pharmacology of olive biophenols in alzheimer's disease prevention and treatment

242 Pages·2015·3.34 MB·English

PHARMACOLOGY OF OLIVE BIOPHENOLS
IN ALZHEIMER’S DISEASE PREVENTION
AND TREATMENT

SYED HARIS OMAR
B.Pharm & M.Pharm (Pharmacology)

A thesis submitted for Charles Sturt University for the degree of
Doctor of Philosophy
MARCH 2015

School of Biomedical Sciences
Faculty of Science
Charles Sturt University
Wagga Wagga, NSW, Australia

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TABLE OF CONTENTS

LIST OF TABLES ................................................................................................................... viii
LIST OF FIGURES ................................................................................................................. viii
CERTIFICATE OF AUTHORSHIP ........................................................................................ x
ACKNOWLEDGMENTS ......................................................................................................... xi
ETHICS, BIOSAFETY AND/OR RADIATION SAFETY APPROVAL ............................ xii
PUBLICATIONS ..................................................................................................................... xiii
ABSTRACT .............................................................................................................................. xiv
LIST OF ABBREVIATIONS ................................................................................................. xvi
CHAPTER 1 ................................................................................................................................ 1
PLANT BIOPHENOLS AND ALZHEIMER’S DISEASE: TOWARDS A HOLISTIC
APPROACH ................................................................................................................................ 1
1.1. ABSTRACT ....................................................................................................................... 2
1.2. INTRODUCTION ............................................................................................................. 3
1.3. BIOPHENOLS ................................................................................................................... 5
1.4. ALZHEIMER’S DISEASE ................................................................................................ 5
1.5. PATHOPHYSIOLOGY OF AD ........................................................................................ 6
1.5.1. The Amyloid Cascade and Modified Amyloid Hypothesis .......................................... 6
1.5.2. Cholinergic Hypothesis ............................................................................................... 8
1.5.3. Mitochondrial Cascade Hypothesis ............................................................................ 9
1.5.4. Oxidative Stress Hypothesis ...................................................................................... 10
1.5.5. Other Hypotheses ...................................................................................................... 11
1.6. PLANTS FOR FIGHTING AD ....................................................................................... 13
1.7. PHARMACOLOGY OF BIOPHENOLS IN AD ............................................................ 16
1.8. CELLULAR AND MOLECULAR LEVEL: BIOCHEMICAL EFFECTS .................... 19
1.8.1. Antioxidant activity ................................................................................................... 19
1.8.1.1. Evidence from in vitro studies ........................................................................... 20
1.8.1.2. Evidence from animal studies ............................................................................ 22
1.8.1.3. Evidence from human studies ............................................................................ 23
1.8.1.4. Assessment of the evidence ............................................................................... 24
1.8.2. Neuroprotective activity ............................................................................................ 26
1.8.2.1. Evidence from in vitro studies ........................................................................... 27
1.8.2.2. Evidence from animal studies ............................................................................ 27
1.8.2.3. Evidence from human studies ............................................................................ 28
1.8.2.4. Assessment of the evidence ............................................................................... 28
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1.8.3. Cholinesterase inhibition activity ............................................................................. 29
1.8.3.1. Evidence from in vitro studies ........................................................................... 29
1.8.3.2. Evidence from animal studies ............................................................................ 30
1.8.3.3. Evidence from human studies ............................................................................ 31
1.8.3.4. Assessment of the evidence ............................................................................... 31
1.8.4. NMDA receptor antagonistic activity ....................................................................... 31
1.8.5. Mitochondrial protective activity .............................................................................. 32
1.8.6. Epigenetic activity ..................................................................................................... 33
1.8.7. Anti-prion activity ..................................................................................................... 34
1.9. TISSUE LEVEL: EFFECTS ON HISTOPATHOLOGY ................................................ 34
1.9.1. Anti-amyloidogenic activity ...................................................................................... 35
1.9.1.1. Evidence from in vitro studies ........................................................................... 35
1.9.1.2. Evidence from animal studies ............................................................................ 39
