Screening Of Active Substances From Rubus Chingii Hu Against Alzheimer’s Disease

Object:The number of patients with Alzheimer’s disease（AD）has greatly increased with the aging of the world population and the growth of average life span.It is urgent to find effective drugs to control and alleviate AD.Literature research shows,The dichloromethane extract of Rubus chingii Hu can improve the learning and memory ability of natural aging rats and kidney yang deficiency dementia rats.In this subject,the dichloromethane extract of Rubus chingii Hu was isolated and the activity of monomers was studied in order to find the active substance basis of its anti-Alzheimer’s disease and verify whether these compounds have medicinal effects.That provides the basis for the development of Anti-Alzheimer’s drug.Methods:1.Extraction and purification of Rubus chingii Hu using traditional and modern chromatography techniques:Using 80%ethanol solvent and under high pressure steam reflux,After the extract was concentrated which sequentially extraction with different solvents and the dichloromethane extract is re-extracted.Use traditional and modern chromatography technology to separated and purificated by silica gel column,ODS column,gel column,preparative column,etc.Use NMR、MS to identify the structure of the resulting monomer compound.2.Preliminary Screening of Active Compounds Using Molecular Docking:Finding Alzheimer’s disease-related receptor proteins through literature and PDB databases and find the 22 target proteins of definite ligands and anti-AD drugs entering clinical phase I.Target protein and chemical formula structure processing and Molecular docking after these import into Discovery Studio 4.0.The compound is a potential active compound when it interacts with the target protein’s docking energy Lib Dock Score is greater than or equal to 0 and it can be used with 5 or more.3.Anti-AD activity study of those compounds using AD in vitro cell model:The H₂O₂ induced PC12 cells to establish H₂O₂ cell injury model.Those active compounds from the molecular docking method was screened for drug concentration without cytotoxicity by the MTT cytotoxicity test.Choose three concentrations of high,medium and low for model administration within a safe concentration range,and add CCK-8 after culture for a period of time to detect cell viability.Result:1.Finally,14 monomer compounds were identified,which were respectively caroten（e1）,β-sitosterol（2）,4-hexane-1-pentadecenene（3）,ethyl heptadecanoate（4）,1H-indole-3-formaldehyde（5）,aurantiamide acetate（6）,p-hydroxybenzaldehyde（7）,trans-p-hydroxycinnamaldehyde（8）,emodin（9）,glycyrrhricin（10）,isoliquiritigenin（11）,β-sitosterol palmitate（12）,palmitic acid（13）,ellagic acid（14）.Among them,compounds 3-5 are obtained from plants of this family for the first time,and compounds 6 and 9-11 are obtained from Rubus palmatum for the first time.2.The results show that these 7 compounds are all potential active compounds and have no obvious bias in the five different types of target proteins.3.The results showed that the safe concentration of each compound was as follows:when the concentration of ethyl heptadecanoate is 20～100μmol/L,the concentration of 1H-indole-3-formaldehyde is 40～80μmol/L,the concentration of aurantiamide acetate is 20～100μmol/L,the concentration of emodin is 40～100μmol/L,the concentration of liquiritigenin is 10～80μmol/L,and the concentration of isoliquiritigenin is 40～100μmol/L.Ethyl heptadecanoate,1H-indole-3-formaldehyde,aurantiamide acetate,emodin and isoliquiritigenin were used at three concentrations of 40μmol/L,80μmol/L and 100μmol/L and glycyrrhricin was used at three concentrations of 20μmol/L,40μmol/L and 80μmol/L.The results show these six compounds have the ability to inhibit the damage of H₂O₂ cells.All of them have anti-AD activity.Conclusion:In this study,This dichloromethane part from Rubus chingii Hu’s anti-AD active part for compound isolation.7 compounds were found for the first time by Rubus chingii Hu including 4-hexane-1-25ene,ethyl heptadecanoate,1H-indole-3-formaldehyde,aurantiamide acetate,emodin,liquiritigenin and isoliquiritigenin.Then,the molecular docking find that they may have anti-Alzheimer’s disease activity.Therefore,experiments were performed on 6 of these compounds with a purity of more than 98%to construct a PC12 cell damage model by H₂O_2.The results showed that ethyl heptadecanoate,1H-indole-3-carboxaldehyde,aurantiamide acetate,emodin,glycyrrhizin,and isoliquiritin all have anti-AD activity.