Anthraquinones Inhibit Insulin Amyloidosis in Crowded Environments

Natural anthraquinones possess a wide range of biological activities, including antibacterial, antiviral, antitumor, and antioxidant effects. However, studies on their ability to inhibit amyloid protein aggregation remain relatively limited. In this study, we used insulin as a model protein to investigate the anti-amyloidogenic potential of several natural anthraquinones. Specifically, the inhibitory mechanisms of five anthraquinones (emodin, anthraflavin, aloe-emodin, alizarin, and purpurin) on insulin amyloid fibrillation were explored in both dilute and crowded environments (PEG 2000 and PEG 4000). Multidisciplinary analytical results demonstrated that all five anthraquinones could effectively inhibit insulin amyloid fibrillation in both dilute and crowded environments. Simultaneously, crowded agents themselves also exhibited inhibitory effects on insulin amyloid aggregation. However, the inhibitory efficacy of anthraquinones was weaker in crowded environments than in dilute solutions, indicating that although crowded agents themselves suppressed insulin aggregation, they may interfere with the regulatory roles of anthraquinones on insulin aggregation behavior. Interestingly, purpurin showed stronger inhibitory activity in crowded environments compared to dilute solutions. Furthermore, fluorescence spectral analysis suggested that the quenching mechanism of insulin by all these anthraquinones was identified as static quenching mode. Molecular simulation studies revealed that anthraquinones could bind to the aggregation-prone regions of insulin via hydrogen bonding and hydrophobic interactions, thereby inhibiting insulin amyloid aggregation. Notably, the inhibitory capacity of these compounds was correlated with their structural features and the binding affinities to insulin. Collectively, this study explored the anti-amyloid activity of anthraquinones, which held significant research value for the development of potential therapeutic agents for amyloid-associated proteinopathies.