Studies On Chemical Constituents,anti-ulcerative Colitis Activity And Pharmacokinetics Of Brachybotrys Paridiformis Maxim. Ex Oliv.

In order to further elucidate the chemical components of Brachybotrys paridiformis Maxim.ex Oliv.,find and develop natural active ingredients,the dissertation focused on the chemical constituents and the anti-ulcerative colitis(UC)effect of Brachybotrys paridiformis and the pharmacokinetics of the active component based on the review of the research progress of Brachybotrys paridiformis and UC.This dissertation mainly includes the analysis,isolation and of chemical constituents of Brachybotrys paridiformis based on bioactivity-oriented separation;the screening of active ingredients against UC based on network pharmacology and molecular docking;the evaluation of the pharmacological effects of the active ingredients based on a mouse UC model induced by Dextran Sulphate Sodium(DSS);the mechanism of the active ingredients against UC based on metabolomics and the pharmacokinetics study of the active ingredients based on ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The following innovative results have been achieved:Ⅰ.The study on the chemical components of Brachybotrys paridiformis1.The study on the chemical components of Brachybotrys paridiformis based on bioactivity-oriented separation(1)Screening the active sites of Brachybotrys paridiformis with anti-inflammatory activityThe ethanol extraction of Brachybotrys paridiformis(EBP)and the extractions of EBP [n-hexane(EBP-1),ethyl acetate(EBP-2),n-butanol(EBP-3),and the remaining aqueous layer(EBP-4)] were evaluated for their anti-inflammatory activities using the RAW264.7 macrophage inflammation model induced by lipopolysaccharide(LPS).EBP-1 and EBP-2,especially EBP-2 were screened out as the anti-inflammatory active sites of Brachybotrys paridiformis according to the levels of NO,TNF-α,IL-6and IL-1β in the cell supernatant.(2)Rapid analysis and identification of the chemical constituents of the anti-inflammatory active sitesBased on Ultra performance liquid chromatography quadrupole-time of flight mass spectrometry(UPLC-Q/TOF-MS)combined with UNIFI platform,the rapid analysis and identification of the chemical constituents of the anti-inflammatory extraction sites(EBP-1 and EBP-2)were carried out.A total of 56 compounds(32components from EBP-1 and 24 components from EBP-2)were identified,including16 organic acids and esters,14 terpenes,6 flavonoids,4 phenylpropanoid,3naphthoquinone,2 alkaloid and 11 other kinds of compounds,indicating that the chemical constituents of EBP-1 and EBP-2 were diverse in structural types.(3)Isolation and identification of chemical constituents of the anti-inflammatory active sites based on chromatographic technologyBased on activity-oriented separation and some technologies such as silica gel column chromatography,recrystallization and high performance liquid chromatography,a total of 43 chemical components were isolated from EBP-1 and EBP-2,among which 38 compounds were identified by analysis of physical and chemical properties and NMR,including gentiopicroside(1),8-O-acetyl shanzhiside methylester(2),geraniol(3),apigenin(4),hyperoside(5),quercitrin(6),epicatechin gallate(7),epicatechin(8),alkannin(9),acetylshikonin(10),isobutyrylshikonin(11),β-acetoxy isovaleryl shikonin(12),isovalerylshikonin(13),deoxyshikonin(14),β,β’-dimethylacrylshikonin(15),3-(4-hydroxy-3,5-dimethoxyphenyl)-1,2-propanediol(16),ethyl rosmarinate(17),luteolin(18),rutin(19),2-hydroxycinnamic acid(20),ursolic acid(21),ferulic acid(22),p-coumaric acid(23),salvianolic acid B(24),syringic acid(25),protocatechuic acid(26),oleanolic acid(27),quinic acid(28),gallic acid(29),methyl gallate(30),daucosterol(31),rosmarinic acid(32),methyl rosmarinate(33),stigmasterol(34),β-Sitosterol(35),quercetin(36),kaempferol(37)and caffeic acid(38).Among them,compounds 1~28 were isolated from Brachybotrys paridiformis for the first time.2.Screening the chemical constituents of Brachybotrys paridiformis with anti-UC effect(1)Screening the potential active ingredients using network pharmacologyThe interaction network of “chemical components of Brachybotrys paridiformis-UC core targets” was established using network pharmacology,and five potential active ingredients including p-coumaric acid,quercetin,caffeic acid,ferulic acid and ethyl rosmarinate(ER)with larger degree values were screened out.These ingredients were predicted to mainly act on four key targets including MPO,TNF-α,IL-6 and IL-1β,and further regulate some signaling pathways such as PI3K-Akt、TNF、HIF-1and MAPK.(2)Confirming the potential active ingredients using molecular dockingFive chemical components four key targets screened by network pharmacology were selected as ligands and receptors respectively for molecular docking.The potential active ingredients were further screened by using binding energy.The results showed that all the selected five chemical components could bind well with the four key targets,among them the binding energy of ER to the four targets was the highest.