Screening, isolation and evaluation of antioxidative compounds from Geranium macrorrhizum, Potentilla fruticosa and Rhaponticum carthamoides Promotor Prof. Dr. E.J.R. Sudhölter, Hoogleraar Organische Chemie, Wageningen Universiteit Co-promotoren Dr. T.A. van Beek, Universitair Hoofddocent, Laboratorium voor Organische Chemie, Wageningen Universiteit Dr. Ir. J.P.H. Linssen, Universitair Docent, leerstoelgroep Productontwerpen en Kwaliteitskunde, Wageningen Universiteit Promotiecommissie Dr. D. Joulain (The Robertet Group, Grasse, France) Prof. Dr. Ir. I.M.C.M. Rietjens (Wageningen Universiteit) Prof. Dr. Ir. P.R. Venskutonis (Kaunas University of Technology, Lithuania) Prof. Dr. R. Verpoorte (Universiteit Leiden) Dit onderzoek is uitgevoerd binnen de onderzoeksschool VLAG Screening, isolation and evaluation of antioxidative compounds from Geranium macrorrhizum, Potentilla fruticosa and Rhaponticum carthamoides Giedrius Miliauskas Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M.J. Kropff, in het openbaar te verdedigen op maandag 20 november 2006 des namiddags te vier uur in de Aula Giedrius Miliauskas Screening, isolation and evaluation of antioxidative compounds from Geranium macrorrhizum, Potentilla fruticosa and Rhaponticum carthamoides Thesis Wageningen University – with summaries in English, Dutch and Lithuanian 2006 ISBN 90-8504-487-1 CONTENT 1. General Introduction 1.1. Problem definition 1 1.2 Aims and scope of the work 2 1.3 Literature survey 4 1.3.1 Classification of antioxidants 4 1.3.2 Methods of antioxidant activity evaluation 9 1.3.2.1 Methods to evaluate primary changes 9 1.3.2.2 Methods to evaluate secondary changes 9 1.3.2.3 Other tests to evaluate radical scavenging or antioxidant activity 10 1.3.3 Sources of natural antioxidants 11 1.4 References 14 2. Screening of radical scavenging activity of some medicinal and aromatic plant extracts 2.1 Introduction 19 2.2 Review of the screened in this study plants 19 2.3 Experimental 21 2.3.1 Reagents 21 2.3.2 Plant material 22 2.3.3 Extraction procedures 22 2.3.4 Radical scavenging assays 23 2.3.4.1 DPPH radical scavenging assay 23 2.3.4.2 ABTS radical cation scavenging assay 23 2.3.4.3 Measuring the amount of phenolic compounds 24 2.3.4.4 Statistical analysis 24 2.4 Results and discussion 25 2.4.1 Evaluation of radical scavenging activity by model tests 25 2.4.2 Determination of the amount of phenolic compounds 28 2.5 References 29 3. Isolation and identification of radical scavengers from Geranium macrorrhizum 3.1 Introduction 31 3.2 Materials and methods 31 3.2.1 Reagents 31 3.2.2 Radical scavenging assays 32 3.2.3 Procedures for isolation of active compounds 33 3.2.4 Structure elucidation of isolated compounds 34 3.2.5 Quantitative comparison of major radical scavenging compounds in different botanical parts 34 3.3 Results and discussion 35 3.3.1 Extraction process and assessment of radical scavenging activity of obtained fractions 35 3.3.2 On-line HPLC–DPPH/ABTS assays for detection of active radical scavengers 36 3.3.3 Isolation, purification and structure elucidation of active compounds 37 3.3.4 Assessment of radical scavenging activity of isolated compounds 40 3.3.5 Quantitative comparison of major radical scavenging compounds in different botanical parts 41 3.4 References 42 4. Isolation and identification of radical scavengers from Potentilla fruticosa 4.1 Introduction 45 4.2 Materials and methods 45 4.3 Results and discussion 46 4.3.1 Assessment of radical scavenging activity of various plant fractions 46 4.3.2 Isolation, purification and structure elucidation of active compounds 48 4.3.3 Assessment of radical scavenging activity of isolated compounds 52 4.3.4 Quantitative comparison of major radical scavenging compounds in different botanical parts 55 4.4 References 56 5. Isolation and identification of radical scavengers from Rhaponticum carthamoides 5.1 Introduction 57 5.2 Materials and methods 57 5.2.1 General Experimental Procedures 57 5.2.2 Plant Material and Extraction Procedures. 58 5.2.3 Radical Scavenging Assays 58 5.2.4 LC-DAD-SPE-NMR Instrumental Setup 58 5.3 Results and Discussion 60 5.4 References 66 6. Practical evaluation of the antioxidative properties and preliminary safety studies of selected plant extracts 6.1 Introduction 67 6.2 Materials and methods 67 6.2.1 Determination of superoxide anion radical scavenging properties 67 6.2.2 Determination of hydrogen peroxide scavenging properties 68 6.2.3 Linoleic acid β-carotene oxidation assay 68 6.2.4 Determination of peroxide value 69 6.2.5 UV absorbance test 70 6.2.6 Measurement of hexanal using static headspace GC (HS-GC) method 70 6.2.7 Evaluation of antioxidative capacity of plant extracts during accelerated oxidation of fermented sausages 71 6.2.7.1 Peroxide value measurements 72 6.2.7.2 Determination of TBARS 72 