Study On Screening Of Active Substances For Improving Learning And Memory Based On Target Metabolomics Of C.elegans

Alzheimer’s disease（AD）is a neurodegenerative disease characterized by learning and memory impairment.At present,it is recognized that excessive aggregation ofβ-amyloid protein(Aβ_1-42)in brain tissue produces neurotoxicity,oxidative damage,mitophagy,and abnormal metabolites in vivo,which are the main causes of AD symptoms such as dementia.Gastrodiae Rhizoma,as a medicinal and food homologous substance,has antioxidant,antidepressant,neuroprotective effects and can alleviate neurological diseases.It is of great significance in the treatment of Alzheimer’s disease,Parkinson’s disease,depression and other neurological diseases,and has good development and application prospects.Metabonomics can effectively identify biomarkers,but there is no systematic report on the study of active substances in Gastrodia elata for improving learning and memory and biomarkers related to its efficacy based on targeted metabolomics.On this basis,the AD models of C.elegans CL4176 and CL2355 were used to study the improvement effect of Gastrodia elata on learning and memory impairment.The active substances for learning and memory improvement were screened byβ-amyloid protein content determination experiment,and the screening results were verified by chemotaxis experiment.On this basis,the effects of six main active components of Gastrodia elata Blume on the content ofβ-amyloid protein in C.elegans were investigated.Taking the content ofβ-amyloid protein as an index,it was found that the active component of Gastrodia elata Blume balisonin E had a significant effect on reducing Aβ-amyloid protein in AD C.elegans.The research results are as follows:（1）The active substances to improve learning and memory in Gastrodia elata were screened by C.elegans paralysis experiment,β-amyloid protein content determination and Na Cl chemotaxis experiment.The results of paralysis experiment showed that the total extract of Gastrodia elata and the positive drug Hup A could delay the paralysis time of AD C.elegans CL4176,and the optimal administration concentrations of total extract of Gastrodia elata and Hup A were 100μg/m L and 8μg/m L,respectively.On this basis,the effects of six main active components in Gastrodia elata on the content ofβ-amyloid protein in C.elegans were investigated.The content ofβ-amyloid protein was used as an indicator to screen out that the active component balisonin E in Gastrodia elata had significant improvement on the symptoms of AD.The recovery effect of the main active components in Gastrodia elata on the learning and memory of AD C.elegans CL2355 was evaluated by C.elegans chemotaxis experiment,and the screening results of the active components in Gastrodia elata were further verified.The results showed that Gastrodia elata could delay the paralysis of AD C.elegans,reduce the content ofβ-amyloid protein in C.elegans,and improve the learning and memory ability,and the effect of Barisin E was significant.（2）Review the related literature of AD,Parkinson and other brain neurological diseases,sorting out the endogenous small molecules of AD diseases.Based on the targeted metabolomics of C.elegans,an efficient and sensitive HPLC-MS/MS method was established to quantitatively determine the contents of GABA,ACH,L-Phe and other small molecules in different groups of C.elegans.Gastrodia elata can make the content of target small molecules related to learning and memory in AD C.elegans back to the healthy level.When the concentration ratio of the analyte to the internal standard was 0.08-16 ng·m L^-1（positive mode）and 0.4-40 ng·m L^-1（negative mode）,there was a good linear relationship between the concentration ratio and the ratio of peak area,and the linear relationship was R~2>0.9950;RSD value of precision was less than 15%;the accuracy RE was-8.25%～3.55%;the extraction recovery was 86.26%-108.73%;the matrix effect was 2.7%～13.46%.（3）Based on the targeted metabolomics model of C.elegans,the biomarkers related to the improvement of learning and memory efficacy of balisonin E were screened.Through the in vivo metabolism experiment of balisonin E on AD C.elegans,according to the content change results of C.elegans samples between groups,PCA and OPLS-DA analysis methods were used to screen out the biomarkers related to the efficacy of balisonin E,such as L-leu,L-Phe and ACH.Balisonin E can make the endogenous small molecules in C.elegans indicating AD symptoms back to the healthy level.