The soybean lipoxygenase-fluorescein reaction may be used to assess antioxidant capacity of phytochemicals and serum

Abstract

Evaluation of the putative beneficial effects of food antioxidants by in vitro assays of the antioxidant capacity (AC) of food extracts sometimes appears questionable. A more realistic evaluation of antioxidant effectiveness may derive from the integration of in vitro assays with ex vivo assays of blood/serum/plasma AC after food intake. The aim of this study was to develop the novel lipoxygenase-fluorescein (LOX-FL) method to assay the AC of both food extracts and serum/plasma. This method was applied on both extracts from the antioxidant-rich dietary cereal supplement, Lisosan G, and serum (seven subjects) for 240 min after the intake of 20 g of supplement. The widely used ORAC and TEAC methods were used for comparison. The new LOX-FL method is based on the reaction between the soybean LOX-1 isoenzyme and FL in the presence of linoleic acid, which undergoes hydroperoxidation, thus generating physiological reactive species, including LOO˙, LO˙, HO˙ and ¹O₂, which are able to quench FL. The quenching rate is slowed by antioxidants; this inhibition may be calibrated in terms of Trolox equivalents to assess the AC. Interestingly, the LOX-FL method discriminated the in vitro AC of four different Lisosan G extracts similarly to the ORAC and TEAC methods. In contrast, only the LOX-FL method was able to highlight a general increase in the serum AC (up to 40% after 30 min) after Lisosan G intake, thus confirming its physiological effectiveness by ex vivo serum assay. This quality of the LOX-FL method probably comes from the ability to highlight simultaneously different antioxidant mechanisms and to effectively show synergy among food phenols and endogenous serum antioxidants.