2.7.2. Reduced Glutathione and Oxidized Glutathione

The contents of GSH and GSSG contents were determined using the 5,5 -dithiobis-(2 nitrobenzoic acid) (DTNB) and glutathione reductase procedure as detailed by Griffith [29], with certain modifications. Frozen fruit tissue (5 g) was homogenized with 10 mL of cold 5% sulphosalicylic acid and passed through two layers of Miracloth (Calbiochem, San Diego, CA, USA). The homogenate was centrifuged at 12,000× *g* for 10 min at 4 ◦C. The extract was neutralized to pH 7.0 using with 7.5 M triethanolamine. Each neutralized solution was divided into 2 assays. One (1 mL) was used to determine the total glutathione content (GSH and GSSG). Another (1 mL) was reacted with 20 μL of 2-vinylpyridine for 60 min at 20 ◦C to allow the derivatization of GSH and the detection of only GSSG in the subsequent assay. The assay was carried out by adding 50 μL of the sample, 150 μL of 125 mM sodium phosphate buffer (pH 6.5) including 6.3 mM EDTA, 700 μL of 0.3 mM NADPH, 100 μL of 0.6 mM DTNB, and 10 μL of 50 units mL−<sup>1</sup> GR in a cuvette with a 1 cm light path. The absorbance was monitored spectrophotometrically at 412 nm (Shimadzu UV-1800, Shimadzu, Japan) for 120 s at a temperature of 30 ◦C. Total glutathione and GSSG contents were estimated from the standard curve of GSH using 25–100 μL.
