Study On Material Basis And Action Mechanism Of Compound Suan-Zao-Ren Granule In The Treatment Of Insomnia

This study take the compound Suan-Zao-Ren granule as the research object,and the high resolution mass spectrometry technology was used to investigate the chemical components in vitro,the serum consitituents and metabolites in vivo,the pharmacokinetics of Suan-Zao-Ren decoction and compound Suan-Zao-Ren granule in rat plasma,and the metabolomics of compound Suan-Zao-Ren granule in the serum and brain homogenize of insomnia model rats.A sensitive and rapid ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry was established to identify the chemical components of compound Suan-Zao-Ren granule in vitro.Multiple data processing menthods like mass defect filtering,product ion filtering and neutral loss filtering could improve the accuracy and efficiency of analytic process.101 compounds were separated and identified,and all the constituents were also assigned to their corresponding individual herbs.11 compounds from Ziziphi Spinosae Semen,22 compounds from Chuanxiong Rhizoma,17 compounds from Anemarrhenae Rhizoma,47 compounds from Glycyrrhizae Radix et Rhizoma and 9 compounds from Poria.The above components could also divided into several groups according the type of compounds:34 compounds from flavones,21 compounds from saponins,13 compounds from phthalides,9 compounds from triterpene acids,7 compounds from organic acids,4 compounds from coumarins,2 compounds from xanthones and other kinds of compounds.The fragmentation patterns of different kinds of components were also illustrated.A sensitive and rapid ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry coupled with multiple data processing methods were developed to analyse the serum components and the relevant metabolites after administration of compound Suan-Zao-Ren granule.Under the optimized conditions,35 prototype compounds were detected in the rat serum,including 7 compounds from flavones,9 compounds from saponins,10 compounds from phthalides,5 compounds from triterpene acids,1 compounds from organic acids,1 compounds from coumarins and 2 compounds from xanthones.33 metabolites in serum of above consitituents were identified.The bioactive compounds mainly underwent the phase Ⅰ metabolic pathways including hydroxylation,hydrolysis,and phase Ⅱ metabolic pathways including sulfate conjugation,glucuronide conjugation,cysteine conjugation,acetycysteine conjugation and glutathione conjugation.A rapid and sensitive ultra-fast liquid chromatography-tandem mass spectrometry method was first developed and fully validated for the simultaneous quantification of seven main active components,spinosin,mangiferin,neomangiferin,ferulic acid,liquiritin,isoliquiritin and liquiritin apioside in rat plasma.The method was also successfully applied to compare the pharmacokinetics of these active ingredients after oral administration of Suan-Zao-Ren decoction and compound Suan-Zao-Ren granule.The separation was achieved on a Venusil MP C18 column and the detection was conducted by the multiple reaction monitoring mode using negative ion mode.The mobile phase consisted with water(containing 0.1%formic acid)and methanol at a flow rate of 0.4ml/min.The results indicated that all the analytes had similar mean concentration-time curves trend between two groups.No significant differences were observed in pharmacokinetic parameters of mangiferin,while the others had significant differences.A systematic and reliable metabolomics based on ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry coupled with statistical data analysis methods were used to find potential biomarkers in rat serum and brain homogenize,and analyze the resistance to insomnia mechanism of compound SuanZao-Ren granule.According to the loading plot of principal component analysisdiscriminant analysis,there were 14 biomarkers were screened from rat serum,including dopamine,kynurenine,5-hydroxyindoleacetic acid,phenylalanine,tryptophan,indole,2-isopropyl-3-oxosuccinate,glycine,melatonin,N-acetyl-Lphenylalanine,prostaglandin D2,serotonin,3-indolepropionic acid,5-hydroxy-Ltryptophan.19 biomarkers were found in brain homogenize,including 2phenylacetamide,tyrosine,dopamine,N-acetylaspartylglutamic acid,adenosine,adenine,phenylalanine,xanthine,xanthosine,xanthurenic acid,tryptophan,indole,phenylethylamine,melatonin,prostaglandin D2,serotonin,anandamide,calcitriol and 5-Hydroxy-L-tryptophan.The score plot of principal component analysis-discriminant analysis showed that the blank control,model,compound Suan-Zao-Ren granule and diazepam group could be separated well.And the compound Suan-Zao-Ren granule and diazepam group could be recovered towards to the blank control group.Furthermore,the study on the metabolic pathways of above biomarkers were also investigated to reveal the relationship between insomnia mechanism and biomarkers.Based on the systemic analysis of relative biomarkers and metabolic pathways,it proved that compound Suan-Zao-Ren granule could relieve the insomnia syndrome in the insomnia model rats.