| Chinese herbal compounds,the essence of Traditional Chinese Medicine in clinical application,have the multiple function ingredients,paths and targets.From the angle of chemical nature,Chinese herbal compound is a systematic and complex interaction system consisting of multiple components of different traditional Chinese medicines,and plays the role of reducing toxicity and enhancing efficacy.Because of the complexity of Chinese herbal compounds,it is still difficult to completely provide modern scientific basis of their effects and interpret the mechanism of their interactions.From the angle of the mechanism,drug interaction divides into the physical and chemical interaction,pharmacokinetic interaction and pharmacodynamic interaction.Pharmacokinetic interactions can lead to the changes on pharmacokinetic or pharmacodynamic intensity or toxicity of Chinese herbal compounds,which are the main reasons for in vivo drug interactions.Current studies of pharmacokinetic interactions about Chinese herbal compounds mainly focused on the determination of blood drug concentration and analysis of the absorption and metabolites of in vivo and in vitro.While the researches of interactions and mechanism in metabolism,distribution and excretion are limited due to the limitation of experimental methods at present.Pharmacokinetic interactions in Chinese herbal compounds contain the interaction effects in absorption,distribution,metabolism and excretion.Metabolic interactions are the most common in pharmacokinetic interactions,and are important and difficult in pharmacokinetics studies of Chinese herbal compounds.Liver,an important organ of the drug metabolism and the main place of biological transformation in body,is rich of enzyme system.Currently,the liver microsomal incubation system in vitro is commonly applied to the in vitro liver metabolism study of Chinese herbal compounds,but there exists the following problems.First,it is only suitable for the study of single or several representative compounds by directly incubating drug and liver microsomes,but not suitable for multicomponent research of Chinese herbal compounds.Second,Chinese medicine extract is a complex system,components are absorbed in intestine and influenced each other,directly incubating drug and liver microsomes do not conform to the actual process of traditional Chinese medicine metabolism in liver.Third,phase I and phase II metabolic pathways are competing with each other.But we generally separately monitor phase I and phase II metabolic pathways,which will lead to some deviation,and can't represent the actual situation of the bioconversion in body.Therefore,to really explore in vivo metabolic process of multicomponents of Chinese herbal compounds,we should objectively analyze the actual operation process of multicomponents and their interactions in vivo.It needs to create a new in vitro model for multicomponent liver metabolism study that is suitable for the complex traditional Chinese medicine system.Based on the above problems,we established a new type of in vitro intestinal absorption linking liver metabolism model to react interactions between multicomponents of Chinese herbal compounds,which in combination with in vitro absorption model of gut sac method and in vitro liver metabolism model of liver microsome incubation method.By using this model,intestinal permeability fluid got by gut sac method is equal to the components of drugs after gastrointestinal absorption and metabolism and before entering the hepatic portal vein.Then,starting phase I and phase II incubation metabolic pathways simultaneously,which can simulate the real absorption and metabolism characteristics and interaction process of drugs in the body.Meanwhile,we established proteomics method of livers of rats induced by Scutellariae radix,Coptidis rhizoma as well as their compatibility for the first time.Through the analysis of differentially expressed proteins,we intuitively observed the regulation at the protein level,and studied effect targets of Scutellariae radix and Coptidis rhizoma,which provided a new evaluation method for in vivo interactions of traditional Chinese medicine.Therefore,the project carried out the researches as follows:1 Established the qualitative and quantitative analysis methods of the main components of Scutellariae radix and Coptidis rhizomeThe test solution of Scutellariae radix and Coptidis rhizome was prepared according to the methods in Chinese Pharmacopoeia.The main components of Scutellariae radix and Coptidis rhizome were identified in full scan mode by