Identification of natural polyphenols and extracts acting as inhibitors of amyloid

Abstract

Identification of natural polyphenols and extracts acting as inhibitors of amyloid beta-derived cellular damage Alzheimer's disease (AD) is an incurable, degenerative and terminal disease that is generally prevalent in the elderly population. The number of individuals with AD is constantly increasing in parallel with life expectancy. Studies have shown that global prevalence of dementia would double every twenty years amounting to approximately 82 million cases by 2040. In Malta, the estimated figure for dementia of 4,072 affected individuals recorded in 2007 is expected to double by 2035 with 50-60% of cases being classified as AD. Despite its prevalence, an efficient treatment for the disease is still lacking. This research study forms part of a large-scale project; the first stage involved the use of a lipsomal model, the results and protocols of which were then applied to the next stage which was carried out by the author in part fulfillment of the Masters in Science in Pharmacology. The primary aim of this second stage of the research was to identify natural plant compounds that act as Inhibitors of amyloid-beta (Aβ) derived cellular damage. To be able to carry out this work it was necessary to establish suitable working protocols that offer technically sound and repeatable results. A number of variables were identified and found to influence the cytotoxicity assay protocol such as cell type, initial seeding density, cell passage number, establishment time of the cells prior to exposure to the test compound, serum,

DMSO and PBS concentrations, phenazine methosulfate concentration and XTT incubation time. These parameters were fine-tuned to meet the requirements for each cytotoxicity assay carried out. A total of seventeen polyphenols and four plant extracts were screened using the XTT assay to obtain cytotoxicity profiles for each. This data together with that obtained from the liposomal stage were used to narrow down the array of natural plant compounds to the four most efficient polyphenols and extracts - nordihydroguiaretic acid, baicalein, black tea and Padina extract. For this study the protein used was the Aβ42 which is one of the three pathological signatures of AD. It is produced by endoproteolysis of the parenteral amyloid precursor protein via the amyloidogenic cleavage pathway. Monomers of amyloid-beta are formed but they readily aggregate to form multimeric complexes. Research has shown that it is the small oligomeric forms (1 to 10 nm) that show greatest toxicity. Working with Aβ peptides was challenging due to their unpredictable aggregation nature. The aggregation protocol established during the lipsomal stage of the project was adopted. This however gave inconsistent cellular toxicity with the result that fresh rather than aggregated amyloid-beta was used since the former offered at least 30% toxicity at a concentration of 5 µM which was suitable to carry out analyses. The basis of this research incorporated two main themes, the natural plant products and the Aβ. These came together in the final stage of the project where the four selected polyphenols and extracts were each combined with the fresh Aβ and applied to the SH-SYSY cells to note any change in cell

viability. A significant increase in cell viability was obtained when using nordihydroguiaretic acid (55%), baicalein (21%) and Padina extract (35%). The results obtained therefore confirm the inhibitory activity of three of the natural plant products against Aβ42 derived cellular damage. These could serve as lead compounds in the next deliverable of the project with the final aim of obtaining new therapeutic agents for the treatment of amyloidogenic diseases. Alzheimer's Disease Amyloid Beta Plant natural products Cytotoxicity