1.Studies On The In Vivo Disposition Of Mangiferin And Its Aglycone,Norathyriol 2.Research On The Anti-Diabetic Material Of Sanxiaotang Based On The In Vivo Disposition Of Its Ingredients

The presented study consists of two parts: studies on the in vivo disposition of Mangiferin and its aglycone,Norathyriol and research on the anti-diabetic material of Sanxiaotang based on the in vivo disposition of its ingredients.Mangiferin is one of the main bioactive ingredients against diabetics in Anemarrhenae rhizome,and it can be deglycosylated to generate norathyriol with more potent anti-diabetes and hypolipidemic effects.However,the underlying mechanism leading to the low oral bioavailability of mangiferin and the disposition of norathyriol in vivo remain unclear.Thus,the presented study aimed to elucidate the in vivo disposition of Mangiferin and Norathyriol through the research on the metabolism,pharmacokinetics,liver first-pass effect and drug-drug interaction,and so on,which can provide significant information for their structural modification and pharmacological mechanism.San Xiao Tang,a twelve-herb combination documented in Yanfangxinbian,consists of Codonopsis pilosula,Atractylodes macrocephala,Angelica sinensis,Poria cocos,Rehmannia glutinosa,Phellodendron chinense,Anemarrhena asphodeloides,Coptis chinensis,Ophiopogon japonicus,Trichosanthes kirilowii or Trichosanthes rosthornii,Scutellaria baicalensis,Glycyrrhiza uralensis,has been clinically used in treatment of diabetes.Previous studies in our lab showed that the ethanolic extract of SXT?SXT-CT?exhibited more potentials against diabetes than its aqueous extract?SXT-ST?,which indicated that there were significant differences between the bioactive ingredients of the two extracts,including the structure and amount the compounds in extracts,as well as their disposition in vivo.Consequently,with the aim of unraveling the differences of bioactive ingredients between the two extracts,we conducted the systemically qualitive and quantitative analysis of the compounds in SXT-CT and SXT-ST,and in the portal vein plasma,liver,systemic plasma and kidney after administration of the two extracts.Based on these results,the compounds which showed significantly differences in pharmacokinetic behaviors after the administration of the two extracts,were chosen to test their antidiabetics effect.Finally,a metabolomics study on changes of endogenous metabolites in the diabetic mice was performed after the administration of the combination of bioactive ingredients,which provided clues for explaining the potential anti-diabetic mechanism.1.Pharmacokinetics,hepatic first-pass effect and drug interaction studies of MangiferinThrough the research on the pharmacokinetics of Mangiferin in the portal vein plasma,liver and systemic plasma,results indicated that hepatic first-pass effect slightly contributed to the concentration of Mangiferin in vivo?16.2%?,and the hepatic accumulation and hepatic metabolism of Mangiferin were limited.Based on those results,the intestinal first-pass effect on the pharmacokinetics of Mangiferin was investigated.By comparing the PK profiles with or without the inhibitors of uridine diphosphosphate-glucuronosyltransferase?UGT?,cytochrome P450?CYP450?,P-gp or in different rat models?normal and germ-free rats induced by antibiotics treatment?.Results suggested those inhibitors exhibited limited impact on the pharmacokinetic behaviors of Mangiferin.Moreover,the amount of Norathyriol derived from Mangiferin via enterobacteria was limited,only accounting for 0.1% of Mangiferin in Mangiferin-incubated enterobacteria samples,which corresponded to its considerably low exposure?concentration of norathyriol < 3 ng/m L?in the systemic plasma after single Mangiferin dosing.These results suggested the poor bioavailability of Mangiferin possibly was not mainly mediated by transporters or metabolic enzymes.The in vitro experiments on temperature-dependent uptake in hepatocyte revealed the Mangiferin uptake was mainly dependent on poor passive transport and not involved in the active transport.Therefore,the poor bioavailability of Mangiferin possibly mainly attributed to its poor membrane permeability and intestinal absorption resulting from its