| Objective:The chemical constituents of Alnus mandshurica(Callier)Hand.-Mazz.were studied and the compounds were identified by modern spectroscopy.The anti-tumor activity of compounds isolated in vitro was verified.Through network pharmacology and molecular docking,the mechanism of anti-tumor activity of A.mandshurica was further predicted,so as to provide reference for clarifying the pharmacodynamic material basis and mechanism of A.mandshurica.Methods:1.Ethanol extract was reflux by 90% ethanol,condensed by decompression to extract the crushed dried stem bark of A.mandshurica.The extract was condensed by decompression to obtain ethanol extract.Three kinds of extracts were obtained by solvent gradient extraction with CH2Cl2,Et OAc and n-Bu OH.The extracts of Et OAc from A.mandshurica were selected to separate the chemical components by a variety of chromatographic separation techniques,and the structure of monomer compounds was identified by spectroscopy.2.MTT method was used to evaluate the anti-tumor effect of A.mandshurica.The cell survival ratio of MDA-MB-231 cell line,A549 cell line and SMMC7721 cell line was determined by extraction and chemical composition of A.mandshurica.3.Combined network pharmacology and molecular docking techniques to investigate the mechanism of action of the active ingredients of A.mandshurica for the treatment of oncological diseases and the prediction of their targets.Results:1.In the process of studying the chemical constituents of ethyl acetate extract of A.mandshurica,a total of 23 chemical constituents were isolated.The structures of the compounds were determined by 1H-NMR and 13C-NMR,Their structures were named as tremuloidine(1),salicoside(2),4-Hydroxy-3-methoxybenzoic acid(3),3,4,5-trihydroxybenzenoic acid(4),3,4-dihydroxyben(5),4-hydroxycinnamic acid(6),eugenol(7),isograndidentatin A(8),isograndidentatin B(9),(+)-gallocatechin(10),hesperetin(11),7-methoxy dihydrokaempferol(12),isosakuranetin(13),dihydrokaempferol-4’-methyl ether(14),3,5-dihydroxy-7,4’-dimethoxyflavanone(15),naringenin(16),chrysin(17),rhamnetin(18),scopoletin(19),isoscopoletin(20),1,7-diphenylhept-4-en-3-one(21),betulinic acid(22),β-daucosterol(23).The isolated compounds were 9 phenols,9 flavonoids,2 coumarins,1 diphenylheptane,1terpenoid and 1 steroid.2.The results of in vitro experiments showed that the extract of Et OAc from A.mandshurica had a higher effect on the survival ratio of tumor cells.Compound 1、4、8、11 and 20 had a lower survival ratio against MDA-MB-231 cells,compound 1、4、8、14、17 and 22 had a lower survival ratio against A549 cells and compound 3、8、11、14、17 and 22 had a lower survival ratio against SMMC7721 cell line.Compound22 has the most obvious effect on A549 cells and SMMC7721 cells.Suggesting that A.mandshurica may have anti-tumor activity.3.Active components of A.mandshurica were screened through network pharmacology and molecular docking.Molecular docking showed that the main active components had good binding activities with core targets.Among them,1751 targets were involved,1751 were enriched to GO biological process,98 were enriched to Molecular function and 87 were cellular component.There were 148 KEGG pathways,including PI3K-Akt signaling pathways.Compounds of A.mandshurica and target protein EGFR were docked to screen out compounds with better activity.The results of isograndidentatin A docking protein were better than that of the original ligand.The docking protein scores of isograndidentatin A and the original ligand were-10.310 and-9.615,respectively.Conclusion:In this study,23 known compounds were isolated from A.mandshurica,among which compounds 2,5-11,15,17,20,21 and 22 were isolated from A.mandshurica for the first time.Compounds 2,8,9 and 20 were isolated from Alnus for the first time,which provided material basis for further research on the pharmacological action and mechanism of A.mandshurica.Compounds 1,3,4,8,11,14,17,20 and 22 showed good anti-tumor activity by in vitro assays.Network pharmacology and molecular docking methods were used to analyze the mechanism of A.mandshurica in treating tumor diseases,which preliminarily revealed that A.mandshurica may have multiple components and synergistically play anti-tumor effects through multiple targets and multiple signaling pathways,providing theoretical basis for further research.The research in this paper,to a certain extent,provides the research basis for the anti-tumor activity of A.mandshurica,and provides a scientific theoretical basis for further rational utilization and development of A.mandshurica. |
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