A Metabonomic And Tryptophan Metabolic Pathway Study On The Intervene Effect Of Total Ginsenoside On Alzheimer Disease

The intervening effect of total ginsenosides on Alzheimer disease was studied based on metabonomic fingerprinting and metabolic profiling.Two types of Alzheimer disease animal models were selected to learn the mechanism of total ginsenosides by liquid chromatography-tandem mass technique.1.A UFLC-MS/MS method was developed for simultaneous determination of ten ginsenosides including ginsenoside monomers Rbl,Rc,Rd,Re,Rf,Rg1,Rg2,Rh1,Ro and pseudoginsenoside F11.Digoxin was chosen as the internal standard in analysis.Gradient elution by 0.1%formic acid water and methanol was conducted adopting Shimazu UFLC system and Venusil MP C18 column.Multiple reaction monitoring was applied in negative ion mode with ESI source by AB 4000 QTRAP mass spectrometry.The linearities of 11 analytes were satisfying in corresponding concentration ranges and met the requirements of method validation.2.A UHPLC-TOF/MS based metabonomic method was established to find potential biomarkers and study the metabolism disturbance.Mouse plasma was pretreated by acetonitrile to achieve protein precipitation.Agilent UHPLC system and ZORBAX Eclipse plus C18 were applied in analysis.After a gradient elution of 0.1%formic acid water and 0.1%formic acid acetonitrile,metabonomic chromatogram was obtained by full scan in positive ion mode with ESI source.3.Amyloid beta 1-42 was injected into lateral ventricle to form acute Alzheimer disease mouse model.Behavior and metabonome discrepancy of model,sham and total ginsenosides administrated mice were studied.Behavior test results revealed that the spatial learning and memory abilities were deficit in model mice,and total ginsenosides could improve cognition abilities in dose-dependent manners.Principle component analysis showed that model and sham were divided into two groups,while metabolic network was re-balanced after drug administration.Accordingly,19 biomarkers were found and identified,including amino acids such as tryptophan,acyl carnitines such as acetyl carnitine,polyunsaturated fatty acids such as docosahexaenoic acid,sphingolipids such as phytosphingosine,and lysophosphatidylcholines.The levels of these metabolites were recovered in different degrees after total ginsenosides administration,which indicated the pharmacologic activity of total ginsenosides was related with calcium ion channel inhibition,fatty acid metabolism promotion,antiinflammation and antioxidation.4.APP/PS1 transgenic mouse model was selected as chronic Alzheimer disease mouse model to learn the discrepancy of behavior and metabonome between model,wildtype and total ginsenosides administrated mice.Morris water maze test revealed total ginsenoside could improve spatial learning and memory abilities in APP/PS1 transgenic mouse model.In principle component analysis,model and total ginsenoside administration group were separated significantly,while administration group was close to control group.15 biomarkers were found and identified including monoacylglycerols,phosphatidylcholines,acyl amino acid,tyrosine and sphingolipids.The levels of these metabolites were all recovered in different degrees in total ginsenosides administration group,which implied the pharmacologic activity of total ginsenosides was related with tyrosine pathway modulation,sphingolipid and phospholipid regulation,and signal transduction.5.A UFLC-MS/MS method was establisheded for simultaneous determination of eleven endogenous metabolite of tryptophan and its downstream products including 5-hydroxytryptophan,serotonin,N-acetylserotonin,melatonin,5-methoxytryptophan,5-hydroxyindoleacetate,kynurenine,kynurenic acid,xanthurenic acid and quinolinic acid.Aminopyrine was selected as the internal standard in analysis.Gradient elution by 0.01%formic acid water and methanol was conducted adopting Shimazu UFLC system and Atlantis T3 column.Multiple reaction monitoring was applied in positive ion mode with ESI source by AB 4000 QTRAP mass spectrometry.The established method was suitable for two biological matrixes including brain and plasma.Pretreatment procedure of brain sample consisted of methanol homogenation,protein precipitation and centrifugation,while plasma sample underwent methanol protein precipitation,centrifugation,supernatant separation,dryness and reconstitution by 0.1%formic acid water.The validated quantification method was conformed to the requirements of biological sample analysis with respect to limit of quantification,linearity,precision,accuracy,recovery,matrix effect and stability.6.The intervening effect of total ginsenosides was evaluated by the established tryptophan pathway analysis method.Acute Alzheimer disease mouse model was formed by intracerebroventricular injection of amyloid beta 1-42.After 28 days total ginsenosides administration,brain and plasma samples were collected and determined In model mice brain homogenate,the levels of serotonin,N-acetylserotonin and 5-hydroxyindoleacetate were up-regulated compared with sham group,while the level of kynurenine was down-regulated.In plasma,the concentrations of 5-hydroxytryptophan and serotonin were higher than sham group,while the concentrations of tryptophan,5-methoxytryptophan,5-hydroxyindoleacetate,kynurenine,kynurenic acid,xanthurenic acid and quinolinic acid were lower than sham group.After total ginsenosides administration,three of four disturbed endogenous components in model mouse brain were readjusted,and all nine disturbed endogenous components in model mouse plasma were rebalanced.All concentration level changes were proved to be significantly by ANOVA.Different dosages of total ginsenosides revealed different degree regulate effect on tryptophan and its downstream products.A significant dose-dependent manner of total ginsenosides could be observed through results,while the effect of high dose group is better than that of positive medicine donepezil.