Study On Chemical Composition And Anti-myocardial Ischemia Effect Of Fresh Berries Of Panax Quinquefolium L.

On the basis of reviewing the research progress on the chemical composition and pharmacological activities of berries from Panax ginseng,Panax notoginseng and Panax quinquefolium L.in Araliaceae,the identification and content determination of fresh berries from American ginseng,the preparation of total saponins,and the evaluation of anti-myocardial ischemia effect of total saponins were carried out.These results have been achieved:1.Study on the chemical composition of fresh berries of American ginseng（1）Rapid identification of chemical composition in fresh berries of American ginsengFresh American ginseng berries produced in Jilin province and Shandong province were taken and manually peeled into peels-pulps,pedicels and seeds.Ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry（UPLC-Q/TOF-MS）technology was used to determine the chemical composition of American ginseng berries（including peels-pulps,pedicels,seeds）.The small molecule chemical compositions（100Da～1500 Da）were quickly identified using the UNIFI natural product analysis platform.By comparison with reference substances,accurate molecular weights and typical fragments,a total of 100 compounds were identified,including 93 in peels-pulps,55 in pedicels,and 21 in seeds.According to the structure types,it included 76 saponins,16organic acids and esters,1 flavonoid,3 amino acids,and 4 other components.The results showed that the three parts of fresh American ginseng berries were rich in chemical substances,and were mainly composed of saponins,but there were certain differences in the types of saponins.In this study,55 and 21 compounds were respectively identified from pedicels and seeds for the first time,which made up for the lack of research on the chemical composition of American ginseng pedicels and seeds.In addition,among the 93components identified from the peel-pulp,55 were identified for the first time.（2）Determination of saponins in fresh berries of American ginsengThe contents of total saponins of 8 batches of fresh peels-pulps,pedicels and seeds were determined by UV-Vis spectrophotometry（UV-Vis）.The measured data showed that the total saponin contents in fresh peels-pulps were 3.95%～4.39%,the total saponin contents in fresh pedicels were 0.21%～0.41%,and the total saponin contents in fresh seeds were 0.31%～0.43%.The results showed that the contents of total saponins in peels-pulps were the highest,and the contents of total saponins in pedicels and seeds were similar.A high-performance liquid chromatography-evaporative light-scattering detector（HPLC-ELSD）method was established for the determination of 11 monomer saponins in fresh peels-pulps,pedicels and seeds.The results of methodological investigation showed that the linear relationship was good（r>0.9997）,and the RSD values of precision,reproducibility and accuracy were all less than 3%.The content of monomer saponins in 8 batches of fresh peels-pulps,pedicels and seeds were determined by the established method.The results showed that the contents of monomer saponins in the three parts were quite different:in peels-pulps,the contents of ginsenosides Rb₃（0.96%～1.25%）,Rg₃（0.59%～0.76%）and PF₁₁（0.44%～0.57%）were higher than other saponins.The contents of ginsenosides Re（0.033%～0.061%）,（20R）-Rg₃（0.035%～0.063%）and Rb₃（0.023%～0.049%）in pedicels were higher than other saponins,while in seeds,the contents of ginsenoside Rb₃（0.089%～0.17%）,PF₁₁（0.031%～0.068%）and Rb₁（0.029%～0.052%）were higher than other saponins.Studies have shown that saponins were existed in three parts,but the contents and compositions were different.Since the seeds are often used in cultivation,the pedicels of American ginseng should be used as a new source of saponins.（3）Preparation and content determination of total saponins in fresh berries of American ginsengBy the investigation of immersion extraction method and reflux extraction method（with the extraction rate of total saponins as the index）,and the investigation of the enrichment and purification of D101,D4020,AB-8 macroporous adsorption resins（with the content of total saponins as the index）,the preparation process of total saponins from peels-pulps（TSPP）,total saponins from pedicels（TSP）and total saponins from seeds（TSS）was screened.The key process parameters were:70%ethanol reflux extraction,AB-8 macroporous resin purification.According to the screened preparation process,8batches of TSPP,TSP and TSS were prepared,and the total saponin contents were61.15%～67.96%,39.37%～62.08%,40.65%～56.39%,respectively.The contents of monomer saponins in the prepared batches of TSPP,TSP and TSS were also determined.The results showed that,similar to the peels-pulps,pedicels and seeds,there were also significant differences in the contents of monomer saponins in TSPP,TSP and TSS.For example:the contents of ginsenoside Rb₃,Rg₃,PF₁₁and Re in TSPP were 17.35%～19.28%,10.62%～11.80%,7.97%～8.85%and 3.89%～4.33%,respec-tively;the contents of ginsenoside Re,（20R）-Rg₃and Rb₃in TSP were respectively7.19%～14.03%,8.61%～15.58%,6.20%～12.10%;the contents of ginsenoside