antioxidant and biological activities of tyrosol, hydroxytyrosol and their esters

160 Pages·2017·1.69 MB·English

ANTIOXIDANT AND BIOLOGICAL ACTIVITIES OF
TYROSOL, HYDROXYTYROSOL AND THEIR ESTERS

by

© Yuanxin Sun

A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for
the degree of Master of Science

Department of Biochemistry
Memorial University of Newfoundland

January 2017

St. John’s Newfoundland and Labrador Canada
ABSTRACT
A series of lipophilic esters of tyrosol and hydroxytyrosol, two naturally occurring phenols in
olive oil with interesting biological properties, were prepared by an enzymatic and simplified
base extraction procedure. Both tyrosol and hydroxytyrosol are hydrophilic molecules with poor
solubility in lipophilic media, resulting in limited usage in foods and limited uptake by the cells
and bioavailability in vivo. The antioxidant activities of esters so produced were evaluated using
the 2,2-diphenyl-1-picrylhydrazyl (DPPH), human low-density lipoprotein (LDL) oxidation, and
DNA strand scission assays. The antiviral properties of selected esters were measured using
hepatitis C virus (HCV) protease and alpha-glucosidase inhibitory activities. Antiglycation by
bovine serum albumin (BSA)-glucose assay, protection effect against oxidative stress, generation
anti-inflammatory products by nitrite assay, and cytotoxic properties by 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of selected esters were also evaluated. All
methods used for the antioxidant activity evaluation indicated that tyrosol (TY) and its
derivatives were less active than the corresponding hydroxytyrosols (HTY), which reflected the
influence of the ortho-diphenolic (catechol) structure of the latter on antioxidant capacity. For
hydroxytyrosol, the introduction of the lipid moiety decreased its antioxidant activity. We
observed the inhibition of HTY saturated fatty acid esters against hydroxyl radical induced DNA
oxidation decreased as alkyl chain length increased. Meanwhile, an unsaturation-dependent
antioxidant effect was observed for TY and HTY esters in DNA strand scission assay, and for
TY esters in DPPH assay. However, in the LDL oxidation assay, the polyunsaturated fatty acid
(PUFA) moiety of TY esters may be oxidized. For antiviral properties of selected esters, most of
the TY derivatives that showed potent inhibition on α-glucosidase were not active against HCV
while HTY esters showed very good HCV protease inhibition, especially HTY caprylate, stearate
II
and oleate esters which displayed 3-fold stronger inhibition than that of embelin (the positive
control). Moreover, it was found that lipophilization by esterification could improve the anti-
inflammatory and antiglycation effects of tyrosol and hydroxytyrosol. These results indicate that
the lipophilic ester derivatives can served as antioxidant ingredients in food, as well as anti-
inflammatory, antiglycation, and antiviral agents, and ingredients in other therapeutic
applications, supplements and natural health products.

III
ACKNOWLEDGEMENTS
I would like to express my sincere thanks to my supervisor Dr. F. Shahidi for his guidance,
patience, direction, personal encouragement throughout the project, and providing me with an
opportunity to get the Master's degree. I would also like to express my appreciation to my
supervisory committee members Dr. V. Booth and Dr. P. Davis. I would also like to thank Dr.
Dayong Zhou for his continued support and help. Thanks are extended to Dr. Chao-Mei Ma,
Min-Hsiung Pan, Mingfu Wang and Xiaoqian Hu for their help with cell line studies. I would
like to extend my thanks to Linda Winsor and Adam Beaton at the Centre for Chemical Analysis,
Research and Training (C-CART), Memorial University of Newfoundland for training me on the
HPLC-MS and FT-IR equipments. I would also like to express my sincere thanks to all my
friends and colleagues in Dr. Shahidi's research group for creating an enjoyable research
environment. Finally, special thanks are extended to my family for their continued support and
help during my studies.