large polarity.2.Absorption,metabolism,and pharmacokinetics profiles of Norathyriol,an aglycone of MangiferinA total of 20 metabolites of norathyriol were identified in rat plasma,urine,feces,bile,heart,liver,spleen,lung and kidney after oral administration through HPLC-Q-TOF-MS/MS,and the proposed metabolic pathways included methylation,glucuronidation,sulfation and glycosylation.The results suggested Norathyriol and its metabolite exhibited extensive tissue distribution,as indicated by that Norathyriol and its metabolite M10,M11 and M12 were detected in all the tested biological samples mentioned above.Furthermore,a large proportion of absorbed norathyriol were subjected to glucuronide and sulfate conjugation after administration of norathyriol,as implicated by that the exposure of Norathyriol conjugations determined after hydrolysis with ?-glucuronidase and sulfatase were 10.4-and 5.1-fold that of determined without enzymic hydrolysis after oral and intravenous administration,respectively.Nevertheless,Norathyriol was well absorbed,as indicated by its absolute bioavailability of 30.4%,greater than that of Mangiferin?1.2%?,consistent with the better permeation ability and Norathyriol not being the substrate of P-gp?the main intestinal efflux transporter?.Compared with Mangiferin,Norathyriol was considered the better drug lead due to its greater bioavailability and stronger anti-diabetics and hypolipidemic effects.3.The analysis of SXT ethanolic?SXT-CT?and aqueous?SXT-ST?extracts components and metabolites after oral administrationBased on the previous finding that the ethanolic extract of SXT?SXT-CT?exhibited more potentials against diabetes than its aqueous extract?SXT-ST?,the in vitro-in vivo study was conducted from the qualitative analysis perspective,in order to explore the difference of chemical constitution between the two extracts.With the aid of HPLC-Q-TOF-MS/MS,and based on summary of the fragmentation patterns of flavones,alkaloids and saponins,the qualitative analysis herb?in vitro?-portal vein plasma?before absorbing into liver?-liver?metabolic organ?-systemic plasma?after eliminating by liver?were performed.A total of 59 compounds from SXT-CT and SXT-ST were identified,including 16 alkaloids,27 flavone,6 saponin and other 10 compounds.Furthermore,the metabolism in rats after oral administration of SXT-CT decoction was carried out.A total of 24 prototypes were detected in vivo: 19 prototypes including 4 alkaloids?Mangnoflorine,Phellodendrine,Berberine and Berberrubine?,11 flavones?Baicalein,Wogonin,Chrysin,Oroxylin A et al.?,and 1 saponin?Timosaponin A3?,were observed in portal vein plasma;17 prototypes including 6 alkaloids?Berberine and Berberrubine,et.al?,10 flavones?Baicalein,Wogonin,Chrysin and Oroxylin A et al.?,and 1 saponin?Timosaponin A3?were detected in liver;10 prototypes including 1 alkaloid?Mangnoflorine?and 9 flavones?Baicalein,Wogonin,Chrysin and Oroxylin A,et al.?were detected in systemic plasma;7 prototypes?all belongs to flavones,including Baicalin,Baicalein,et al.?were detected in kidney.Besides,there were 25 metabolites detected in vivo after administration of SXT-CT,mainly derived from the metabolism of flavones and alkaloids,including the hydroxylation,methylation and glucuronidation of flavones and demethylation,hydrogenation and glucuronidation of alkaloids.In summary,the compound detected in vivo mainly consisted of protypes belonging to flavones,alkaloids and timosaponin and the metabolites derived from the metabolism of flavones and alkaloids,which indicated that the compounds belonging to flavones,alkaloids and timosaponin types were possibly the bioactive material base of SXT-CT.By comparing the ingredients identified in SXT-CT and SXT-ST and after administration of the two extracts,there were no significant difference regarding of the number,type and in vivo distribution of compounds.Therefore,only from the qualitative analysis perspective,it was impossible to deduce the reason why SXT-CT elicited stronger anti-diabetic effect than SXT-ST.4.The determination of ingredients of SXT-CT and SXT-ST and the pharmacokinetics in rats after oral administrationAmong 18 quantitatively determined compounds,Berberine?1.00,relative ratio to Berberine?exhibited