| Objective: Firstly,D.nobile samples were investigated to examine the influence patterns of external environmental factors on quality control marker of Chinese Pharmacopoeia.Secondly,in accordance with the studies on high-throughput secondary metabolomics,the influence of the external environment on the broad-spectrum material base of D.nobile was evaluated.Then,determinants of secondary metabolite synthesis would be identified and the dominant chemical composition would be excavated.Finally,the network pharmacology method was used to predict the potential effects on pharmacological efficacy,which laid the foundation for the improvement of the quality standard from the analysis-oriented to the efficacy-oriented.Methods: 1.Collection of D.nobile samples under different environmental factors: Samples of D.nobile were collected according to the collecting place,altitude and light.The stems of fresh D.nobile were separated,cut into sections,dried,crushed into powder and screened through No.3 sieve.2.Determination of quality control marker in Chinese Pharmacopoeia of D.nobile: According to Chinese Pharmacopoeia(2015),the content of dendrobine in D.nobile was detected.The sample was extracted by 0.05% formic acid-methanol solution and determined by GC.Naphthalene was used as the internal standard,and the flame ionization detector(FID)was used as the detector.To screen the main environmental factors that affected the content of dendrobine in D.nobile.3.Secondary metabolics analysis of D.nobile:UPLC-Q/TOF-MS was used for high-throughput detection of the secondary metabolite spectrum of D.nobile.The structure of the dominant chemical component was speculated using literature and database.4.Predictive analysis of the efficacy of dominant chemical components in D.nobile by network pharmacology: The possible target proteins of the dominant chemical components were screened by network pharmacology method.The enrichment analysis of disease,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway were used to explore the possible pharmacological effects and mechanisms.Results: 1.Samples collection: A total of 146 samples of D.nobile were collected in Chishui,Guizhou and Haikou,Hainan based on the three factors including collecting place,altitude and light.2.Altitude was the key factor affecting the content of dendrobine in D.nobile: In the samples collected this time,it was found that the effects of the collecting place and light on the content of dendrobine were not significant.The content of dendrobine in D.nobile grown at 692 m was significantly higher than other altitudes(P<0.05).3.Altitude was also a key factor affecting the profile of other secondary metabolites in D.nobile: The secondary metabolite spectrum of each sample was established using UPLCQ/TOF-MS technology.PCA analysis showed,in the samples collected this time,collecting place,light had no significant effect on the secondary metabolic product profiles.However,the profiles of the samples grown at altitude of 692 m had significant difference from 44 m,338 m and 508 m.4.Excavation of the dominant chemical composition of imitation wild D.nobile at altitude of 692 m: The results of HRMS and OPLS-DA suggested that there were 11 different components in samples grown at altitude of 692 m compared with 44 m,338 m,508 m.And there were 12 different components in samples grown at altitude of 692 m compared with all altitude in Chishui,Guizhou.There were 9 common components in the two screening results,which may be the dominant chemical components of D.nobile grown at altitude of 692 m.The 9 components were identified by combining high-resolution molecular weigh,secondary mass spectrometry fragment information,databases and literatures.The presumed results showed 5 of them were alkaloids,2 were amino acid derivatives,1 was aliphatic organic acid and 1 has not been identified.5.The efficacy predictions of newly discovered dominant chemical composition: The targets of 9 dominant chemical components were acquired using the Swiss Target Prediction database.One of all had no relevant targets.A total of 225 targets were selected.The network diagram of targets and dominant chemical components was builted.The threshold was set as degree≥50.3 key characteristic chemical compounds were screened,including 1-[[(1,1-Dimethylethoxy)carbonyl] amino]-α-hydroxy-3-methoxycyclobutaneac etic acid,2-Methyl-1H-indol-7-yl-β-D-mannopyranoside and 6-hydroxydendrobine.225 targets were used to conducted PPI analysis.The threshold was set as degree≥20.5 key targets were screened out,including MAPK1,EGFR,F2,MAPK8,and CHRM2.Meanwhile,225 targets were used to perform disease enrichment and KEGG pathway analysis.They may have the potential effects on neurological diseases,metabolic diseases,tumors and other diseases through neuroactive ligand-receptor interaction,calcium signaling pathway,c AMP signaling pathway,cholinergic synapse,serotonergic synapse and so on.Conclusion: In the practice of imitating wild planting mode of D.nobile,planting place and light have no significant effect on the quality of D.nobile.But the influence of altitude on the quality of D.nobile should be fully considered.At the same time,the eight dominant chemical components were selected,which could be used as an important reference index for the formulation of D.nobile. |
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