Short communicationDevelopment of a simple 96-well plate method for evaluation of antioxidant activity based on the oxidative haemolysis inhibition assay (OxHLIA)
Highlights
► A 96-well plate method for a cell-based antioxidant assay was developed. ► Inhibition of oxidative haemolysis by antioxidants was monitored by turbidity. ► This assay is simple to use, with good precision and reproducibility.
Introduction
Dietary antioxidants, such as polyphenols, play an important role in disease prevention (Fang et al., 2002, Osawa and Kato, 2005), and many methods have been developed to assess the antioxidant capacity of foods and chemical compounds in vitro (Aruoma, 2003, Magalhães et al., 2008, Pérez-Jiménez et al., 2008, Prior et al., 2005). Each method has advantages and disadvantages, and it is therefore essential to evaluate antioxidant activities using a combination of several methods (Aruoma, 2003, Magalhães et al., 2008, Pérez-Jiménez et al., 2008, Prior et al., 2005). Recently, cell-based methods have been preferably performed in combination with chemical methods to screen for antioxidant activities of foods and food components (Barros et al., 2008, Hsu et al., 2010, Satoh et al., 2005, Tai et al., 2010, Takebayashi et al., 2008). We have recently reported a cell-based assay, named oxidative haemolysis inhibition assay (OxHLIA) (Takebayashi, Chen, & Tai, 2010a). OxHLIA is based on inhibition of free radical-induced membrane damage in sheep erythrocytes by antioxidants. For details, peroxyl radicals generated from 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (Niki, 1990) attack biomembranes of erythrocytes and eventually cause haemolysis (Sato, Kamo, Takahashi, & Suzuki, 1995). This haemolysis is inhibited by antioxidant activities of tested samples (Takebayashi et al., 2010a). The advantages of OxHLIA are: (1) it uses peroxyl radicals as pro-oxidants and erythrocytes as oxidizable targets so that the results obtained reflect biologically relevant radical-scavenging activity compared to other in vitro methods using non-natural radicals (such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay (Brand-Williams, Cuvelier, & Berset, 1995)) and non-natural oxidizable targets (such as an oxygen radical absorbance capacity (ORACFL) assay (Prior et al., 2003)); and (2) it reflects microlocalization of antioxidants to the biomembranes of erythrocytes (Takebayashi et al., 2010a). The original OxHLIA was performed in a test-tube format, and the degree of haemolysis was determined by absorbance of the supernatant after centrifugation. This made it difficult to evaluate large numbers of samples at the same time. Thus, in this study, we first attempted to determine the degree of haemolysis by measuring the turbidity of the erythrocyte suspension without centrifugation, and we then developed OxHLIA in a 96-well plate format. There have already been some methodological studies (Blasa et al., 2011, González et al., 2010, Honzel et al., 2008, Niki, 1990, Niki et al., 1988) on cell-based antioxidant assays using erythrocytes, and the method described here was comprised of the best mix of these earlier works with ingenious modifications. This plate method enabled evaluation of large numbers of samples in small quantity at the same time with satisfactory precision and reproducibility.
Section snippets
Cells and chemicals
Sheep erythrocytes were obtained from Nihon Seibutsu Zairyou Center (Tokyo, Japan). To reduce the influence of individual differences in sheep (Takebayashi et al., 2007), pooled erythrocytes from four sheep were used.
Ascorbic acid, glutathione (reduced form) and AAPH were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), uric acid, hesperetin, (+)-catechin and 3-methyl-1-phenyl-2-pyrazoline-5-one (edaravone) were
Comparison of OxHLIA by the absorbance method and turbidity method in a test-tube format
The original OxHLIA is a useful in vitro method for evaluating the biologically relevant antioxidant activities (Takebayashi et al., 2010a). In this method, the degree of haemolysis is determined from the concentration of haemoglobin in the supernatant after centrifugation by measuring the absorbance at 524 nm. Those centrifugation steps make it difficult to assess large numbers of samples at the same time. Hence, in this study, we first showed that the degree of haemolysis could be determined
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