6.2.7.3 Determination of hexanal amount 73 6.2.8 Preliminary safety evaluation studies 73 6.2.8.1 Investigation of the enzymatic oxidation of extracts 74 6.2.8.2 Protective effects of extracts against singlet oxygen caused erythrocyte hemolysis 74 6.2.8.3 Mutagenicity evaluation of plant extracts (somatic mutations and recombinations test in Drosophila melanogaster wing cells) 75 6.3 Results and Discussion 75 6.3.1 Antioxidant activity evaluation of selected extracts from Geranium macrorrhizum 75 6.3.1.1 Determination of superoxide anion and hydrogen peroxide scavenging properties 76 6.3.1.2 Linoleic acid-β-carotene coupled oxidation assay 78 6.3.1.3. Determination of peroxide value (PV) 79 6.3.1.4 UV absorbance test 80 6.3.1.5 Effect of extracts on the formation of hexanal 81 6.3.2 Antioxidant activity evaluation of selected extracts from Potentilla fruticosa 83 6.3.2.1 Determination of superoxide anion and hydrogen peroxide scavenging properties 83 6.3.2.2 Linoleic acid-β-carotene coupled oxidation assay 84 6.3.2.3 Determination of peroxide value 85 6.3.2.4 UV absorbance test 86 6.3.2.5 Measurement of hexanal using static head space GC (HS-GC) method 87 6.3.3 Evaluation of the activity of selected extracts from G. macrorrhizum and P. fruticosa during the oxidation of fermented sausages 88 6.3.3.1 Determination of peroxide value 88 6.3.3.2 Determination of TBARS 90 6.3.3.3 Measurement of hexanal formation 91 6.3.4 Comparison of antioxidant activities between Geranium and Potentilla extracts 93 6.3.5 Preliminary safety evaluation of chosen plant extracts 93 6.3.5.1 Enzymatic oxidation of polyphenols from plant extracts with peroxidase and tyrosinase 94 6.3.5.2 Protective effects of plant extracts against singlet oxygen caused erythrocyte hemolysis 95 6.3.5.3 Mutagenicity evaluation of plant extracts (somatic mutations and recombinations test in Drosophila melanogaster wing cells) 96 6.4 References 97 7. Hyphenated on-line method (HPLC-DAD-MS-DPPH/ABTS) for detection of radical scavenging compounds in complex extracts 7.1 Introduction 101 7.2 Materials and methods 101 7.2.1 On-line HPLC-DAD-MS-DPPH/ABTS analytical setups 102 7.3 Results and Discussion 103 7.3.1 Development of the experimental setup 103 7.3.2 Evaluation of the hyphenated LC-DAD-MS-DPPH/ABTS methods with Ginkgo biloba and Rosmarinus officinalis extracts 104 7.4 References 110 8. A comparison between analytical and semi-preparative columns for LC-SPE-NMR 8.1 Introduction 113 8.2 Materials and Methods 115 8.2.1 Reagents 115 8.2.2 LC-DAD-SPE-NMR instrumental setup 116 8.2.3 Efficiency testing 117 8.3 Results and discussion 117 8.4 References 129 9. Conclusions and general discussion 131 Summary 135 Samenvatting 141 Sąntrauka 147 Abbreviations 153 Acknowledgements 155 Curriculum vitae 157 List of publications 158 Chapter one GENERAL INTRODUCTION 1.1 Problem definition Electron transfer is one of the most fundamental processes in chemistry. The transfer of an electron from a donor (reducing species) to an acceptor (oxidizing species) results in a change in properties of both components participating in the reaction. In general oxidation can be defined as a conversion of a chemical substance into another having fewer electrons [1]. Food molecules (lipids, proteins, carbohydrates) are widely involved in oxidation reactions; these reactions also play an important role in the ageing processes of living organisms. Oxidation reactions can be induced by so called reactive oxygen species (ROS), which is a general term encompassing various reactive substances. Basically these are free radicals - organic substances with one or more unpaired electrons, such as hydroxyl (OHx), peroxyl (ROOx), alkoxy (ROx), x x¯ peroxonitrite (ONOO ), superoxide anion (O2 ) and others. Other important ROS include singlet 1 oxygen ( O2), ozone (O3), nitrogen dioxide (NO2) and hypochlorous acid (HOCl). Oxidation can also be induced and accelerated by metal ions, phagocyte derived ROS and some other reactive substances [2]. ROS are continuously generated in living organisms and are used in various beneficial physiological reactions, e.g. in signal transduction pathways in the body, immune defense, enzyme catalyzed oxidation [3], however in many cases ROS are harmful. Oxidative stress, caused by ROS in the living cell is associated with numerous diseases, like coronary heart disease, cancer, cataracts and age-related macular degeneration and ageing [4]. ROS are the major cause of lipid containing food products deterioration, commonly defined as rancidity. Significant changes can occur in product odour, taste, colour, texture, nutritive value. Progressing oxidation results in complete spoilage of foods. Although protective mechanisms exist both in living cells (e.g. enzymes: superoxide dismutase, glutathione peroxidase) and in foods (e.g.