Oxidized epigallocatechin gallate inhibited lysozyme fibrillation more strongly than the native form

Abstract

Epigallocatechin gallate (EGCG), the most abundant flavanoid in green tea, is currently being evaluated in the clinic due to its benefits in the treatment of amyloid disorders. Its anti-amyloidogenic effect has been attributed to direct interaction of the intact molecule with misfolded polypeptides. In addition, antioxidant activity is also involved in the anti-amyloidogenic role. The detailed molecular mechanism is still unclear and requires further investigation. In the present study, the kinetics of EGCG oxidation and the anti-amyloidogenic effect of the resultant oxidation substances have been examined. The results indicate that EGCG degrades in a medium at pH 8.0 with a half-life less than 2h. By utilizing lysozyme as an in vitro model, the oxidized EGCG demonstrates a more potent anti-amyloidogenic capacity than the intact molecule, as shown by ThT and ANS fluorescence, TEM determination, and hemolytic assay. The oxidized EGCG also has a stronger disruptive effect on preformed fibrils than the native form. Ascorbic acid eliminates the disruptive role of native EGCG on the fibrils, suggesting that oxidation is a prerequisite in fibril disruption. The results of this work demonstrate that oxidized EGCG plays a more important role than the intact molecule in anti-amyloidogenic activity. These insights into the action of EGCG may provide a novel route to understand the anti-amyloidogenic activity of natural polyphenols.

Keywords: Amyloid fibrils; Anti-amyloidogenic activity; Cytotoxicity; Epigallocatechin gallate; Lysozyme; Oxidation.

Graphical abstract

Fig. 1

Analysis of EGCG oxidation. (A) HPLC analysis of the kinetics of EGCG oxidation at 37 °C and pH 8.0. EGCG (12.7 min) and its oxidation substances (10.2 min) were detected at 280 nm. Time-dependent changes of peak area of EGCG and the oxidation substances are shown in (B) and (C), respectively. (D) UV-spectra of native EGCG (E) and the oxidation products (E12h).

Fig. 2

Kinetics of lysozyme fibrillation in the absence and presence of 50 µg/mL native EGCG or oxidized EGCG at 12 h. Incubation temperature was 65 °C. (A) ThT assay. Open squares, lysozyme (Ly) only; open triangles, Ly+E; filled squares, Ly+E12h. (B) Dose-dependent inhibitory effects of EGCG and oxidized EGCG on lysozyme fibrillation. Asterisks represent p<0.01 (n=3). (C) ANS assay. Same symbols are used as in (A).

Fig. 3

TEM images of lysozyme assemblies. Samples were prepared by incubating lysozyme (1 mg/mL) at 65 °C for 6 days in the absence (A) and presence of 50 µg/mL EGCG (B) and oxidized EGCG at 12 h (C). Scale bar represents 200 nm.

Fig. 4

Fibril-disrupting effects of EGCG and oxidized EGCG. Mature fibrils were prepared specifically by incubating lysozyme (10 mg/mL) at 65 °C for 6 days in the absence of EGCG. For details see materials and methods section. (A) Amorphous aggregates were induced by co-incubating preformed fibrils with EGCG at 37 °C for 1 h. The final concentration of mature lysozyme fibrils (Ly F) was 1 mg/mL; EGCG and oxidized EGCG, 50 µg/mL; VC (ascorbic acid), 4 mM. (B) Quantitative determination of the conversion of lysozyme fibrils to amorphous aggregates. The conversion of fibrils to amorphous aggregates was expressed as the percentage of lysozyme deposited. Asterisk represents p<0.05 (n=3).

Fig. 5

Hemolytic assay. Hemolysis of erythrocytes was induced by incubating (37 °C) the cell suspensions (1%, v/v) with lysozyme assemblies (0.2 mg/mL) for 3 h. Lysozyme assemblies were prepared by incubating lysozyme (1 mg/mL) at 65 °C for 6 days in the absence and presence of 50 µg/mL native or oxidized EGCG. Asterisks represent p<0.01 (n=3). The control solution showed no hemolytic effect (data not shown).

Scheme 1

Molecular structure of EGCG.