Isolation, identification and activity of natural antioxidants from sweet grass (Hierochloe odorata), costmary (Chrysanthemum balsamita) and horehound (Marrubium vulgare), cultivated in Lithuania Audrius Pukalskas Promotor Prof. Dr. Ae. de Groot, Hoogleraar Bio-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 Prof. Dr. Ir. M.A.J.S. van Boekel (Wageningen Universiteit) Prof. Dr. G.J. de Jong (Universiteit Utrecht) Prof. Dr. Ir. I.M.C.M. Rietjens (Wageningen Universiteit) Prof. Dr. R. Verpoorte (Universiteit Leiden) Dit onderzoek is uitgevoerd binnen de onderzoeksschool VLAG Isolation, identification and activity of natural antioxidants from sweet grass (Hierochloe odorata), costmary (Chrysanthemum balsamita) and horehound (Marrubium vulgare), cultivated in Lithuania Audrius Pukalskas 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 1 december 2008 des namiddags te vier uur in de Aula Audrius Pukalskas Isolation, identification and activity of natural antioxidants from sweet grass (Hierochloe odorata), costmary (Chrysanthemum balsamita) and horehound (Marrubium vulgare), cultivated in Lithuania Thesis Wageningen University – with summaries in English, Dutch and Lithuanian 2008 ISBN 978-90-8504-982-1 Contents 1. Introduction ..................................................................................................................................... 3 1.1. Oxidation and Autoxidation. .................................................................................................... 3 1.2. Autoxidation of Lipids. ............................................................................................................ 3 1.3. Natural antioxidants.................................................................................................................. 5 1.4. Natural sources of antioxidant compounds. ............................................................................. 6 1.5. Methods for evaluation of antioxidants.................................................................................... 7 1.6. The aims of this study............................................................................................................... 9 1.7. References .............................................................................................................................. 11 2. Preliminary Screening of the Antioxidant Activity of some Plant Extracts in Rapeseed Oil* ..... 19 2.1. Introduction ............................................................................................................................ 19 2.2. Materials and Methods ........................................................................................................... 20 2.2.1. Materials.......................................................................................................................... 20 2.2.2. Methods ........................................................................................................................... 21 2.3. Results and Discussion ........................................................................................................... 23 Additive ............................................................................................................................................. 26 Without additive ........................................................................................................................ 26 2.4. Conclusions ............................................................................................................................ 27 2.5. References .............................................................................................................................. 28 3. Antioxidant Activity of Extracts from Sweet Grass (Hierochloe odorata), Costmary (Chrysanthemum balsamita) and Horehound (Marrubium vulgare) Obtained By Different Extraction and Fractionation Procedures*......................................................................................... 30 3.1. Introduction ............................................................................................................................ 30 3.2 Materials and Methods ............................................................................................................ 31 3.2.1. Materials.......................................................................................................................... 31 3.2.2. Methods ........................................................................................................................... 31 3.3. Results and Discussion. .......................................................................................................... 35 3.3.1. Preliminary fractionation. Deodorisation. ....................................................................... 35 Sample ............................................................................................................................................... 37 Sample ............................................................................................................................................... 42 3.3.2. Activities of fractions obtained after partitioning............................................................ 43 3.4. Conclusions ............................................................................................................................ 47 3.5. References .............................................................................................................................. 48 4. Isolation of Radical Scavengers from Sweet Grass, Costmary and Horehound ........................... 50 4.1. Introduction ............................................................................................................................ 50 4.2. Materials and Methods ........................................................................................................... 51 4.2.1. Chemicals. ....................................................................................................................... 51 4.2.2. Preparation of Plant Extracts........................................................................................... 51 4.2.3. DPPH Assay. ................................................................................................................... 52 4.2.4. ABTS Assay. ................................................................................................................... 52 4.2.5. HPLC – DPPH Conditions and Instrumentation............................................................. 53 4.2.6. Isolation of radical scavengers from sweet grass. ........................................................... 54 4.2.7. Isolation of Radical Scavengers from Costmary ............................................................. 55 4.2.8. Isolation of Active Compounds from Horehound........................................................... 60 4.3. Results and Discussion ........................................................................................................... 61 4.3.1. Sweet grass ...................................................................................................................... 61 4.3.2. Costmary.......................................................................................................................... 68 4.3.3. Horehound ....................................................................................................................... 75 4.4. Conclusions. ........................................................................................................................... 82 4.5. References .............................................................................................................................. 84 5. Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-NMR system for the rapid identification of antioxidants in complex plant extracts*............................................. 88 5.1. Introduction ............................................................................................................................ 88 5.2. Experimental........................................................................................................................... 89 5.2.1. Materials and chemicals .................................................................................................. 89 5.2.2. Sample preparation.......................................................................................................... 89 5.2.3. HPLC-RSD-DAD-SPE-NMR conditions and instrumental setup .................................. 89 5.3. Results and discussion............................................................................................................ 91 5.4. Conclusions ............................................................................................................................ 98 5.5. References ............................................................................................................................ 100 6. Influence of extraction solvents on the yield of antioxidants from sweet