(3)Confirmation of anti-UC active components using LPS-induced RAW264.7 cell modelThe LPS-induced RAW264.7 cell model was used to evaluate the anti-inflammatory effects of the five potential active ingredients including coumaric acid,quercetin,caffeic acid,ferulic acid and ER.The results showed that all the five compounds could reduce the levels of pro-inflammatory cytokines NO,TNF-α,IL-1βand IL-6 in a dose-dependent manner,among them ER exerted the strongest anti-inflammatory activity,which was consistent with the results of molecular docking.Ⅱ.Study on the anti-UC activity of ER1.Effects of ER on DSS-induced UC miceThe UC model was established by DSS induction and used to investigate the anti-UC activity of the the screened active ingredients(ER).The results showed that after ER intervention,the levels of inflammatory factors and myeloperoxidase in GU mice were remarkably reduced,and the disease activity index and colon tissue damage in mice were also effectively improved or restored,indicating that ER exerted good anti-UC effect.2.Studies on the anti-UC metabolomics of ERA UPLC-Q/TOF-MS-based metabolomics approach was applied to identify the endogenous metabolites and the metabolic pathways closely related to the protective effect of ER on UC.A total of 28 potential metabolites(thromboxane A2,20-hydroxyleukotriene B4,prostaglandin E2,arachidonic acid and linoleic acid)involved in 6 metabolic pathways(linoleic acid metabolism,arachidonic acid metabolism,α-linolenic acid metabolism,glycerophospholipid metabolism,retinol metabolism and steroid hormone biosynthesis)were re-regulated by ER treatment.Ⅲ.Study on the pharmacokinetic of rats oral administered ER1.Determination of "plasma concentration-time curve" of rats intragastrical administered ERA UPLC-MS/MS quantitative method for the determination of ER in rat plasma was established for the first time,with a linear range of 1~2500 ng/ml,and the specificity,accuracy,precision,stability,extraction recovery and matrix effects were all meet the requirements of pharmacokinetic studies.The plasma concentration-time curves of different concentrations of ER(7.5,15,30 mg/kg)was determined and the parameters were obtained for the first time.The results showed that ER was rapidly absorbed after oral administration,and ER was detected in the plasma at the first sample collection time point for all the three doses,the Tmax of plasma concentration was 0.19-0.25 h;ER was also eliminated rapidly,the t1/2 was 1.58~1.86 h,the CL rate was 42.54~46.07 L·h-1·kg-1,the MRT(0-t)was1.62~1.89 h;the extravascular distribution was extensive,and the Vd was100.25~118.61 L/kg.In addition,the kinetics of ER in rats was a linear process,showing the same trend as the dose dependence in the pharmacodynamic studies.2.Determination of the absolute bioavailability of ER in ratsThe plasma concentration-time curve of intravenous administration of ER(2mg/kg)in rats was determined and the parameters such as t1/2,AUC,CL and Vd were obtained for the first time.The results showed that ER rapidly cleared,the t1/2 was1.54±0.17 h,the CL was 3.90±0.72 L·h-1·kg-1.Taking the AUC(0-t)of intravenous administration of ER as a reference,the absolute bioavailability of intragastric administered ER was 8.76%.3.Study on the biotransformation of intragastric administered ER in ratsA UPLC-Q/TOF-MS method combined with the UNIFI metabolite analysis platform was used to rapidly analyze and identify the main metabolites in the plasma,urine and feces of rats after intragastric administration of ER(30 mg/kg).A total of 10 metabolites,including 4 phase I metabolites and 6 phase II metabolites were identified.The phase I metabolic reactions mainly included dehydration,hydrogenation,water addition,the phase II metabolic reactions mainly included methylation,sulfation and glucuronidation.In conclusion,the chemical constituents of Brachybotrys paridiformis were deeply studied,ER with good anti-UC activity was screened out,and the absorption and biotransformation of ER in rats were clarified.This dissertation could provide scientific basis for elucidating the chemical constituents and expanding the medicinal uses of Brachybotrys paridiformis,and provide theoretical support for further developing ER.The innovations of this thesis were as follows:1.Analyzed and identified the chemical components of Brachybotrys paridiformis based on UPLC-MS for the first time.2.A total of 28 compounds were isolated from Brachybotrys paridiformis for the first time.3.ER was screened out with good anti-UC effect for the first time,and its mechanism was explored using metabolomics.4.The pharmacokinetic study of ER was carried out for the first time.