Preparation And Analysis Of Polygala Medicinal Materials And Nerve Cell Exosome

Purpose:1.We analyzed the material basis and possible mechanism of action of Polygalae Radix（P.R.）for the treatment of Alzheimer’s disease（AD）through network pharmacology to provide reference for the study of the mechanism of action and the active ingredients of P.R.2.Identification of chemical constituents of P.R.and preparation and analyzation of exosome-like nanoparticles from P.R.（PRELNs）were carried out to parse the material basis of PRELNs and explore the application values of PRELNs in the prevention and treatment of neurological diseases.3.Conducting research on cellular exosome sample pretreatment materials.Based on the study of the protective effect of the constituents in P.R.on Aβ_1-42-induced SH-SY5Y cells,the application practice of exosome preparation in different groups was carried out,the process of exosome isolation and preparation was optimized.Methods:1.Ingredients of P.R.were obtained through literatures and databases such as TCMID,Pubchem and FAFdrugs4.Targets of these ingredients were gained from Swiss,TCMSP and SEA databases.AD-related targets were acquired from the Dis Ge Net database.The intersection targets of the components and AD were selected by the Venn diagram.The protein interaction（PPI）network was constructed on the STRING database,and the core targets of disease treatment were built by the Cytoscape software.Enrichment analysis of GO and KEGG pathways was performed by David database.The gene mapping was carried out through the KEGG platform.The component-target-pathway network was constructed by Cytoscape software to screen out the potential active ingredients and treatment mechanism of P.R.treatment of AD.Finally,molecular docking between key active ingredients and key targets was performed using Autodock Vina software.2.The chemical components identification of P.R.was carried out by high performance liquid chromatography-mass spectrometry（HPLC-MS）.PRELNs were extracted and identified by ultrahigh-speed centrifugation and HPLC-MS.The characterizations of PRELNs were judged by transmission electron microscopy and Zetasizer Nano-ZS.The total protein content was determined by BCA method.The molecular weight distributions of proteins were measured via sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）.HPLC-MS was used to analyze the chemical constituents from PRELNs.3.The successful synthesis of Fe₃O₄@PDA-Ti⁴⁺was proved by transmission electron microscopy,Fourier transform mass spectrometer,inductively coupled plasma emission spectrometer and other instruments.The dosage of dopamine hydrochloride and titanium sulfate was optimized to determine the preparation conditions of Fe₃O₄@PDA-Ti⁴⁺materials.To find the optimal capture conditions of Fe₃O₄@PDA-Ti⁴⁺nanomaterials,the volume of medium,the amount of Fe₃O₄@PDA-Ti⁴⁺material,the incubation time of material and medium,and the elution time of ammonia water were optimized.Results:1.The results showed 66 active ingredients,377 AD intersection targets,including 17potential active components and 56 core targets.GO enrichment analysis yielded 556 items（P<0.05）.KEGG pathway analysis enriched 23 pathways（P<0.05）,which involve the pathway of PI3K-Akt,MAPK,Fox O and so on.Molecular docking results showed that the key active components had good binding activity to the key targets of AD treatment.2.33 and 39 compounds were identified from P.R and its lyophilized powder respectively.PRELNs were saucer-like vesicles with a particle size of 153.6 nm and a Zeta potential of-9.2m V.The protein concentration measured by BCA method was 1.22 mg/m L and the molecular weight distributions of proteins varied from 10 k Da to 170 k Da.A total of 31 ingredients were characterized from the PRELNs,13 of which did not exist in the lyophilized powder of P.R.3.The Aβ_1-42-injured concentration was determined to be 10μmol/L and the highest concentration for the lyophilized powder of P.R was 7.5μg/m L.The Fe₃O₄@PDA-Ti⁴⁺treatment conditions were screened and optimized as followed:2 mg of Fe₃O₄@PDA-Ti⁴⁺material was added into 5 ml cell culture supernatant medium,and the mixture was incubated on a shaker with gently shaking for 5 minutes at room temperature.Then the material was isolated from the mixture via magnet and continuously washed twice with PBS buffer.Finally,the material was eluted in a 10%ammonia aqueous solution for 5 min.Collecting 10%ammonia eluent for future useConclusions:1.P.R.treats AD with a multi-component,multi-target,and multi-channel integrated action method,to regulate phosphorylation,cell apoptosis,and resist inflammatory.2.The results indicated that PRELNs contain Tenuifoliose A,Onjisaponin B,PolygalasaponinⅩⅩⅩⅡand other ingredients,which may be the basis of its active functions,and 13ingredients may have special application value.3.The preparation conditions of Fe₃O₄@PDA-Ti⁴⁺nanomaterials separation were determined to achieve the efficient preparation of exosomes from different groups of SH-SY5Y cell.