1.9.1.3. Evidence from human studies ............................................................................ 41
1.9.1.4. Assessment of the evidence ............................................................................... 42
1.9.2. Anti-tau aggregation activity .................................................................................... 42
1.9.2.1. Evidence from in vitro studies ........................................................................... 43
1.9.2.2. Evidence from animal studies ............................................................................ 45
1.9.2.3. Assessment of the evidence ............................................................................... 45
1.10. FUNCTIONAL LEVEL: EFFECTS ON COGNITION AND BEHAVIOUR .............. 46
1.10.1. Evidence from in vitro studies ................................................................................. 46
1.10.2. Evidence from epidemiological studies ................................................................... 46
1.10.3. Evidence from animal studies ................................................................................. 47
1.10.4. Evidence from Clinical studies ............................................................................... 49
1.10.5. Assessment of the evidence ..................................................................................... 51
1.11. CONCLUSIONS ............................................................................................................ 53
CHAPTER 2 .............................................................................................................................. 57
ANTI-ALZHEIMER’S DISEASE ACTIVITIES OF OLIVE BIOPHENOLS: THE
KNOWN & THE UNKNOWN (TOWARDS A RATIONALE FOR THESE STUDIES) . 57
2.1. Mediterranean Diet: where it all began ............................................................................ 58
2.2. Olive Biophenols: the chemistry ...................................................................................... 58
2.3. Olive biophenols: the antioxidant activity ....................................................................... 59
2.4. Olive biophenols and AD ................................................................................................. 60
2.5. The aim & objectives of the current study ....................................................................... 62
2.5.1. To characterize some commercially available olive biophenol extracts and to ....... 62
compare their phenolic composition. .................................................................................. 62
2.5.2. To evaluate the in vitro antioxidant activity of olive biophenols at the .................... 62
molecular level and compare individual biophenols with phenolic extracts. ..................... 62
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2.5.3. To evaluate the in vitro inhibitory activity of olive biophenols against a ................. 62
number of enzymes involved in AD and compare individual biophenols with .................... 62
phenolic extracts. ................................................................................................................ 62
2.5.4. To evaluate the in vitro anti-amyloid activities of olive biophenols and .................. 62
extracts. ............................................................................................................................... 62
2.5.5. To evaluate the neuroprotective activities of olive biophenols and extracts at ........ 62
the cellular level against various neurotoxic substances. ................................................... 62
2.5.6. To evaluate the capability of olive biophenol extract to exert anti-AD activities ..... 62
in vivo using a genetically-modified mouse model of AD. .................................................. 62
CHAPTER 3 .............................................................................................................................. 63
PHENOLIC COMPOSITION OF OLIVE BIOPHENOL EXTRACTS AND IN VITRO
ANTIOXIDANT ACTIVITY IN COMPARISON WITH INDIVIDUAL OLIVE
BIOPHENOLS .......................................................................................................................... 63
3.1. Abstract ............................................................................................................................ 64
3.2. Introduction ...................................................................................................................... 65
3.3. Materials and Methods ..................................................................................................... 67
3.3.1. Chemicals and plant extracts .................................................................................... 67
3.3.2. Sample preparation ................................................................................................... 67
3.3.3. Determination of total phenol content in commercial extracts ................................. 68
3.3.4. Determination of total flavonoid content in commercial extracts ............................. 69
3.3.5. HPLC-DAD-online ABTS radical scavenging analysis ............................................ 69
3.3.6. Liquid Chromatography-Mass spectrometry (LC-MS) ............................................. 70
3.3.7. Superoxide radical (SOR) scavenging assay ............................................................ 71
3.3.8. In vitro H O scavenging assay ................................................................................. 71
2 2
3.3.9. Ferric reducing/antioxidant power (FRAP) assay .................................................... 72
3.3.10. Statistical analysis .................................................................................................. 72
3.4. Results and discussion ..................................................................................................... 73
3.4.1. Total phenol and total flavonoid contents ................................................................. 73
3.4.2. HPLC-DAD-ABTS analysis and LC-MS ................................................................... 74