HPLC-LTQ-Orbitrap-MS.29 flavonoids were identified in Scutellariae radix,which contained 16 flavonoid glycosides and 13 flavonoid aglycones.12 alkaloids were identified in Coptidis rhizome.The contents of 6 main components of Scutellariae radix and Coptidis rhizome were determined by the developed HPLC method,respectively,which all met the pharmacopoeia standards.2 Evaluation of multicomponent absorptive characteristics and interactions of Scutellariae radix and Coptidis rhizomaThe compounds of Scutellariae radix and Coptidis rhizoma in the intestinal permeability fluid were identified and determinated by LC-MS/MS.The results showed that the flavonoids and alkaloids were absorbed in the whole intestine,and they have a longer absorption window,Except for baicalin,we found 28 compounds in the intestinal permeability fluid of Scutellariae radix.HPLC analysis methods were established to analyze the main components of Scutellariae radix and Coptidis rhizoma.We studied the absorbing properties of 22 flavonoids and 10 alkaloids by calculating the main composition of the intestinal absorption kinetics parameters and analyzing by the SPSS software.The results showed that Scutellaria flavonoids and Coptis alkaloids are widely absorbed in the gut,but the absorption site of them were different.The small intestine is the main absorption site of flavonoid aglycones,but colonic absorption is poor.The flavonoid aglycones easily absorbed than flavone glycosides,which may related to the properties of them or flavonoid aglycones were easily metabolized than flavone glycosides.The absorption of alkaloids is poor,which may be related to the activity of P-gp.Because of the interactions of the components,the absorption of each component is better than that of the monomer.The effects of the compatibility of Scutellariae radix and Coptidis rhizoma on the contents of flavonoids and alkaloids in rats were studied by using the model of intestinal sac in rats which induced by gastric perfusion.It was found that the absorption characteristics of Scutellaria flavonoids and Coptis alkaloids are changed after induction;the absorption of Scutellaria flavonoids compounds was inhibited by Coptidis rhizome,scutellaria-coptis can promote its absorption.The results showed that after the compatibility of Scutellariae radix and Coptidis rhizoma,is conducive to the play of the efficacy of Coptidis rhizoma.Both of Scutellariae radix and scutellaria-coptis can promote the absorption of alkaloids,and the effect of scutellaria-coptis was stronger,and thus further verified that after the combination of Scutellariae radix and Coptidis rhizoma,the efficacy is better than medication alone.3 Evaluation of multicomponent metabolic characteristics and interactions of Scutellariae radix and Coptidis rhizomaLiver microsome incubation method in vitro was used to incubate intestinal permeability fluid of Scutellariae radix and Coptidis rhizoma obtained from rat intestinal sac method with rat liver microsomes.HPLC analysis method was established to determine the intestinal permeability fluid of Scutellariae radix and Coptidis rhizoma in the rat liver phase ? and phase? metabolism system,and explored phase ? and phase ? metabolism kinetics characteristics of the chemical compositions of Scutellariae radix and Coptidis rhizoma in rat liver.According to the results,phase ? metabolism of Scutellariae flavonoids is mainly the hydrolysis reaction,flavonoid glycosides component concentrations reduced,and flavonoid glycosides metaclass concentrations increased.phase ? metabolism is mainly the glucuronic acid binding reaction,flavonoid glycosides component concentrations increased,and flavonoid glycosides metaclass concentrations reduced.phase ? metabolism of Coptidis alkaloids is mainly the hydrolysis reaction,and each component presents different metabolic state.In phase ? metabolism,glucuronic acid binding reaction takes place in every component,and the concentration of them decreased along with the time.After rats were induced,by using the rat liver microsome incubation model,the effects of compatibility of Scutellariae radix and Coptidis rhizoma on phase ? and phase ? metabolic kinetics of the components in intestinal permeability fluid of Scutellariae radix and Coptidis rhizoma in liver microsomes were determined,respectively.The results showed that Coptidis rhizoma and scutellaria-coptis all can inhibit metabolism of Scutellariae flavonoids and the effect of Coptidis rhizoma was stronger.Scutellariae radix and scutellaria-coptis all can inhibit the metabolism of the Coptidis alkaloids,and