the highest amount in SXT-CT,followed by Baicalin?0.57?,Baicalein?0.12?,Timosapinin A3?0.07?and Wogonoside?0.05?and the amount of the other 13 compounds were lower than 5% of that of Berberine.In vivo,after administration of SXT-CT,Baicalin and Wogonoside exhibited the highest exposures both in the portal vein and systemic plasma;Timosaponin A3?1?exhibited highest exposure in the liver,followed by Wogonin?0.56?,Oroxylin A?0.41?,Baicalein?0.36?,Berberine?0.36?and its metabolite,Thalifendine?0.43?;Wogonin?1?,Oroxylin A?0.50?and Baicalein?0.48?exhibited high exposures in the kidney.Given diabetes and diabetic nephropathy were both comprehensive metabolic diseases,which may involve of many effect targets,the compounds with high exposure in the plasma and target organs should be both taken into consideration.In the two extracts,the amounts of Berberine,Baicalein and Timosaponin A3 in SXT-CT were higher than those in SXT-ST.Although Baicalin exhibited lower amount in SXT-CT than in SXT-ST,its amount was considerably high in SXT-CT,only second to that of Berberine.By comparing the quantitative results from the pharmacokinetics after administration of the two extracts in rats,the AUC of Baicalin in the portal vein plasma on SXT-CT treatment was 1.7-fold that on SXT-ST treatment?p <0.05?;the AUC of Timosaponin A3,Baicalin,Berberine and its metabolites,Berberrubine,Thalifendine and Columbamine in the liver on SXT-CT treatment were 1.8-,3.4-,2.4-,3.2-and 5.4-fold those that on SXT-ST treatment?p <0.05,0.01,0.001?;the AUC of Oroxyloside in the systemic plasma on SXT-CT treatment was 86-fold X that on SXT-ST treatment?p <0.001?.Based on the above qualitative and quantitative results in vivo and in vitro,Baicalin,Berberine and Timosaponin A3 might be the critically potential bioactive ingredients in SXT-CT,which were considered the major as the major target compositions for the pharmacological screenings and mechanism research of fighting against diabetes and diabetic nephropathy.5.Study on the protective effects of bioactive combination of ingredients from San Xiao Tang against diabetes and diabetic nephropathy in db/db mice by HPLC-Q-TOF-MS/MS-based metabolomicsBased on the mentioned above results,the anti-diabetics effects of the combination of Baicalin,Berberine and Timosaponin A3,as well as the combination of Baicalin,Berberine and the aglycone of Timosaponin A3,Sarsasapogenin were evaluated in ICR mice and db/db mice.The results indicated that the two combinations reduce the blood glucose of ICR mice and the corresponding area under curves of glucose concentration over time?p <0.05?in oral glucose tolerance test?OGTT?.The latter combination significantly reduced fasting blood glucose at the 2^nd,4^th,7^th,8^th,9^th and 10^th week?p <0.05,0.01?and postprandial blood glucose at the 4^th,5^th,8^th,9^th and 10^th week?p <0.05,0.01?compared with model group,and reduces the blood glucose of db/db mice and the corresponding area under curves of glucose concentration over time?p <0.01?in OGTT at the 11^th week.The combination reduced the volume of urine and the amount of microalbumin?p <0.05?over 24 h at the 12^th week.The results indicated that the combination of Baicalin,Berberine and Sarsasapogenin exhibited anti-diabetes and beneficial effect on diabetic nephropathy.A metabolomics study on endogenous metabolites change in the urinary of db/db mice after administration of the combination was performed through HPLC-Q-TOF-MS/MS.The resulting data were subjected to multivariate analysis?partial least squares discriminant analysis and orthogonal partial least squares discriminant analysis?and a total of fourteen metabolites were identified,which returned to normal levels after treatment of combination of the ingredients through the comparison of their peak areas among the three groups.By searching Metaboanalyst and KEGG database,these metabolites were involved in several metabolic pathways,such as Phenylalanine metabolism,Phenylalanine,tyrosine and tryptophan biosynthesis,Valine,leucine and isoleucine biosynthesis,and Glycine,serine and threonine metabolism,which provided clues for explaining the potential anti-diabetic mechanism of the bioactive ingredients and combination from Sanxiaotang.