The Inhibitory Effect Of Polyphenol Compounds And Ascorbic Acid On Protein Amyloid Fibrosis

Proteins with correct folding states play important roles in the physiological functions of human beings.Under certain conditions,however,protein can form amyloid aggregates through a misfolding pathway,resulting in a variety of neurodegenerative diseases,such as Parkinson's disease,Alzheimer's disease and Huntington's disease.In the process of amyloid formation,a protein undergoes changes in the secondary structures,including a reduction of a-helices,an increase of ?-sheets,and an exposure of inner hydrophobic moieties.These structural alternations result in the formation of intermolecular aggregates and assembling into amyloid fibrils.Many factors such as temperature,pH,ion strength and some small cosolutes influenced the formation of amyloid fibrils.According to the literatures,some natural polyphenols possess the ability to inhibit protein amyloid fibrillation,depolymerize mature fibrils or transform the fibrils into amorphous aggregates.As a consequence,the fibrillar cytotoxicity is reduced.However,the molecular mechanism in the anti-amyloidogenic role of polyphenols is still unclear and remains to be explored.It has been widely accepted that polyphenols mainly interacted with amyloid fibrils in the form of their intact molecules during the process of amyloid inhibition or fibril depolymerization.Their unstability and oxidation under common experimental conditions is frequently neglected.Our previous research data showed that the auto-oxidative products of natural polyphenols had a stronger anti-amyloidogenic activity than the native molecule.Therefore,it is of great significance to further explore the auto-oxidation properties and anti-amyloidogenic activities of natural polyphenols for the purpose of designing and screening new drugs.In the present study,the auto-oxidation and unstability of epigallocatechin-3-gallate(EGCG),gallic acid and ascorbic acid under normal experimental conditions have been analyzed and evaluated.By utilizing bovine insulin and lysozyme as model proteins,the anti-amyloidogenic roles of these compounds and their oxidative products have been examined.The inside molecular mechanisms have also been explored.Experimental methods and results1.Amyloid fibrillation of bovine insulin and lysozymeUnder the experimental conditions of this study,bovine insulin and lysozyme can transforme into amyloid fibrils.The changes in the second structure of protein during amyloid fibrillation were monitored by thioflavin(ThT)fluorescence and circular dichroism(CD)spectroscopy.The surficial hydrophobicity was probed with ANS fluorescence,and the fibrillar morphology was observed under transmission electron microscopy(TEM).The results showed that protein fibrillation was accompanied with an increase in the beta-sheet structure and a decrease in alpha helix of the protein.The exposed hydrophobic moieties combined with the probe,leading to an enhancement in ANS fluorescence.TEM examinations demonstrated the fibrillar morphology formed by insulin and lysozyme.2.The effects of EGCG and its oxidative products on protein amyloid fibrillationEGCG is unstable and easily oxidizied under normal aqueous circumstances.The present study explored the unstability and oxidation of EGCG in solutions at pH 6.5?9.0.The anti-amyloidogenic roles of EGCG and its oxidative products EGCGox were evaluated.The results showed that EGCG could inhibit the fibril growth,and the EGCGox formed under variety pH conditions demonstrated stronger inhibitory role than the native form.Of the EGCGox formed under different pH conditions,the substances formed at a weak alkaline pH showed a satisfactory inhibitory effect on protein fibrillation.Further examinations were performed to detect the anti-amyloidogenic role of EGCGox formed at pH 7.4.The resultant data suggested that the EGCGox had a strong anti-amyloidogenic role,including the inhibition of fibril formation and degradation of the preformed fibrils.3.The effect of gallic acid on protein amyloid fibrillationHigh performance liquid chromatography and mass spectrometry(HPLC-MS)has been performed to analyze the EGCGox formed at pH 7.4.The MS data indicated that the major oxidation product of EGCG had a relative molecular weight of 170,corresponding to the polyphenolic compound gallic acid.Using lysozyme as a model protein,the anti-amyloidogenic role of gallic acid has been examined.The results suggested that gallic acid inhibit lysozyme fibrillation and depolymerized the existed amyloid fibrils.Moreover,gallic acid was not stable and oxidized easily in aqueous solution at pH 7.4.The resultant oxidative products showed a stronger anti-amyloidogenic role than gallic acid.4.The effect of ascorbic acid on protein amyloid fibrillationAscorbic acid,namely vitamin C(Vc),an important vitamin and biological antioxidant,has been demonstrated to be associated with amyloid-related diseases and capabled of inhibiting amyloid aggregation of polypeptides.The unstability and auto-oxidation of Vc under normal physiological conditions are closely related to biological activity of this substance.Therefore,it is important to analyze the autoxidation behavior of Vc and explore the effect of Vc autoxidative products on protein amyloid fibrillation.In the present study,utilizing lysozyme and bovine insulin as an in vitro model,we determined the kinetics of Vc auto-oxidation,and investigated the anti-amyloidogenic activities of Vc and its oxidative products.The results showed that Vc was unstable in aqueous solution.Vc and its oxidative products inhibited protein fibrillation and disrupted preformed mature amyloid fibrils,with an activity positively correlated to the degree of Vc degradation.We suggest that the auto-oxidative products were the actual active forms in anti-amyloidogenic actions of Vc.ConclusionEGCG,gallic acid and Vc possess the ability to inhibit protein amyloid fibrillation and depolymerize the existed fibrils.In the present study,the stability of these compounds and their anti-amyloidogenic roles were evaluated.The resultant data indicated that EGCG was oxidizable under a neutral or weak alkaline circumstance.All oxidative products formed at different pH showed stronger anti-amyloidogenic effects than native EGCG.Gallic acid,the main oxidative product of EGCG,also exhibited inhibitiony effect on protein amyloid fibrillation.Gallic acid was unstable.Its oxidative products also showed a stronger inhibitory effect on protein fibrillation than the native molecule.Ascorbic acid was proned to auto-oxidation and forms a variety of compounds upon oxidation.Of the oxidative products,dehydroascorbic acid and erythritone exhibited anti-amyloidogenic effects more strongly than ascorbic acid.The results of the present study revealed that the anti-amyloidogenic roles of natural reductants were closely related to their auto-oxidation properties.Novel anti-amyloidosis medicines can be designed or screened on the basis of the auto-oxidative products,rendering new choices to the treament of neurodegenerative diseases.