Study On Quality Control Of Flavonoids Part From SZS And The Mechanism Of Depression-relieving And Spirit-quieting From Its Compatibility

Objective: To establish method of Semen Ziziphi Spinosae flavonoids for quality control;To study the effect of different compatiblility and ratio of the Semen Ziziphi Spinosae flavonoids on behavior and antidepressant mechanism in depressed and insomnia mice.The pharmacological components of the flavonoids of Semen Ziziphi Spinosae were indentified by the spectral-efficiency correlation analysis,and the pharmacological components were isolated from the Semen Ziziphi Spinosae flavonoids;The main components of the depression-relieving and spirit-quieting mechanism were investigated.The effective dose and the biological information of the transcriptome were analyzed,which laid a foundation for clarifying the effective components and mechanism of depression-relieving and spirit-quieting.Methods: Fingerprints of the flavonoids of Semen Ziziphi Spinosae were established by high performance liquid chromatography(HPLC),and the characteristic components of flavonoid fingerprints were identified by HPLC-MS.The content of two main flavonoids was determined by HPLC.To study the effect of different flavonoid compatibility drugs on the antidepressant effect of mice,the levels of Glu,GABA and BDNF in the brain were measured by enzyme-linked immunosorbent assay(ELISA),and the ratio of flavonoids compatibility were analyzed.The method of high performance countercurrent chromatographic was used to separate the flavonoids from Semen Ziziphi Spinosae.To observe the effect of different combinations of monomer components on depressed and insomnia mice,the levels of Glu,GABA and BDNF in the brain of depressed mice were determined by enzyme-linked immunosorbent assay(ELISA).The change contents in the initial study show drug molecules existed interactions in vitro.The differential gene expression of brain tissue in mice was analyzed by high-throughput sequencing technique.The main signal pathways of differentially expressed genes were enriched by pathway enrichment.Results: Fingerprints of the flavonoids from Semen Ziziphi Spinosae were established by HPLC.Ten peaks were selected as the reference peak.Ten common peaks were calibrated for ten batches of samples.Eight of flavonoids and one of alkaloid were identified by HPLC-MS.The content of two major flavonoids in different batches was mersured.The content of spinosin was determined to be greater than 161.53 mg/g and the content of 6’’’-feruloylspinosin should be greater than 52.71 mg/g.The analysis of the behavioral indexes of depressive and insomnia mice showed that the mindle dose of alkaloid-flavonoids(1:14)and the low dose of alkaloid-flavonoids(1:12)could play an antidepressant and anti-insomnia effect.The results showed that the mindle dose of alkaloid-flavonoids(1:14)and the low dose of alkaloid-flavonoids(1:12)groups could significantly improve the symptoms of depression and insomnia mice,the effects related to balance brain Glu and GABA levels,increased brain-derived neurotrophic factor.According to the fingerprint and the efficacy index of the Spearman correlation analysis,the gray correlation analysis and the multivariate regression analysis showed that the flavonoids of Semen Ziziphi Spinosae were such as vicenin II,glucosylvitexin,spinosin,6’’’-p-hydroxybenzoylspinosin and 6’’’-ferulylspinosin and alkaloid component such as magnoflorine has a strong correlation with the efficacy index.Based on the nature of Semen Ziziphi Spinosae flavonoids,N-butyl alcohol-ethyl acetate-water(2:3:5)were chosen as solvent system,and three compounds were isolated and identified,the compound 1was magnoflorine,the compound 2 was spinosin,the compound 3 was6’’’-feruloylspinosin.Three-component compatibility group and four-component compatibility group were able to improve the symptoms of depressive and insomnia mice.The three-components group,magnoflorine 28mg/kg+spinosin64mg/kg+6’’’-ferulylspinosin 24mg/kg,its ratio of the two neurotransmitters in the compatibility groups was closer to mice of the blank group.The results of HPLC analysis showed that the 6’’’-ferulylspinosin was added in relation to the compatibility of magnoflorine and spinosin can make peak area value of magnoflorine and spinosin get decline.High-throughput sequencing analysis showed that four genes,ENSMUSG00000090698,ENSMUSG 00000081824,ENSMUSG00000062202 and ENSMUSG00000026516 had different expression,and they included four metabolic pathways:ribosome biogenesis in eukaryotes,oxidative phosphorylation,parkinson’s disease and non-alcoholic fatty liver disease(NAFLD).Conclusion: According to the qualitative characteristics of the fingerprints of the flavonoids of Semen Ziziphi Spinosae and its quantitative information,the quality control standard of the flavonoids was established,which provided the scientific basis and standard for the standardization of the flavonoids.The results of pharmacodynamics showed that the low dose group of alkaloids-flavonoids(1:12)and middle dose group of alkaloid-flavonoids(1:14)could significantly improve the symptoms of depressive and insomnia mice,due to the balance of brain Glu and GABA levels,increased brain-derived neurotrophic factor.Based on the spectrum-effect correlation analysis to confirm six component were related to effects of depression-relieving and spirit-quieting.with the method of high-speed countercurrent chromatography,three components can be isolated from the Semen Ziziphi Spinosae flavonoids.The results showed that the recoveries were high,the operation was simple and the separation was large.The effect of antidepressant and anti-insomnia of the three-component combination group was better than that of the four-component combination group,and its mechanism might be related to regulating different expression of four genes.