Studyonthe Pharmacodynamic Substance Basisof Ginseng-Ziziphi Spinosaein Semenand Their Neuroprotective Effects On ROT Induced Parkinson’s Disease Model

The drug pair is the compatibility combination of two traditional Chinese medicines based on the seven emotions and the law of"opposites complement each other,opposites must complement each other,opposites make complement each other,and opposites make complement each other".In the clinical practice of traditional Chinese medicine,two drugs are often used at the same time,which is simpler than the compound of multiple traditional Chinese medicine,and has better curative effect than single traditional Chinese medicine,and can also reduce toxicity and side effects.Therefore,the study of the compatibility characteristics of drug pairs is not only conducive to understanding the rules of prescription of traditional Chinese medicine,but also provides valuable reference for clinical practice of traditional Chinese medicine.Ginseng-ziziphi spinosae semen drug pair is the accumulation of experience in clinical use of traditional Chinese medicine,which has been used for thousands of years.Ginseng can calm the nerves and nourish the mind,replenish the spleen and lungs,regulate the central nervous system,and treat neurasthenia and neurodegenerative psychosis.Ziziphi spinosae semen can calm the mind,improve people’s memory,and treat insomnia,forgetfulness,inattention,and heart deficiency.The combination of the two drugs has the effect of calming the nerves and tonifying the body.It is a common drug pair for the treatment of central nervous system diseases.Objective:In this paper,the ginseng-ziziphi spinosae semen drug pair was used as the research object,and the optimal compatibility ratio of ginseng-ziziphi spinosae semen for neuroprotection was obtained by mathematical correlation analysis and activity screening.On this basis,modern analytical technology and biological activity research were used to study the material basis and mechanism of the compatibility ratio in the treatment of parkinson’s disease,providing a theoretical basis for the better development of the traditional drug pair of ginseng and ziziphi spinosae semen.Methods:1.Based on data mining of ancient and modern prescriptions,the compatibility characteristics and extensional analysis of ginseng-ziziphi spinosae semen drug pair:In this study,excel and spss modeler software were used for data mining of the ancient and modern prescriptions of the ginseng and ziziphi spinosae semen medicinal pair.Apriori algorithm was used to analyze the compatibility characteristics of the ginseng and ziziphi spinosae semen medicinal flavor,such as the processed varieties,the applied dose,the compatibility ratio and the efficacy of the same traditional Chinese medicine,to obtain the commonly used compatibility ratio and the dosage.The compatibility characteristics and medication rules of the ginseng-ziziphi spinosae semen drug pair were revealed,which has guiding significance for further rational application and development of drugs and clinical practice.2.Based on the combination of entropy weight method and grey correlation degree method,the best compatibility ratio of ginseng and ziziphi spinosae semen for neuroprotection was selected:In this study,the grey correlation analysis method was used to establish a correlation analysis between the neuroprotective activity and the content of chemical components of different compatibility of ginseng and jujube seed,and to systematically and comprehensively evaluate the relationship between the activity and the components.To further clarify the functional characteristics of natural products with multi-components and multi-targets,and optimize the best compatibility ratio of ginseng and ziziphi spinosae semen medicine for neuroprotective activity.3.Study on the material basis of the effect of the best compatible extract of ginseng and ziziphi spinosae semen on neuroprotection in the treatment of parkinson’s disease:The chemical components of the best compatible extract of ginseng and ziziphi spinosae semen for neuroprotection were studied by high performance liquid chromatography,liquid chromatography-mass spectrometry and other modern analytical techniques;At the same time,the Agilent-DAD-HPLC chromatograph was used to establish its HPLC fingerprint,which provided a theoretical basis for the further development and application of the ginseng-ziziphi spinosae semen drug pair.4.Study on the mechanism of the best compatible extract of ginseng and ziziphi spinosae semen on neuroprotection in the treatment of parkinson’s disease:First of all,the LC-MS data obtained from the experiment were applied to the target prediction of network pharmacology,and the active components and potential targets of the best compatible extract of ginseng and ziziphi spinosae semen for neuroprotection in the treatment of parkinson’s disease were obtained.Furthermore,the molecular docking technology was used to predict the potential mechanism of the treatment of Parkinson’s disease with the best compatible extract of ginseng-ziziphi spinosae semen for neuroprotection.On this basis,we used rotenone to induce the establishment of parkinson’s model in vitro and in vivo,and used modern biological techniques such as flow cytometry,immunofluorescence,enzyme-linked immunosorbent assay,morphological research,animal behavior,immunohistochemistry,protein blotting and real-time fluorescence quantification to study the mechanism of its treatment of parkinson’s disease.Results:1.Based on data mining of ancient and modern prescriptions,the compatibility characteristics and extensional analysis of ginseng-ziziphi spinosae semen drug pair:The data mining method was used to retrieve and contain 598 prescriptions containing ginseng and jujube seed,which originated from 162 medical books.The prescriptions included mainly originated from the Song,Ming and Qing dynasties,and were classified into six diseases,including the internal medicine of traditional Chinese medicine,gynecology