3.4.3. SOR scavenging activities ......................................................................................... 80
3.4.4. H O radical scavenging activities by olive biophenols ........................................... 84
2 2
3.4.5. Iron (III) reducing power in FRAP ........................................................................... 85
3.5. Conclusion ....................................................................................................................... 87
CHAPTER 4 .............................................................................................................................. 89
MODULATION OF SOME ENZYMES INVOLVED IN ALZHEIMER’S DISEASE BY
OLIVE BIOPHENOLS ............................................................................................................ 89
4.1. Abstract ............................................................................................................................ 90
4.2. Introduction ...................................................................................................................... 91
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4.3. Materials and methods ..................................................................................................... 94
4.3.1. Beta-secretase (BACE-1) inhibition assay ................................................................ 94
4.3.2. Acetylcholinesterase and butyrylcholinesterase inhibition assay ............................. 95
4.3.3. Determination of tyrosinase enzyme kinetic parameters .......................................... 96
4.3.4. Tyrosinase inhibition assay ....................................................................................... 96
4.3.5. Histone deacetylase (HDAC) inhibition assay .......................................................... 97
4.3.6. Statistical analysis .................................................................................................... 97
4.4. Results and discussion ..................................................................................................... 98
4.4.1. BACE1 enzyme inhibition by olive biophenols ......................................................... 98
4.4.2. AChE and BChE inhibition by olive biophenols ..................................................... 100
4.4.3. Tyrosinase and L-DOPA Kinetics ........................................................................... 105
4.4.4. Tyrosinase inhibition assay ..................................................................................... 106
4.4.5. HDAC inhibitions by olive biophenols .................................................................... 108
4.5. Conclusion ..................................................................................................................... 110
CHAPTER 5 ............................................................................................................................ 114
ANTI-AMYLOID PROPERTIES OF OLIVE BIOPHENOLS ......................................... 114
5.1. Abstract .......................................................................................................................... 115
5.2. Introduction .................................................................................................................... 116
5.3. Material and methods ..................................................................................................... 118
5.3.1 Aβ fibril preparation.............................................................................................. 119
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5.3.2. Acetylcholinesterase and butyrylcholinesterase induced Aβ fibril preparation ... 119
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5.3.3. Aβ fibril formation and aggregation inhibitory assay .......................................... 119
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5.3.3.1 Transmission electron microscope (TEM) imaging .......................................... 120
5.3.3.2. Thioflavin-T (ThT) fluorometric assay ............................................................ 120
5.3.3.3. Congo red binding assay .................................................................................. 121
5.3.3.4. HPLC analysis of Aβ with or without olive biophenols ................................ 121
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5.3.4. In situ copper and amyloid binding assay............................................................... 122
5.3.5. Statistical analysis .................................................................................................. 122
5.4. Results and discussion ................................................................................................... 122
5.4.1. The effect of olive biophenols on Aβ aggregation (TEM) ..................................... 122
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5.4.2. ThT fluorometric assay of Aβ fibril inhibition by olive biophenols ...................... 124
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5.4.3. Congo red assay of Aβ inhibition by olive biophenols ......................................... 126
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5.4.4 Effect of olive biophenols on Aβ fibril formation (HPLC analysis) ....................... 127
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5.4.5. Copper-chelation activity with Aβ ........................................................................ 135
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5.5. Conclusion ..................................................................................................................... 136
CHAPTER 6 ............................................................................................................................ 139
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NEUROPROTECTIVE EFFECTS OF OLIVE BIOPHENOLS AGAINST SOME
NEUROTOXIC SUBSTANCES ............................................................................................ 139
6.1. Abstract .......................................................................................................................... 140
