The Research Report On Anti-toxoplasma Gondii Of Effective Substance Basis From Acer Tegmentosum Maxim In Changbai Mountain Area

OBJECTIVE:Study the chemical constituents in Acer tegmentosum Maxim and perform quality analysis,and to explore the anti-Toxoplasma gondii effect of the chemical constituents of A.tegmentosum.Based on the research methods of network pharmacology,the mechanism of the treatment of T.gondii was explored,and the chemical components of A.tegmentosum produced multiple signal pathways against T.gondii through multiple targets and multiple pathways.Using molecular docking methods,combined with network pharmacology,the isolated A.tegmentosum compound and key proteins were docked,and natural compounds with potential anti-T.gondii effects were screened.Method:1.The dried stem bark of A.tegmentosum was extracted with 90%Et OH under reflux,and the extract was distilled under reduced pressure to obtain an Et OH extract.Using solvent gradient extraction method,using PE,CH₂Cl₂and Et OAc for extraction in sequence,and vacuum distillation to obtain three extracts.A variety of chromatographic separation techniques are used to separate and purify the obtained PE,CH₂Cl₂and Et OAc extracts of different polarities.The structure is identified by the NMR spectrum(¹H-NMR and ¹³C-NMR)of the monomer compound and its physical and chemical properties.2.The content of salidroside and tyrosol,the chemical components separated from A.tegmentosum,was determined by high performance liquid chromatography（HPLC）(Venusil ASB C₁₈column:5μm,4.6×250 mm,mobile phase:15%methanol-water,Flow rate:1 m L·min^-1,temperature in the chromatographic column:40°C).3.Using the tetrazolium salt（MTT）colorimetric method to detect the anti-T.gondii effect of the monomeric compound isolated from A.tegmentosum,through human gastric epithelial cells（GES-1 cells）and GES-1 after infection with T.gondii.Calculate the selectivity index（SI）of the IC₅₀value of the cells to evaluate the protective effect of the single compound of A.tegmentosum against T.gondii.4.Predict the active ingredients in A.tegmentosum and sites targeted by T.gondii on the basis of network pharmacology.And combine the active ingredients in A.tegmentosum with sites targeted by Heat shock protein 70（TgHSP70）protein through molecular docking to ensure an active ingredient that plays a stronger role in anti-T.gondii activity and docking.Result:1.In this experiment,18 compounds were obtained from the Et OAc extract of A.tegmentosum and identified as tyrosol（1）,salidroside（2）,β-D-galactopyranoside（3）,dihydroconiferyl alcohol（4）,methyl（p-hydroxyphenyl）acetate（5）,4-hydroxybenzoic acid（6）,gallic acid（7）,ethyl gallate（8）,4-hydroxyphenylacetic acid（9）,2-（4-hydroxyphenyl）ethyl3,4,5-trihydroxybenzoate（10）,6-hydroxy-7-methoxycoumarin（11）,fraxetin（12）,asculetine（13）,scopoletin（14）,（+）-gallocatechin（15）,quercetin（16）,1,4-benzenedicarboxylic acid（17）,2-ethylhexyl phthalate（18）.The isolated compounds include 10 phenol compounds,4coumarin compounds,2 flavonoids,and 2 benzoic acid lipids.Compounds 3,4,5,6,8,9,10,11,12,13,17,18 are compounds isolated from this plant for the first time.Compounds 3,4,5,17,18 are the first compounds isolated from this Acer.2.The content of salidroside and tyrosol in the Et OH extract of A.tegmentosum was determined by content as 3.1227 mg/g and 1.0900 mg/g.The contents of salidroside and tyrosol in the water extract of A.tegmentosum were 2.14744 mg/g and 0.5458mg/g.3.In vitro experiment（MTT）,the compound 6’-O-galloylsalidroside,quercetin,grayanoside A,dihydroconiferyl alcohol,（-）-epigallocatechin-3-O-gallate,6-hydroxy-7-methoxycoumarin has a larger value than spi,It shows that these compounds are less cytotoxic in GES-1 cells.The IC₅₀（μM）of the nine tested compounds are all higher than the spi value,indicating that the compound is more toxic to T.gondii.Among them,the SI of quercetin,grayanoside A and6-hydroxy-7-methoxycoumarin is better than spi,indicating that these compounds may have better anti-T.gondii activity than spiramycin.4.Through network pharmacology and molecular docking,19 active components of A.tegmentosum were screened,involving 446 targets;molecular docking showed that the main active components have good binding activity with core targets.Enriched to1504 GO biological process,58 molecular function,31 cellular component;124KEGG pathways,including TNF,IL-6 and other signaling pathways.By molecularly docking the 19 compounds isolated from A.tegmentosum with the target protein TgHSP70,the compounds with better activity were screened out.Among them,the result of better activity and better docking effect is（+）-gallocatechin（-5.778）>asculetine（-5.682）>salidroside（-5.562）>2-（4-hydroxyphenyl）ethyl 3,4,5-trihydroxybenzoate（-5.392）>gallic acid（-5.310）.The anti-T.gondii activity of five monomercompounds（+）-gallocatechin,asculetine,salidroside,2-（4-hydroxyphenyl）ethyl 3,4,5-trihydroxybenzoate,and gallic acid obtained by molecular docking was evaluated and evaluated The ability of the monomer to bind to the TgHSP70 protein was evaluated.Conclusion:In this study,18 monomer compounds were separated from the Et OAc layer of the Et OH crude extract of A.tegmentosum,which provided a basis for further research on the mechanism of A.tegmentosum and the development and utilization of the medicinal plant resources.The in vitro experiment proved that the extract of A.tegmentosum has anti-T.gondii activity.Using network pharmacology and molecular docking methods to explore the mechanism of the treatment of T.gondii,this study initially reveals that A.tegmentosum may have multiple components,multiple targets,and multiple signaling pathways to play a role in the treatment of T.gondii.It provides a basis for in-depth research.This study has increased our understanding of the chemical composition of A.tegmentosum and provided a reliable theoretical basis for the development and application of A.tegmentosum.