Study On The Isolation,Extraction And Activity Evaluation Of The Effective Components Against Ischemic Stroke In Medicinal Plants Puerariae Flos And Ligusticum Chuanxiong

In China,Puerariae flos(P.Flos)and Ligusticum chuanxiong(CX)are traditional medicinal plants demonstrating a profound theoretical foundation and applications.Currently,P.Flos is mainly assessed in diabetic research,while CX is primarily aimed at regulating the nervous system.Stroke,as a type of blood circulation disorder,has a significant influence on the daily life of patients.Reportedly,P.Flos and CX have specific effects in ischemic brain injury and secondary peroxidation brain injury,however,few reports are available on its active ingredients and mechanism of action.Therefore,the isolation,purification,activity screening,and evaluation of the anti-ischemic-stroke active components in P.Flos and CX need to be further investigated.Utilizing the medicinal plants P.Flos and CX as the research objects and employing the UF-LC-MS activity screening technology,the active components,and their structures were identified.Additionally,the protective effects of the P.Flos active ingredients were assessed using PC12 cells,while the active components in CX were evaluated in rat liver microsomal metabolites.Furthermore,the purification of P.Flos and the effective constituents in CX were assessed using the activity-oriented,application of HSCCC combined with Semi-preparative HPLC.The active components in P.Flos and CX were analyzed using chromatography and mass spectrometry.The research was performed as follows:First,three high-purity active ingredients were isolated from P.Flos using HSCCC.Following preliminary identification,the active ingredients were identified as Puerarin,Tectorigenin,and Genistin,with purities of 90.60%,99.00%,and 91.73%,respectively.Three potential lactate dehydrogenase(LDH)inhibitors and three potential ?-glucosidase inhibitors were screened by UF-LC-MS.The results indicated that isoflavones in P.Flos failed to demonstrate significant inhibitory activities against xanthine oxidase(XOD).Furthermore,the MTT assay was used to investigate the protective effect of the P.Flos extract on PC12 cells damaged by 1-methyl-4-phenylpyridinium(MPP+).Compared with the cell survival rate of the MPP+ injury group that of the P.Flos extract group was significantly increased,with the P.Flos extract demonstrating protective effects on damaged PC12 cells.Second,the two chemical categories(mainly phthalein and phenolic acids)present in the CX extract were rapidly identified by the UPLC-Q-Exactive technique.HSCCC and semi-preparative HPLC were used to separate and purify the crude extracts of CX,and four active components were obtained,namely Ligustrazine,Ferulic acid,Z-ligustilide,and 3-Butylidenephthalide,with a purity of 90.60%,99.24%,97.01% and 97.86%,respectively.Two potential LDH inhibitors and three potential superoxide dismutase(SOD)inhibitors were screened from CX using UF-LC-MS.Additionally,based on the in vitro metabolic system of rat liver microparticles(CYP450),the metabolites and metabolic pathways of CX derived Ferulic acid and Z-ligustilide in rat liver cells were analyzed using the UPLC-Q-Exactive technique.The results demonstrated that,in rat CYP450,the stable metabolic processes of Ferulic acid were gluconylation,hydroxylation,and decarboxylation,and the stable metabolic processes of Z-ligustilide were decarbonylation,demethylation,and decarboxylation.In summary,this paper systematically studied the separation and purification,structure identification,activity screening,and mechanism of action of the active components in the medicinal plants P.Flos and CX,and successfully separated,identified,and simultaneously evaluated the active components in P.Flos and CX.