EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex

Abstract

Accumulated evidence indicates that the interconversion of iron between ferric (Fe(3+)) and ferrous (Fe(2+)) can be realized through interaction with reactive oxygen species in the Fenton and Haber-Weiss reactions and thereby physiologically effects redox cycling. The imbalance of iron and ROS may eventually cause tissue damage such as renal proximal tubule injury and necrosis. Many approaches were exploited to ameliorate the oxidative stress caused by the imbalance. (-)-Epigallocatechin-3-gallate, the most active and most abundant catechin in tea, was found to be involved in the protection of a spectrum of renal injuries caused by oxidative stress. Most of studies suggested that EGCG works as an antioxidant. In this paper, Multivariate analysis of the LC-MS data of tea extracts and binding assays showed that the tea polyphenol EGCG can form stable complex with iron through the protein Ngal, a biomarker of acute kidney injury. UV-Vis and Luminescence spectrum methods showed that Ngal can inhibit the chemical reactivity of iron and EGCG through forming an Ngal-EGCG-iron complex. In thinking of the interaction of iron and ROS, we proposed that EGCG may work as both antioxidant and Ngal binding siderphore in protection of kidney from injuries.

Fig. 1

a the structure of EGCG. b Binding assay of tea extracts of Loose tea and Jingwei Fu tea (data normalized to Ent)

Fig. 2

Multivariate analysis of LC–MS data. a A paired comparison of OPLS-DA analysis of samples according to various extracts of two teas: OPLS-DA score plots derived from HPLC–MS spectra (R²X = 0.709, R²Y = 0.527, Q² = 0.617). b S-plot generated from OPLS-DA models of the six extracts of two types of tea shows covariance w and correlation p (corr), w[1] ≥ |0.1|, p (corr) ≥ |0.5|, the variables (p<0.01, t test) was identified as the metabolites related to grouping. c The contribution score of H2O fractions and esp. EGCG (normalized to average). d EGCG relative concentration in the six extracts (normalized to average). (Color figure online)

Fig. 3

Association of EGCG with Ngal and iron and releasing iron in acid condition. a Paper chromatography of free Fe³⁺, Fe³⁺ + Ent, Fe³⁺ + EGCG and Fe³⁺ + tea water infusion (tea). b UV spectrum of apoNgal (black line), Ngal–Ent–iron (green line), Ngal–Cat–iron (blue line), and Ngal–EGCG–iron (red line) complex. c Dose responsive curve of EGCG binding iron through Ngal (Ngal 10 μM, EGCG 1.0, 5.0, 10, 30, 50, 100 μM, normalized to Ent). d Dissociation of iron from the complex in acid condition: pH responsive curve of EGCG binding iron through Ngal (washing buffer pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, normalized to Ent). (Color figure online)

Fig. 4

The binding sites at the protein Ngal: The formation of Ngal–siderophore–iron complex (Ent, Cat, EGCG, black bar) can be inhibited by 50 fold red Enterobactin (FeEnt, grey bar), and the site specific mutant (K125 and K134 to Alanine, white bar) of Ngal cannot form a complex with EGCG, Ent, Cat and iron. Data was normalized to Ent

Fig. 5

EGCG reducing power and prooxidant capacity can be inhibited by Ngal. a UV detected the conversion of Fe³⁺ to Fe²⁺ by EGCG (red line) can be inhibited by adding Ngal (green line), p = 0.039 through 10 min, two-tailed t test, n = 4. b Conversion of HPF to fluorescein (Ex 490 nm, Em 515 nm) was detected in the presence of EGCG, Fe³⁺ and H₂O₂ (red line), but the addition of Ngal blocked the reaction (green line; HPF + EGCG + FeCl₃ ± Ngal: p<10⁻⁵, n = 3, two-tailed t test) (each experiment contained Fe³⁺, which was not shown on the figure legends). (Color figure online)