A novel high-throughput image based rapid Folin-Ciocalteau assay for assessment of reducing capacity

Abstract

The aim of the presented work was to develop a novel high-throughput rapid Folin-Ciocalteau assay for the quantification of reducing capacity based on image scanner (Image-F-C assay). The original rapid F-C assay using a 96-well plate was improved by adding a neutralization step that stabilizes the formed color, enabling image acquisition using a flatbed scanner. The effects of reaction volume, scanning orientation and model of flatbed scanner were assessed based on linearity, sensitivity and reproducibility of gallic acid standard curve. Euclidean distance calculated from R (Red), G (Green) and B (Blue) values was chosen, based on linearity and sensitivity, in order to quantify the reducing capacity. An in-house program using free ImageJ macro language was written to calculate automatically the RGB values of each well. The Image-F-C assay is linear within the range of 0-20 mg L-1 of gallic acid (R2 ≥ 0.9939). The reducing capacity of herbal infusions using the new method was: tymus (251 ± 11), digestive (166 ± 9), sen (88 ± 7), chamomile (54 ± 3), green tea (615 ± 12), lemon black tea (143 ± 4), coffee (576 ± 20), and fruit juices Biofrutas mediterraneo (939 ± 35), pineapple juice (520 ± 27) and apple juice (226 ± 17) and an inter-day relative standard error < 8% was observed. Bland-Altman and correlation analyses showed that there were no significant differences between the reducing capacity values measured by the new Image-F-C and the original rapid F-C assay.