| Trifolium pratense L,Medicago sativa and Stellera chamaejasme L is a traditional Chinese herb plants,has extensive pharmacological effects,the main active ingredient of flavonoids,as anti-inflammatory antimicrobial,antitumor and improve cardiovascular system components,has been concerned by many researchers,therefore,this thesis aimed at three kinds of medicinal plants in the filtration and separation and purification methods of the flavonoid active ingredient research work,as a medicinal plant Trifolium pratense L,Medicago sativa and Stellera chamaejasme L flavonoids ingredients in the development and application of experimental basis.Medicinal plants based on Trifolium pratense L,Medicago sativa and Stellera chamaejasme L as the research object,established the evaluation at the same time flavonoids-chemical composition an enzyme called cyclooxygenase 2,fatty oxidase and the activity of lactate dehydrogenase inhibitors such as mass spectrum detection method,and the rat liver microsomal metabolism in vitro measurement combined with ultrafiltration mass spectrometry,flavonoids ingredients in medicinal plants for the pharmacological activity research and purification,the concrete research content is as follows:First of all,nine chemical constituents in red clover were identified by liquid mass spectrometry(LC-MS).Seven potential cyclooxygenase-2(COX-2)inhibitors were screened from Trifolium pratense L by ultrafiltration.Screening results oriented,adopting counter current chromatography(CCC)combined with type half the preparation of high performance liquid chromatography(HPLC)(Semi-preparative HPLC)technology was isolated from Trifolium pratense L to seven kinds of flavonoids ingredients,identified as daidzin,ononin,daidzein,sissotrin,formononetin,irilone,biochanin A,the purity of 97.59%,94.31%,80.15%,97.45%,98.35%,99.55% and 92.24%.Secondly,we took Stellera chamaejasme L as the research object,using orthogonal experimental method to separate and extract the flavonoids from Stellera chamaejasme L The optimal extraction process was determined as follows: ultrasonic time of 2.0 h,extraction times of 2,liquid-solid ratio of 35:1,ethanol concentration of80%,and optimized extraction rate of 26.56 mg/g.LC-MS technique was used for rapid structural identification of six chemical constituents from chamaejasme chamaejasme.They were daphnoretin,isopimpinellin,chamaechromone,neochamaejasmine A,chamaejasmine,isochamaejasmine.Five potential lipoxygenase(LOX)inhibitors were screened by ultrafiltration technology from Stellera chamaejasme L,which were daphnoretin,isopimpinellin,chamaechromone,neochamaejasmine A,chamaejasmine.With activity as the guide,five flavonoids were isolated from Stellera chamaejasme L by counter current chromatography(CCC)combined with semi-preparative liquid chromatography.They were identified as daphnoretin,isopimpinellin,chamaechromone,neochamaejasmine A,chamaejasmine.Their purities were as follows: 96.8%,90.75%,91.41%,93.98% and 98.91%,at the same time,based on the rat liver microsomal(CYP450)in vitro metabolic system,and USES the UPLC-Q-Exactive technology,the effective components in the chamaechromone metabolites in rat liver cells and metabolic pathways are studied,results show that in CYP450 in rats,chamaechromone a stability in the metabolic process of ring crack,methylation and hydrogenation,etc.Finally,response surface combined with genetic algorithm was used to optimize the extraction process of flavonoids from Medicago sativa.The structure of four flavonoids in Medicago sativa was identified by LC-MS.Lactate dehydrogenase(LDH)was selected as the biological target molecule,and three potential LDH inhibitors were screened from Medicago sativa,which were quercetin,genistein and formononetin,respectively.The inhibitory strength of the three LDH inhibitors compared with 1.0 U/m L was 38.19%,43.50% and 33.44%,respectively.Three flavonoids were isolated and purified by counter current chromatography with the purities of 94.35%,90.18% and 90.52%,respectively.In conclusion,through the above research work,the related combined technology with mass spectrometry and chromatography as the core was established to study the flavonoids in Trifolium pratense L,Stellera chamaejasme L and Medicago sativa from the perspectives of chemistry,biology and mathematics.The purpose of separation,identification and evaluation of flavonoids in three medicinal plants was realized. |
