Discussion
These observations suggest that aromatic small molecules of CE may directly insert into amyloidogenic core of early aggregates and inhibit amyloid fibril formation by disrupting the pi-pi interactions.
Methods
Acidic pH and high temperatures were used to drive the protein towards amyloid formation. Lysozyme was dissolved at 2 mg/mL in 50mM glycine buffer (pH 2.5), and then incubated at 57 °C for the specified durations while stirred gently by Teflon magnetic bars. Various techniques including thioflavin T, fluorescence, Congo red absorbance assay and AFM micrography were used to characterize the HEWL fibrillation processes.
Results
In the absence of CE typical amyloid fibrils (like amyloids formed in Alzheimer disease) became evident after 48 h of incubation. Upon incubation with various extract concentrations in the range of 0.1-1 mg/ml, formation of fibrillar assemblies were significantly inhibited (P<0.05). AFM analysis and MTT assay also confirmed the role of the extract in amyloid inhibition. Our studies showed that the presence of CE did not have any effect on protein stabilization and thus directly interact with amyloid structure and inhibit formation of these structures. Furthermore, a docking experiment showed that a pi-pi interaction may occur between the aromatic component of cinnamaldehyde and W62. Interestingly, W62 is one of the principal aromatic residues that interact with glycine amide, which is an aggregation suppressor of HEWL.