Antioxidant action of 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid, an efficient aldose reductase inhibitor, in a 1,1′-diphenyl-2-picrylhydrazyl assay and in the cellular system of isolated erythrocytes exposed to tert-butyl hydroperoxide

Figures & data

Figure 1 Compounds studied.

Figure 2 Continual absorbance decrease of ethanolic solution of DPPH radical (50 μmol/l) in the presence of 200 μM concentration of the compounds tested at λ_max = 518 nm. (•) Compound 1 and (○) compound 2. The curves represent results from two typical experiments.

Table 1 DPPH test

Compound	Absorbance decrease (−ΔA/30 minutes)
1	0.219±0.005
2	0.009±0.001
Melatonin	0.022±0.006

Antiradical activity of 1 and 2 in comparison with melatonin.

The ethanolic solution of DPPH radical (50 μM) was incubated in the presence of the compound tested (200 μM). Absorbance decrease at 518 nm was determined during the first 30-minute interval. Results are mean values ± SD from at least three measurements.

Figure 3 Stoichiometry of DPPH scavenging by compound 1. Concentration dependence of DPPH absorbance decrease in the presence of increasing concentrations of 1. C_DPPH = 50 μM, time of reaction 24 hours. Results are mean values ± SD from five experiments.

Figure 4 (A) Hemolysis curves induced by t-BuOOH. Erythrocyte suspensions (1.5%) were incubated with 250 μM t-BuOOH in the presence of 100 μM (-•-) or 1 mM (-▪-) 1. Control incubations (-Δ-). Each value is the mean ± SD from four to eight experiments. (B) Kinetics of t-BuOOH quenching by compound 1. t-BuOOH (250 µM) was incubated in the absence (-Δ-) or presence of 100 µM (-•-) or 1 mM (-▪-) compound 1 under the conditions of the hemolytic experiment omitting the erythrocytes. Results are mean values ± SD from three experiments.

Figure 5 Time course of uptake of compound 1 by isolated rat erythrocytes. Compound 1 (A) 100 μM, (B) 1 mM was incubated with washed red blood cells (hematocrit of 20%) for indicated time intervals at 37°C. Experimental points represent mean values ± SEM from four independent experiments.

Figure 6 Effect of compound 1 on osmotic fragility of rat erythrocytes. Erythrocyte suspensions (0.4% hematocrit) in 10 mM phosphate buffer containing increasing concentrations of NaCl were incubated at 37°C for 1 hour in the absence (Δ) or presence (•) of compound 1 (250 μM). Experimental points represent mean values ± SD from at least three independent experiments.