 5 1.3. Research Contributions and Impact to Society . 6 Chapter 2: Extraction of Natural Antioxidant from Rice Bran . 7 2.1 Executive Summary . 7 2.2 Literature Review . 8 2.2.1 Rice and Rice Brans . 8 2.2.2 Extraction and Analysis of Antioxidants . 10 2.2.3 Rice Antioxidants . 15 2.2.3.1 Polyphenols . 16 2.2.3.1.1 Phenolic Acids in Rice . 19 2.2.3.1.2 Flavonoids in Rice . 19 2.2.3.1.3 Anthocyanins in Rice . 21 2.2.3.1. 4 Heath Benefits of Polyphenols . 21 2.2.3.2 Gamma-Oryzanol . 24 2.2.3.2.1 Health benefits of γ-Oryzanol . 24 2.2.3.3 Vitamin E . 27 2.2.3.3.1 Health Benefits of Vitamin E (Tocotrienols) . 28 2.3 Research Aims and Objectives . 31 2.4 Experimental Design. 31 2.4.1 Materials and Chemicals . 31 2.4.1.1 Rice Samples . 31 2.4.1.2 Chemicals . 33 2.4.2 Rice Sample Treatment and Preparation of Rice Bran Sample . 34 2.4.3 Methodology . 35 2.4.3.1 Simple Solvent Extraction . 35 2.4.3.3 Determination of Total Flavonoid Content . 36 VIII 2.4.3.4 Determination of Total Anthocyanin Content. 37 2.4.3.5 Determination of Total Gamma Oryzanol (γ-Oryzanol) Content . 38 2.4.3.6 Determination of Vitamin E Content . 39 2.4.3.7 Statistical Analysis . 39 2.4.4 Results and Discussion . 40 2.4.4.1 Determination of Total Phenolic Content . 40 2.4.4.2 Determination of Total Flavonoid Content . 44 2.4.4.3 Determination of Total Anthocyanin Content. 46 2.4.4. 4 Determination of Total Gamma Oryzanol (γ-Oryzanol) Content . 48 2.4.4.5 Determination of Vitamin E Content . 50 2.4.5 Conclusion . 54 Chapter 3: Bioactivity Studies of Natural Antioxidants Derived from Rice Bran of Different Sarawak Local Rice Varieties . 57 3.1 Executive Summary . 57 3.2 Literature Review . 58 3.2.1 Reactive Oxygen Species (ROS) and Oxidative Stress . 58 3.2.2 Oxidative Stress Related Disease . 60 3.2.2.1 Cardiovascular Diseases . 61 3.2.2.1.1 Atherosclerosis . 62 3.2.2.1.2 Myocardial Infarction (MI) and Myocardial Reperfusion Injury . 66 3.2.3 Antioxidants . 69 3.2.3.1 Endogenous antioxidant . 70 3.2.3.1.1 Superoxide Dismutase (SOD) . 70 3.2.3.1.2 Catalase (CAT) . 71 3.2.3.1.3 Glutathione Peroxidase (GPx) . 72 3.2.3.2 Exogenous Antioxidants . 73 3.3 Research Aims and Objectives . 75 3.4 Experimental Design. 75 3.4.1 Materials and Chemicals . 75 3.4.2 Test Samples . 76 3.4.3 Methodology . 76 3.4.3.1 In Vitro Chemical-Based System . 78 3.4.3.1.1 DPPH Free Radical Scavenging Assay . 78 3.4.3.1.2 Trolox Equivalent Antioxidant Capacity (TEAC) Assay . 78 3.4.3.1.3 Statistical Analysis . 79 3.4.3.2 In Vitro Cell Culture-Based System . 80 IX 3.4.3.2.1 Cell Culture and Growth Curve Study . 80 3.4.3.2.2 Cell cytotoxicity Assay . 81 3.4.3.2.3 Induction of Oxidative Stress . 82 3.4.3.2.4 Endogenous Antioxidant Enzyme Activity Studies . 82 3.4.3.2. 5 Endogenous Antioxidant Enzyme Gene Expression Studies . 83 3.4.3.2. 6 Statistical Analysis . 85 3.4.4 Results and Discussions . 86 3.4.4.1 In Vitro Chemical-Based System . 86 3.4.4.1.1 DPPH Free Radical Scavenging Assay . 86 3.4.4.1.2 Trolox Equivalent Antioxidant Capacity (TEAC) Assay . 93 3.4.4.2 In Vitro Cell Culture-Based System . 98 3.4.4.2.1 Morphology and Growth of H9c2(2-1) Cardiomyocytes . 98 3.4.4.2.2 Cell Cytotoxicity Assay (RBE) . 101 3.4.4.2.3 Cell Cytoxicity Assay (Hydrogen Peroxide) . 109 3.4.4.2.4 Cell Viability Assay (Rice Bran Extract + Hydrogen Peroxide) . 112 3.4.4.2. 5 Effects of Different Treatments on Activities and Gene Expression of Endogenous Cellular Antioxidant Enzymes in H9c2(2-1) Cells . 118 (A) Superoxide Dismutase (SOD) . 119 (i) Effects of RBE on total SOD enzymatic activity and gene expression of SOD2 in H9c2(2-1) Cells . 119 (ii) Effects of H2O2 on total SOD enzymatic activity and gene expression of SOD2 in H9c2(2-1) Cells . 121 (iii) Effects of H2O2 on total SOD enzymatic activity and gene expression of SOD2 in H9c2(2-1) cells pre-treated with RBE . 123 (B) Catalase (CAT) . 129 (i) Effects of RBE on total enzymatic activity and gene expression of CAT in H9c2(2-1) cells . 129 (ii) Effects of Hydrogen peroxide (H2O2) on total enzymatic activity and gene expression of CAT in H9c2(2-1) cells . 131 (iii) Effects of H2O2 on total enzymatic activity and gene expression of CAT in H9c2(2-1) cells pre-treated with RBE . 133 (C) Glutathione Peroxidase (GPx) .