Anti-amyloid Fibrosis Of EGCG And Its Oxidation Products

Undern certain circumstances,protein or polypeptides are able to assemble into amyloid fibrils which deposits in cell membranes and ultimately induces apoptosis.Numerous investigations have demonstrated that some natural polyphenolic compounds could inhibit amyloid formation and disrupt preformed amyloid fibrils,transforming amyloid fibrils into amorphous aggregates.Epigallocatechin-3-gallate(EGCG),the main biologically active component in green tea,owns pharmacological effects in many aspects,including inhibition of protein fibrillation and attenuation of fibrillar cytotoxicity.EGCG is currently being evaluated in the clinic for treating neurodegenerative diseases.In the present study,the chemical stability of EGCG under a neutral or weak alkaline medium was evaluated.By utilizing lysozyme and bovine insulin as in vitro model proteins,the anti-amyloidogenic activities of EGCG and its oxidative products have been determined and compared.The results indicated that EGCG can quickly transform into stable products in a neutral or alkaline medium,and the oxidized form exhibited a greater effect in inhibiting protein fibrillation than the intact molecule.The chemical structures of oxidized species have been characterized by mass spectrometry.It is generally considered that the anti-amyloidogenic capacity of EGCG is mainly due to the non-covalent interactions between its intact molecule and polypeptide chains.The instability of EGCG and the role of its oxidative products are usually neglected.The importance of this study is to reveal firstly that the oxidative products are the main components in the anti-amyloidogenic activity of EGCG.This study may provide a novel led structure for the treatment of amyloid diseases.Methods and results:1.Kinetics of fibril growth of lysozyme and insulinThe growth kinetics of amyloid fibrils was monitored by using two fluorescent probes thioflavin(ThT)and 8-anilino-l-naphthalenesulfonic acid(ANS).ThT assay detected the growth curves of protein fibrillation,and ANS fluorescence probed the surficial hyfrophobicity of amyloid species.The changes in second structure and morphology were detected by CD and TEM.The results show that the conformation of protein was changed along with the incubation,including a decrease of a-helices,increases of ?-sheet structure and surfacial hydrophobicity.The TEM images showed that the mature lysozyme and insulin fibrils had a typical amyloid morphology.Fibril growth was accelerated by agitation and co-incubation with salt.2.The chemical stability of EGCGHPLC,UV spectra,LC-MS and MS/MS have been adopted to determine the oxidative process of EGCG.The results showed that EGCG was unstable in a weak alkaline medium and degraded to oxidized products.UV spectra suggested that the oxidative products still possessed a phenolic structure.Upon incubation for 12 h,two stable oxidative products were generated,with m/z ratios of 169 and 225,respectively.The m/z 169 ion corresponds to the deprotonated gallic acid.The structure of m/z 225 ion was further analyzed with MS/MS and a possible degradation pathway of EGCG was suggested.3.Inhibition of protein fibrillation by EGCG and its oxidized productsBy utilizing lysozyme and insulin as in vitro models,the inhibition of amyloid fibrillation by EGCG and its oxidized products have been investigated.The results show that the oxidized EGCG demonstrates a more potent anti-amyloidogenic role than intact molecule;and the inhibitory capacity was positively correlated with the degree of EGCG oxidation.In addition,the inhibitory role of EGCG on amyloid formation decreased with acceleration of fibril growth,whereas the role of oxidized products kept unchanged.NBT assay showed that oxidized EGCG formed more quinoproteins than the native form,suggesting that the formation of quinone structure plays a key role in amyloid inhibition.4.The fibril-disruptive roles of EGCG and oxidized EGCGEGCG and its oxidized products were mixed respectively with matured lysozyme fibrils prior to incubation and centrifugation.The supernatants were subject to quantitative analysis of protein for evaluation of fibril-disruptive activities.The results indicated that oxidized EGCG had 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 data of Zeta-potentiometric and ANS assays showed that oxidized EGCG decreased the surfacial charge of the protein and caused fibril aggregation.In aggregation process,the hydrophobic region of fibril is encased in the interior of the precipitate,which led to an attenuation of ANS fluorescence.5.Hemolysis induced by lysozyme assemblies prepared in the absence and presence of an inhibitorThe hemolytic effects increased with the growth of fibrils,namely,the oligomers and protofibrils exhibit cytotoxicity while mature fibrils show a weak or no effect.The oxidized EGCG-generated lysozyme assemblies are less cytotoxic than those created in the presence of native EGCG.This fact may be attributed to the hydrophobic interactions between cell membranes and oxidized EGCG-generated lysozyme assemblies.The hydrophobicity of the assemblies,probed by ANS fluorescence,decreased by oxidized EGCG.Therefore cytotoxicity of the assemblies lowered.6.Effects of flavanols on protein fibrillationEGC,ECG and C,having a similar basic structure with EGCG,were selected to investigate the oxidation process and anti-amyloid acticity for a comparison with EGCG and evaluation of the structure-activity relationship.The results of UV spectra and HPLC suggested that the quantity and position of hydroxyl group on the B-ring and D-ring are important in the oxidation process and anti-amyloidogenic activity of a flavanol.Furthermore,the D-ring is the sole moiety in the formation of oxidative products with strong inhibitory effect on amyloid fibrillation.ConclusionsIn conclusion,EGCG is susceptible to oxidation in air under neutral or weak alkaline pH.The half-life of EGCG is less than 2 h under the experimental conditions of this study.The oxidized EGCG demonstrates a more potent anti-amyloidogenic role than the intact molecule.The oxidized EGCG transforms to quinone or quinonoid substances which covalently bind to polypeptide chains,resulting in the inhibition of amyloid growth.Meanwhile,the oxidized EGCG also has a stronger disruptive effect on mature lysozyme fibrils than the native form.Ascorbic acid eliminates the disruptive effect of native EGCG on lysozyme fibrils,suggesting that oxidation is a prerequisite in the fibril-disruptive role of EGCG.In comparison with four flavanols ployphenols,the B-ring and D-ring play a key role in oxidation process and anti-amyloidogenic activities.