Antioxidants and Human Health: Validation of a leaf disc assay to assess antioxidants status and total phenolic content in edible leafy vegetables

Abstract

Abstract Background: Dietary antioxidants - especially in leafy vegetables – have consistently been linked to reducing the risk of developing chronic diseases. However, human intervention studies have often failed to prove these benefits. Despite this, there is interest in increasing the consumption of plant-based diets. Researchers have tended to measure and manipulate antioxidant levels and phenolic content in fresh and frozen leafy vegetables to promote health. However, appropriate assessment of plant antioxidant status is needed. Objective: To validate a new 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) leaf disc assay for measuring and comparing antioxidant status and phenolic content across different edible leafy vegetables to help studying food quality and gives a meaningful comparison among foods to improve antioxidant intakes in plant based diets by using non-destructive method. Additionally, to determine the effect of defrosting on plant antioxidant status by performing a pilot study, using ABTS leaf disc assay. Review method: Six different plants; baby red-leaf lettuce, basil, rocket, red chard, spinach and basil were included. ABTS reagent was used to determine antioxidant status for both fresh and frozen leaf discs (expressed as %of scavenging activity). While Folin-Ciocalteu colorimetric assay was used to estimate the phenolic content (expressed as mg gallic acid equivalents per g fresh weight of leaf disc). Result: TAA ranged from 2.04% to 8.84±0.53% which was the highest in baby red-leaf lettuce and the lowest in basil. While phenolic content varied from 0.03±0.00 to 0.46±0.001mg GAE/g fresh weight of leaf discs which was the highest also in baby red-leaf lettuce and lowest in red chard, while the TPC was not detectable in basil. A positive correlation was observed between TAA and TPC (r2=0.64). 24 hours of leaf disc freezing had no deleterious effect on TAA or TPC compared to fresh leaf disc, instead it had significantly higher TAA and TPC than fresh spinach (p<0.001). Conclusion: ABTS leaf disc assay appears to be a promising, rapid, simple and non-destructive method to measure and compare TAA and TPC for use in studying plant health, food quality, and stress tolerance. More experiments should be performed with differing antioxidant levels, or with plants grown under stress - as this would more fully test the durability of this assay for use in the assessment of plant food quality.