| The ginseng plant Panax ginseng C.A.Mey.is abundant in the triterpenoid ginsenosides,which can be divided into dammarane tetracyclic triterpenes and oleanolic acid pentacyclic triterpenes according to the parent nucleus structure of the aglycone.Among the various medicinal parts of ginseng,Dammarane saponins are the main ones,which can be divided into protopaanaxadiol saponins and protopanaxatriol saponins according to whether or not there is a hydroxyl group attached to the C-6 position.In this thesis,the novel biological activities of Protopanaxatriol Saponins(PTS)and aglycone PPT(Protopanaxatriol)against Ulcerative Colitis(UC)and Cerebral Ischemia-Reperfusion Injury(CI/RI)were studied,as well as their mechanism of action and pharmacokinetics were investigated.The following innovative results:Ⅰ.The study on the pharmacological effects of PTS against UC1.Determination of monomeric saponins in PTSThe contents of ginsenosides Rg1,Re,Rf,Rh1,Rg2,F1 and PPT in PTS were determined by high performance liquid chromatography with ultraviolet detector(HPLC-UV),which were 19.68%,30.21%,2.87%,8.41%,1.56%,3.28%and 4.15%,respectively,with the total contents accounting for 70.16%of PTS.2.Effect of PTS on LPS-induced RAW264.7 macrophagesA model of LPS-induced inflammation in RAW264.7 macrophages was established,and the anti-inflammatory activity of PTS was evaluated using the amount of inflammatory factors released from the cell supernatant as an indicator.The results showed that PTS(25,50 and 100 μg/ml)reduced NO,TNF-α,IL-1β and IL-6 levels in a dose-dependent manner,indicating that PTS has a good protective effect against inflammatory injury.3.Effect of PTS on DSS-induced UC in miceA DSS-induced mice UC model was established,and the therapeutic effects of PTS(25,50 and 100 mg/kg)on UC mice were investigated using macroscopic index scores,histopathology,and inflammatory factor levels in the colon and serum as indicators.The results showed that PTS significantly slowed down the weight loss,reduced the disease activity index,improved the pathological damage of the colon,and reduced the levels of inflammatory factors and myeloperoxidase(MPO)in UC mice,indicating that PTS has good anti-UC activity.4.Metabolomics study of PTS anti-UCA UPLC-Q/TOF-MS-based metabolomics technique was used to detect metabolite changes in the serum and colon of mice and to explore the biomarkers and metabolic pathways closely related to the anti-UC effect of PTS.The results revealed that PTS could exert anti-UC activity through the modulation of 29 biomarkers,including xylulose,riboflavin and 19-hydroxytryptophan,and the 11 metabolic pathways involved in riboflavin metabolism,arachidonic acid metabolism and etc..5.Investigating the mechanism of action with PTS against UC based on network pharmacology and molecular docking techniquesA "PTS-UC-intersection target" network was constructed using network pharmacology techniques,and molecular docking techniques were used to dock the main components with key targets to predict the potential mechanism of PTS against UC.The results showed that PTS may exert anti-UC activity by affecting key targets such as STAT3,PIK3CA,VEGFA,AKT1 and FGF2,thereby regulating EGFR tyrosine kinase inhibitor resistance,PI3K-Akt,VEGF and other signalling pathways.The binding energies of the seven major components of PTS to the above five key targets were all less than-6.4 kcal/mol,indicating good binding effects.Ⅱ.The study on their anti-CI/RI activity and mechanism of PTS and PPT1.Effect of PTS on CI/RI ratsA middle cerebral artery occlusion/reperfusion(MCAO/R)model was established using the suture-occluded method.The protective effects of PTS(25,50 and 100 mg/kg)on CI/RI rats were evaluated using neurological deficit scores,cerebral infarct volumes,histopathological patterns,oxidative and inflammatory factor levels as indicators.The results showed that medium and high doses of PTS significantly reduced neurological damage,brain infarct volume,attenuated neuronal damage,improved cell morphology,increased the number of Nisssl bodies,regulated the content of oxidative factors and reduced the level of inflammatory factors,having anti-CI/RI biological activity.2.Effect of major components in PTS on OGD/R-PC12 cellsThe protective effects of Rg1,Re,Rf,Rh1,Rg2,F1 and PPT(6.25,12.5,25 μM)on OGD/R-PC12 cells were evaluated using the OGD/R-PC12 cell model,which simulates in vitro cerebral ischemia-reperfusion injury,using cell survival,oxidation and inflammatory factors as indicators.The results showed that the seven components significantly increased cell survival,reduced MDA,TNF-α and IL-6 levels and increased SOD levels.They had good protective effects against in vitro cerebral ischemia-reperfusion injury.Among them,PPT(12.5 μM)had the strongest activity and was better than the positive control drug nimodipine.3.Effect of PPT on CI/RI ratsA middle cerebral artery occlusion/reperfusion(MCAO/R)rat