| Oxidative stress is closely bound up with various diseases,including Alzheimer Disease(AD),Parkinson’s Disease(PD)and Huntington’s Disease(HD)three major neurodegenerative diseases.Acrylamide is a neurotoxin that can induce oxidative stress injury,frequently found in the fried foods.This kind of food is an integral part of Chinese traditional processing delicacy.Thus,the development of an effective inhibition of acrylamide oxidative damage of natural antioxidants is of great importance.Semen Ziziphi Spinosae(SZS)as a traditional Chinese medicine,has a variety of pharmacological activities,such as sedation and depression.The latest research shows that flavonoids prepared from SZS behave obvious antioxidant activity,which is a kind of natural antioxidant potential,with good development prospects.However,the structure-activity relationship of SZS flavonoids and its effect and mechanism on cell antioxidant protection were not reported yet.Therefore,ethanol heating reflux extraction method combined with a variety of chromatographic technology was used to prepare flavonoids from SZS,and HPLC-MS/MS was used to analyse.The antioxidant activity of flavonoids was stuied by using a variety of antioxidant activities in vitro,and the structure-activity relationship of different compounds was studied.N1E-115 cells model was used to study the protective effect of 6-CS and its mechanism of SZS flavonoids on oxidative damage of N1E-115 cells induced by acrylamide.The main results are as follow:(1)The SZS flavonoids were extracted from SZS by ethanol refluxing extraction and purified by column chromatography.After analysis of HPLC-MS/MS method,the final confirmation of the compounds were spinosin(S),isovitexin-2’’-O-β-D-glucopyranoside(IVG),6’’’-feruloylspinosin(6-FS),6’’’-p-coumaroylspinosin(6-CS),isovitexin(IV)and swertisin(ST).(2)The results of antioxidative of SZS flavonoids in vitro are as follows: TRC assay: IV> 6-CS> IVG> 6-FS> ST> S,ABTS assay:6-CS> IV> 6-FS > IVG> ST> S,DPPH assay: 6-CS> 6-FS > IV> IVG> ST> S,O2-.assay: 6-CS> IV> 6-FS> IVG> ST> S.LOS assay: 6-CS> IV> 6-FS> IVG > S > ST.The results showed that the 6 flavonoids have significant antioxidant activity.And the flavonoids behave different antioxidant activities evaluated by the different methods and the general rules are as follows: the antioxidant capacity was in direct proportion to the level of hydroxylation,which was inversely proportional to the level of glycosylation,and the ortho substitution of a ring could signicantly enhance the antioxidant activity of –OH,6’’’-feruloyl-and 6’’’-p-coumaroyl is conducive to improve its antioxidant activity.(3)N1E-115 cells were used as the model to investigate the protective effect of 6-CS on acrylamide induced oxidative damage by the method of molecular fluorescene probe and chemiuminescence.The results showed that acrylamide can significantly induce oxidative damage in N1E-115 cells(5mmol/L acrylamide treatment for 24 h,cell viability decreased to 64.51%),and 10 μmol/L,20 μmol/L and 50 μmol/L 6-CS could significantly inhibit the cell oxidative damage induced by acrylamide(cell viability values were 73.21%(10 μmol/L),80.12%(20 μmol/L)and 89.05%(50 μmol/L));In addition,acrylamide can significantly induce N1E-115 cells reactive oxygen accumulation,decreased the mitochondrial membrane potential,glutathione content decreased.And the three concentrations of 6-CS can protect cell by significantly reducing the intracellular ROS level,recovering the level of intracellular reduced glutathione and mitochondrial membrane potential and clearance of intracellular malondialdehyde.(4)Western Blot test results showed that acrylamide can facilitate LC3II/I protein levels by oxidative stress induced by N1E-115 cells,inhibit the expression of Tim23 protein,promote the expression of p62 protein,inhibiting the protein level of Bcl-2/Bax,induce autophagy and apoptosis.10 μmol/L,20 μmol/L and 50 μmol/L 6-CS can effectively restore the level of LC3II/I,Bcl-2/Bax protein and Tim23 protein expression level.The results showed that 6-CS had a significant inhibitory effect on acrylamide induced autophagy and apoptosis,and the protective effect of acrylamide on the oxidative damage. |
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