IV
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................... II
ACKNOWLEDGEMENTS ........................................................................................................ IV
TABLE OF CONTENTS ............................................................................................................. V
LIST OF TABLES ..................................................................................................................... VIII
LIST OF FIGURES ..................................................................................................................... IX
LIST OF ABBREVIATION........................................................................................................ XI
CHAPTER 1. INTRODUCTION ................................................................................................ 1
CHAPTER 2. LITERATURE REVIEW .................................................................................... 5
2.1 Tyrosol and Hydroxytyrosol ................................................................................................. 5
2.1.1 Structures and Derivatives .............................................................................................. 6
2.1.2 Sources............................................................................................................................ 9
2.2 Fatty Acids .......................................................................................................................... 12
2.2.1 Saturated fatty acids...................................................................................................... 12
2.2.2 Monounsaturated fatty acids ......................................................................................... 13
2.2.3 Polyunsaturated fatty acids ........................................................................................... 13
2.3 Lipid oxidation .................................................................................................................... 15
2.3.1 Mechanism of lipid oxidation ....................................................................................... 15
2.3.2 Factors affecting lipid oxidation ................................................................................... 19
2.3.3 Influence of lipid oxidation .......................................................................................... 20
2.4 Mechanisms of action of antioxidants ................................................................................. 21
2.5 Measurement of antioxidant activity ................................................................................... 25
V
2.5.1 Radical scavenging assays ............................................................................................ 26
2.5.2 Antioxidant evaluation in a model system ................................................................... 33
2.6 Bioactivities ......................................................................................................................... 38
2.6.1 Antiviral ........................................................................................................................ 39
2.6.2 Anti-inflammatory ........................................................................................................ 43
2.6.3 Anticancer ..................................................................................................................... 46
2.6.4 Other activities .............................................................................................................. 48
CHAPTER 3. MATERIAL AND METHODS ......................................................................... 51
3.1 Materials .............................................................................................................................. 51
3.2 Methods ............................................................................................................................... 52
3.2.1 Synthesis of tyrosol and hydroxytyrosol fatty acid esters ............................................ 52
3.2.1.1 Extraction and purification of DHA ...................................................................... 52
3.2.1.2 Preliminary screening ............................................................................................ 53
3.2.1.3 General procedure for enzymatic esterification ..................................................... 54
3.2.1.4 Purification and identification of tyrosol and hydroxytyrosol esters ..................... 54
3.2.2 Antioxidant evaluation ................................................................................................. 55
3.2.2.1 DPPH radical scavenging activity ......................................................................... 55
3.2.2.2 Cupric ion-induced human low-density lipoprotein oxidation .............................. 56
3.2.2.3 DNA strand scission assay ..................................................................................... 57
3.2.3 Antiviral Activities ....................................................................................................... 58
3.2.3.1 HCV protease inhibitory activity ........................................................................... 58
3.2.3.2 Determination of inhibitory activity against alpha-glucosidase ............................ 59
3.2.4 Cell Culture and Cytotoxic Assay ................................................................................ 60
3.2.5 Determination of ROS generation in HepG2 Cells ...................................................... 60
3.2.6 Nitrite assay .................................................................................................................. 61
3.2.7 Bovine Serum Albumin (BSA)-Glucose Assay ........................................................... 62
3.3 Statistical method ................................................................................................................ 62
CHAPTER 4. RESULTS AND DISCUSSION ......................................................................... 63
VI
4.1 Synthesis of tyrosol and hydroxytyrosol fatty acid esters ................................................... 63
4.1.1 Enzyme screening ......................................................................................................... 63
4.1.2 Mole ratio effect ........................................................................................................... 66
4.1.3 Comparison of the enzymatic and chemical methods .................................................. 67
4.1.4 Syntheis of TY and HTY fatty acid esters .................................................................... 69
4.2 Antioxidant evaluation ........................................................................................................ 70
4.2.1 DPPH radical scavenging activity ................................................................................ 70
4.2.2 DNA strand scission assay ........................................................................................... 79
4.2.3 Cupric Ion-Induced Human LDL oxidation ................................................................. 88
4.3 Antiviral Properties ............................................................................................................. 96
4.3.1 HCV Protease Inhibition .............................................................................................. 96
4.3.2 α-Glucosidase inhibitory activity ................................................................................. 98
4.4 The Effect of Tyrosol, Hydroxytyrosol and their Esters on the Viability of HepG2 ........ 100
4.5 Protection on HepG2 against t-BuOOH induced oxidative stress .................................... 103
4.6 Inhibition of LPS-induced nitrite production by EPA and DHA esters of tyrosol and
hydroxytyrosol ........................................................................................................................ 107
4.7 Antiglycation effects by BSA-glucose assay .................................................................... 111
CHAPTER 5 .............................................................................................................................. 115
SUMMARY AND RECOMMENDATIONS .......................................................................... 115
REFERENCES .......................................................................................................................... 117