model was established using the suture-occluded method.The protective effects of PPT(5,10 and 20 mg/kg)on CI/RI rats were evaluated using neurological deficit scores,cerebral infarct volume,blood-brain barrier integrity,histopathological morphology,oxidative and inflammatory factor levels as indicators.The results showed that PPT significantly reduced neurological injury and brain edema,brain infarct volume,repaired the blood-brain barrier,attenuated neuronal damage,improved cell morphology,increased the number of Nisssl bodies,regulated oxidative factor content and reduced inflammatory factor levels.It showed strong anti-CI/RI activity.4.Effect of PPT on apoptosis,autophagy and angiogenesis-related proteins in OGD/R-PC12 cellsAn OGD/R-PC12 cell model was established and the expression of PI3K/AKT pathway-related proteins was measured.The results showed that PPT significantly increased the expression of p-PI3K/PI3K,p-AKT/AKT and Bcl-2/Bax proteins;decreased the expression of cleaved-Caspase 9/Caspase 9,cleaved-Caspase 3/Caspase 3 mitochondrial apoptosis proteins;promoted the expression of autophagy-related proteins p-mTOR/mTOR and angiogenesis-associated protein HIF-1α.The molecular docking results of PPT with key proteins such as Akt,HIF-1α,and PI3K showed that the binding energy was less than-7.0 kcal/mol.In conclusion,PPT may regulate apoptosis,autophagy and angiogenesis-related proteins in OGD/R-PC12 cells through the PI3K/Akt pathway,and exert protection against cell injury.5.Metabolomics study of PPT against CI/RIA UPLC-Q/TOF-MS-based metabolomics technique was used to explore the biomarkers and their metabolic pathways associated with CI/RI treatment with PPT by detecting metabolite changes in serum and brain tissues of rats.The results showed that PPT could exert anti-CI/RI effects by back-regulating 19 potential biomarkers including L-phenylalanine,2-aminonaphthalene and ursodeoxycholic acid,involving a total of five metabolic pathway pathways,such as phenylalanine,tyrosine and tryptophan biosynthesis.Ⅲ.The pharmacokinetic study to the rats orally administration with PPTA UPLC-MS/MS method was developed for the determination of PPT in biological samples.The LLOQ,linear range,specificity,accuracy,precision,dilution reliability,extraction recovery,matrix effect and stability of the method met the requirements of pharmacokinetic studies.The drug concentration-time profiles(10,20 and 40 mg/kg),tissue distribution,excretion and metabolism of the rats orally administration with PPT were determined.1.Plasma drug concentration-time curvesThe plasma levels of PPT are low,with Cmax 243.94,391.93 and 572.55 ng/ml,respectively;absorption in vivo is rapid,with Tmax 0.92-1.00 h;elimination is slow,with t1/2 7.31-9.00 h,CL 9.07-14.08 L/kg·h-1 and MRT(0-t)9.22-9.53 h;extravascular distribution is high,with Vd 95.86-182.46 L/kg.The pharmacokinetics of PPT in vivo was linear and there were no gender differences.2.Tissue distributionThe results of the tissue distribution study with PPT(20 mg/kg)orally administered to rats showed that the drug concentration with PPT in tissues was significantly higher than the plasma drug concentration,and was widely distributed in tissues from 1.5 h to 9 h of administration.At 1.5 h and 3 h,PPT was mainly distributed in the small intestine,lung and heart,and concentration peaked at 3 h.The PPT concentration peaked in brain tissue at 9 h.It is suggested that its anti-brain ischemia-reperfusion injury is related to the distribution of PPT in the brain.3.ExcretionOrally administered with PPT(20 mg/kg)to rats,the maximum excretion rate via fecal samples was reached at 4-8 h,and at 8-12 h via urine samples,with the cumulative excretion in 72 h of 49.05±2.07%and 3.81±0.21%for fecal and urine samples,respectively.The total cumulative excretion was 52.86%.The higher cumulative fecal excretion may be related to the low water solubility of PPT.4.MetabolismPPT is extensively metabolized in vivo and a total of 13 metabolites were identified in plasma,fecal and urine samples from rats orally administered with PPT(20 mg/kg),including 3 phase Ⅰ metabolites and 10 phase Ⅱ metabolites.In summary,this thesis screened the novel bioactivities of PTS and aglycone PPT,as well as preliminarily explored its mechanism of action and elucidated the pharmacokinetic behaviour of PPT in vivo.It provides a scientific basis for further research and development of innovative drugs for PTS and PPT,as well as provides candidates of natural source for UC and CI/RI.The innovation points of this thesis are as follows:1.The pharmacological action of PTS against ulcerative colitis was studied;2.The pharmacological effects of PTS and PPT against cerebral ischaemia-reperfusion injury was explored;3.The pharmacokinetics studies of PPT was conducted and more systematic pharmacokinetic parameters were obtained. |
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