Rb₃,PF₁₁and Rb₁in TSS were 12.46%～20.57%,5.71%～9.42%,2.17%～4.23%,respectively.2.Study on the anti-myocardial ischemia effect of total saponins from fresh berries of American ginseng（1）Effects of TSPP,TSP and TSS on the survival rates of H₂O₂-induced H9c2cardiomyocytesThe H₂O₂-induced H9c2 cardiomyocyte injury model was established,and the effects of TSPP,TSP and TSS on the survival rates of cardiomyocytes were determined by MTT assay.The results showed that,compared with model group,TSPP,TSP and TSS could significantly improve the survival rates of cardiomyocytes,and showed a protective effect on injured cardiomyocytes.Among them,the survival rates of H9c2cardiomyocytes were 97.9%～119.6%after TSPP intervention at concentrations of 20 and40μg/ml,which were higher than that of H9c2 cardiomyocytes with TSP and TSS intervention（88.2%～108.5%,84.6%～96.1%）.TSPP showed the strongest cardiomyocyte survival-enhancing effect.（2）Effect of TSPP on acute myocardial ischemia model ratsA rat model of acute myocardial ischemia（MI）was established by ligating rat coronary arteries.With ST segment offset,infarct sizes,myocardial three enzymes（CK,AST and LDH）,superoxide dismutase（SOD）and malondialdehyde（MDA）as indicators,the effects of TSPP on myocardial ischemia rats were evaluated.The results showed that intragastric administration of TSPP（50,100 mg/kg）could significantly improve the abnormal elevation of ECG ST,reduce the size of myocardial infarction,inhibit the release of CK,AST and LDH,increase SOD activity,reduce MDA production of MI rats.Compared with the positive control drug Ginkgo Tablets,TSPP has the advantages of quick onset and long-lasting effect.Studies have shown that TSPP has a good anti-myocardial ischemia effect.（3）Exploring the mechanism of TSPP against myocardial ischemia based on network pharmacologyTaking 67 saponins identified from TSPP by UPLC-Q/TOF-MS as"candidate compounds","TSPP-MI core target"network was constructed.The topological analysis results showed that:There were 39 saponins whose Degree values were higher than the average.Among them,ginsenosides Re,Rb₃,Rg₃and PF₁₁have higher contents in TSPP,and acted on 46,51,44 and 33 targets,respectively.Therefore,they might be the active ingredients against myocardial ischemia.There were 64 MI targets whose Degree values were higher than average.Among them,the degrees of SOD1,LDHA,CKB,GOT2 and ROS1 were 51,43,46,30,and 52,respectively,which may be the key targets of myocardial ischemia.Among them,SOD1,LDHA,CKB,GOT2 targets have been determined as SOD,LDH,CK,MDA in TSPP-intervened acute myocardial ischemia model rats.In addition,it was also predicted that the anti-myocardial ischemia effect of TSPP might be exerted by regulating signaling pathways such as PI3K-Akt,HIF-1,JAK-STAT,TNF,VEGF,proteoglycans in cancer,MAPK and Rap1.The study provides a scientific basis for further exploring the mechanism of TSPP anti-myocardial ischemia（4）Molecular docking of key active ingredients with key targetsMolecular docking technology was used to further study the interaction and recognition patterns between potential active components(ginsenosides Re,Rb₃,Rg₃and PF₁₁)and key targets（SOD1,LDHA,CKB,GOT2 and ROS1）.Molecular docking results showed that the four ginsenosides had good binding activities to five MI targets.The binding energy was-9.8 kcal/mol～-7.2 kcal/mol,which means the ligands and receptors have the good binding effects.In conclusion,through the research of this paper,the chemical composition of fresh fruits of American ginseng was enriched,the lack of research on chemical composition and biological activity of American ginseng pedicels and seeds was made up.And it was proved that American ginseng pedicels is the new source of ginsenosides.In addition,TSPP,TSP and TSS all showed the good myocardial protective effect,among which,TSPP have the strongest effect.The research results of this paper could provide scientific data and theoretical support for the further research and development of American ginseng berries.The innovations of this dissertation are as follows:1.A total of 55 and 21 chemical components were respectively identified from pedicels and seeds for the first time,which made up for the lack of research on American ginseng pedicels and seeds.It is proved that American ginseng pedicels is a new source of saponins.2.Among the 93 components identified from the peel and pulp,55 components were identified for the first time.3.An HPLC-ELSD method for the simultaneous determination of 11 ginsenosides in fresh American ginseng berries was established,and the content of monomer saponins in TSPP,TSP and TSS were determined.4.The total saponins from pedicels and seeds were prepared for the first time,and the effects on the survival rate of H9c2 cardiomyocytes induced by H₂O₂were evaluated.It was preliminarily proved that TSP,TSS also had the good anti-myocardial ischemia effects.5.Based on network pharmacology and molecular docking,the mechanism of TSPP against myocardial ischemia was explored for the first time.6.The key active components against myocardial ischemia in TSPP were screened out as ginsenosides Re,Rb₃,Rg₃and PF₁₁.