Studies On Screening And Metabolomics-based Mechanisms Of The Active Components In Schisandra Chinensis For Treating Alzheimer's Disease

Alzheimer's disease(AD)is a degenerative central nervous system disease,characterized by generalized dementia such as memory loss,cognitive dysfunction,speech disorder and mood swings.AD is the most common cause of dementia.It is necessary and urgent to find a new therapeutic approach and develop new drugs for AD.The pathogenesis of AD is complicated,with the cause for most Alzheimer's cases is still mostly unknown.Till now,available treatments offer relatively small symptomatic benefit,especially lack of effective treatment drugs for AD.Therefore,novel drugs with high active and low-toxic are in demand for prevention and treatment of AD,which has become the research hotspot in AD.The dried and mature fruit of Schisandra chinensis(Mulanaceae)is a source of medicinal herbs with mild medicinal properties that generally applied on the lungs,heart and kidneys.It has been proved that Schisandra chinensis could be applied for replenishing vital energy,invigorating the kidney and benefiting the heart,and even calming down the nerves.Even though our understanding of AD has increased dramatically over recent years and the therapeutic effect of Schisandra chinensis on AD has been proved the pharmacodynamic components of Schisandra chinensis and its mechanism of action are still unclear.Based on the pathogenesis of AD,this paper explored the pharmacodynamic material basis and mechanism of Schisandra chinensis in the treatment of AD.The main research contents include the following aspects:1.Preparation and composition analysis of each component in Schisandra chinensisFirstly,different components in Schisandra chinensis were successfully isolated and purified by modern extraction and purification methods,obtaining polysaccharides,lignans,volatile oils and organic acids.Then the compositions incomponents were identified by mass spectrometry.(1)The extraction of Schisandra polysaccharides by cold water extraction and alcohol precipitation was optimized according to L9(33)orthogonal table.The optimal extraction conditions were that,polysaccharides extracted 2 times with a sample-to-solvent ratio 1:30(w/v)for 90 minutes.The content of polysaccharides in Schisandra chinensis was determined by phenol-sulfuric acid method.Extraction rate and purity were calculated to be 7.079%and 58.65%,respectively.After acid hydrolysis and derivatization,the results of GC-MS analysis showed that the Schisandra polysaccharide is composed of arabinose,glucose,galactose,mannose,ribose,xylose,glucuronic acid,rhamnose and fucose.Finally,the relative molecular weights of <5×103,5×103~1×104,1×104~1×105,1×105~5×105,5×105~1×106 and >1×106 Da were obtained by dialysis membrane method.The results showed that the relative molecular mass of Schisandra chinensis was mainly distributed in the range of <5×103 and >1×105 Da.They accounted for24.96% and 23.76% of the total polysaccharide,respectively.(2)Flash extraction was applied to get the crude extract of Schisandra lignans,and then further purified by AB-8 macroporous resin to obtain purified lignans.The purity of lignans in Schisandra was 87.11%,mesured by chromotropic acid-sulfuric acid reaction.(3)The volatile oil in Schisandra chinensis was extracted by traditional distillation extraction method,and the yield was 1.392%.Then,GC-MS was used to analyze the components,and 48 compounds were identified,including 27 terpenoids,4 aromatic compounds(2 aromatic ethers,2 aromatic hydrocarbons),and 10 aliphatic compounds(9 fatty alcohols,1 fatty ether),4 esters and 3 ketones.(4)The Schisandra chinensis powder was added with 30 volumes of distilled water,and extracted twice at 10? for90 minutes.The supernatant was collected and the organic acid components were obtained after concentration,alcohol precipitation,supernatant collection,concentration and lyophilization.The purity and the extraction yield of the organic acid component of Schisandra chinensis was 58.83% and 27.61%,respectively.They were determined by ZD-2A automatic potentiometric titration technique.The above experimental results provided a pharmacodynamic basis for the subsequent screening and mechanism of action of Schisandra in the treatment of Alzheimer's disease.2.Screening of active components of Schisandra chinensis in the treatment of Alzheimer's diseaseIn this study,the active components of Schisandra chinensis against AD were screened by both in vivo and in vitro experiments.Firstly,Al Cl3 and D-galactose were used to establish AD model in mice.The effects of various components of Schisandra chinensis on memory acquisition and consolidation ability,neural cell protection,A?deposition and tau protein phosphorylation in AD mice were evaluated by jumping platform behavior experiment,HE staining and immunohistochemical experiment.Then,A?25~35 induced PC12 cell apoptosis model was applied for further screened and evaluated the active components of Schisandra chinensis.The protective ability of each component was detected by MTT assay.The morphological changes were also detected under inverted microscope.The effects of various components on the expression of Caspase-3,Bcl-2 and Bax in apoptosis model were estimated by Western blotting.The results showed that Schisandra polysaccharides and lignans could significantly improve the cognitive ability of AD mice,and increase the survival of apoptosis in A?25~35 induced PC12 cells,indicating their anti-AD effects.It was determined that polysaccharides and lignans are the active components of Schisandra in the treatment of Alzheimer's disease.Because Schisandra polysaccharides were firstly found to have significant anti-AD effects.Therefore,we focus on it as a research object,and deeply studied its mechanism in treatment of AD.3.Study on Stablishment,Evaluation and UPLC-Q-TOF/MS-Based Metabolic Profiling of A?25~35 induced