Evaluation of the effects of amyloid ß aggregation from seaweed extracts by a microliter-scale high-throughput screening system with a quantum dot nanoprobe.

Inhibitors of amyloid ß (Aß) aggregation have the potential to serve as lead compounds for anti-Alzheimer's disease (AD) agents because Aß aggregation is a key step in AD pathogenesis. Recently, we developed a novel microliter-scale high-throughput screening (MSHTS) system for Aß aggregation inhibitors that applied fluorescence microscopic analysis with quantum dot nanoprobes, and attempted to comprehensively screen the inhibitors from spices using this system (Ishigaki et al., PLoS One, 8, e72992, 2013). In this study, we tried to evaluate the inhibitory activities of 11 seaweed extracts on Aß aggregation using the MSHTS system. The half-maximal effective concentration (EC50) of the ethanolic extracts from all seaweeds exceeded 4.9 mg/ml, indicating that the extracts inhibit Aß aggregation although this activity was significantly lower than that displayed by members of the Lamiaceae, a family of herbal spices that showed highest activity among 52 spices tested in our 2013 study. On the other hand, the EC50 of boiling water extracts was 0.013-0.42 mg/ml which was comparable with the EC50 of the extracts from the Lamiaceae family. These results suggest that the extraction efficiency of the inhibitors by boiling water extraction was higher than that by ethanolic extraction. Moreover, analysis of fluorescence micrographs, which were obtained from the MSHTS system, revealed that the morphology of the Aß aggregates coincubated with boiling water extracts differed from control aggregates, suggesting that the MSHTS system is also useful for screening substances that affect the morphology of aggregates.

A microliter-scale high-throughput screening system with quantum-dot nanoprobes for amyloid-ß aggregation inhibitors.

The aggregation of amyloid ß protein (Aß) is a key step in the pathogenesis of Alzheimer's disease (AD), and therefore inhibitory substances for Aß aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aß aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50). We attempted to comprehensively screen Aß aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aß aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Saturejahortensis, summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aß aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology.