| As a classic herb-pair,Anemarrhenae rhizome(Zhimu)combined with Phellodendri chinensis cortex(Huangbai)has been always used to treat diabetes.The compatibility of TCM is aimed at efficacy enhancement and toxicity reduction via changing the process of pharmacokinetic and metabolic profiles of effective ingredients in vivo.In order to clarifying the change of the vivo process of compounds and the relevant compatibility mechanisms when Zhimu and Huangbai combined and orally administrated to rats,we introduced “4-step progress”,by which the determination of herb and the pharmacokinetic study in portal venous plasma(before entering the liver),liver(major metabolic and therapeutic target)and systemic plasma(after hepatic metabolism)could be carried out in rats after oral administration.Combined with the results of enteric bacteria and primary hepatocytes metabolism in vitro,we attempted to clarifying the relationships of pharmacological activities and vivo process,as well as compatibility mechanisms of the combination of Zhimu-Huangbai herb-pair at metabolic and pharmacokinetic levels to some degree.The main achievements were as follows:1.The material base exploration of Zhimu by application of “4-step progress”A HPLC-MS/MS method for simultaneous determination of seven components of Zhimu(including three xanthones and four saponins)had been developed and validated,and then was applied to “4-step progress” investigation of Zhimu decoction.The quantitative results of Zhimu decoction were as follows: Timosaponin B2(1)> Mangiferin(0.33)> Timosaponin B3(0.24)> Neomangiferin(0.05)> Timosaponin A3(0.005);the order of AUC of ingredients in portal vein plasma was Mangiferin(1)> Norathyriol(0.38)> Timosaponin B2(0.29)> Timosaponin B3(0.22)> Timosaponin A3(0.13)> Neomangiferin(0.01),inconsistent to those in the herb decoction,and Mangiferin and its metaboliteNorathyriol were of the highest exposure in portal vein plasma,whereas Neomangiferin exhibited an extremely low exposure;Timosaponin B2 and Timosaponin B3 were not detected in liver,but Timosaponin A3 showed a large volume of collection,and the ranking order of exposure of ingredients in liver was Abstract Shanghai Normal University Master of Philosophy Timosaponin A3(1)> Norathyriol(0.02);the order of AUC of ingredients in systemic plasma was Mangiferin(1)> Timosaponin B2(0.15)> Timosaponin B3(0.10)> Norathyriol(0.09)> Timosaponin A3(0.08)> Neomangiferin(0.01),where Mangiferin displayed the topmost exposure.Combining the above-mentioned information and results from primary hepatocyte and enteric bacteria metabolism,the in vivo dynamic processes of active compounds of Zhimu could be reasonable to be deduced,as following: Timosaponin B2 and Timosaponin B3 had a great scale exposure in both portal vein and systemic plasma,together with their huge liver extraction ratio(ER)of 67.5% and 70.4%,respectively,and this could be likely to attribute to the hepatic extraction(such as bile extraction),in consideration of no accumulation and metabolism of those saponins happened in liver;Timosaponin A3 was originated from deglycosylation of saponins(such as Timosaponin B2 and Timosaponin B3)on the effect of enteric bacteria and highly accumulated in liver;Mangiferin displayed the topmost exposure in portal vein and systemic plasma,accompanying the liver extraction ratio(ER)of 35.6%,and the sizable exposure of Mangiferin was mainly contributable to the direct absorption from herb decoction,as well as part from the metabolism of Neomangiferin;Norathyriol,the metabolite of Neomangiferin and Mangiferin on the effect enteric bacteria,displayed certain accumulation in the liver.2.The material base exploration of Huangbai by application of “4-step progress”A HPLC-MS/MS method for determination of eight components(including three prototypes and five metabolites of alkaloids)had been developed and validated,then we applied it to “4-step progress” investigation of Huangbai decoction.The amount order of main compounds in Huangbai decoction was Berberine(1)> Phellodendrine(0.23)> Magnoflorine(0.15),and the contents of berberine metabolites were extremely tiny,less than 5% of that of prototype;the order of AUC of ingredients in portal vein plasma was Magnoflorine(1)> Phellodendrine(0.74)> Berberine(0.46)> Berberrubine(0.12)> Thalifendine(0.05),and it was inconsistent to that of the herbal decoction;All alkaloids accumulated in liver,and Berberine was of the maximum expousure,followed by its metabolites Thalifendine and Jatrorrhizine,as follows: Berberine(1)> Abstract Shanghai Normal University Master of Philosophy Berberrubine(0.96)> Thalifendine(0.87)> Jatrorrhizine(0.68)> Magnoflorine(0.57)> Phellodendrine(0.55)> Demethyleneberberine(0.12)> Columbamine(0.08);the order of AUC of ingredients in systemic plasma was Magnoflorine(1)> Berberine(0.25)> Thalifendine(0.09)> Berberrubine(0.07)> Phellodendrine(0.05),where