the effect of scutellaria-coptis was stronger.This suggests that the metabolic rate is reduced after compatibility,which may inhibit the conversion of drug toxic material or reduce metabolism of effective substance,and is conducive to play drug effectiveness.4 Evaluation of multicomponent metabolic interactions of Scutellariae radix and Coptidis rhizomaAccording to the complex characteristics of Chinese herbal compounds,a model of the combination of in vitro gut sac method and liver microsomes starting the metabolic reaction of phase ? and phase ? simultaneously was established,which can simulate the process of the absorption and metabolism of Scutellariae radix and Coptidis rhizoma in vivo.To study the characteristics of the metabolism of flavonoids in rats,the analysis method of HPLC was established to analyse the metabolism of flavonoids and alkaloids in liver.The results showed that the metabolism of flavonoids and alkaloids were different from the metabolism in phase ?and phase ? reaction system.But the phase ? metabolism was the main metabolic pathway in vivo.The effects of scutellaria-coptis on the hepatic metabolism of Scutellariae radix and Coptidis rhizoma in rats were investigated.The results showed that the metabolism of Scutellariae radix can be inhibited by Coptidis rhizoma and scutellaria-coptis,the effect of Coptidis rhizoma was stronger.Scutellariae radix and scutellaria-coptis all can inhibit the metabolism of the Coptidis alkaloids,and the effect of scutellaria-coptis was stronger.5 Evaluation of the liver functional protein in Scutellariae radix,Coptidis rhizoma as well as their compatibility by proteomicsAn optimal proteomics approach was establish to analyze the protein profiling in rat liver tissue treated with Scutellariae radix and Coptidis rhizoma as well as their compatibility.78 differential expressed proteins between the experimental groups and the control group were found and identified by 2-DE and MALDI-TOF-MS analyses.This finding indicated Scutellariae radix and Coptidis rhizoma as well as their compatibility can assuredly influence the expression of the proteins in rat liver.To validate the proteomic analysis results,glutathion peroxidase,catalase and betaine homocysteine methyl transferase were selected and confirmed by western blotting.The results of triplicate western blots consist with the proteomic analysis results.By querying the relational databases,the identified 78 differential expressed proteins were summarized.These proteins involved in oxidative stress,lipid metabolism,energy metabolism,signal transduction and cytoskeleton.In summary,based on the ideas of compound multicomponents and their interaction,the induced in vivo and gut sac method in vitro were chosed for the first time to study the characteristics absorption and interaction mechanism of multicomponent of Scutellariae radix and Coptidis rhizoma.In view of the problem existed in the liver metabolism of Chinese herbal compounds in vitro.For the first time,the classical rat liver microsomal incubation method was optimized to evaluate the multicomponent of Scutellariae radix and Coptidis rhizoma by using intestinal permeability and liver microsomal incubation in vitro.According to the complex characteristics of Chinese herbal compounds,a new type of in vitro intestinal absorption linking liver metabolism model was established for the first time,which could really simulate the in vivo absorption and metabolism characteristics and interaction process between Scutellariae radix and Coptidis rhizome,which provided the research basis for in vivo study.Meanwhile,proteomics method of livers of rats induced by Scutellariae radix,Coptidis rhizoma as well as their compatibility were established for the first time to intuitively observe the regulation at the protein level and study effect targets of Scutellariae radix and Coptidis rhizoma,which will be useful to evaluate their compatibility mechanism.In vivo intestinal absorption linking liver metabolism model established in our study makes the results of in vitro experiments better consistent with results of in vivo,which reduce the difficulty of the Chinese herbal compounds researches greatly,and enhanced the pertinence and effectiveness.The method provides the references to evaluation of the interactions between multiple components of Chinese herbal compounds in vivo.Meanwhile,it is an important exploration for establishing pharmacokinetic research system in conformity with the characteristics of traditional Chinese medicine. |
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