and pediatrics.The applied dose of ginseng formula is mainly distributed in the range of 11-15 g,21-30 g and 31-50 g,and that of ziziphi spinosae semen formula is mainly distributed in the range of 16-30 g,both of which are strongly related to the efficacy of calming and tonifying the nerves.The correlation between the dose range and efficacy shows that the two drugs can be appropriately added or decreased according to the main symptoms of clinical medication.The statistical analysis of the frequency of the matching of ginseng and ziziphi spinosae semen showed that there was a certain correlation between the efficacy of the prescription and the matching of the two,which was mainly divided into five compatibility ratios of 1:1,1:2,2:1,3:2 and 1:4,with the highest frequency of 1:1.At this time,the target prescription had a strong effect of calming and tonifying.2.Based on the combination of entropy weight method and grey correlation degree method,the best compatibility ratio of ginseng-ziziphi spinosae semen for neuroprotection was selected:Taking the commonly used compatibility ratio（1:1,1:2,2:1,3:2,1:4）of ginseng-ziziphi spinosae semen obtained through data mining as the research object,the quality evaluation model of ginseng-ziziphi spinosae semen with different proportions was established based on the comprehensive evaluation of the content of nine components,antioxidant activity in vitro,cell antioxidant activity,loss of cell excitability induced by glutamic acid and cytotoxicity induced by rotenone.After weighting the 16 quality characteristic indexes of the ginseng-ziziphi spinosae semen ratio,the ginseng-ziziphi spinosae semen ratio（1:1）was selected as the best ratio for neuroprotection.3.Study on the pharmacodynamic substance basis of the best compatible extract of ginseng and ziziphi spinosae semen medicine for neuroprotection:HPLC-MS was used to study the chemical constituents of ginseng-ziziphi spinosae semen medicinal pairs with the best compatibility ratio for neuroprotection.A total of 56 compounds were identified,including jujuboside A,juboside B,ginsenoside Rg₁,ginsenoside Rb₁,ginsenoside Rg₆,flavonoid compound spironocin,polyphenol compound neochlorogenic acid,triterpenoid compound oleanolic acid,fatty acid compound linoleic acid,stearamide,etc.HPLC was used to establish the fingerprint of the best compatible extract by using the similarity evaluation software of traditional chinese medicine chromatographic fingerprint and the multi-point correction method.Through the processing of the HPLC chromatogram of 12 batches of the best compatible extracts of ginseng and ziziphi spinosae semen for nerve protection,a total of17 common peaks were marked.Through comparison of retention time and chromatographic behavior,11 compounds including spironocin,swertisin,ginsenoside Rg₃,ginsenoside Rg₁,ginsenoside Re,ginsenoside Rb₁,jujuboside A,ginsenoside Rb₂,ginsenoside Rb₃,jujuboside B,and ginsenoside Rd were identified by comparison of reference substance.The pharmacodynamic material basis of ginseng-ziziphi spinosae semen seed neuroprotective extract was basically clarified.4.Study on the mechanism of the best compatible extract of ginseng and ziziphi spinosae semen on neuroprotection in the treatment of parkinson’s disease:In this study,through network pharmacology and molecular docking,the best compatible extract of ginseng and ziziphi spinosae seme for neuroprotection in the treatment of parkinson’s disease was obtained.The potential targets of action were AKT1,TNF,TP53,CASP3,PTGS2.Pathway analysis showed that the treatment of parkinson’s disease with the best combination of ginseng and ziziphi spinosae semen on neuroprotection mainly involves MAPK signal pathway,PI3K-Akt signal pathway,apoptosis signal pathway,neurotrophin signal pathway,etc.The study of cell model in vitro showed that the extract of drug pair can play a role in repairing oxidative stress damage by reducing ROS level in SH-SY5Y cells after ROT modeling,inhibiting lipid oxidation,and improving the activity of antioxidant enzymes.At the same time,it can reduce TNF-α、IL-6 and IL-1βThe expression of inflammatory factors plays an anti-inflammatory role;The m RNA of the key factors in the pathway was determined by RT-q PCR.The results showed that the best compatible extract of ginseng and ziziphi spinosae semen for neuroprotection could inhibit the expression of apoptotic protein Caspes3by regulating the expression of PI3K and Akt in the PI3K-Akt pathway,and play a role in the treatment of parkinson’s disease.In vivo animal model research shows that the best compatibility extract of ginseng and ziziphi spinosae semen medicine for neuroprotection can effectively slow down the expression of oxidative stress and inflammatory factors in the serum of rats in the model group,and increase the content of neurotrophin and dopamine in the brain of rats in the model group.The results of protein immunoblotting experiments indicate that the best compatible extract of ginseng and ziziphi spinosae semen medicine for neuroprotection can play a therapeutic role in Parkinson’s disease by acting on the PI3K/Akt pathway.The above research results indicate that the best combination extract of ginseng and ziziphi spinosae semen medicine for neuroprotection has a therapeutic effect on Parkinson’s disease through multiple pathways,directions,and targets,which is in line with the overall regulation of traditional Chinese medicine treatment characteristics.Conclusion:In this experiment,data mining and grey correlation analysis were used to obtain the best compatibility ratio of ginseng and jujube seed medicine for neuroprotection.On this basis,modern biotechnology research such as mass spectrometry,HPLC fingerprint,immunohistochemistry,western blot,etc.were used to preliminarily discuss the best compatibility of ginseng and ziziphi spinosae semen medicine for neuroprotection and the mechanism of action,which laid a theoretical foundation for better development and utilization of ginseng and ziziphi spinosae semen medicine.