Study On The Therapeutic Material Basis Of American Ginseng Berry In Treatment Of Ulcerative Colitis

Ulcerative colitis（UC）is challenging to cure,has a high relapse rate,and a high rate of malignancy,making it a precancerous lesion for colon cancer.Existing drugs mainly treat UC through anti-inflammatory or immunomodulatory means,but they have limitations such as poor efficacy and numerous adverse reactions.American ginseng berry,the mature fresh fruit of Panax quinquefolium L.,is rich in active ingredients such as ginsenosides and possesses biological activities such as anti-myocardial ischemia,anti-inflammation,and anti-colon cancer.Therefore,this thesis proposes the application of American ginseng berry in the prevention and treatment of ulcerative colitis to reduce the incidence of colon cancer.Based on a review of research progress on the fruits of the Panax genus and anti-ulcerative colitis research,this study explores the pharmacological effects of American ginseng berry against ulcerative colitis and its material basis.The following innovative results were obtained:1.Study on the anti-ulcerative colitis effects of American ginseng berry（1）Effect on lipopolysaccharide（LPS）-induced epithelial barrier damage in Caco-2cells:An LPS-induced Caco-2 cell epithelial barrier damage model was established to evaluate the ameliorative effects of the 70%ethanol extract of American ginseng berry on inflammation and barrier damage.The results showed that the extract could increase the transepithelial electrical resistance（TEER）value of Caco-2 cells,decrease the levels of pro-inflammatory factors（TNF-α,IL-6,and IL-1β）,and enhance the expression of tight junction proteins.（2）Effects on dextran sulfate sodium（DSS）-induced ulcerative colitis in miceA DSS-induced UC mouse model was used to evaluate the effects of the 70%ethanol extract of American ginseng berry on ulcerative colitis.The results showed that the extract（50.0,100.0,200.0 mg/kg）could increase mouse body weight,decrease disease activity index（DAI）scores,increase colon length,decrease spleen index,decrease pro-inflammatory factors（TNF-α,IL-1β,IL-6）and myeloperoxidase（MPO）levels,increase anti-inflammatory factor IL-10 levels,alleviate intestinal injury,restore tight junction protein levels,and modulate the abundance,diversity,and structure of the gut microbiota in a dose-dependent manner.These findings indicate that the 70%ethanol extract of American ginseng berry can effectively alleviate the inflammatory response in ulcerative colitis mice,protect the intestinal mucosal barrier,and modulate the gut microbiota,exhibiting potent anti-ulcerative colitis effects.（3）Study of the effects of American ginseng berry intervention in UC mice based on metabolomics of colonic contentsUsing an UPLC-Q-TOF/MS-based metabolomics technology,the effects of the 70%ethanol extract of American ginseng berry on endogenous metabolites and related metabolic pathways in mouse colonic contents were investigated.The results showed that after intervention with the extract,the levels of 10 metabolites,including hydroxybutyric acid,were significantly reversed,and these 10 metabolites were primarily involved in 5 metabolic pathways,including butyrate metabolism.2.Chemical composition analysis of American ginseng berries（1）Metabolomics study of different parts of American ginsengBased on UPLC-Q-TOF/MS technology combined with multivariate statistical analysis,an untargeted metabolomics study of various parts of American ginseng（root,stem and leaves,fruit,seeds）was conducted.Principal component analysis showed that the secondary metabolite profiles of American ginseng berry differed significantly from those of the root,stem and leaves,and seeds.Through orthogonal partial least squares discriminant analysis,S-plot visualization,database queries,and reference compound comparisons,14characteristic components with higher contents in the fruit part than in the other three parts were identified,including pseudo-ginsenoside RT4,ginsenoside Rk1,and ginsenoside Rk2.