The apparent health benefits of a Mediterranean diet have been attributed to the consumption of unprocessed extra virgin olive oil (EVOO), which contains a high content of polyphenols having antioxidant and anti-inflammatory properties. Phenolic compounds from a range of dietary sources have also been found to reduce the rate of protein self-assembly into amyloid fibrils associated with Alzheimer’s disease (AD) and other protein misfolding disorders.
Amyloid fibrils are nanoscale fibrous structures formed by the self-assembly of certain proteins into repeating arrays of β-strands stabilized by intermolecular hydrogen bonds. Amyloid is associated with over 30 human diseases and can occur systemically or in localized areas such as around the brain as observed in Alzheimer’s disease. AD is associated with the assembly of amyloid-β (Aβ) peptides into β-sheet rich amyloid fibrils and the aggregation of microtubule-associated protein tau into neurofibrillary tangles in the brain. There is a clinical need to find a cure for the disease, or a treatment option which improves the quality of life for patients with Alzheimer’s disease. The phenolic compounds oleuropein and oleocanthal from extra virgin olive oil (EVOO) have previously been shown to individually reduce the accumulation of amyloid fibrils associated with Alzheimer’s disease (AD), either by inhibiting amyloid formation (oleuropein) or by promoting amyloid clearance (oleocanthal). EVOO contains many other compounds, but as to date, no such evaluation of EVOO phenol mixtures when isolated from the fatty acid component of olive oil has been reported. This thesis aims to examine mixed and individual polyphenols isolated from olive oil and explore their ability to modulate amyloid formation by Aβ and tau.
Chapter 1 focuses on the current literature around natural phenolic compounds derived from EVOO. Also, this chapter discusses the literature around amyloid diseases with a particular focus on Alzheimer’s disease including its pathologies and overview of pharmacological and non-pharmacological treatment approaches of AD. Chapter 2 discusses the main techniques used for analysis and characterisation of polyphenols found in natural products. Also, this chapter illustrates techniques used for investigation and analysis of amyloid β (proteins). Chapter 3 shows preparation and implementation of a methodology for the expression, purification and characterisation of labelled (15N) and unlabelled, amyloid proteins. In Chapter 4, different methods of extraction of phenols from EVOO are evaluated to optimise the yield and range of phenols obtained in the extracted mixture. Mixtures are extracted from Greek and Saudi Arabian EVOO and analysed in detail using chromatographic and magnetic resonance methods. Over 30 different compounds are identified, several of which are quantified and shown to be present in different concentrations in the two EVOO extracts. Chapter 5 describes a range of methods that are used to test the effects of the phenolic mixtures in vitro on the aggregation of the 40-amino acid Aβ40 peptide and a repeat-domain fragment of tau. Thioflavin T fluorescence and circular dichroism measurements show that the Greek extract reduces the rate of tau aggregation only at very high phenolic concentrations (> 100 µg/mL). By comparison, Greek and Saudi extracts exert similar effects on Aβ40 aggregation at much lower concentrations (< 20 µg/mL). Transmission electron microscopy (TEM) indicates that the extracts reduce fibril deposition of Aβ40 after the end-point of aggregation is reached. An HPLC procedure combined with TEM, dynamic light scattering, and solid-state NMR reveals that most compounds in the extracts bind to pre-formed Aβ40 fibrils, which generates soluble Aβ oligomers that are mildly toxic to SH-SY5Y cells. Much higher (500 µg/mL) extract concentrations are required to remodel tau filaments into oligomers and no binding of phenolic compounds to the pre-formed filaments is observed. It is concluded that the entire phenolic profiles of different EVOO samples are similarly capable of modulating Aβ40 aggregation and fibril morphology in vitro at relatively low concentrations but are much less efficient at modulating tau aggregation.
Chapter 6 describes the synthesis and characterization of solid lipid nanoparticles (SLNs) with the aim of enhancing the in vivo delivery of polyphenol mixtures in the treatment of amyloid diseases and other pathologies. The method was used to successfully prepare SLNs having a homogeneous size distribution and a high efficiency of polyphenol encapsulation from the mixtures prepared in Chapter 4. Animal experiments are being investigated in future work to increase absorption, bioavailability, and circulation times of EVOO polyphenols in vivo.
It is estimated that around 3 M tonnes of olive oil are consumed worldwide annually. The results described in this thesis have increased understanding of the potentially neuroprotective benefits of EVOO consumed in the Mediterranean diet and laid the foundations for future in vivo applications of EVOO polyphenol mixtures for disease testing.