Alzheimer's DiseaseA? is a polypeptide produced by proteolytic hydrolysis of amyloid precursor protein that considered as a common pathway leading to dementia for a variety of reasons.The model of brain injury caused by injection of A? is similar to human senile dementia.In order to further study the mechanism of action of Schisandra polysaccharides in the treatment of AD,the AD model was established by injecting A?25~35 into the bilateral hippocampus of experimental rats.It was evaluated by behavioral,pathological and immunohistochemical experiments.At the same time,urine metabolic profile of A?25-35-induced Alzheimer's disease rats at 2,4 and 8weeks after surgery was studied by using UHPLC-Q-TOF-MS.The results showed that the AD model induced by A?25~35 could be successfully replicated 8 weeks after surgery.Urine metabolomics studies showed that 45 endogenous metabolites were identified as potential biomarkers of AD.Analysis of related metabolic pathways indicated phenylalanine and tyrosine metabolism,tryptophan metabolism,arginine and proline metabolism,citric acid cycle,vitamin B6 metabolism,fatty acid and glycerophospholipid metabolism,taurine and hypotaurine metabolism,purine and pyrimidine metabolic pathways were disrupted in A?25~35 induced AD rats.UHPLC-TQ-MS was used to quantify eight representative biomarkers of the above eight metabolic pathways.The results show that the trend of quantitative metabolite content is consistent with the results of non-targeted metabolomics,indicating that the results of endogenous markers obtained by non-targeted metabolomics are reliable.According to the biological significance of the metabolic profile,the pathogenesis of AD is mainly caused by abnormal metabolism of amino acids,changes in the two-way communication system of the central nervous system and gastrointestinal tract caused by intestinal microflora imbalance,energy metabolism inhibition,oxidative stress damage and the loss of neuroprotective substances.The metabolomics study served as a foundation for the investigation of the treatment mechanism of Schisandra polysaccharide against AD.4.Effect of Schisandra Polysaccharide on Neurotransmitters in hippocampus of AD rats induced by A?25~35Firstly,to evaluate the effect of Schisandra polysaccharides on cognitive ability in AD rats,morris water maze behavioral experiments were used in A?25~35 induced AD rats.Then,The HE staining and immunohistochemistry experiments were used to investigate the treatment of Schisandra polysaccharides on AD.Subsequently,the activities of GSK-3?,NOS,SOD and Ach E were also examined using the ELISA kit.Finally,a method for the determination of neurotransmitters by ultra performance liquid chromatography-quadrupole mass spectrometry(UHPLC-TQ-MS)was established.Based on the quantitative methods,we measured the content of neurotransmitters in the hippocampus of each group.The results showed thatSchisandra polysaccharide could significantly reduce memory acquisition and memory consolidation in AD rats.The deposition of A? and phosphorylation of tau protein in brain were reduce by Schisandra polysaccharide treatment.Inhibited activation of microglia and astrocytes,combined with reduced Ach E,GSK-3? and NOS levels and increase SOD activity were observed after treated with Schisandra polysaccharide.The content of serotonin,norepinephrine,dopamine,acetylcholine,taurine,?-aminobutyric acid and glycine in the hippocampus of AD rats was significantly decreased,whereas the levels of glutamic acid and aspartic acid were decreased.After treatment with Schisandra polysaccharides,the levels of the 9neuroactive substances were partially adjusted to normal levels.The results indicated that Schisandra polysaccharide could protect the central nervous system by regulating the content of nerve active substances in the hippocampus of AD rats,thus improving the learning and memory ability of AD rats.5.Investigation on the mechanism of Schisandra polysaccharides on the treatment of Alzheimer's disease based on metabolomicsUHPLC-Q-TOF-MS combined with PCA analysis was applied to investigate the urinary metabolomics of AD rats treated with Schisandra polysaccharides.The whole mechanism of the treatment of AD by Schisandra polysaccharides was discussed from the perspective of metabolomics.Metabolomics studies found that a total of 38 metabolites were finally identified as potential biomarkers of AD,and all of them had a significant recovery compared with AD model after Schisandra polysaccharides administration.Seventeen compounds were identified in positive ion mode and eleven compounds were identified in negative ion mode.These biomarkers primarily affect tryptophan,tyrosine,phenylalanine,taurine and hypotaurine,arginine and proline,purine and pyrimidine,niacin and nicotinamide,niacin and nicotinamide,and lysine metabolic pathways.Metabolomics studies have shown that Schisandra polysaccharides plays a role in protecting the central nervous system,regulating intestinal microbial metabolism,enhancing energy metabolism,and promoting antioxidant effects by regulating the levels of endogenous metabolites in related pathways.In summary,based on modern separation and purification techniques,polysaccharides,lignans,volatile oils and organic acids in Schisandra were purified.Combined with mass spectrometry,the main compositions in these components were identified.The significant active components of AD are screened by in vivo and in vitro experiments.UHPLC-TQ-MS and UHPLC-Q-TOF-MS techniques were applied to reveal the pathogenesis of AD and the therapeutic mechanism of Schisandra polysaccharides against AD from the perspective of pharmacodynamics and metabolomics.The experimental results of this paper provide a theoretical basis for further elucidating the pharmacodynamic basis and therapeutic mechanism of Schisandra in the treatment of AD,which also lay a foundation for the development of new drug for the treatment of AD.