ANTIOXIDANT PROPERTIES OF FLAXSEED LIGNANS USING IN VITRO MODEL SYSTEMS A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Pharmacy and Nutrition of the University of Saskatchewan Saskatoon, Saskatchewan Canada By Farah Hosseinian Copyright Farah Hosseinian April 2006 All Rights Reserved The author claims copyright. Use shall not be made of the material contained herein without proper acknowledgment, as indicated on the copyright page. i PERMISION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis, in whole or in parts, should be addressed to: Head College of Pharmacy and Nutrition University of Saskatchewan 110 Science Place Saskatoon, SK S7N 5C9 Canada ii 1.0 ABSTRACT The major objectives of this study were to investigate the antioxidant properties of flaxseed lignans secoisolariciresinol (SECO 2) and secoisolariciresinol diglycoside (SDG 1) and their major oxidative compounds using 2,2'-azobis(2- amidinopropane) dihydrochloride (AAPH 47) in an in vitro model of lipid peroxidation. This investigation was facilitated by the structural elucidation of the major oxidative compounds and the ability of flaxseed lignans to delay the onset of oxidation in two model systems. This study showed that SECO 2 oxidation occurs at the aromatic (4-OH) and aliphatic (9-OH) hydroxyl groups. Conversely for SDG 1, only compounds derived from the oxidation of aromatic hydroxyl groups were obtained because the 9-OH position is glucosylated. SECO 2 oxidation with AAPH 47 showed that the intermediate 2a is most likely involved in the generation of early-forming (48 and 52) and 2c for the formation of late-forming (49, 50 and 51) oxidation compounds. Compound 48 is formed from dimerization of 2a that is converted to 52 and then to 51. Compound 50 was formed by the addition of a carbon-centre free radical of AAPH (AP radical) to 2c. Compounds 50 and 51 trap carbon-centered AP radicals supporting SECO 2 as a chain-breaking antioxidant and AAPH 47 as a proper model for study of SECO 2 oxidation in vitro. iii SDG 1 oxidation with AAPH 47 indicated that intermediates 1b and 1c are most likely involved for the formation of early forming compounds (55 and 58) and 1a leads to the late forming compounds (56 and 57). Compound 55 is a result of dimerization. Compound 56 may be directly formed via intermediate radical 1a by adding AP free radicals. Compound 56 was a stable non-radical compound that could trap AP free radicals, thereby supporting SDG 1 as a chain-breaking antioxidant. Hydrogen abstraction from 4-hydroxyl yielded the radical 1a and hydroxyl radical addition to 1a yielded 57. Compound 58 formed from the addition • of OH or H2O to 1c. This study demonstrated that AAPH 47 produces carbon-centred AP radicals upon thermal decomposition and mimics the formation of lipid peroxyl radicals. Interaction of carbon-centred AP radicals with SECO 2 and SDG 1 provides a good model to study the antioxidant reactions of SECO 2 in vitro. The relative antioxidant capacity of the flaxseed lignans versus BHT 17, in two model systems, was determined. The stoichiometric ratio for SECO 2 and SDG 1 were 1.5 and 1.1-1.2, respectively, compared to BHT 17 (2.0). The induction time by Rancimat analyzer measured inhibition of autoxidation mediated by flaxseed lignans SECO, SDG and SDG polymer in comparison with BHT 17. The induction time data demonstrated that SECO 2 protected canola oil better than either SDG 1 or SDG polymer 3. iv These results are important for better understanding about the chemistry behind flaxseed lignan antioxidant activities. This study provided useful evidence that flaxseed lignans can be used as natural antioxidants. v This thesis is dedicated to……… ….my parents my brother, Vahid my dear husband, Javad and my loving children, Hamed and Saeed vi ACKNOWLEDGEMENTS I would like to sincerely thank my supervisor Dr. Ed. S. Krol for his very kind and generous support, academic advice, as well as his patience, understanding, expertise and criticism in the preparation of this manuscript. I would like to thank the other members of the advisory committee, Drs. J. Alcorn, A.D. Muir, N.D. Westcott and M. Foldvari for their helpful and valuable suggestions during my research work. Sincere thanks to Dr. D. Gorecki, Dr. M. Foldvari and B. Juurlink for their positive encouragement for final preparation of this manuscript. I also thank my parents for their support and understanding. A special thank you goes to my husband Javad, and to my loving children, Hamed and Saeed, for their help, encouragement, and patience during my program. Sincere thanks and appreciation is also extended to Mrs. Krista Thompson and Mrs. Kendra Fesyk for their technical support, Mrs. Sandra Northrup for providing SECO and Mr. Brock Chatson from PBI for providing use of the NMR. I would also to thank Dr. Susan Marles for her useful suggestions during my work and Dr. M. Reaney for the use of the Rancimat apparatus in his lab. Lastly, sincere thank you and gratitude to all of my friends for their support during my stay in Saskatoon living far from my family. I would also to thank the HSURC and SFDC for their financial support and Agriculture and Agri-Food Canada (Saskatoon Research Centre) for the use of the facilities during my program. vii Table of Contents……………………………………………………………....Page PERMISION TO USE.............................................................................................. ii 1.0 ABSTRACT..................................................................................................iii ACKNOWLEDGEMENTS ................................................................................... vii 2.0. LITERATURE REVIEW ............................................................................ 1 2.1. Flaxseed, general description...................................................................... 1 2.2. Flaxseed oil................................................................................................. 1 2.3. Flaxseed mucilage....................................................................................... 2 2.4. Flaxseed lignans.......................................................................................... 2 2.4.1. Properties of SECO 2 and SDG 1 ....................................................... 5 2.4.1.1. Optical rotation of SECO 2 and SDG 1 .......................................... 6 2.5. Mammalian lignans..................................................................................... 8 2.5.1. Conversion of plant lignans to mammalian lignans............................ 8 2.5.2. Role of gut flora in the oxidation of plant lignans to mammalian lignans................................................................................................. 9 2.6. Hepatic metabolism of mammalian lignans.............................................. 12 2.6.1. Phase I metabolism ........................................................................... 12 2.6.2. Phase 2 metabolism........................................................................... 12 2.7. Flaxseed lignan uptake and urinary levels ................................................ 15 2.8. Flaxseed lignan uptake and plasma levels ................................................ 16 2.9. Flaxseed health benefits............................................................................ 17 2.9.1. ALA in flaxseed - health benefits ..................................................... 18 2.9.1.1. ALA: Animal studies .................................................................... 18 2.9.1.2. ALA: Human studies .................................................................... 18 2.9.1.3. Mechanism of action..................................................................... 20 2.9.2. Mucilage - health benefits................................................................. 20 2.9.3. Flaxseed lignans – health benefits .................................................... 21 2.9.3.1. Animal studies.............................................................................. 22 2.9.3.2. Human studies............................................................................... 23 2.9.3.3. In vitro studies............................................................................... 25 2.9.3.4. Correlation between in vitro production and in vivo excretion of lignans........................................................................................... 26 2.10. Lignan- mechanism of effect ................................................................ 27 2.10.1. Non-hormonal mechanisms .............................................................