6.2. Introduction .................................................................................................................... 141
6.3. Material and methods ..................................................................................................... 142
6.3.1. Aβ fibril preparation............................................................................................. 143
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6.3.2. Cell culture .............................................................................................................. 143
6.3.3. H O -induced toxicity in SH-SY5Y cells and neuroprotective potential ................. 144
2 2
6.3.4. Aβ induced SH-SY5Y cell toxicity and olive biophenols treatment ...................... 144
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6.3.5. L-DOPA-induced SH-SY5Y toxicity and protection by olive biophenols ................ 144
6.3.6. Copper induced SH-SY5Y toxicity and protection by olive biophenol .................... 145
6.3.7. Cell viability assay .................................................................................................. 146
6.4. Results and discussion ................................................................................................... 146
6.4.1. Neuropreventive effect against H O -induced oxidative stress ............................... 146
2 2
6.4.2. Aβ induced SH-SY5Y toxicity and protection by olive biophenols ....................... 148
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6.4.3. L-DOPA toxicity and SH-SY5Y protection by olive biophenols .............................. 150
6.4.4. Copper induced SH-SY5Y cells toxicity and protection by olive biophenols .......... 153
6.5. Conclusion ..................................................................................................................... 155
CHAPTER 7 ............................................................................................................................ 160
ANTI-ALZHEIMER’S ACTIVITY OF OLIVE BIOPHENOLS IN VIVO ..................... 160
7.1. Abstract .......................................................................................................................... 161
7.2. Introduction .................................................................................................................... 162
7.3. Materials and methods ................................................................................................... 163
7.3.1. Materials ................................................................................................................. 163
7.3.2. Animals ................................................................................................................... 164
7.3.3. Diet .......................................................................................................................... 164
7.3.4. Behavioral analysis ................................................................................................. 164
7.3.4.1. Novel Objection Recognition in mice .............................................................. 165
7.3.4.2. The light and dark test ...................................................................................... 167
7.3.4.3. Barnes maze test............................................................................................... 167
7.3.5. Histology ................................................................................................................. 169
7.3.5.1. Amyloid plaque burden counting ..................................................................... 170
7.3.6. Blood sampling and plasma collection ................................................................... 171
7.3.6.1. Biochemical analysis ........................................................................................ 171
a. Plasma cholesterol determination ......................................................................... 171
b. Plasma triglyceride determination ........................................................................ 171
c. Plasma glucose determination ............................................................................... 172
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7.3.7. Statistical analysis .................................................................................................. 172
7.4. Results and discussion ................................................................................................... 172
7.4.1. Novel object recognition ......................................................................................... 173
7.4.2. Light and dark test .................................................................................................. 173
7.4.3. Barnes test ............................................................................................................... 174
7.4.4. Amyloid plaque burden ........................................................................................... 175
7.4.5. Biochemical analysis............................................................................................... 176
7.4.5.1. Serum cholesterol level .................................................................................... 176
7.4.5.2. Plasma triglyceride level .................................................................................. 177
7.4.5.3. Plasma glucose level ........................................................................................ 177
7.5. Conclusion ..................................................................................................................... 178
CHAPTER 8 ............................................................................................................................ 181
GENERAL CONCLUSIONS AND FUTURE DIRECTIONS ........................................... 181
8.1. Concluding remarks ....................................................................................................... 182
REFERENCES ........................................................................................................................ 187

