Study On The Effect Of Ethanol Extract Of Tartary Buckwheat Buds Against Oxidative Stress Via Nrf2-ARE Pathway

Tartary buckwheat is a dicotyledon plant of Polygonaceae which is rich in polyphenols and has the pharmacological effects of reducing blood sugar and blood lipids.It is pointed out that many pharmacological effects of tartary buckwheat are related to its antioxidant activity.After germination,the antioxidant activity of tartary buckwheat has been increased significantly,but the protective mechanism of high antioxidant activity has not been clarified.Therefore,based on the analysis of polyphenols content and antioxidant activity,the effects of ethanol extracts from tartary buckwheat sprouts?TBSE?on the protein expressions and activities of major antioxidant enzymes in damaged cells and the relationship between the protective effect and Nrf2-ARE signaling pathway were investigated to clarify the specific mechanism by establishing oxidative damage models with hydrogen peroxide?H₂O₂?and acrylamide?Ala?.Furthermore,it provides a theoretical basis for widening the utilization of buckwheat products.The main results are as follows:1.The determination of high performance liquid chromatography shows that the TBSE mainly contains six phenolic substances,namely chlorogenic acid,caffeic acid,syringic acid,rutin,ferulic acid and quercetin.TBSE has quinone oxidoreductase induction activity and intracellular antioxidant activity to some extent.2.TBSE can resist oxidative damage of HepG2 cells induced by hydrogen peroxide.MTT colorimetric method was used to screen out the non-cytotoxic concentration of TBSE and the concentration of 5?g/mL,10 ?g/mL.and 20 ?g/mL are proper.The proper concentration of quercetin in positive control group is 2.5 ?M,5 pM and 10?M.The oxidative damage concentration of hydrogen peroxide is 400 ?M,Compared with the blank group,the cell survival rate of H₂O₂-injured group decline significantly,while the cell survival rate increase significantly after pretreatment with TBSE and quercetin for 24 hours.3.TBSE can reduce ROS level of HepG2 cells induced by H₂O₂,maintain the stability of mitochondrial membrane potential and protect cells from apoptosis induced by H₂O₂.Treatment with H₂O₂ could induce the increase of MDA content and decrease the activities of SOD,CAT and GST enzymes.But this trend is antagonized after pretreatment with TBSE and quercetin for 24 hours.Western Blot test shows that TBSE could up-regulate the expression of antioxidant enzymes NQO1 and HO-1 and promote nuclear transfer of Nrf2 in HepG2 cells injured by H₂O₂.4.TBSE can resist oxidative damage of Hepalclc 7 cells induced by acrylamide?Ala?.MTT results showed that the proper concentration of extract is 5 ?g/mL,10 ?g/mL and 20 ?g/mL.The proper concentration of quercetin in positive control is 5 ?M,10 ?M and 20 ?M.The proper concentration of Ala was 10 mM for oxidative damage.Compared with the blank group,the cell survival rate of Ala-injured group decline significantly,while the cell survival rate increase significantly after pretreatment with TBSE and quercetin for 24 hours.5.TBSE can reduce ROS level of Hepalclc 7 cells induced by Ala,maintain the stability of mitochondrial membrane potential.Treatment with Ala could induce the increase of MDA content and decrease the activities of SOD,CAT and GST enzymes.But this trend is antagonized after pretreatment with TBSE and quercetin for 24 hours.Western Blot test shows that TBSE could up-regulate the expression of antioxidant enzymes NQO1 and HO-1 and promote nuclear transfer of Nrf2 in Hepalclc 7 cells injured by Ala.In conclusion,TBSE has significant antioxidant effect,and can resist oxidative stress injury of cells.Its protective mechanism is related to the nuclear transfer of Nrf2 in Nrf2-ARE signaling pathway.