The Study On Extraction And The Mechanism Of Inhibiting AChE Of Biatractylolide

Objective: To extract biatractylolide from Chinese traditional medicine Zhejiang Atractylodes macrocephala, and explore mechanism of biatractylolide inhibition of ACh E. Provide a theoretical basis for the further development of biatractylolide become anti-AD drug.Methods:1. Extraction and separation of biatractylolide: the raw Zhejiang Atractylodes macrocephala were dip extracted with ethyl acetate.Using chromatographic separation technology into chemical ingredients of ethyl acetate extract, the molecular structure of the compounds were confirmed by 1H-NMR, 13C-NMR. Isolated a series of compounds including biatractylolide.2. Docking the biatractylolide and ACh E protein molecules under the molecular docking software MOE, clear molecular site of action among each other.3. The test of Biatractylolide inhibiting ACh E activity: using the method of Ellman quantitative evaluation the ACh E inhibitory activity of biatractylolide, getting different concentrations biatractylolide of ACh E inhibition rate. Draw the concentration-response curve, to fit a curve equation, and the IC50 concentration is calculated.4. The research of biatractylolide inhibiting ACh E expression: the ACh E-S and GSK-3β were transfected to GSK-3β knockout MEF cells and 293 T cells, adding different concentrations of biatractylolide,cultured cells after 24 h, by Western blot to observe MEF cells ACh E expression levels and expression levels of ACh E in 293 T cells.The result:1. Ten compounds initially isolated from the ethyl acetate extract of Atractylodes macrocephala, and the structure are confirmed respectively, atractylenolide I(3000 mg), atractylenolide II(800 mg),atractylenolide III(7000 mg), biatractylolide(480 mg), dandelion terpene acetate(240 mg), juniper camphor(180 mg), octacosanic acid(50 mg). β-sitosterol(380 mg), heptadecanoic acid(600 mg),7-α-hydroxy-β-sitosterol(9.8 mg).2. Molecular docking studies show biatractylolide can be combined with Trp279 by H-π(3.31 ?) role of PAS active sites and Trp84 forming H-π(3.88 ?) role of CAS active sites of ACh E protein, biatractylolide binds to ACh E stability. RMSD = 1.14 ? shows good stability of the Molecular docking system.3. Biatractylolide can effectively inhibit the activity of ACh E hydrolysis of ACh, biatractylolide and ACh E inhibition rate have a certain dose-effect relationship within a certain range of concentrations, built a dose-response curve equation: y=-0.33x2+9.14x+6.99(R2= 0.9874), IC50 value is 14.1685 μM.4. Biatractylolide has a dose-dependent manner to downregulate the expression of ACh E of MEF cells and 293 T cells.Conclusion: Biatractylolide has been isolated from the ethyl acetate extract, and its yield was higher than that reported in the literature. The mechanism of biatractylolide inhibiting ACh E is not only through binding to ACh E, but also the reduced ACh E expression may be by inhibiting the activity of GSK3β.