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LIST OF TABLES
1.1. Plants with outstanding anti-AD and neuroprotective potential………………………14
3.1. Major peaks identified in olive extracts……………………………………………….79
3.2. SOR scavenging, H O scavenging and FRAP value of olive biophenols………….....86
2 2
7.1. Novel objection recognition task in mice…………………………………………….165
7.2. Barnes maze task in mice…………………………………………………………….168
7.3. Staining protocol of brain sample……………………………………………………170

LIST OF FIGURES
1.1. Schematic diagram illustrating amyloid pathology……………………..………...…7
1.2. Schematic diagram illustrating mitochondrial hypothesis…………………..……...10
1.3. Schematic diagram illustrating oxidative stress hypothesis…………………..…….11
1.4. Chemical structures of important biophenols with anti-AD activities…………..….17
1.5. A holistic approach is required to evaluate the anti-AD properties…………..……19
1.6. Tau pathology and the role of biophenols…………………………………….……43
2.1. Chemical structure of the major olive biophenols………………………….……...61
3.1. Chemical structure of non-flavonoid and flavonoid olive biophenols…………….68
3.2. Calibration curve of gallic acid and quercetin……………………………………73
3.3. HPLC-DAD-ABTS chromatograms of olive biophenols…………………….........78
3.4. SOR scavenging activities of non-flavonoids olive biophenols……………………81
3.5. SOR scavenging activities of flavonoids olive biophenols………….…...………...82
3.6. SOR scavenging activity of olive extracts…………………………….….………..83
3.7. Standard curve for ferric reducing antioxidant power assay and non-flavonoids,
flavonoids, and extracts………………………………………………….….…....86
4.1. BACE1 inhibition by olive biophenols………………………………….….…….99
4.2. Dose dependent inhibition of AChE and BChE by galantamine..…….……..….101
4.3. Dose dependent inhibition of AChE by olive biophenols…………….……..…..102
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4.4. BChE inhibition by olive biophenols………………………………….……..…104
4.5. Michaelis-Menten curve and Lineweaver-Burk plot for L-DOPA…………...105
4.6. Tyrosinase inhibition by kojic acid and quercetin……………………………106
4.7. Tyrosinase inhibition by equimolar flavonoids mixture………………..…….107
4.8. Standard curve of HDAC standard…………………...……………...............108
4.9. HDAC inhibition by olive biophenols…………………………..……………109
5.1. Aβ fibril formation and inhibition………………………………………….123
42
5.2. Thioflavin T assay…………………………………………………………...125
5.3. Congo red assay………………………………………………………….…127
5.4. HPLC-Chromatograms of Aβ and non-flavonoid olive biophenols.………130
42
5.5. HPLC-Chromatograms of Aβ and flavonoid olive biophenols……………132
42
5.6. HPLC-Chromatograms of Aβ with extract olive biophenols………...........134
42
5.7. Copper chelating activity of Aβ ………………….......................................136
42
6.1. H O induced SH-SY5Y cell toxicity………………………………………..147
2 2
6.2. SH-SY5Y cells morphology with or without Aβ …………………………..148
42
6.3. Aβ induced SH-SY5Y toxicity and protection by olive biophenols……….149
42
6.4. L-DOPA induced SH-SY5Y toxicity………………………………………..150
6.5. Olive biophenols and L-DOPA induced SH-SY5Y toxicity………………...151
6.6. Copper induce SH-SY5Y cells toxicity……………………………………..154
7.1. Novel object recognition task………………………………………….......173
7.2. Light and dark test…………………………………………………………174
7.3. Barnes maze test…………………………………………….......................175
7.4. Amyloid plaque burden and olive biophenol protection…………………..175
7.5. Cholesterol level in APP and wild mice………………………………..176
swe
7.6. Triglyceride level in APP and wild mice……………………………….177
swe
7.7. Glucose level in APP and wild mice……………………………………178
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CERTIFICATE OF AUTHORSHIP

“I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material previously published or written by another
person nor material which to a substantial extent has been accepted for the award of any
other degree or diploma at Charles Sturt University or any other educational institution,
except where due acknowledgment is made in the thesis. Any contribution made to the
research by colleagues with whom I have worked at Charles Sturt University or elsewhere
during my candidature is fully acknowledged.

I agree that this thesis be accessible for the purpose of study and research in accordance
with the normal conditions established by the Executive Director, Library Services or
nominee, for the care, loan and reproduction of theses.”

SYED HARIS OMAR

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