Background: Identifying agents that inhibit amyloid beta peptide (A beta) aggregation is the ultimate goal for slowing Alzheimer's disease (AD) progression. This study investigated whether the glycoside asiaticoside inhibits A beta(1-42) fibrillation in vitro. Methods: Fluorescence correlation spectroscopy (FCS), evaluating the Brownian diffusion times of moving particles in a small confocal volume at the single-molecule level, was used. If asiaticoside inhibits early A beta(1-42) fibrillation steps, more A beta s would remain free and rapidly diffuse in the confocal volume. In contrast, "weaker or no inhibition" permits a greater number of A beta s to polymerize into oligomers, leading to fibers and gives rise to slow diffusion times in the solution. Trace amounts of 5-carboxytetramethylrhodamine (TAMRA)-labeled A beta(1-42) in the presence of excess unlabeled A beta(1-42) (10 mu M) was used as a fluorescent probe. Steady-state and kinetic-Thioflavin T (ThT) fluorospectroscopy, laser-scanning fluorescence microscopy (LSM), and transmission electron microscopy (TEM) were also used to monitor fibrillation. Binding of asiaticoside with A beta(1-42) at the atomic level was computationally examined using the Molegro Virtual Docker and PatchDock. Results: With 1 h of incubation time for aggregation, FCS data analysis revealed that the diffusion time of TAMRA-A beta(1-42) was 208 +/- 4 mu s, which decreased to 164 +/- 8.0 mu s in the presence of asiaticoside, clearly indicating that asiaticoside inhibited the early stages A beta(1-42) of fibrillation, leaving more free A beta s in the solution and permitting their rapid diffusion in the confocal volume. The inhibitory effects were also evidenced by reduced fiber formation as assessed by steady-state and kinetic ThT fluorospectroscopy, LSM, and TEM. Asiaticoside elongated the lag phase of A beta(1-42) fibrillation, indicating the formation of smaller amyloid species were impaired in the presence of asiaticoside. Molecular docking revealed that asiaticoside binds with amyloid intra-and inter-molecular amino acid residues, which are responsible for beta-sheet formation and longitudinal extension of fibrils. Conclusion: Finally, asiaticoside prevents amyloidogenesis that precedes neurodegeneration in patients with Alzheimer's disease.