Improved superoxide-generating system suitable for the assessment of the superoxide-scavenging ability of aqueous extracts of food constituents using ultraweak chemiluminescence

In the interest of developing a simple and rapid ultraweak chemiluminescence assay for assessing the superoxide (O₂^-)-scavenging activities of various aqueous extracts of food constituents, a specific and stable O₂^--generating system was sought. Reported herein is the obtainment for the first time of a specific and stable O₂^--generating system consisting of methylglyoxal (MG), a reactive 2-oxo aldehyde and arginine, which has been shown to produce much steadier lucigenin-based chemiluminesence (LBCL) than the conventional xanthine/xanthine oxidase system running in parallel and monitoring by an ultraweak chemiluminescence analyzer. Upon mixing of MG and arginine in a phosphate-buffered saline solution, pH 7.4, steady, time-dependent increments of LBCL can be visually observed. The plateau of LBCL can be reached in approximately 10 min and retained in a steadily stable state thereafter without fluctuation for the next 15 min. The lucigenin-based LBCL generation was shown to be specific since it could be effectively inhibited by active bovine SOD, but not by heat-inactivated enzyme or catalase. Conversely, the xanthine/xanthine oxidase system can merely produce a LBCL peak rapidly but decay instantaneously. To illustrate the application of the proposed method for assessing the O₂^--scavenging ability of various food extracts, namely, Prunus mume (A), Lilum lancifolium (B), Creataegus pinnatifida (C), Tremella fuciformis (D), Fortunella margarita (E), and Scutellaria baicalensis (F), we used the following protocol: 12 min after monitoring of LBCL, 1 mg/mL of each of the test compounds was added to the assay system and various degrees of sudden drop of LBCL values were observed, indicating differences in O₂^--scavenging abilities exerted by these food extracts that can be visually compared. Consequently, the percentages of inhibition of LBCL versus the concentrations of a test compound can be constructed. It follows that the concentration needed to inhibit 50% of LBCL (IC₅₀) of a test compound can be extrapolated from the curve. Using this approach, we were able to obtain the IC₅₀ values of various compounds to be tested and the order of inhibitory efficiency of the above-mentioned food extracts was ranked, being A > B > C > D > E > F, respectively.