The Anti-amyloid Fibrosis Effect Of Flavonoids And Their Oxidation Products

Proteins or polypeptides,under some circumstances,are able to transform into fibrils through amyloidosis.These amyloid aggregates deposit in human tissues,resulting in damage and dysfunction of the organ.The deposition of protein amyloid is one of important characteristics in many human diseases.There are more than 20 human diseases that are known to be associated with amyloid fonnation of proteins,for instance,amyloid-? peptide in Alzheimer's disease,a-synuclein in Parkinson's disease,and lysozyme in systematic amyloidosis.Although these diseases have not been effectively treated at present,a large number of studies have shown that some small molecular compounds possess the abilities to inhibit protein amyloid fibrillation and degrade mature fibrils.Among the small molecules,natural occurring flavonoids have attracted extensive attention.Natural flavonoids have a basic structure of C6-C3-C6 backbone,deriving from flavones(2-phenylchromone)as the mother nucleus.Their chemical properties and biological activities are mainly determined by the number and location of hydroxyl groups and other functional groups in the molecule.Inhibiting the formation of amyloid fibrils,depolymerizing mature fibrils and transforming the fibrils into amorphous aggregates are included in the anti-amyloidogenic effects of flavonoids.The anti-amyloidogenic mechanism of flavonoids is very complex.Numerous studies have so far attributed the inhibition of fibril growth and disaggregation of mature fibrils to the interactions between polypeptide and intact flavonoids.The susceptibility to oxidation of these chemicals under common experimental conditions has been usually neglected.Recent studies have shown that some flavonoids are oxidized quickly under in vitro physiological conditions.Their biological activities are related not only to the intact molecules,but also to the oxidation process and oxidative products.In order to evaluate systematically the effect of flavonoids against protein amyloid fibrillation,quercetin and its three structure-related flavonols,myricetin,kaempferol,and morin,and four corresponding flavanonols,taxifolin,dihydromorin,dihydromyricetin,and dihydrokaempferol,were selected as target molecules in this study.The research works included analyzing the oxidation behavior of these compounds,evaluating their anti-amyloidogenic roles by utilizing lysozyme as an in vitro model,and exploring their structure-activity relationship in anti-amyloidogenic activity.Methods and results:1.The stability of flavonoidsHPLC has been adopted to determine the oxidation kinetics of flavonoids at different pH.The results showed that the selected flavonols except morin and dihydromorin were unstable in a weak alkaline medium.The half-lives of flavonoids were related to the number and position of hydroxyl groups on the B-ring and double bond structure on the C-ring.Morin and dihydromorin,with a resorcinol structure on the B-ring,were relatively stable.2.Kinetics of fibril growth of lysozymeIn this study,thioflavin T(ThT)fluorescence was used to detect the growth process of lysozyme fibrils,8-aniline-1-sodium benzenesulfonate(ANS)was used to detect the exposure of hydrophobic domains,circular dichroism(CD)was used to detect the changes in protein secondary structure,and transmission electron microscopy(TEM)was used to characterize the morphology of lysozyme amyloid aggregates.The results showed that amyloid fonnation was characterized by a decrease in a-helix,an increase in ?-sheets,and an exposure of hydrophobic region of the protein.At the final stage of amyloid formation,typical fibrillar morphology can be observed under TEM.3.The effects of flavonoids on lysozyme amyloid fibrillationThe inhibitory roles of flavonol,flavanonol and their oxidative products on fibril growth were determined and compared by using the lysozyme amyloidogenic system.The results demonstrated that all flavonoids were able to inhibit lysozyme fibrillation.Flavonols had stronger inhibitory effect than flavanonols.All the oxidative products inhibited amyloid formation more effectively than their intact molecules.The anti-amyloidogenic effect of flavonoids was closely related to the number and position of hydroxyl groups on the B-ring.Myricetin,with a pyrogallic structure on the B-ring,and its oxidative products are the most potent compounds in amyloid inhibition.4.The fibril-disruptive roles of flavonoids and their oxidative productsFlavonoids and their oxidative products were added into mature lysozyme fibrils.ThT fluorescence,Bradford assay and TEM were utilized to detect the degradation of the fibrils.The results showed that flavonoids were able to disaggregate mature lysozyme fibrils.The number and position of hydroxyl groups on the B-ring are correlated with the fibril-disruptive ability of the molecules.The more hydroxyl groups on the B-ring,the stronger the fibril-disruptive role of the molecules.Flavonoid with adjacent hydroxyl groups on the B-ring possessed stronger fibril-disruptive role than those with a resorcinol structure.More effective role in fibril disaggregation was observed after oxidation of a flavonoid molecule.5.The interactions between flavonoids and lysozymeIn order to further understand the molecular mechanism inside the anti-amyloidogenic activity of flavonoids,the interactions between flavonoids and lysozyme were analyzed by using intrinsic fluorescence and simulated molecular docking technology.The results of intrinsic fluorescence showed that flavonoids had a quenching effect on protein fluorescence,and the quenching potency was positively correlated with the anti-amyloidogenic activity,indicating that a stable complex formed by lysozyme and flavonoid molecule.According to the structure of complexes obtained by molecular docking,it can be concluded that flavonoids are docked in a chimerism state into the hydrophobic cavity of lysozyme.Hydrogen bond and van der Waals forces between flavonoids and lysozyme were main binding forces in the lysozyme-flavonoid complex.The greater the binding energy was,the more stable the complex formed,and the stronger an anti-amyloidogenic role achieved.Conclusions:In this study,eight flavonoids with a similar chemical structure were selected to investigate their stability at different pH and the anti-amyloidogenic effects of intact molecules and oxidative adducts.The results showed that all flavonoids played an inhibitory role on lysozyme amyloid fibrillation,and the oxidative adducts showed a stronger activity than intact flavonoids.The oxidation rate of flavonoids was closely related to the position and number of hydroxyl groups on the B-ring.Flavonoids with catechol and pyro gallic groups were easy to be oxidized,and those with resorcinol structure were relatively stable.The anti-amyloidogenic effect of flavonoids was also related to their structure.Compounds with catechol and pyrogallic moieties on the B-ring and those with a double bond on the C-ring demonstrated a strong activity on lysozyme fibrillation.The binding of a flavonoid with lysozyme resulted in the formation of a stable complex.The more stable the complex was,the stronger the anti-amyloidogenic activity obtained.This study for the first time explored systematically the stability and anti-amyloidogenic role of flavonoids.It was found that the anti-amyloidogenic activity of a flavonoid was enhanced after oxidation.This study provided a new rout in searching for novel medicines for the treatment of amyloid diseases,and the inside molecular mechanisms are still remained to be further explored.