Development of a Method Suitable for High-Throughput Screening to Measure Antioxidant Activity in a Linoleic Acid Emulsion

Abstract

An improved system for measuring antioxidant activity via thiobarbituric acid reactive substances and ferric thiocyanate assays is reported, on the basis of oxidation of a linoleic acid (LA) emulsion. Oxidation times were reduced from 20 h to 5 h by increasing the reaction temperature from 37 °C to 50 °C and with an acceptable precision of <10% coefficient of variation (CV). Antioxidants varying in polarity and chemical class—250 µM Trolox, quercetin, ascorbic acid and gallic acid—were used for method optimisation. Further reductions in reaction time were investigated through the addition of catalysts, oxygen initiators or increasing temperature to 60 °C; however, antioxidant activity varied from that established at 37 °C and 20 h reaction time—the method validation conditions. Further validation of the method was achieved with catechin, epicatechin, caffeic acid and α-tocopherol, with results at 50 °C and 5 h comparable to those at 37 °C and 20 h. The improved assay has the potential to rapidly screen antioxidants of various polarities, thus making it useful in studies where large numbers of plant extracts require testing. Furthermore, as this assay involves protection of a lipid, the assay is likely to provide complementary information to well-established tests, such as the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.

Keywords

• thiobarbituric acid reactive substances
• ferric thiocyanate
• high-throughput screening
• bioactivity
• plant extracts
• method validation
• lipid oxidation
• peroxidation