In Vitro Study On The Mechanism Of The Effect Of The Alcohol Extract From The Root Of Anacyclus Pyrethrum On Cough Variant Asthma

Objective: To explore the quality control index of Anacyclus pyrethrum root,optimize the extraction process of flavonoids from the alcohol extract of Anachi root,and study the mechanism of the effect of Anacyclus pyrethrum root alcohol extract on lymphocytes in patients with CVA.Methods: The quality standard of Anacyclus pyrethrum was studied by microscopic identification,thin layer identification,content determination and impurity examination.The content of flavonoids was determined by ultraviolet spectrophotometry as the evaluation index.The extraction time,times and solvent of total flavonoids were determined by single factor and orthogonal test.The effects of different concentrations of EEAP and the positive control drug(prednisone acetate)on lymphocyte proliferation in patients with CVA were detected by CCK-8 method.The contents of IL4,IL-5,IL-6,IL-13,IFN-?,GM-CSF,SOD and MDA in the culture medium were measured by ELISA,and the expression of TLR4 and NF-?B(P65)mRNA in CVA lymphocytes of each group were detected by RT-PCR.The expression levels of TLR4,NF-?B(P65),?-catenin,?-?-catenin,GSK-3?,?-GSK-3?,4-HNE and TRX in CVA lymphocytes were detected by WB method.Results: 1 EEAP contains chlorogenic acid,anthocyanin and alkaloids.The average recovery of flavonoids is 96.36%,which is 2.0%.The optimum extraction conditions of flavonoids from alcohol extract were as follows:ethanol concentration 65%,ratio of material to liquid 1:10(g:mL),extraction time 1 h,extraction times 3 times,and the content of flavonoids was 22.23 mg/g.2 EEAP could promote the proliferation of lymphocytes and decrease the concentration of IL-4,IL-5,IL-6,IL-13,GM-CSF,TNF-? and MDA.3 EEAP could inhibit the expression of TLR4,NF-?B(P65)mRNA,and EEAP could inhibit the protein expression of TLR4,NF-?B(P65),?-catenin,?-GSK-3? and 4-HNE.3 EEAP could increase the concentration of IFN-? and SOD(P < 0.05),while EEAP could inhibit the expression of TLR4,NF-?B(P65),?-catenin,?-GSK-3? and 4-HNE.Promote the protein expression of?-?-catenin,GSK-3? and Trx.Conclusion: 1 The quality standard of Anacyclus pyrethrum root has been established preliminarily,which provides the experimental basis for the further utilization and development of the root,and the extraction process of flavonoids from the alcohol extract of the root has been optimized by orthogonal test.And the process is stable and feasible.2 EEAP can improve and relieve the symptoms of CVA,and its effect may be to inhibit the release of inflammatory factors and the production of oxidative stress by inhibiting the TLR4/NF-?B signaling pathway and Wnt signaling pathway.Thus play its role in the treatment of CVA.