High-Throughput Superoxide Anion Radical Scavenging Capacity Assay Hongxun Tao, †,∥ Jinge Zhou, †,∥ Tao Wu,* ,† and Zhihong Cheng* ,‡ † Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, The Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China ‡ Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China * S Supporting Information ABSTRACT: A high-throughput superoxide anion radical (O 2 •− ) scavenging capacity assay based on the xanthine oxidase/ xanthine reaction system was developed and validated in the present study. The reaction conditions including detection wavelength, concentrations of reactant components, reaction temperature, reaction time, pH, terminator reagent, and sample dissolving solvents were optimized. The accuracy and reliability of the assay were assessed by evaluation of linearity (r 2 =0 .9513−0.9957), precision (intraday RSD 1.13−4.05% and interday RSD 2.13−5.62%), accuracy (95.64−97.42% recovery), and stability (RSD 2.62−6.19%), as well as comparison with the conventional colorimetric method. The EC 50 values obtained by the current method and the conventional assay were highly correlated (r > 0.99). This high-throughput O 2 •− scavenging assay may be used for screening and estimating potential superoxide anion radical (O 2 •− ) scavengers, especially food extracts and natural products with a very small amount of test material. KEYWORDS: xanthine oxidase, superoxide anion radical, high throughput, antioxidant activity ■ INTRODUCTION Free radicals play an important role in physiology and pathology as mediators of many biochemical events. Excessive free radical production is associated with numerous chronic disease conditions, such as cancer, 1 cardiovascular diseases, 2 diabetes mellitus, 3 gastric ulcer, 4 and neurological disorders. 5 In recent years, antioxidants have attracted a great deal of attention as potential agents for the control of diseases associated with oxidative damage. Superoxide anion radicals (O 2 •− ), one of the most important reactive oxygen species (ROS), are produced as byproducts of metabolism, particularly in mitochondrial respiration, 6 and serve as a progenitor for other toxic ROS such as hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO − ), and hydroxyl radicals (HO • ). This leads to a growing interest in the discovery and development of valuable nutraceuticals and functional foods capable of scavenging O 2 •− . A number of O 2 •− scavenging assay protocols have been developed, including an electron spin resonance (ESR) method, 7−14 a high-perform- ance liquid chromatography (HPLC) method, 15−17 an ultra- high-performance liquid chromatography and triple-quadrupole mass spectrometry (UHPLC-MS) method, 18 and a thin layer chromatography (TLC) bioautographic method. 19 Among these, the ESR, HPLC, and UHPLC-MS methods have been shown to possess good specificity and accuracy, but require relative expensive instrumentation. TLC bioautography is an ideal method for the estimation of antioxidants present in complicated extracts and can be easily adopted in high-throughput analysis. However, this method is generally developed and used in qualitative ways. Compared to these methods, spectrophotometry is one of the most widely used techniques in biomedicinal analysis 20−25 due to its inherent ease of application and no requirement of expensive equipment. The conventional spectrophotometric methods are performed manually in cuvettes, which generally require a large sample quantity. These requirements are not compatible with the capabilities of most food chemistry laboratories charged with routine analysis of pure antioxidants. Therefore, a sensitive and high-throughput assay is needed to facilitate the rapid and automated screening of the O 2 •− scavengers. Superoxide anion radicals can be generated in vitro by many methods such as the hydrogen peroxide degradation system in alkaline dimethyl sulfoxide (DMSO), 11,26 the phenazine methosulfate (PMS)/β-nicotinamide adenine dinucleotide (NADH) system, 14,27,28 the riboflavin irradiation system, 17,19,29 and the xanthine/xanthine oxidase (X/XO) sys- tem. 12,18−23,28,30 Among these the X/XO system is a classical enzymatic method based on XO oxidation of xanthine giving O 2 •− , which subsequently reacts with a pale yellow tetrazolium salt, nitroblue tetrazolium chloride (NBT), to yield a purple formazan by monitoring its absorbance at 560 nm. In this study, a high-throughput method in a 96-well microplate format for O 2 •− scavenging capacity assay based on the X/XO system was developed and validated. In addition, this high-throughput assay was used to estimate the O 2 •− scavenging activity of some pure compounds and food extracts. Received: May 11, 2014 Revised: August 27, 2014 Accepted: September 3, 2014 Published: September 3, 2014 Article pubs.acs.org/JAFC © 2014 American Chemical Society 9266 dx.doi.org/10.1021/jf502160d | J. Agric. Food Chem. 2014, 62, 9266−9272 Downloaded via MACAU UNIV on April 25, 2021 at 05:53:31 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.