| The research content of this paper belongs to the national traditional Chinese medicine industry research project?201407002?,the forward-looking joint research project of industry-university-research cooperation in Jiangsu province?by2015008-04?and and the key research and development plan of Ningxia?east-west cooperation project??2017BY079?.The thesis is divided into four chapters and the description of each chapte are summarized as follows:Chapter one,Literature ReviewThe researches on the source,variety and medicinal parts of Astragalus membranaceus plant resources was carried out.The researches of the medicinal records of the aerial part of A.membranaceus and recent researches of the chemical constituents and pharmacological activities of aerial part of Astragalus membranaceus was reviewed.The research patents of the flower of A.membranaceus were summarized,and the resources exploitation and utilization of the flowers of A.membranaceus were put forward in order to provide scientific basis and reference for the system utilization and industrialization development of A.membranaceus resources.Chapter two,The analysis and evaluation of the chemical constituents of the flower of A.membranaceus var.mongholicus?AMF??1?In order to clarify the composition and distribution characteristics of primary metabolites in AMF,ultra-high performance liquid chromatography?UPLC-TQ/MS?,gas chromatography-mass spectrometry?GC-MS?,ultraviolet-visible spectrophotometry?UV?,high performance liquid chromatography and BCA kit methods were used to analyze and evaluate the primary metabolites of AMF including nucleosides,amino acids,fatty acids,sugars?polysaccharides monosaccharides and oligosaccharides?and soluble protein components respectively.The results showed that there were 6 nucleosides?0.28%?and 15 free amino acids?0.65%?in AMF,contained abundant polysaccharides?4.69%?,fructose?4.55%?,glucose?0.87%?,and Sucrose?0.11%?and water soluble proteins?31.48%?.Besides,eight fatty acids were also identified,among which nutmeg acid,palmitic acid and oleic acid were the main constituents.?2?UV,UPLC-TQ/MS,and GC-MS methods were used to determine the secondary metabolites of AMF including flavonoids,phenolic acids,saponins and volatile components were analyzed and evaluated.The results showed that total flavonoids?0.52%?,total phenolic acids?3.83%?and total saponins?1.62%?were detected.The content of hyperoside?725.7±6.6,?g g-1?and isoquercitrin?449.6±1.7?g·g-1?in flavonol acid was relatively high,the content of mangiferin?0.33±0.002?g·g1?was the lowest.In addition,thirty-two oxygen-containing derivatives volatile compounds were identified which can contribute to further development and utilization.?3?UPLC-TQ/MS method was used to analyze the distribution of 25 kinds of flavonoids,phenolic acids and saponins,22 kinds of free amino acids and nucleosides in different parts of A.membranaceus and A.membranaceus.var.mongholicus from different areas in July.The results showed that there were remarkable differences in dihydroflavones,isoflavane,isoflavones,flavones,pterocarpans,phenolic acid and saponins contents,not only in different organs of the same species,but also in the same organ of different species.In addition,the contents of the same type of compounds in two different species plants are significantly different.In addition,the contents of total nucleosides and amino acids in the flowers were the highest in Mazichuan,Minxian County,Gansu Province,and the main component was asparagine.It provides scientific support for the utilization of resources of and A.membranaceus.var.mongholicusChapter three,biological activity evaluation and resource value discovery of AMF?1?In this section,the antioxidant activities of the five enrichment sites of AMF isolated from macroporous resin were firstly screened by free radical scavenging method based on 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid?ABTS?2,2-diphenyl-M-picrylhydrazyl?DPPH?,and Fe3+reduction capacity?FRAP?.The results showed that ME?50%eluent?had a strong DPPH free radical scavenging activity?IC50=35.10±0.2?gmL-1?,ABTS free radical scavenging activity?IC50=22.02±2.70 ?g mL-1?.The reduction ability of ME was the highest parts in FRAP method?3.00±0.13,?mol Fe2+g-1?.The antioxidant capacity was close to that of Trolox positive control group.51 compounds were detected in ME,and a total of 31 compounds including four phenolic acids,nineteen flavonoids,three isoflavones,two pterocarpans and three saponins were identified using UPLC-QTOF-MS in ME.UV and UPLC-TQ/MS methods were used to analyze the contents of total phenols and total flavones in five parts.The results showed that the total phenolic acid content?1.87±0.03 mg·g-1?and total flavonoid contents in the ME site were the highest?36.40±1.23 mg g-1?.Hyperoside?16.29±0.20 mg·g-1?was the main component of ME,followed by rutin?6.10=10.08mg·g-1?.Isorhamnetin-3-O-glucoside?4.97±0.05 mg·g-1?and astragalin?4.81±0.03 mg g-1?.The correlation analysis between chemical constituents and antioxidation showed that there is a strong correlation between antioxidant capacity and TPC,TFC in vitro,among which caffeic acid and calycosin-7-O-?