Magnoflorine displayed the topmost exposure.Combining the above-mentioned information and results from primary hepatocyte and enteric bacteria metabolism,the in vivo dynamic processes of active compounds of Huangbai could be reasonable to be deduced,as following:,Phellodendrine underwent a strong hepatic passed effect(96.1%),and this could be highly correlated with its hepatic accumulation and metabolism;Magnoflorine displayed considerable exposure in the portal vein plasma-liver-systemic plasma,and its liver extraction ratio(ER)was only 47.5%,which may be relevant to its hepatic accumulation,but not hepatic metabolism;Berberine was absorbed into portal vein plasma,suffering from a serious hepatic first pass effect(71.5%),and then entered into the systemic plasma.Simultaneously,Berberine could be widely metabolized into Berberrubine,Thalifendine,Demethyleneberberine,Jatrorrhizine and Columbamine on the effect of enteric bacteria and hepatic enzymes,and all of those metabolites accumulated a lot in the liver to various degree,as well as Berberrubine,Thalifendine were detected in the plasma.To sum up,the systemic investigation on the effective components group of Zhimu and Huangbai decoction by application of “4-step progress” in vivo and in vitro was successfully performed,and it is aimed at clarifying the in vivo dynamic processes of bioactive components(both prototype and metabolite)and the material base in Zhimu and Huangbai from a view of metabolic and pharmacokinetic point comprehensively,which could lay a solid foundation for the further research on manifesting the material basis of Zhimu-huangbai herb-pair,as well as the corresponding compatibility mechanisms.3.The material base exploration of Zhimu-Huangbai by application of “4-step progress”Through the comparison of the results between single herb and herb-pair by application of “4-step progress”,there was no significant difference on the content of ingredients between single and combined herb group.After oral administration in rats,compared to the sole herb treatment,the exposure of saponins had changed Abstract Shanghai Normal University Master of Philosophy in both portal vein plasma and systemic plasma,where the exposures of Timosaponin B2 were increased by 67% and 149%,respectively,but those of Timosaponin B3 were reduced by 41% and 29%,respectively,as well as those for Timosaponin A3 decreased by 51% and 22% respectively,but with an augmented accumulation of Timosaponin A3 by 57% in liver.The co-administration leaded to decreased exposure of xanthones: in portal vein plasma and systemic plasma,the corresponding decreased AUCs of Neomangiferin was by 40% and 23% for Neomangiferin,54% and 58% for mangiferin,and both 82% for Norathyriol,respectively,whereas increased accumulation of Norathyriol by 57% appeared in liver.In contrast,the combination treatment could result in the increased AUC of all alkaloids in vivo on different levels: in portal vein plasma,liver and systemic plasma,the respective increased AUC was by 64%,181% and 195% for Phellodendrine,110%,31% and 67% for Magnoflorine,234%,233% and 169% for Berberine,respectively.Besides,when rats taking Zhimu and Huangbai together,Berberrubine and Thalifendine,the metabolites of berberine,displayed an increased exposure both in portal vein plasma and systemic plasma,and particularly,the hepatic accumulation of Thalifendine,Demethyleneberberine and Columbamine also augmented in different degrees.Hence,the above changes were probably owed to the drug-drug interaction of multiple components derived from Zhimu and Huangbai in terms of metabolic enzymes and transporters.Through the application of “4-step progress”,the content and exposure of prototypes and metabolites in vitro and in vivo was successfully compared between single and combined herb treatment,and consequently,there was quite limited change on the content of ingredients between the single and combined herb treatment,but those in vivo espouse of prototypes and metabolites did change: the exposure of xanthones decreased in portal vein plasma and systemic plasma,where all alkaloids and Timosaponin B2 improved,but Timosaponin B3 reduced diversely;it was notable for the metabolites of xanthones,saponins and alkaloids showed various degree improvement of hepatic accumulation.Combined with pharmacological investigations,it is reasonable to deduce the combination of Zhimu and Huangbai could not only increase the exposure of prototypes with antidiabetic activity(such as Magnoflorine,Berberine)in the bloodstream and liver,but also promote the hepatic accumulation of metabolites exerting hypoglycemic potentials(Timosaponin A3,Norathyriol,metabolites of Berberine),leading to the Abstract Shanghai Normal University Master of Philosophy possible efficacy enhancement in diabetes management. |
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