（2）Isolation and identification of chemical components from American ginseng berryUsing techniques such as large-pore adsorption column chromatography,silica gel column chromatography,ODS column chromatography,Sephadex LH-20 gel column chromatography,medium-pressure liquid chromatography,and semi-preparative high-performance liquid chromatography,combined with molecular networking,57compounds were isolated and identified from the 70%ethanol extract of American ginseng berry.These included 48 triterpenoids,3 steroids,4 phenylpropanoids,1 phenolic acid,and 1phenyl glycoside.Among them,compounds 1,2（tetracyclic triterpenoids）and compound 3（phenyl glycoside）were previously unreported new compounds.Compounds 53,54,and 55（phenylpropanoids）were isolated from Panax plants for the first time,and 28 compounds including compounds 10,14-20,23,25,26,28,29,33-38,40,42,45-52 were isolated from American ginseng berry for the first time.（3）Determination of saponin contents in the 70%ethanol extract of American ginseng berryThe total saponin content was determined by UV-visible spectrophotometry,and the content of major monomeric saponins above 0.01%was determined by HPLC-ELSD.The results showed that the total saponin content was 60%;dammarane-type monomeric ginsenosides Rb1（3.07%）,Rb2（2.81%）、Rb3（19.66%）、Rc（1.47%）、Rd（4.49%）、Re（3.52%）、Rg1（0.87%）、Rg3（17.71%）、notoginseoside R2（3.93%）;as well as oleanane-type monomeric saponin PF11（6.38%）.In summary,the 70%ethanol extract of American ginseng berry contains not only dammarane-type saponins but also a certain proportion of oleanane-type saponins.2.Study on the anti-ulcerative colitis effect and pharmacokinetics of pseudo-ginsenoside（1）Semi-synthesis of the oleanane-type active component pseudo-ginsenoside RT4 from American ginseng berryIn a preliminary experiment,8 isolated oleanane-type monomer saponins were screened for anti-inflammatory activity in LPS-induced RAW264.7 cells,and RT4 exhibited the best anti-inflammatory effect.However,its natural abundance in American ginseng berry is relatively low,making it difficult to obtain large quantities through isolation and extraction.Therefore,in this dissertation,ginsenoside Re extracted from fresh ginseng berries（with a ginsenoside Re content of 2.6%）was used as the starting material.Re was subjected to alkaline degradation to prepare Rh1,which was then oxidized and cyclized in a semi-synthetic process to prepare RT4 with a yield of 20.7%and a purity of 98.0%,achieving the directed synthesis of a trace natural product.（2）Effects of pseudo-ginsenoside RT4 on LPS-induced epithelial barrier damage in Caco-2 cellsAn LPS-induced Caco-2 cell epithelial barrier damage model was used to evaluate the effects of RT4 on barrier damage.The results showed that RT4 could dose-dependently.increase cellular TEER values,decrease pro-inflammatory factors（TNF-α,IL-6,and IL-1β）levels,and improve the expression and distribution of tight junction proteins.（3）Effects of pseudo-ginsenoside RT4 on DSS-induced ulcerative colitis in miceA DSS-induced ulcerative colitis mouse model was used to evaluate the effects of RT4on ulcerative colitis.The results showed that RT4（10.0,20.0,40.0 mg/kg）could increase mouse body weight,decrease DAI scores,increase colon length,decrease spleen index,improve intestinal barrier function,decrease pro-inflammatory factors TNF-α,IL-1β,and IL-6 levels,increase anti-inflammatory factor IL-10 levels,increase short-chain fatty acid levels,alleviate intestinal injury,improve the state of intestinal mucosal epithelial cells,correct gut microbiota dysbiosis,and restore the abundance of the gut microbiota in a dose-dependent manner.In summary,RT4 can effectively alleviate intestinal inflammation,restore gut microecological balance,and protect against intestinal injury.