Polyphenols And Lysozyme Fiber Studies, The Role Of Human Erythrocytes

Tea polyphenols,known as catechins,usually account for 30%～42%of the dry weight of solids in brewed green tea,is the main biologically active compound of tea.It is the general name of flavanol,catechins,flavonol and phenolic acids.Of these catechins,EGCG is the most abundant (accounting for 50%～75%of the catechins) and the most biologically active compound.Tea polyphenolic compounds are known to be strong antioxidants.However,several studies reported the oxidative effects of tea polyphenols,suggesting that tea polyphenols had a dual function of anti-oxidant and pro-oxidant potentials.In the current study,we evaluated the effects of tea polyphenols(EGCG,ECG,EGC,EC and C) on erythrocyte in the presence and absence of lysozyme fibril.The results suggest that two of these tea polyphenols(EGCG and EGC) significantly altered the redox status of erythrocyte.In the absence of external stress,EGCG and EGC act as the pro-oxidant in erythrocyte.This was indicated by changing cellular morphology,inducing GSH consumption and quinoprotein formation,modifying membrane lipids and proteins particularly applied at high doses.Their oxidative activity had a direct relationship to their EGCG or EGC concentration and exposure time.EGCG acts as a pro-oxidant in erythrocyte and utilizes a mechanism similar to that of 1,4-benzoquinone.In the presence of lysozyme fibril,EGCG and EGC act as the anti-oxidant in erythrocyte.Methods and Conclusions:Part One.The influence of Tea polyphenols on the cytotoxicity of lysozyme fibril1.The cytotoxicity of lysozyme fibril on erythrocyteGhost membrane was separated of erythrocyte treated with lysozyme fibril followed with SDS-PAGE to detected alteration of membrane protein.The result showed that lysozyme fibril induced ghost membrane protein aggregation.2.The influence of EGCG on the cytotoxicity of lysozyme fibrilCompared to the cytotoxicity of lysozyme fibril on erythrocyte,the treatment of erythrocytes with lysozyme fibril and EGCG resulted in the less ghost membrane protein aggregation.The SDS-PAGE results indicated EGCG protected the ghost membrane protein form the cytotoxicity of lysozyme fibril.3 The effect of Tea polyphenols on morphological change of erythrocyte which induced by lysozyme fibril Compared to the control sample,the treatment of erythrocyte with lysozyme fibril resulted in aggregation of cells and in the formation of global vesicles with various sizes.EGCG and EGC can relieve these morphological change of erythrocyte.Part Two.The internation of Tea polyphenols and human erythrocyte membrane1.The influence of Tea polyphenols on the membrane protein pattern of erythrocyteGhost membrane was separated of erythrocyte treated with Tea polyphenols,followed with SDS-PAGE to detected alteration of membrane protein.The result showed that EGCG and EGC induced ghost membrane protein aggregation.These protein aggregation can totally diminished by adding vitamin C.Whether EGCG acts pro-oxidant effects or not is highly dependent upon its concentration.EGCG alone have no effects on the ghost membrane at concentrations below 1μg/ml, while increasing it higher than 1μg/ml induced ghost membrane protein produced aggregation.The concentration had a positive relationship to their oxidative activity.2.Glutathion level of erythrocyte in the presence of Tea polyphenolsEllman method was used to determine the GSH content of erythrocyte.The result showed that Tea polyphenols caused significant inducing GSH consumption,the order was EGCG＞EGC>>ECG≈EC≈C.GSH plays a key role in protecting the red cell from oxidative damage.The consumption of GSH is mainly attributed to radical species which form the EGCG and EGC oxidized.3.The influence of Tea polyphenols on the membrane phospholipids of erythrocyteEGCG attacked the lipid bilayers of cellular membrane and induced alternations of membrane properties including membrane fluidity,enzymatic and trsport activities,and membrane intactness and morphology.High performance liquid chromatographic(HPLC) analyses showed decreases of three major membrane phospholipids PS,PE and PC of erythrocyte under treatment of EGCG.4.The effect of Tea polyphenols on erythrocyte morphologyCompared to the control sample,the treatment of erythrocytes with EGCG and EGC resulted in morpholocical alternation and aggregation of cells.ECG,EC and C almost have no effects on erythrocyte morphology.5 The effect comparison of EGCG and 1,4-benzoquinone on erythrocyteGhost membrane was separated of erythrocyte treated with EGCG and 1,4-benzoquinone, followed with SDS-PAGE to detected alteration of membrane protein.The result showed that EGCG and 1,4-benzoquinone induced ghost membrane protein aggregation.These protein aggregation can totally diminished by adding vitamin C.Ellman method was used to determine the GSH content of erythrocyte.The result showed that EGCG and 1,4-benzoquinone both caused significant inducing GSH consumption.HPLC analyses showed decreases of three major membrane phospholipids PS, PE and PC of erythrocyte under treatment of EGCG and 1,4-benzoquinone.These result provide strong evidence that EGCG and EGC acts as a pro-oxidant on erythrocyte and utilizes a mechanism similar to that of 1,4-benzoquinone.6.The effect comparison of different Tea polyphenols on erythrocyteBased on the results as above,we concluded that EGCG and EGC significantly altered the redox status of erythrocyte;ECG,EC and C almost have no effects on erythrocyte.This phenomenon could be explained by the structural differences of the tea polyphenols.