ANTIOXIDANT PROPERTIES OF FLAXSEED LIGNANS USING IN VITRO MODEL SYSTEMS A ...

257 Pages·2006·0.97 MB·English

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.
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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

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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.
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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 H O to 1c.
2
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.
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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.

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This thesis is dedicated to………

….my parents
my brother, Vahid
my dear husband, Javad and
my loving children, Hamed and Saeed

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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.
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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..............................................................27
2.10.1.1. Antioxidant activity......................................................................27
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2.10.1.2. Hormonal mechanisms..................................................................31
2.11. Free radicals-physiology.......................................................................34
2.11.1. Reactive oxygen species-sources......................................................35
2.11.2. Reactive oxygen species-classification.............................................36
2.11.3. Enzymatic formation of ROS............................................................37
2.12. Reactive oxygen species-mechanism....................................................38
2.12.1. Superoxide anion (•O  )....................................................................38
2
2.12.2. Hydroxyl radical (•OH).....................................................................39
2.12.3. Hydrogen peroxide (H O )...............................................................40
2 2
2.12.4. Singlet oxygen..................................................................................41
2.12.5. Peroxyl and alkoxyl radicals.............................................................41
2.13. Reactive nitrogen species (RNS)..........................................................42
2.13.1. Nitric oxide and nitric dioxide..........................................................42
2.13.2. Peroxynitrite......................................................................................42
2.14. Protein oxidation...................................................................................42
2.14.1. DNA strand breaks............................................................................43
2.14.2. Nucleic acid oxidation......................................................................43
2.15. Lipid Peroxidation in humans...............................................................44
2.15.1. Lipid Peroxidation in foods..................................................................45
2.15.2. Antioxidant in food systems-important factors for measuring
antioxidants effectiveness.....................................................................46
2.15.3. Synthetic antioxidants in foods.............................................................54
2.16. Enzymic detoxification of xenobiotics in the body..............................55
2.16.1. Superoxide dismutase (SOD)............................................................56
2.16.2. Catalase.............................................................................................56
2.16.3. Glutathione peroxidase (GPx)...........................................................56
2.16.4. Glutathione (GSH)............................................................................57
2.16.5. Glutathione S-transferases (GSTs)....................................................57
2.16.6. Glutathione reductase........................................................................58
2.17. Non-enzymic detoxification of xenobiotics in the body.......................59
2.17.1. Tocopherols.......................................................................................59
2.17.2. Ascorbic acid....................................................................................59
2.18. Plant-derived antioxidants.....................................................................60
2.18.1. Flavonoids.........................................................................................60
2.18.3. Flavonol............................................................................................61
2.18.3.1. Quercetin.......................................................................................61
2.18.3.2. Catechins.......................................................................................62
2.18.3.3. Proanthocyanins............................................................................67
2.18.4. Mechanism of action.........................................................................68
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