Analysis Of Components Of Ginkgo Biloba Extract And Study On The Regulatory Effect Of Its Active Components On One-carbon Metabolism In Lung Cance

Ginkgo biloba has the functions of astringing the lungs,relieving asthma,promoting blood circulation and removing blood stasis,and relieving pain.The active ingredients ginkgolides contained in the extract have the therapeutic effect on cancer and the regulation of one-carbon metabolism of lung cancer.The research topics are concerned.In order to grasp the pharmacodynamic material basis of Ginkgo biloba extract(GBE)and understand its metabolic mechanism on lung cancer,this study used LC-MS/MS technology and combined with network pharmacology to analyze the ginkgolide chemical constituents of GBE.Furthermore,metabolomics method was used,combined with cell proliferation experiments,it was found that ginkgolide could inhibit the growth of lung cancer cells by regulating the one-carbon metabolic pathway.The research content of this dissertation mainly includes the following four parts:1.Chemical composition and functional analysis of Ginkgo biloba extractFirst,the chemical composition analysis of GBE was carried out by LC-MS/MS technology.Through high-resolution MS and MS/MS spectral analysis,combined with standard product comparison,database search,literature comparison and mass spectrometry analysis,a total of Identification of 44 chemical constituents,including 31 flavonoids,5 esters(including ginkgolides A,B,C,bilobalide and methylparaben)and 8 organic acids(including ginkgolic acid),salicylic acid,caffeic acid,vanillic acid,and coumarin,etc.),and the structures of 15 compounds were confirmed by standard products.In addition,28 constituents in GBE were predicted to have lung cancer therapeutic activity by network pharmacology method,of which 10 compounds were consistent with the identified constituents(4 ginkgolides,5 flavonoids and 1 phenolic acid).It is found that ginkgolide A,ginkgolide C and ginkgolide B had higher drug similarity(DL)values,while bilobalide had the highest oral bioavailability(OB)value,it was speculated that ginkgolides has certain potential for the treatment of lung cancer.Further through the action targets of these active ingredients and lung cancer-related targets,a"drug-functional ingredient-target gene-disease" network was established,and the pathways related to lipid metabolism,JNK/SAPK signaling pathway,MAPK signaling pathway and TNF signaling pathway were screened out.They also closely related 20 key target genes.The results suggest that the functional components in GBE may affect lung cancer by regulating multiple pathways,and provide a molecular basis for subsequent studies on the metabolic regulation of active components in GBE.Further quantitative analysis of the above four ginkgolides in GBE and ginkgolide injection(GLs)at dosed concentrations were carried out to provide a reference for discussing the effect of ginkgolide on lung cancer treatment.2.Effects of Ginkgo biloba extract and its active components on the growth and proliferation of lung cancer cellsOn the basis of the above research,combined with literature research,this study selected four ginkgolides with certain potential in the treatment of lung cancer[ginkgolide A(GA),ginkgolide B(GB),ginkgolide C(GC),bilobalide(BB)]as the key investigation object,the effect of GBE and GLs whose main components are these four ginkgolides on the proliferation activity of A549 lung cancer cells was explored by CCK-8 experiment.The results showed that both GBE and GLs had a significant inhibitory effect on the proliferation of A549 lung cancer cells in a certain concentration-dependent manner,and the median lethal rates were about 270 μg/mL and 150 μg/mL,respectively.Combined with the quantitative results,the contents of GA,GB,GC and BB in GBE of 270 μg/mL and GLs of 150 μg/mL were 21.61 μg/mL,1.93 μg/mL,3.08 μg/mL,43.08 μg/mL and 27.51 μg/mL,2.67μg/mL,5.83 μg/mL,70.10 μg/mL,respectively.The contents of 4 ginkgolides in GLs of 150 μg/mL were higher than that of GBE of 270 μg/mL.It was inferred from the analysis that although other components in GBE and ginkgolides jointly affected the proliferation of lung cancer cells,increasing the dosage of 4 ginkgolides,especially BB and GC,which can also effectively inhibit the proliferation of lung cancer cells.At the same time,GA,GB,GC and BB also had a certain inhibitory effect on the proliferation of A549 cells,but when the dose reached 100 μg/mL,the cell survival rate was still more than 50%.The above results showed that ginkgolides can inhibit the proliferation of lung cancer cells,and the combined effect of four ginkgolides is better than that of four ginkgolides monomer alone.This result provides valuable information for ginkgolides to be used in the treatment of lung cancer.3.Study on the metabolic regulation of ginkgolide on A549 lung cancer cellsIn this study,a non-targeted metabolomic study was performed on A549 lung cancer cells before and after administration of GLs using LC-MS analysis methods in positive and negative ion detection modes.Through multivariate statistical analysis,54 differential metabolites,including lipids,purines and amino acids,were found and identified.The results of metabolic pathway enrichment analysis showed that glutathione metabolism,amino acid metabolism(including tryptophan metabolism,aspartate and glutamate metabolism,cystine and methionine metabolism,arginine and proline metabolism,and Amino sugar and nucleotide sugar metabolism),lipid metabolism(including sphingolipid metabolism,glycerophospholipid metabolism),purine metabolism and folic acid cycle were all changed,and the changes in purine metabolism and glutathione metabolism were more significant.Therefore,it is inferred that ginkgolides affect the proliferation of A549 lung cancer cells mainly by regulating the one-carbon metabolic pathway.4.The regulation of ginkgolide on one-carbon metabolism of A549 lung cancer cellsIn this study,a targeted metabolomic analysis method based on LC-MS/MS technology was first established for the one-carbon metabolic pathway closely related to lung cancer and its 21 metabolites.By comparing the A549 cell extracts between the GLs-administered group and the blank control group,16 metabolites were found to have significant changes,especially purine,glutathione,tetrahydrofolate and adenosine metabolites.The results suggest that GLs may inhibit AHCY,leading to the down-regulation of adenosine and the up-regulation of S-adenosylmethionine,S-adenosylhomocysteine and glutathione.Therefore,the regulation increased cellular immunity in response to ROS-induced oxidative stress damage,and achieve the effect of treating lung cancer.It is also possible that folate cycle is regulated by DHFR,MTHFD and MTHFR,which ultimately inhibits the growth and proliferation of A549 cells.The above results provide a new molecular basis and theoretical reference for revealing the regulatory effect of GLs components on the one-carbon metabolic pathway of A549 lung cancer cells.In this study,by combining network pharmacology and metabolomic approaches,we found that GLs in GBE can inhibit the proliferation of A549 cells.GLs have significant regulatory effects on metabolic pathways related to one-carbon metabolism,such as purine metabolism,GSH metabolism,and methionine cycle.Targeted studies of one-carbon metabolism have shown that GLs may induce the upregulation of SAM,SAH and GSH by inhibiting AHCY in response to ROS-induced oxidative stress damage,and may also regulate folate cycle through DHFR,MTHFD and MTHFR,which ultimately inhibit the growth and proliferation of A549 cells.