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Plants 2022, 11, 2379 14 of 18 4.4. Non-Enzymatic Antioxidants 4.4.1. Total Phenolics Fresh plant samples were ground in 80% acetone and were centrifuged at 8000 rpm for 10 min. The supernatant (100 μL) was reacted with 100 μL Folin Ciocalteu’s phenol reagent and 5 mL of Na2CO3 (20%). The volume was then raised to 10 mL with dH2O. After vigorous shaking, absorbance of samples was observed at 750 nm [74]. 4.4.2. Ascorbic acid (AsA) For AsA estimation, plant samples were completely homogenized in 6% TCA solution and were centrifuged at 8000 rpm for 10 min. The reaction solution consisted of 4 mL supernatant, 2 mL of 2% dinitrophenylhydrazine and one drop of thiourea (10%) solution, mixed in test tubes. The reaction solutions were then water bathed for 20 min at 100 ◦C followed by cooling at room temperature. The test tubes were then shifted to ice and 5 mL of 80% H2SO4 was mixed slowly in these tubes. The absorbance of samples was recorded at 530 nm [75]. 4.4.3. α-Tocopherol (Vitamin E) The α-tocopherol contents were assayed by grinding plant samples in a mixture of petroleum ether:ethanol (2:1.6 v/v). The homogenized material was centrifuged at 8000 rpm for 20 min. The supernatant (1 mL) was mixed with 200 μL of 2% 2,2-dipyridyl and placed in dark adopted incubation for 5 min till the final red coloration of solution. The volume of solution was raised to 4 mL with dH2O and absorbance of the samples was read at 520 nm [76]. 4.4.4. Glutathione Contents Plant samples were homogenized in 10% TCA solution, centrifuged for 12 min at 12,000 rpm and supernatant was removed for the estimation of total glutathione, reduced glutathione (GSH) and oxidized glutathione (GSSG) contents. To assay total glutathione, 200 μL supernatant was added 600 μL sodium phosphate buffer (125 mM, pH 7.5), 1 mL GR enzyme (10 units mL−1), 200 μL DNTB (6 mM) and 1 mL of NADPH (0.3 mM) solution. For GSH assay, 200 μL supernatant was added to 1.4 mL sodium phosphate buffer (125 mM, pH 7.5) and 200 μL DNTB (6 mM). The mixtures were water bathed with stirring at 30 ◦C for 10 min followed by an immediate ice bath before the reading was taken. The absorbance for total glutathione and GSH was taken at 412 nm [77]. The GSSG concentration in samples was worked out by subtracting GSH from total glutathione. 4.4.5. Proline For proline estimation, plant samples (0.5 g) were grinded in 10 mL 3% sulfosalicylic acid solution and were filtrated with Whatman filter paper. A total of 2 mL of extract sample was added to 2 mL ninhidrin and 2 mL of glacial acetic acid solution in test tubes, which were water bathed at 100 ◦C for 60 min followed by immediate cooling in ice. After cooling, 4.0 mL of toluene was poured into these test tubes, mixed vigorously and kept at room temperature until two layers were formed. The absorbance of the upper colored layer was taken at 520 nm [78]. 4.5. Hydrogen Peroxide (H2O2) and Malondialdehyde (MDA) To assay H2O2 contents, the plant material (0.25 g) was homogenized in 5 mL TCA (0.1%) solution and centrifuged for 15 min at 12,000 rpm. The supernatant (0.5 mL) was mixed with 0.5 mL sodium phosphate buffer and 1 mL of potassium iodide (KI) solution in test tubes. Test tubes were vortexed and absorbance was read at 390 nm [79]. The MDA contents were estimated using Heath and Packer [80] methodology. Then, 1 mL of supernatant (same as used in protein estimation) was mixed to 1 mL TBA (0.5%) solution prepared in 20% TCA solution in test tubes and were water bathed for 30 min at 95 ◦C. The tubes were then ice bathed for 5 min followed centrifugation at 6000 rpm. ThePDF Image | Silicon-Induced Mitigation of NaCl Stress in Barley
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