Research On The Discovery Of Lead Compounds With Neuroprotective Effects Based On The Herbal Pair Of Radix Dioscorea

Background:Neurodegenerative disease,such as Alzheimer’s disease(AD),Parkinson’s disease(PD)and so on,which widely exist in old-aged groups and seriously threat to their health and life.So,the method of studying the treatment of these neurodegenerative diseases is current research hotspot.Presently,in the terms of pathogenesis and theory of treatment at neurodegenerative disease,people have already made a great progress.However,there are still lack of effective treatment prescription and medicine in clinical.At present,there are existed the treatment of neurodegenerative diseases,such as electro acupuncture,western medicine,gene therapy and so on.But the general effect is not very well and costly.Moreover,they are bad for human’s body.Drug treatment is currently the main method of such diseases at home and abroad.For this reason,finding a safe and efficient drug is still an important direction in treating such disease.Research methods:This research based on the theory of couplet medicine and the combination principle.We choose Dioscoreae Spongipsae Rhizoma couplet medicine as study objects.Dioscoreae Spongipsae Rhizoma and Alpinia oxyphylla couplet medicines are originated from a traditional Chinese prescription Bixie Fenqing Drink,Yangshi Jiacang Prescription.Modern pharmacology researches showed that both Dioscoreae Spongipsae Rhizoma and Alpinia oxyphylla exists the activity of neuroprotection.Diosgenin and amino acid are effective component in Dioscoreae Spongipsae Rhizoma and Alpinia oxyphylla respective.In clinical,diosgenin showed the activity of neuroprotection.Furthermore,amino acid is an important active component of Alpinia oxyphylla and takes the effects of healthcare and tonifying.Hence,this study is under the guidance of theory of TCM,based on the theory of couplet medicine and the combination principle.We chose diosgenin as a basic core and introduced the amino acids into its C-3 hydroxyl through the esterification reaction and deprotection reaction,a series of diosgenin-amino acids derivatives had obtained.In the aspect of bioactivity evaluation,cell model and quail chorioallantoic membrane model were being used by evaluating the derivative’s neuroprotective activity.Through optimization of experiment operation,a new method,quail chorioallantoic membrane model,is tentative established based on the improvement and development of chicken chorioallantoic membrane(CAM)model.Cell bioactivity evaluation is mainly selected H9c2 and SH-SY5Y.MTT assay was used to evaluate the cell toxicity and neuroprotective activity of derivatives.In addition,Giemsa and DAPI stain method was used to observe the morphology variation of SH-SY5Y after drug administration.The quail chorioallantoic membrane experiment is further illustrated the effect of neuroprotective activity after drug administration.Research results:In terms of chemistry,eighteen diosgenin-amino acids derivatives were obtained by chemical combination and their structures was determined by 1H-NMR、13C-NMR and HR-MS.Then the melting point and optical rotation were measured.The determination of optical rotation displayed that the configuration of those derivatives didn’t changed.In the aspect of cell activity,H9c2 and tert-butyl hydroperoxide induced SH-SY5Y was used to evaluate the cell toxicity and neuroprotective activity of derivatives respectively.The result of combining screen showed that DG-15 displayed the good neuroprotective activity and low cell toxicity.As for the quail chorioallantoic membrane model evaluation,the result showed that DG-15 can promote the generation of microvascular.Which indicated DG-15 had a positive effect of cardiovascular and neuroprotection.Conclusion:This research totally got eighteen diosgenin-amino acids derivatives,eight of them are new.At the same time,bioactivity evaluation of those derivatives carried out.Through MTT experiment,we found that derivatives with protective radical cannot capable of being target compound owing to their high cell toxicity.Eventually,DG-15 was selected as target compound because of its low cell toxicity and good neuroprotective activity.It’s cell toxicity lower than diosgenin.Furthermore,the neuroprotective activity is better than diosgenin and positive drug edaravone.Further stain results showed that DG-15 take an obvious protection effect of tert-butyl hydroperoxide induced SH-SY5Y no matter from cell’s morphology and amount.Result of quail chorioallantoic membrane model evaluation also demonstrated DG-15 can advance the generation of microvascular.The amount of microvascular of low dose groups(19.5±9.59)is apparently more than blank group(9±3.90).In summary,this study is based on theory of couplet medicine,through combinating the effective components of similar pharmacological action to realize the effects of synergism and attenuation.In addition,based on the model of CAM,we preliminary established a new evaluation method,quail chorioallantoic membrane model,to solve the deficiency of CAM.For example,embryonated egg may cause the experimental instability owing to their too many varieties.But quail embryo’s egg could reduce such differential because of their single species relatively.The tentatively establishment of new model can bring an idea for other drug experimental researches.