Use of pyrogallol red and pyranine as probes to evaluate antioxidant capacities towards hypochlorite

Abstract

Hypochlorite is a strong oxidant able to induce deleterious effects in biological systems. The goal of this work was to investigate the use of PGR and PYR as probes in assays aimed at evaluating antioxidant activities towards hypochorite and apply it to plant extracts employed in Chilean folk medicine. The consumption of PGR and PYR was evaluated from the decrease in the visible absorbance and fluorescence intensity, respectively. Total phenolic content was determined by the Folin Ciocalteau assay. PGR and PYR react with hypochlorite with different kinetics, being considerably faster the consumption of PGR. Different stoichiometric values were also determined: 0.7 molecules of PGR and 0.33 molecules of PYR were bleached per each molecule of added hypochlorite. Both probes were protected by antioxidants, but the rate of PGR bleaching was too fast to perform a kinetic analysis. For PYR, the protection took place without changes in its initial consumption rate, suggesting a competition between the dye and the antioxidant for hypochlorite. Plant extracts protected PYR giving a PYR-HOCl index that follows the order: Fuchsia magellanica ≈ Marrubium vulgare ≈ Tagetes minuta > Chenopodium ambrosoides ≈ Satureja montana > Thymus praecox. Based on both the kinetic data and the protection afforded by pure antioxidants, we selected PYR as the best probe. The proposed methodology allows evaluating an antioxidant capacity index of plant extracts related to the reactivity of the samples towards hypochlorite.

Figure 1

Probe spectroscopic changes induced by HOCl. (A) Changes in the UV-visible spectrum of PGR (30 µM) induced by hypochlorite (15 µM). Line a and b before and after 5 min of hypochlorite addition, respectively; (B) Changes in the UV-visible spectrum of PYR (50 µM) mediated by 100 µM of hypochlorite. Lines a–e: increasing times up to 30 min of incubation; (C) Changes in the fluorescence spectrum of PYR (5 µM) mediated by hypochlorite (10 µM). Lines a–e: increasing times up to 30 min of incubation.

Figure 2

Kinetic profiles of PGR and PYR consumption induced by hypochlorite. (A) Consumption of PGR followed by visible spectroscopy at 540 nm. PGR (60 µM) was incubated with hypochlorite at 20; 30; 60; and 100 µM. The arrow indicates the time in which hypochlorite was added; (B) PYR consumption followed by fluorescence technique (460 and 510 nm for λ_ex and λ_em, respectively). PYR (5 µM) was incubated with hypochlorite at 5; 10; 20; and 50 µM. Controls experiments (PGR or PYR solution in the absence of hypochlorite).

Figure 3

Dependence of the consumed probe with hypochlorite concentration. (A) Dependence of the PGR consumed with hypochlorite concentration. [PGR] = 15; 30; and 60 µM. (B) Dependence of PYR consumed with hypochlorite concentration. [PYR] = 5; 10; and 50 µM.

Figure 4

Dependence of initial consumption rate of PYR with hypochlorite (A) or PYR initial concentrations (B). Log-Log plots.

Figure 5

Effect of ferulic acid on the consumption of PGR or PYR induced by hypochlorite. (A) Kinetic profiles of the reaction between PGR (15 µM) and hypochlorite (10 µM) in the absence and presence of ferulic acid at 100 and 300 µM. The arrow indicates the time at which hypochlorite was added. (B) Kinetic profiles of the reaction between PYR (5 µM) and hypochlorite (10 µM) in the absence and presence of ferulic acid at 0.5; 5 and 25 µM. Controls experiments (PGR or PYR plus ferulic acid in the absence of hypochlorite).

Figure 6

Dependence of ∆PGR₀/∆PGR_XH with the [XH]/[PGR] ratio. XH = Trolox, gallic acid, and ferulic acid.

Figure 7

Effect of plant extracts (PE) on the hypochlorite-mediated consumption of PYR. (A) Kinetic profiles of the reaction between PYR (5 µM) and hypochlorite (10 µM) in the absence and presence of F. magellanica at 0.2; 1; 2 and 10 µg/mL. (B) Dependence of ∆Probe₀/∆Probe_PE with [PE]/[PYR] ratio. F. magellanica; M. vulgare; C. ambrosoides; and S. montana. PE concentrations correspond to µg of dried extract/mL.

Figure 8

Dependence of the PYR-HOCl index with total phenol content of plant extracts.