In Vitro Antioxidant versus Metal Ion Chelating Properties of Flavonoids: A Structure-Activity Investigation

Abstract

Natural flavonoids such as quercetin, (+)catechin and rutin as well as four methoxylated derivatives of quercetin used as models were investigated to elucidate their impact on the oxidant and antioxidant status of human red blood cells (RBCs). The impact of these compounds against metal toxicity was studied as well as their antiradical activities with DPPH assay. Antihemolytic experiments were conducted on quercetin, (+)catechin and rutin with excess of Fe, Cu and Zn (400 μM), and the oxidant (malondialdehyde, carbonyl proteins) and antioxidant (reduced glutathione, catalase activity) markers were evaluated. The results showed that Fe and Zn have the highest prooxidant effect (37 and 33% of hemolysis, respectively). Quercetin, rutin and (+)catechin exhibited strong antioxidant properties toward Fe, but this effect was decreased with respect to Zn ions. However, the Cu showed a weak antioxidant effect at the highest flavonoid concentration (200 μM), while a prooxidant effect was observed at the lowest flavonoid concentration (100 μM). These results are in agreement with the physico-chemical and antiradical data which demonstrated that binding of the metal ions (for FeNTA: (+)Catechin, KLFeNTA = 1.6(1) × 106 M-1 > Rutin, KLFeNTA = 2.0(9) × 105 M-1 > Quercetin, KLFeNTA = 1.0(7) × 105 M-1 > Q35OH, KLFeNTA = 6.3(8.7) × 104 M-1 > Quercetin3'4'OH and Quercetin 3OH, KLFeNTA ~ 2 × 104 M-1) reflects the (anti)oxidant status of the RBCs. This study reveals that flavonoids have both prooxidant and antioxidant activity depending on the nature and concentration of the flavonoids and metal ions.

Fig 1. Basic structure of flavonoids and general chemical structure of different flavonoids.

Fig 2. Chemical structures of quercetin, rutin, (+)-catechin and of the four polymethylated analogues of quercetin (the thick grey colour points out the potential bidentate binding sites).

Fig 3. Absorption spectrophotometric titration of 3,5,7-tri-O-methyl-quercetin (quercetin3’4’OH).

(A) Absorption spectra, (B) absorption electronic spectra, and (C) complex formation evolution as a function of the [FeNTA]₀ of the FeNTA complex with quercetin3’4’OH. Solvent: CH₃OH/H₂O (80/20 by weight); pH = 7.4 (Hepes buffer); T = 25.0(2°C; l = 1 cm. [Quercetin 3’4’OH]₀ = 2.89 × 10⁻⁵ M. (D) Electrospray mass spectra of quercetin3’4’OH ferric complex (noted LH₂) in the presence of NTA. Solvent: CH₃OH, capillary voltage = 4000 V. [L.FeNTA]_tot = 5 × 10⁻⁵ M; Negative mode; Fragmentor = -100 V.

Fig 4. DPPH scavenging activity of studied flavonoids and the standard: ○-Quercetin, ■-Rutin, ▲-Quercetin 3’,4’OH,▼-Catechin, □-Ascorbic acid, ◄-Quercetin 3 OH, ►-Quercetin 3,5 OH, ●-Quercetin 5 OH.

The average error on the inhibition percentage was estimated to be 4% for all the examined concentrations.

Fig 5. Hemolysis of a 5% human RBCs treated with metal ions (400 μM) in the absence or the presence of flavonoids (200 μM and 100µM) under air atmosphere at 37°C.

The significance of the differences between treated RBCs and control was determined by the Student t-test: *P < 0.05. **P < 0.01.

Fig 6. Oxidant status of a 5% human RBCs treated with metal ions (400 μM) in the absence or the presence of flavonoids (200 μM and 100µM) under air atmosphere at 37°C.

The significance of the differences between treated RBCs and control was determined by the Student t-test: *P < 0.05. **P < 0.01.

Fig 7. Antioxidant status of a 5% human RBCs treated with metal ions (400 μM) in the absence or the presence of flavonoids (200 μM and 100 μM) under air atmosphere at 37°C.

The significance of the differences between treated RBCs and control was determined by the Student t-test: *P < 0.05. **P < 0.01.