Extraction、the Structure Modification Of Atractylenolide And Protective Effect On PC12Cells

Rhizoma Atractylodis macrocephala,the dried rhizome of Atractylodes macrocephala Koidz,belongs to Compositae,sweet and wind,with the efficacy of strengthing spleen and replenishing Qi,eliminating dampness,promoting urination,hidroschesis and calming fetus,mainly produced in Zhejiang,Anhui,Hunan and Jiangxi.Atractylodes lactones are the main active ingredients with the effects of anti-inflammatory,anti-tumour,regulating gastrointestinal function,promoting the absorption of nutrients and other pharmacological activity. Therefore it is necessary to study on the extraction and separation, and on basis of this,the structural modifaction was performed to investigate the pharmacological activity of atractylenolide III and its derivatives.ObjectiveIn this study,Rhizoma Atractylodis macrocephalae was taken as the research object to search for the appropriate way of extracting and isolating atractylenolideIII,and structural modifaction was performed on the atractylenolide III to obtain derivative compounds,then their protective effect on PC12cells were detected.MethodsExtraction and isolation of atractylenolide:Rhizoma Atractylodis macrocephalae was soaked by ethyl acetate at room temperature,then the extract liquid was leaching and reduced pressure distilled to obtained the fluid extract,atractylenolide III was isolated by using repetitious silica gel column chromatography and the structure was identified by means of UV, IR,MS,’H-NMR and13C-NMR.Structural modifaction of atractylenolide:Select the appropriate reactant and catalyst to make the dehydration and acylation,using thin layer chromatography (TLC) to detect the reaction is complete or not.Cells activity detection:PC12cells apoptosis model was established by the serum limitation-induced cell death for3days, then divide the cells into the control group,model group and dosing group. Applied in treatment factors3days later, MTT analysis and Annexin V/PI flow cytometry were used to detect the pharmacological activity of atractylenolide Ⅲ and its derivatives.ResultsRhizoma Atractylodis macrocephalae was soaked by ethyl acetate at room temperature and pure atractylenolide Ⅲ was isolated by using repetitious silica gel column chromatography,the appearance characters and solubility were consistent with the reports,the structure was identified by means of UV, IR, MS,1H-NMR and13C-NMR,and the yield coefficient was0.06%.Atractylenolide III was dehydrated into derivative L1with concentrated sulfuric acid and acetic anhydride,and reacted with acetic anhydride to get acetylation derivative L2by using pyridine as catalyst,their structures were identified by means of UV, IR, MS,1H-NMR and13C-NMR.MTT analysis showed the OD value of dosing group with atractylenolide III and compound L1was increased compared with the model group,while the OD value of dosing group with compound L2was not changed obviously,and flow cytometry results also indicated dosing group with atractylenolide Ⅲ and compound L1could reduce the apoptosis rate of PC12cells,while dosing group with compound L2did not show activity obviously.ConclusionThe extraction method used in the study without crushing and sifting, soaked at room temperature,the solvent consumption is small,atractylenolide Ⅲ was obtained after separation and purification with high purity,this method was processed simply,operated conveniently and low cost, it’s suitable for the large-scale extraction and separation.Atractylenolide Ⅲ was dehydrated into derivative L1with concentrated sulfuric acid and acetic anhydride,the production rate could exceed90%,and the reaction was rapid,but the quantity of concentrated sulfuric acid should be strictly controled, and shaken well in time to avoid carbonation.Atractylenolide Ⅲ reacted with acetic anhydride to get acetylation derivative L2by using pyridine as catalyst,the reaction was easy to proceeded at room temperature and the production rate could exceed95%,but the amount of acetic anhydride should be excess.The results of MTT analysis and Annexin V/PI flow cytometry showed atractylenolide III and compound L1could improve the PC12cells activity,reduce the apoptosis rate of PC12cells,while compound L2did not show effective activity,this difference may related with the-OH in the molecular structure.