（4）Pharmacokinetic study of RT4 in UC miceAn UPLC-MS/MS method was established for the determination of RT4 levels in mouse plasma.After oral administration of RT4（10.0,20.0,40.0 mg/kg）,the plasma concentration-time curves showed linear pharmacokinetics,consistent with the dose-dependent trend observed in the pharmacological studies.The peak time(T_max)was 1-2h,the elimination half-life(t_1/2)was 4.7-7.2 h,indicating rapid absorption and slow elimination of RT4.The apparent volume of distribution（V_d）was 61.9-71.6 L/kg.Within 1-2h after oral administration,RT4 was mainly distributed in the stomach,small intestine,and colon,while from 2-4 h,it was mainly distributed in the colon,indicating widespread and rapid distribution with specific localization in the colon tissue.In summary,in UC mice,RT4exhibited slow elimination and selective distribution in the colon.（5）Pharmacokinetic study of RT4 in healthy rats（1）Absorption and bioavailabilityAn UPLC-MS/MS method was established for the determination of RT4 and its major metabolite 24S-ocotillol（OC）levels in rat plasma.After oral administration of RT4（5.0,10.0,20.0 mg/kg）to rats,the plasma concentration-time curves of the parent drug RT4 and its metabolite OC were determined.The results showed that the T_maxof RT4 was 1.9-2.6 h,the t_1/2was 7.0-9.9 h,and the V_dwas 45.7-54.7 L/kg.The T_maxof the metabolite OC was8.0-8.7 h.It can be seen that before 8 h,the parent drug RT4 was mainly present in the plasma,while from 8-24 h,both the parent drug and the metabolite OC were present in the plasma,and after 24-60 h,the metabolite OC was mainly present in the plasma.After intravenous administration of RT4（2.0 mg/kg）,the t_1/2was 7.7 h.Using the intravenous AUC_（0-t）as a reference,the absolute bioavailability of oral RT4 was calculated to be 17.2%.（2）Tissue distributionThe distribution of RT4 and OC in various tissues of rats after oral administration of RT4（10.0 mg/kg）was studied.At 4 h after administration,RT4 reached the highest concentration in most tissues,which was consistent with the plasma pharmacokinetic trend.From 1-2 h,RT4 was mainly distributed in the stomach,small intestine,and colon,while from 2-4 h,RT4 was mainly distributed in the colon.The metabolite OC was also widely distributed in the stomach,large intestine,and colon,with concentrations higher than in plasma.In summary,compared to the pharmacokinetic behavior in UC mice and healthy rats,RT4 exhibited similar peak times,but the elimination half-life was significantly shortened,and the apparent volume of distribution was increased in the disease state,indicating that RT4 could distribute more rapidly and extensively to the colon to exert its pharmacological effects under disease conditions.（3）BiotransformationUsing UPLC-Q-TOF/MS technology and the UNIFI metabolite analysis platform,a total of 13 metabolites were identified from rat plasma,bile,urine,and feces,including24S-ocotillol（OC）,3 phase I metabolites,and 9 phase II metabolites.In summary,this dissertation completed studies on the pharmacological effects of American ginseng berry against ulcerative colitis and the material basis of their efficacy,providing two candidate drugs,the 70%ethanol extract of American ginseng berry and pseudo-ginsenoside RT4,for the treatment of ulcerative colitis.It also enriched the research on the chemical components of American ginseng berry,expanded the pharmaceutical applications of American ginseng berry,and achieved the directed synthesis of a trace natural product.The main innovations are as follows:（1）The 70%ethanol extract of American ginseng berry was found to exhibit anti-ulcerative colitis bioactivity by reducing inflammation,protecting the intestinal mucosal barrier,and modulating the gut microbiota.（2）A total of 57 compounds were isolated and identified from the 70%ethanol extract of American ginseng berry,including 3 new compounds,3 compounds isolated from Panax plants for the first time,and 28 compounds isolated from American ginseng berry for the first time.（3）Pseudo-ginsenoside RT4,which exhibited potent anti-ulcerative colitis effects,was selected from the characteristic oleanane-type components of American ginseng,and its directed high-efficiency semi-synthesis and pharmacokinetic evaluation were completed.