VII
LIST OF TABLES
Table 2-1. Bond dissociation energies (BDE) of substituted phenols………………………..….25
Table 2-2. Major methods used for measuring oxidation products………………………..…….35
Table 4-1. Effect of enzyme type on the synthesis of tyrosol DHA ester…………….…………64
Table 4-2. Effect of mole ratio of substrates on synthesis of tyrosol DHA ester…………..……67
Table 4-3. Reaction yields for some phenolic fatty acid esters………………………………….70
Table 4-4. DPPH Radical-Scavenging Activities of Tyrosl (TY) and Hydroxytyrosol (HTY)
Esters…………………………………………………………………………...……….76
Table 4-5. Inhibition of supercoiled DNA scission induced by peroxyl and hydroxyl radicals by
tyrosol, hydroxytyrosol and their fatty acid esters…………………….………………..85
Table 4-6. Inhibition against human LDL cholesterol oxidation by tyrosol, hydroxytyrosol and
their esters incubated at 37 °C for 15 h………………………………………...……….91
Table 4-7. Inhibitory effect (IC50) of TY, HTY and their derivatives against HCV protease…..98
Table 4-8. Inhibitory effect (IC50) of TY, HTY and their derivatives against a-glucosidase….101
Table 4-9. The effect on cell viability by (a) TY and its esters treatment; (b) HTY and its esters
treatment…………………………………………………...………………………….103

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LIST OF FIGURES

Figure 2-1. Chemical structures of tyrosol and hydroxytyrosol…………………………………..6
Figure 2-2. Synthesis of hydroxytyrosol alkyl esters by a three-step method…………….………7
Figure 2-3. Synthesis of hydroxytyrosol lipophilic fatty acid esters by a chemical method……...7
Figure 2-4. Synthesis of hydroxytyrosol lipophilic fatty acid esters by an enzymatic method…...8
Figure 2-5. Chemical structure of ALA, EPA and DHA…………………………………..…….15
Figure 2-6. Lipid autoxidation pathways…………………………………...……………………17
Figure 2-7. The possible pathways of secondary oxidation of oleic acid…….………………….18
Figure 2-8. The possible generation pathways of MDA……………………………………...….18
Figure 2-9. Cyclic compound formation from linoleic acid by Diels-Alder reaction at high
temperatures…………………………………………………………………………….20
Figure 2-10. Conjugative resonance stabilization of phenoxyl radical…………………………..24
Figure 2-11. The chemical structure of fluorescein…………………………………...…………29
Figure 2-12. The chemical structure of DPPH radical…………………………………………...29
Figure 4-1. High-performance liquid chromatography (HPLC) chromatograms of starting
materials (a, b) and resultants (c) in the synthesis of tyrosol DHA ester at 280 nm…...65
Figure 4-2. Chemical structures and mass spectrometric data of tyrosol DHA ester……………65
Figure 4-3. General structures of synthesized fatty acid esters.....................................................79
Figure 4-4. Signal intensity of DPPH (0.18 mM) and TY butyrate with DPPH (1 mM) as
observed by EPR..............................................................................................................71
Figure 4-5. DPPH scavenging capacity of saturated fatty acid esters in µmol trolox
equivalents/mmol……………………………………………………………………….77
IX
Figure 4-6. DPPH scavenging capacity of unsaturated fatty acid esters in µmol trolox
equivalents/mmol………………………………………………………………...……..78
Figure 4-7. (a) Agarose gel electrophoresis of inhibition of hydroxyl radical-induced DNA
scission by tyrosol and tyrosol esters (10 uM); (b) peroxyl radical-induced DNA
scission by tyrosol and tyrosol esters (10 uM)…………………………………………80
Figure 4-8. DNA retention (%) of saturated fatty acid esters in hydroxyl and peroxyl radical
induced oxidative scission…………………………………………………..………….86
Figure 4-9. DNA retention (%) of unsaturated fatty acid esters in hydroxyl and peroxyl radical
induced oxidative scission……………………………………………………………...87
Figure 4-10. The inhibition of saturated fatty acid esters against formation of conjugated dienes
at 234 nm against human LDL oxidation over a 15-hour period………………………94
Figure 4-11. The inhibition of unsaturated fatty acid esters against formation of conjugated
dienes at 234 nm against human LDL oxidation over a 15-hour period……….………96
Figure 4-12. Effect of TY, HTY and their esters on intracellular reactive oxygen species (ROS)
generation…………………………………………………………..…………………108
Figure 4-13. Effects of tyrosol and its derivatives compound on LPS-induced NO production in
RAW264.7 macrophages………………………………………...……………………111
Figure 4-14. Effects of hydroxytyrosol and its derivatives compound on LPS-induced NO
production in RAW264.7 macrophages……………………………...……………….112
Figure 4-15. Relative content of fluorescent AGEs of aminoguanidine (AG), tyrosol (TY) and
lipophilized TY derivatives…………………………………………..……………….114
Figure 4-16. Relative content of fluorescent AGEs of aminoguanidine (AG), hydroxytyrosol
(HTY) and lipophilized HTY derivatives……………………………………………..115
X