Studies On Chemical Constituents And Anti-COPD Activity Of Celastrus Orbiculatus Thunb.

Based on the review of the research progress of Celastrus orbiculatus Thunb.(COT)and the prevention and treatment of chronic obstructive pulmonary disease(COPD),the chemical constituents and anti-COPD activity in vitro from different parts of COT were compared comprehensively.Furthermore,the chemical components,anti-COPD effect and mechanism of COTS(extract of Celastrus orbiculatus Thunb.stem),the active medicinal part of COT,were investigated deeply.The following innovative results have been achieved:1.Comparative studies on substance composition and anti-inflammatory activity from different parts of COT(1)Comparative studies on chemical constituents from different parts of COTThe screening analysis of chemical components and non-targeted metabolomics were carried out for the first time:(1)A total of 120 compounds were identified by using UPLC-Q/TOF-MS combined with UNIFI platform,including 92,56 and 32 components from root,stem and leaf of COT,respectively.There were only 15 shared compounds in three parts.The results showed that the chemical constituents were diverse in structural types,such as sesquiterpenoids,diterpenoids,triterpenoids,flavonoids,steroids and phenylpropanoids.For example,triterpenoids were the main identified components from root,triterpenoids and sesquiterpenoids were the main identified constituents from leaf,while triterpenoids and flavonoids were the typical identified compounds from stem.(2)Non-targeted metabolomics was used to compare the contents of the chemical components in three parts of COT,and a total of 26 potential chemical markers were characterized,including 13,8 and 5 from root,stem and leaf of COT,respectively.The above results revealed the structural diversity of secondary metabolites of COT and provided scientific data to clarify the pharmacological fundamental substances.(2)Comparative studies on volatile substances from different parts of COTA total of 218 volatile substances were identified from the root,stem and leaf of COT by using the method of HS-SPME combined GC-MS.Among them,97 volatile substances were identified from root,mainly containing aldehydes(18.47%),alcohols(17.48%),acids(14.81%)and esters(11.44%);67 volatile substances were identified from stem,mainly containing aldehydes(28.82%),hydrocarbons(26.94%),acids(9.78%)and esters(9.76%);141 volatile substances were identified from leaf,mainly containing hydrocarbons(26.94%),alcohols(15.20%),aldehydes(12.99%),ketones(10.61%).Except the 29 shared volatile substances in three parts of COT,and there were 46,20 and 96 characteristic substances were identified in root,stem and leaf,respectively.The results suggested that there were obvious differences in volatile substances of three medicinal parts of COT.(3)Comparative studies on trace elements from different parts of COTThe contents of the trace elements in the root,stem and leaf of COT were determined with the method of ICP-MS.The results indicated that the root,stem and leaf of COT were all rich in Sr,Zn,Mn,B,Cr and Cu.But there were still some differences on the contents of trace elements among the three parts.(4)Comparative studies on effects of different parts of COT on inflammatory injury induced by CSE in A549 cellsTo establish the cigarette smoke injury model in vitro,A549 cells were stimulated by CSE.The effects of 80%methanol extracts of root,stem and leaf of COT on CSE-induced inflammatory injury in A549 cells were evaluated for the first time.The results showed that all the three medicinal parts of COT had protective effects on inflammatory injury in CSE-induced A549 cells to a certain extent.Among them,the extract of stem had the strongest effect,which could significantly reduce the levels of TNF-?,IL-1? and IL-6 in CSE-induced A549 cells in a dose-dependent manner.2.Studies on chemical constituents and anti-COPD activity of COTS(1)Studies on chemical constituents from COTSA total of 45 constituents were isolated from 80%methanol extract of COTS by using macroporous resin,normal-phase and reversed-phase silica gel column chromatography and sephadex column chromatography,and 40 of the isolated compounds were identified with the analysis of physical and chemical properties,NMR and HR-MS,including 15 flavonoids,6 steroids and 4 triterpenoids,4 diterpenoids and 11 other compounds.Among them,compounds 1 and 2 were the new compounds,and compounds 3-28 were isolated from COT for the first time.(2)Studies on anti-COPD activity of COTSThe method of nasal smoking was used to established the CSE-induced COPD mice model,and the COPD mice were given COTS by intragastric administration for 2 weeks aiming at evaluating the effect of COTS on COPD.As a result,compared with model group,the high-dose group and the medium-dose group COTS could increase the body weight of COPD mice,reduce the static compliance and decrease the level of pro-inflammatory factor IL-6.Besides,the high-dose group could also increase the level of SOD,reduce the functional residual capacity,lower the level of pro-inflammatory factors IL-1? and TNF-?,reduce the content of MDA and the airway resistance,improve the pathological injury of lung tissue.The above results could prove that COTS could exert anti-COPD effect in a dose-dependent manner.3.Studies on anti-COPD mechanism of COTS(1)Studies on anti-COPD metabolomics of COTSBased on UPLC-Q/TOF-MS technology,the serum and urine metabolomics approaches were applied to explore the latent mechanism of the anti-COPD effect of COTS.The endogenous metabolites and related metabolic pathways were investigated.The results showed that the contents of endogenous metabolites in serum of COPD model mice were significantly changed compared with the normal group.After intervention with COTS,the levels of 20 endogenous metabolites,including cortisone,arachidonic acid,tryptophan,thiamine,retinyl ester,PC(18:1(9Z)e/2:0)and Se adenosylselenomethionine were significantly re-regulated.As a result,it could be concluded that COTS exerted anti-COPD activity through the regulation of 7 relevant metabolic pathways,including steroid hormone biosynthesis,arachidonic acid metabolism,tryptophan metabolism,thiamine metabolism,retinol metabolism,ether lipid metabolism and selenoamino acid metabolism.(2)Studies on anti-COPD serum pharmacochemistry of COTSBased on the method of UPLC-Q-TOF/MS combined with multivariate statistical analysis,the migration components in serum of COPD mice after the intragastric administration of COTS were studied for the first time.Then,further verification of the migration components in serum of COPD mice was carried out by comparison with retention time of reference standards,precise molecular weight and characteristic MS fragmentation patterns reported in literature,along with the results of the global profiling analysis,16 migrating components were identified such as quercetin,celastrol,daturanolone and dehydrodiisoeugenol,etc.(3)Studies on anti-COPD network pharmacology of COTSBased on the global profiling analysis,isolation,identification and serum migration components analysis of COTS,the method of network pharmacology was further performed in the present study.The interaction network of“COTS chemical components-COPD core targets" was established,the targets of 95 components in COTS were predicted,among them 42 compounds with the degree higher than the average value were screened,including quercetin,celastrol,daturanolone and dehydrodiisoeugenol.These active components exerted anti-COPD effects by acting on the key targets such as PTGS2,ALOX5,AR,ESR1,NOS2 and HMGCR,and the signal pathways such as PI3K-Akt?Sphingolipid?TNF and Toll-like receptor were further regulated.The molecular docking results also indicated that the typical active components(quercetin,celastrol)had the good interaction with above six key targets.In conclusion,based on the comparative study of chemical constituents and in vitro anti-COPD activities of root,stem and leaf of COT,the chemical constituents and biological activities of COTS were deeply studied in this paper,which could provide scientific basis for elucidating the chemical constituents and anti-COPD activity and mechanism of COTS.This paper could also provide theoretical support for further developing COTS.