Study On Metabolic Transformation Of Ziziphi Spinosae Semen Saponins By Intestinal Micro Flora In Vitro

Objective: In this study,the total saponin of Ziziphi Spinosae Semen(ZSSS)was investigated,and its composition was fully characterized based on UPLC-Q-Orbitrap-MS.The metabolic transformation process of intestinal microflora in vivo was simulated,and an intestinal bacteria culture system with stable metabolic ability was established.Optimize the sample pretreatment method,analyze the prototype components,metabolic conversion products before and after the transformation products of ZSSS(ZSSST),and speculate the possible metabolic transformation pathways.Use high-throughput sequencing technology to analyze the regulation of ZSSS to the intestinal microflora of insomnia.The sleep-improving activity of the conversion products of ZSSS was evaluated at the animal level,and the possibility of the metabolites of ZSS was its potential effector.Methods: 1.Preparation of ZSSS and characterization of chemical composition.Taking ZSS as materials,ethanol extraction and macroporous resin isolation and purification method was used to obtain ZSSS.The purity of ZSSS was determined by UV spectrophotometry.The chemical compositions of ZSSS were characterized by LC-MS/MS.2.Intestinal microflora to metabolize and transform the ZSSS in vitro.Using human fresh feces as the source of intestinal microflora,the OD 600 value of bacterial solution and the conversion ability of ZSS saponin components were used as evaluation indicators.The cultivation time and culture medium were investigated and screened to establish an intestinal microflora with the ability to maintain stable metabolism in vitro culture system.On the basis,the method of sample pretreatment was optimized.UPLC-Q-Orbitrap-MS was used to identify the prototype components and metabolites,and to clarify the metabolic effect of normal and insomniac intestinal microflora on ZSSS.Analyze and compare the metabolic rate and metabolic reaction pathways,and provide a basis for clarifying the effect substances of ZSSS.3.Study on the regulating effect of ZSSS on intestinal microflora.The intestinal microflora of normal people,the intestinal microflora of insomnia patients and the ZSSS intervened in the intestinal microflora of insomnia patients were applied to high-throughput sequencing by Illumina MiSeq sequencing platform,and the diversity and bacteria of different samples were analyzed according to the sequencing results species composition and species differences,and analyze the correlation between intestinal microflora and ZSS saponins,so as to screen out the differential bacterial strains of normal and insomniac patients,and clarify the role of ZSSS in regulating intestinal microflora to improve insomnia.4.Study on the ZSSST for improve sleep activity in mice.The macroporous resin was used to purify the ZSSST,and the content of jujuboside A and B in the ZSSST was determined by HPLC-ELSD method.The pentobarbital sodium mouse model was used to evaluate the ZSSS before and after transformation to improve sleep activity,and to explore the effective part of ZSS.Results: 1.In this study,a total of 35 g of saponin was prepared with a purity of 45.75%,and jujuboside A and B were 11.83% and 6.71% respectively.Based on UPLC-Q-Orbitrap-MS,a total of 20 saponins were identified,of which 17 were tetracyclic triterpenes and 3 were pentacyclic triterpenes.2.Establish a culture system of intestinal microflora with the ability to maintain stable metabolism with a culture time of 9 h and a medium of GAM.The metabolic conversion samples of the ZSSS were adsorbed by SPE C18 solid phase extraction cartridges,washed with 40% methanol after water washing,and then eluted with 100% methanol.The 100% methanol eluent was evaporated to dryness and then analyzed.By UPLC-Q-Orbitrap-MS,a total of 38 metabolites of saponin were identified,among which 34 metabolites were identified in the intestinal microflora of normal people,36 metabolites were identified in the intestinal microflora of patients,and sugar radical hydrolysis reaction is the main metabolic pathway,followed by glycosyl reaction,deoxygenation reaction and decarboxylation reaction in the transformation of normal human intestinal microflora,and deoxygenation reaction and oxidation reaction can occur in the intestinal microflora of patients.3.Compared with the intestinal microflora of normal people,the intestinal microflora alpha diversity of patients with insomnia decreased,and after the intervention of ZSSS,the diversity increased significantly.At the phylum level,Firmicutes,Proteobacteria,Actinobacteria and Bacteroidetes,which account for the main abundance,all have callbacks(P<0.001).At the gene level,15 genera were significantly up-regulated and 7 genera were significantly down-regulated after the intervention of the ZSSS.Through LefSe and random forest analysis,Turicibacter and Clostridium were used as biomarker species in the insomniac patient group,and Dorea and Bacteroides as the normal group biomarker species.The correlation between the intestinal microflora with significant differences and the saponin prototype and metabolites was analyzed to clarify that the intestinal microflora and the saponin have mutual effects.4.The intestinal microflora was transformed with 9.00 g ZSSS.The converted samples were purified with macroporous resin to prepare 7.03 g ZSSST.The results showed that the ZSSS and ZSSST reduce the mice's autonomous activity,prolong the sleep time of mice with pentobarbital sodium threshold dose,and reduce the sleep latency of mice.The differences were statistically significant,and both the latency and sleep time Present dose dependence.It showed that the ZSSS and ZSSST had sedative and hypnotic effects,there was no statistical difference between the two,but the overall ZSSST had better effect than the ZSSS.Conclusion: This study establishes a ZSS saponin identification strategy based on UPLC-Q-Orbitrap-MS.From the perspective of metabolic transformation of intestinal microflora,establish an intestinal bacterium with a stable metabolic capacity culture system.Optimized sample preparation method,and studied prototype components,metabolites and metabolic law before and after the conversion of ZSSS;using high-throughput sequencing technology to analyze the effect of ZSSS on intestinal microflora of insomnia,screening in order to effectively distinguish the normal and insomnia marker bacteria.From the animal level,the activity evaluation of the ZSSST was made clear that the metabolite of ZSSS is a potential effective component.This study provides a basis for the determination of the effective substance basis of ZSS saponins based on intestinal microflora and the targeted treatment of insomnia.