-D-glucoside had the strongest correlation with antioxidant activity.The results showed that ME of AMF had certain antioxidant activity.In addition,based on the oxidative stress injury model of HT22 cells induced by glutamate,the apoptotic activity of HT22 cells in different parts,as well as rhamnocitrin,astragalin,kaempferol,isomucronulatol-7-O-glucoside,soyasaponin I and rutin under glutamate toxicity were inhibited by antioxidant activity.The results showed that the activity of ME was the strongest among the five parts,and there was a concentration correlation.In addition,all the compounds inhibited the apoptosis of HT22 cells induced by glutamic acid in varying degrees,in which rhamnocitrin was the most active monomer,followed by kaempferol,and rutin was weak.rhamnocitrin and kaempferol inhibited apoptosis more than Trolox.Based on the metabolomics technology of UPLC-TOF/MS method,the differential metabolites in HT22 cells interfered by glutamate were analyzed.After administration of ME,selenite metabolism and sphingolipid metabolism pathway may be affected,which provides a basis for studying the molecular mechanism and drug action mechanism of L-glutamic acid-induced HT22 cell injury.?2?In this section,The oxidative damage in vivo model of HELF cells induced by hydrogen peroxide,the protective effects of different parts and monomers of quercetin,caffeic acid,kaempferol,isoquercitrin,protocatechuic acid and hyperoside on oxidative damage of HELF cells induced by hydrogen peroxide were studied.The results showed that DT had the strongest protective effect on HELF cells damaged by hydrogen peroxide.And the intensity of action is dependent on the concentration.In addition,the activity of caffeic acid and quercetin was the strongest under the condition of equal concentration for 6 monomers.Based on the invitro model of pulmonary fibrosis induced by intratracheal instillation of bleomycin in c57BL/6 mouse was established.The polysaccharides parts?DT?,freeze-dried powder of 10%eluent?LE?,ME and freeze-dried powder of 90%eluent?HE?were given to mouse for 21 days.Serum,urine and lung tissue samples were collected and biochemical indexes were determined and histopathological analysis of lung was carried out.The results showed that the weight and lung coefficient of mouse in polysaccharide DT group had the tendency to return to normal group,and could significantly reduce the contents of MDA,HYP,TGF-?i and TNF-?,improve the level of T-AOC,and thus play the role of prevention and treatment of pulmonary fibrosis.This conclusion was consistent with the conclusion in vivo model in section one.Based on the metabolomics technology of UPLC-TOF/MS method,the mechanism of intervention of AMF of DT,LE,ME and HE extract on pulmonary fibrosis in mouse was studied.Results 23 endogenous metabolites?serum:11 and urine:12?were identified in serum and urine samples of mouse.Seven relative metabolic pathways were constructed from serum,including Phenylalanine,tyrosine and tryptophan biosynthesis;arachidonic acid metabolism;terpenoid skeleton biosynthesis;pyruvate metabolism;tyrosine metabolism;tryptophan metabolism and cysteine and methionine metabolism.These influence values of terpenoid skeleton biosynthesis pathway were 0.50,0.35,0.25,0.18,0.14,0.11 and 0.11,respectively.Five relative metabolic pathways were constructed from urine,including D-glutamine and D-glutamic acid metabolism;methane metabolism pathway;biotin metabolism;alanine,aspartic acid and glutamic acid metabolism and steroid biosynthesis.The influence values of the two pathways were 15 0.40,0.30,0.26 and 0.18,respectively.After intervention,8 markers had a certain degree of callback?P<0.05?,and a total of six metabolic pathways,including Phenylalanine,tyrosine and tryptophan biosynthesis,cysteine and methionine metabolism,pyruvate metabolism,tyrosine metabolism,D-glutamine and D-glutamic acid metabolism and alanine,aspartic acid and glutamic acid metabolism,were involved in the sample.It has been proved that AMF can improve the pulmonary fibrosis by bleomycin-induced in mouse.Based on the theory of pulmonary intestinal axis in traditional Chinese medicine,the regulation mechanism of DT,LE,ME and HE extracts of AMF on the diversity of intestinal flora in mouse with pulmonary fibrosis was studied by 16s rDNA sequencing.Compared with the control group,the results showed that the intestinal microflora in the pulmonary fibrosis model group had different changes from the level of door classification to the level of genus classification.Compared with the control group,the relative abundance of Cyanobacteria,Erysipelotrichaceae,Ruminiclostridium1,Ruminococcus1,Butyricimonas,and Gastranaerophilales in the model group mouse increased significantly?P<0.05?.The relative abundance of RuminococcaceaeUCG-005 and RuminococcaceaeUCG-004 decreased significantly?P<0.05?.The results showed that there was a certain degree of intestinal microflora imbalance in pulmonary fibrosis mouse.The drug group?except RuminococcaceaeUCG-005?could improve the relative abundance of the above intestinal flora in the model group.It is suggested that the protective effect of AMF on pulmonary fibrosis in mouse may prevent and treat pulmonary fibrosis by correcting the disturbance of microflora and then affecting the immune balance in vivo.Based on the analysis of the correlation with metabolomics,the relationship between intestinal bacteria and potential biomarkers was clarified,providing support for clinical diagnosis,medical treatment and pathophysiological research. |
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