A Micromethod for Polyphenol High-Throughput Screening Saves 90 Percent Reagents and Sample Volume

摘要

There is ample evidence that polyphenols are important natural substances with pronounced antioxidative properties. This study aimed to develop a fast and reliable method to determine total polyphenol content (TPC) in foodstuffs and human samples. The microtitration format offers the advantage of low sample volumes in the microlitre range, facilitating high-throughput screening with 40 samples simultaneously. We accordingly adjusted the so-called Folin-Ciocalteu method to a microtitre format (polyphenols microtitre-PPm) with 90% reduction of reagents. The assay was standardized with gallic acid in the range between 0.1 and 3 mM, using a 20 mu L sample volume. The intra-assay coefficient of variation (CV) was less than 5%, and inter-assay CV was in the range of 10%. Wavelength was measured at 766 nm after two hours of incubation. This micromethod correlates significantly with both the classical Folin-Ciocalteu method and High-Performance Thin-Layer Chromatography (HPTLC) (r(2) = 0.9829). We further observed a significant correlation between PPm and total antioxidants (r(2) = 0.918). The highest polyphenol concentrations were obtained for red, blue, and black fruits, vegetables, and juices. Extracts of red grapes could be harvested almost sugar free and might serve as a basis for polyphenol supplementation. Beer, flour, and bread contained polyphenol concentrations sufficient to meet the minimal daily requirement. We conclude that PPm is a sensitive and reliable method that detects polyphenols even in samples diluted 10-fold. The literature strongly recommends further investigations on the effects of polyphenol uptake on human and animal health.