Yizhi Capsule On Aβ-Induced Rat Model Of Alzheimer’s Disease And The Chemical Constituents Of Intervention

Objective:To "beta amyloid cascade hypothesis" as the breakthrough point, Using β-amyloid peptide (Aβ1-42) inject to intracerebroventricular establish AD rats model. using Yizhi to intervene, to explore the therapeutic effects of Yizhi on Alzheimer’s disease. using UPLC-MS Yizhi capsule for nine kinds of effective component quantitative analysis, which is useful for quality control of Yizhi capsule.Methods:1. The effects of Yizhi on behavior in AD model ratsUsing Morris water maze to test latency and total distance of swimming rats. Westernblot detection between each groups in the rat hippocampus Aβ, Tau protein expression changes.2. The effects of Yizhi on the activity of ChAT,AchE in AD model ratsUsing thio-D-choline assay to detect ChAT activity in serum and brain tissue, using colorimetric detection to detect AchE Activityin brain tissue and serum.3. The effects of Yizhi on the activity of neurons related gene protein in AD model ratsWesternblot detection between each groups in the rat hippocampus IL-18,Caspase-3,Bcl-2,Bax protein expression changes.4. Quantitative analysis Analysis of 9 active Active constituents Constituents in Yizhi capsule Capsule by UPLC-MSTo develop an UPLC-MS method for simultaneous determination of nine active compounds in Yizhi Capsule. including Protocatechuic aldehyde, Chlorogenic acid, Syringin,4-Dicaffeoylquinic Acid, Echinacoside, Verbascoside, Rosmarinic acid, Salvianolic acid B, Salvianolic acid A. The chromatographic separation was performed on a Waters Acquity UPLC BEH C18 column (2.1mm×100mm,1.7μm)with gradient elution of acetonitrile and 0.1% aqueous formic acid solution. at a flow rate of 0.4ml·min-1. The temperature was set as 40℃ and injection volume was 5μL. The monitoring of all analytes was achieved under the negative ionization mode.Results:1. The changes of water maze behavior in different groups1.1 Latency:Compared with the normal group,there was no significant difference in the sham group. compared with the sham group,the escape latency time of the model group was prolonged significantly. During the hidden platform test, compared with the model group, there had significant difference in each treatment group.1.2 Total distance:Compared with the normal group,there was no significant difference in the sham group. compared with the sham group,the total distance of the model group was prolonged significantly. In the first day, Yizhi capsule low-dose group compared with model group, the total distance swimming no statistical significance, while the rest of the treatment group were shortened in the hidden platform within experimental 1-5d total distance swimming, and there had statistical significance.1.3 The changes of Aβ、Tau protein expression in Hippocampus of rats in different groups The sham-operated group compared with the normal group showingnosignificant difference;compared with normal group, sham-operated group Tau protein and Aβ were significantly higher, and there are statistical significance; compared with model group, the phosphorylated Tau protein and Aβ were significantly reduced.2.The changes of the ChAT, AchE activity in different groupsCompared with normal group, there were no significant difference in the sham-operated group; Compared with the sham-operated group,the AchE activity was significantly increased and the ChAT activity was significantly decreased in the model group; Compared with model group, the AchE activity was significantly decreased and the ChAT activity was significantly increased in each treatment group.3. The changes of IL-18, Caspase-3, the Bcl-2, Bax protein expression in Hippocampus of rats in different groupsThe sham group compared with the normal group IL-18, Caspase-3, Bcl-2, Bax were no significant difference in protein expression; Compared with the sham group, IL-18, Caspase-3, Bcl-2, Bax protein expression levels were significantly increased in the model group, Each treatment group compared with the model group, IL-18, Caspase-3, Bax protein reduced, Bcl-2 protein levels were increased with a significant difference.4. Yizhi capsules measurement results nine kinds of active ingredient contentSystem investigated factors affecting the extraction efficiency of the extraction solvent, extraction time, etc, finally determine the 75% methanol ultrasonic extraction for 30 min. All calibration curves showed that nine kinds of active ingredients in the detection range of good linearity, recoveries were 97.71%～101.06% with the RSD less than 3.44%.Conclusion:1. Yizhi capsule can significantly improve the learning and memory function obstacle to the AD model rats, neurofibrillary tangles protein and its role in vivo by reducing tau hyperphosphorylation induced neuronal necrosis caused reduce cholinesterase the active protection cholinergic system function, reduce inflammatory cytokines IL-18, Bcl-2 gene expression related apoptosis Bax,, in order to reduce apoptotic neuronal cells.2. TheUPLC-MS analytical method was simple, rapid, sensitive and high accuracy, And applied to the determination of five batches of samples Yizhi Capsule, which is useful for quality control of Yizhi capsule.