Analysis Of Different Components In Different Parts Of Acanthopanax Senticosus And Protective Mechanism Of Hyperoside In The Oxidative Damage

The incidence of neurodegenerative diseases is increasing year by year with the occurrence of aging,especially Alzheimer’s disease,（AD）,Parkinson’s disease,（PD）and amyotrophic lateral sclerosis（（ALS）,）.It is predicted that in the near future,Alzheimer’s disease will become the third leading cause of death in the elderly after cancer and heart disease.Although the annual medical costs and expenditures for neurodegenerative diseases are huge,there is no specific treatment for the disease.Oxidative stress injury,as one of the breakthroughs of neurodegenerative diseases,has attracted wide attention in recent years.The imbalance of oxidation and antioxidant system in vivo is the cause of oxidative stress.Oxidative stress damage will produce a large number of free radicals.Neurons that are easy to be damaged are very sensitive to free radicals,so the improvement of oxidative stress injury is closely related to the treatment of neurodegenerative diseases.Acanthopanax senticosus is rich in various saponins,flavonoids,polysaccharides and alkaloids.Phenolic acids are widely used in the study of central nervous system diseases such as cerebral ischemia-reperfusion injury,platelet aggregation,depression,Parkinson’s disease and cerebellar atrophy.However,the material basis and mechanism of its efficacy are not clear.This experiment started with the experiment in vitro,establish the model of oxidative stress injury induced by H₂O₂ in PC12 cells,and then study the relationship between Acanthopanax senticosus and the protective effect of oxidative stress injury.The experiment is carried out from the following aspects.1 intervention effects of different concentrations of Methanolextract and water extract of Acanthopanax senticosus on oxidative stress injury of PC12 cells induced by H₂O₂.Using rat pheochromocytoma cell PC12,which is highly similar to neuron in morphology and function,as the experimental object,the oxidative stress injury model of H₂O₂ was established.By using the method that exogenous MTT can react with living cells to form blue-purple crystal,which can reflect the relative survival rate of cells,firstly,the median lethal concentration of H₂O₂ was determined to be 400 μ m / L as the experimental model concentration.Then the optimal dosages of 30% Methanolextract,70% Methanolextract,95% Methanolextract and water extract of Acanthopanax senticosus were determined.PC12 cells were injured by 400 μ m / L H₂O₂ for 12 hours after the corresponding concentration of drugs were given in advance according to the optimal dose.,it was detected that except that 95% Methanolextract had no protective effect on H₂O₂-injured PC12 cells,the other concentrations of Methanolextract and water extract had different degrees of protective effect.Among them,70% Methanolextract has the most obvious protective effect on H₂O₂ oxidative stress injury.So,in the next step,we will use Ultra High-Performance Liquid Tandem Chromatography Quadrupole Time of Flight Mass Spectrometry（UHPLC-QTOF-MS）to analyze the metabonomics of 70% Methanolextract of Acanthopanax senticosus.2 Analysis of the correlation between the differential markers of Acanthopanax senticosus in different parts and their antioxidant stress injury.Traditional Acanthopanax senticosus has more studies on its roots,but there are also related studies on pharmacodynamics of Acanthopanax senticosus seeds and Acanthopanax senticosus leaves.In this chapter,UHPLC-QTOF-MS was used to analyze and identify the chemical composition of 10 batches of Acanthopanax senticosus extracted by 70% alcohol in different parts and screen the different components in different parts（rhizome,seed,leaf）as differential markers.The results of anti-H₂O₂ oxidative damage of PC12 cells in all batches of samples from different parts of Acanthopanax senticosus were tested by MTT colorimetric method.Finally,the results of different markers in different parts of Acanthopanax senticosus were correlated with the results of anti-H₂O₂ oxidative damage in different parts of Acanthopanax senticosus by Pearson correlation analysis to find the material basis related to antioxidant stress injury in different parts of Acanthopanax senticosus.Through identification and analysis,11 substances such as Syringic acid,Eleutheroside E and Quercetin in Acanthopanax senticosus roots and stems and 19 substances such as Chlorogenic acid,Hyperoside and Astragalin in Acanthopanax senticosus seeds were identified.19 substances such as p-Coumaric acid,L-Phenylalanine and Azelaic acid in Acanthopanax senticosus leaves were significantly related to anti-H₂O₂ oxidative stress damage to PC12 cells.The effects of 11 of these substances on oxidative stress injury or antioxidation have been reported to varying degrees in previous studies,including Astragalin,Chlorogenic acid,p-Coumaric acid,Quercetin and Rutin have protective effects on PC12 cells induced by H₂O₂.Among these differential markers,which are significantly related to the protection of H₂O₂-induced PC12 cells,We selected hyperoside,a flavonoid component with high content in Acanthopanax senticosus,which is significantly related to antioxidant stress injury in Acanthopanax senticosus seeds and Acanthopanax senticosus leaves,to explore its protective effect on H₂O₂-induced PC12 cells and its related mechanism.3 Study on the Protective Mechanism of Hyperin on PC12 oxidative stress damage induced by H₂O₂.Hyperoside,as one of the main flavonoid active substances in Acanthopanax senticosus,has the highest content in leaves,and it has been reported in varying degrees in the treatment of oxidative damage and the reduction of inflammation and apoptosis.therefore,it is of great development value as a monomer drug with the potential of nerve repair.In this chapter,the protective effect of hyperin on H₂O₂-damaged PC12 cells and its mechanism were studied for the first time.Using MTT colorimetric method,the optimal concentration of hyperin in protecting PC12 from oxidative stress damage was determined to be 50 μm / L.Before and after treatment with 50 μm / L hyperin,the expressions of pi3 k,Akt,LC3 Bm RNA and protein were significantly up-regulated,while the expressions of p53,Bax,Caspase 3,NF-κB,i NOS,p62,m RNA and protein were significantly down-regulated.pi3k/Akt pathway participates in the regulation of cell proliferation,differentiation and growth,and can protect nerve cells from toxic injury.The activation of this pathway will lead to cascade phosphorylation of Akt and inhibit oxidative stress injury.At the same time,the activation of Akt will activate the expression of downstream factors of this signal pathway,such as NF-κB,p53,Bax and so on.At the same time,At the same time,NF-κB regulates the synthesis of calmodulin independent enzyme i NOS,the release of cytochrome C and the activation of Caspaserelated proteins.,while oxidative damage leads to stress response leading to the increase of p53 expression.The activation of p53 leads to the upregulation of its downstream gene Bax and then activates the inherent apoptotic pathway.pi3k/Akt pathway not only participates in the regulation of apoptosis,but also clears protein aggregation in neurodegenerative diseases by regulating autophagy,co-localization with autophagy selective substrate protein p62 and LC3 B,and immunoprecipitation is involved in the formation of autophagosomes.From the above experiments,it can be verified that the protective effect of hyperin on H₂O₂ damage is mainly achieved by regulating the pathways related to autophagy and apoptosis,and apoptosis and autophagy are closely related to each other and jointly regulate cell survival and growth.In this experiment,the differential chemical components in different parts of Acanthopanax senticosus were screened and identified by metabonomics technology,and the correlation between the metabonomic results of different chemical components and the corresponding oxidative damage protection results of the samples of this part was analyzed.to find the differential markers of different parts of Acanthopanax senticosus and its efficacy-related material basis,and to explore the mechanism of Acanthopanax senticosus in the treatment of oxidative stress injury from a new point of view.It provides a reference idea and research basis for the treatment direction of neurodegenerative diseases and the therapeutic mechanism of Acanthopanax senticosus.