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 2 h. 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.

The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG

“…The effects of potential inhibitor compounds on the process of amyloid fibril formation are often determined by analysing the kinetics of aggregation [15,29,30,32,33,[63][64][65][66][67][68] and/or the maximum ThT intensity [29,30,32,33,65,[67][68][69][70][71] in the absence and presence of the compound. The effects of EGCG and EGCG ox on the process of amyloid fibril formation by both insulin and α-synuclein performed under distinct environmental conditions were assessed using both aforementioned approaches (Figures 1 and S7), and the conclusions are presented in (Table 1).…”

“…Second, specific environmental conditions may induce modifications of compounds ( Figure 6C) [28][29][30]80]. For example, EGCG is not stable at neutral pH and oxidises within several hours.…”

“…Such conformational variability, also known as polymorphism, is thought to be a generic property of amyloid proteins, and has been proposed to be able to cause distinct disease phenotypes [1,26,27]. Moreover, the effects of potential therapeutic agents can vary depending on the conditions in which they are assayed (i.e., pH, temperature, buffer, interfaces, agitation, and others) [12], possibly due to chemical modifications of potential therapeutic molecules [28][29][30]. Since the environmental conditions under which aggregation of amyloid proteins is performed can vary between distinct studies, the search and assessment of potential inhibitors becomes extremely challenging, as the results may lead to diverse conclusions.…”

“…Taken together, EGCG appears to be a "universal" inhibitor of amyloid fibril formation, suggesting that this molecule could be used as a therapeutic agent for the prevention and treatment of amyloid-related disorders. However, EGCG is not stable at neutral or alkaline pH [45][46][47][48], where it is susceptible to auto-oxidation, resulting in the formation of numerous products [28] which may differently affect amyloid aggregation [29,30,49].…”

Flavonoids with Vicinal Hydroxyl Groups Inhibit Human Calcitonin Amyloid Formation

“…[31,38] On the other hand, EGCG has also been indicatedt ou ndergo auto-oxidation to form superoxides and quinones. [39,40] Palhano et al demonstrated that EGCG is fully oxidized after an incubation period of 6h in pH 7.4 phosphate buffer at 25 8C. [24] Oxidized EGCG may covalently bind to the peptide via Schiff base formation and/orr eaction with sulfhydryl groups in cysteiner esidues.…”