Studies On The Mechanisms Of Ding-Zhi-Xiao-Wan For Treating Alzheimer's Disease And Screening Active Components In Acanthopanax Scenticosus

Alzheimer's disease?AD?is a neurological disease that ofter occurs in the elderly,the main clinical symptoms includes cognitive dysfunction,memory loss,loss of self-care ability associated with the personality and emotional change.At present,the pathogenesis of AD has not been clarified,there is no drug to cure the disease so far.The development of anti-AD drug based on target is slow,and the clinical treatment effect is not good.For the multi-pathogenesis of AD,we need find a comprehensive treatment.With the development of analytical techniques,studies has shown that traditional Chinese medicine can exert its effect of treating AD through multiple pathways and multiple targets.Therefore,this paper starts from the Ding-Zhi-Xiao-Wan?Radix ginseng,Radix polygala,Poria cocos,Acorus tatarinowii Schott?for the treatment of AD,establishes an AD rat model,and explores the overall mechanism of DZXW in the treatment of AD from the perspective of metabonomics.Provide theoretical and experimental basis for the clinical research of DZXW.This paper includes the following main contents:?1?Establishment of AD rat model:A rat model of AD was established by bilaterally injection A?_25-35 into the rat hippocampus.The injection site was selected:3.00 mm posterior to fonticuli quadrangularis,2.0 mm in the middle line,and 2.8 mm subdural.The flow rate of the microsyringe was set at 1?L/min,injection 5 min.The contralateral brain was treated with the same method.One week after the operation,intragastric administration was started.DZXW:5.5 g/kg/d,Donepezil:0.167 mg/kg/d.Control group and model group were given equal amount of distilled water.After 30days administration,the Morris water maze trains for 5 days and records the results.?2?Study on the urinary metabolomics in AD rats:after the behavioral experiment,12h urine of rats was collected.The urinary metabolomics study was carried out based on UPLC-Q-TOF-MS technology combined with multivariate analysis.?3?Based on the technology of UPLC-TQ-MS,we developed a quantitative method,and examined the intra-and inter-day precision,accuracy,limit of quantification,limit of detection,matrix effects and recovery of this method.The potential biomarkers of urine in rats were studied quantitatively.?4?Based on the results of the previous study in our group,the anti-oxidative properties of Acanthopanax root 30%ethanol eluting fraction was the strongest in vitro,and can improve the symptoms of PD model mice.In order to study the material basis for the active action of this components,we use UPLC-Q-TOF-MS/MS analysis technique to identify the composition of the substances.In addition,MAO-B was selected as the target protein,and the small molecule active compounds which was combined with MAO-B was selected by affinity ultrafiltration spectrum technology.The experimental results:The behavioral experiments showed that the rat model was successful,and DZXW could improve learning and memory ability of AD rats.Urinary metabonomics results:a total of 28 endogenous biomarkers were identified,including taurine,uric acid,citric acid,creatinine,cysteine,etc.The relative content of these biomarkers after administration was adjusted toward the direction of normal rats,indicating that DZXW improves the cognitive mechanism of AD rats:antioxidative stress,regulation of amino acid metabolism,energy metabolism,protection of neurons,improvement of intestinal Flora metabolism.15 potential biomarkers were quantified in the urine.The results of non-targeted metabolomics were verified by quantitative metabolomics.A total of 19 compounds,including organic acid compounds,coumarins and lignins,were identified in the 30%Ethanol elution component of Acanthopanax root.In vitro enzyme inhibitory activity showed that half maximal inhibitory concentration of Acanthopanax senticosus on MAO-B was 1.611 mg/mL,indicating that this component had the activity of inhibiting of MAO-B.The results of affinity ultrafiltration analysis showed that there were 9 compounds combined with MAO-B,and the substance with the highest binding strength was calculated based on the peak area as Acanthoside E.