Study On The Metabolism Of RB-2 And RB-4 In Radix Bupleurum

Rationales: Depression is a major human blight,which is usually to have chronicity and recurrence.Antidepressants with definite curative effect have already been in clinical use.Meanwhile,side effect caused by toxicity and poor tolerance tends to be exacerbated.Therefore,traditional Chinese medicine owing to low toxicity and mild response has becoming a perfect candidate in the field of drug discovery.Previous research in our group has already demonstrated that polyacetylenes from Radix Bupleuri is the antidepression effective component.The mean serum concentration-time profiles of RB-2 and RB-4 in rats showed a fast distribution phase followed by a relatively slow elimination phase and double peaks after i.g.administration.Further research about in-vitro effect of polyacetylenes on antidepressant activity,the result demonstrated that polyacetylenes potently inhibit monoamine of rat brain synaptosomes,with a better potency than their corresponding specific inhibitors.So,as a candidate for polyacetylenes,it is indispensable to study the metabolic characteristics.When studying the characteristic about metabolism both in vivo and in vitro,the following question would be clarified: the categories of metabolic and the pathway of metabolism,the interaction of enzyme and compound,also the properties of pharmacology and toxicology.It will be provide some scientific evidence for drug discovery and drug in clinical use.However,the metabolic characteristics of polyacetylenes from Radix Bupleuri remains unclarity,the research about metabolic characteristics of polyacetylenes appears more prominent.Objective: To identify the main endosomatic metabolites of RB-2 and RB-4 from Radix Bupleuri;to elucidate the metabolic pathways;to find the CYP450 enzymes isoforms employed in the metabolism;to study the influenc of polyacetylenes for the CYP450 enzymes isoforms;to evaluate the metabolic characteristics of polyacetylenes from Radix Bupleuri;to provide a scientific evidence for the development of antidepressant drugs.Methods: 1.The control group and two test groups were divided at random using male ICR mice.The RSLC-Q Extractive Orbitrap-MS analysis technology and Compound Discoverier anlysis software were employed to investigate the fragmentation and retention time of polyacetylenes as the base of identification.100 mg·kg-1 polyacetylenes was in intragastric administration.After gavage,the serum,urine,feces and bile sample were pretreated and analyzed.To detect the mass information of metabolites,the speculation of metabolites in metabolic pathway and structure were identified in vivo.2.The mice liver microsomes were prepared using differential centrifugation.The incubation system contained polyacetylenes RB-2/RB-4,regeneration system and liver mircosomes.Mixing them together incubated.The analysis method was the same as in vivo.The metabolic pathway of polyacetylenes in human or mice liver mircosomes in vitro was speculated in the same progress as those in vivo.The difference between in vivo/in vitro and in MLMs/in HLMs was also compared.3.In the assay of ascertainment of CYP450 enzyme isoform,the types of metabolism were selected in HLMs detected.CYP 450 selective chemical inhibitors and human recombinant CYP isozymes were used to investigate the effects on the metabolism of polyacetylenes in HLMs.Selective chemical inhibitors which FDA was optimized were used,that is ?-naphthoflavone(CYP1A2),sertraline(CYP2B6),montelukast(CYP2C8),sulfaphenazole(CYP2C9),ticlopidine(CYP2C19),quinidine(CYP2D6),clomethiazole(CYP2E1)and ketoconazole(CYP3A4).Using these two methods,the phenotypes of CYP450 enzyme were investigated.4.Established a MRM quantitative analysis method in combination with UHPLC-MS/MS,Seven selective substrates,phenacetin for CYP1A2,efavirenz for CYP2B6,paclitaxel for CYP2C8,tolbutamide for CYP2C9,dextromethorphan for CYP2D6,chlorzoxazone for CYP2E1,testosterone for CYP3A4,were used for evaluation the activity of CYP450 enzymes.Results: 1.Compare to blank samples,the reaction of oxidation should be the major metabolic pathway for RB-2 in Phase?with 4 types of which oxidation,oxidation combined oxidation,desaturation combined oxidation,desaturation combined oxidation following oxidation.In phase?,the conjugational groups usually combine with metabolite of phase?(always glucuronide conjugation).The same process also occurred in RB-4.The difference between two of compounds was the type of metabolites,which RB-4 contained oxidation,oxidation combined oxidation,desaturation combined oxidation following oxidation.The excretion of polyacetylenes was mainly followed with feces.2.Ultimately,a total of 4 potential metabolites of RB-2 and RB-4 were investigated in HLMs,respectively.In phase?,the reaction of oxidation should be the major metabolic pathway for polyacetylenes in vitro.The conjugational groups usually combine with metabolite of phase?(always glucuronide conjugation)in phase?.The reaction of oxidation was detected both in vivo and in vitro.The disparity between to two different bioenvironments in metabolite was the degree of oxidation,which the reaction of oxidation was more completely in mice.Only glucuronide conjugation was identified while others none.The two types of oxidation were unique in liver microsomal(hydration combined oxidation following oxidation,hydration following oxidation combined glucuronide conjugation).Two genus also was analysis.The result showed that there had the same metabolic pathway both in MLMs and in HLMs.However,the format of hydration combined oxidation following oxidation was unique in HLMs and other special format in vitro was only existed in MLMs.3.The result showed that the special selective CYP450 inhibitor sulfaphenazole(CYP2C9),montelukast(CYP2C8)and clomethiazole(CYP2E1)were inhibited the metabolic rate of polyacetylenes.Sertraline(CYP2B6)and ketoconazole(CYP3A4)were influenced the generation of metabolite for polyacetylenes in part.Meanwhile,the result using human recombinant CYP isozymes showed that CYP2C9 and CYP2E1 were potential CYP450 enzyme isoform for RB-2 and RB-4,next were CYP2B6 and CYP3A4.4.Quantitative analysis method using UHPLC-MS/MS was established to evaluate the activity of CYP450 enzymes by analysis the reduction of selective substrates.When the concentration of RB-2 or RB-4 was 100 ?mol·L-1,the polyacetylenes can inhibit CYP1A2 faintly.However,both RB-2 and RB-4 had no obvious inhibitory effects on the other isoforms of CYP2B6,CYP2C8,CYP2C9,CYP2D6,CYP2E1 and CYP3A4.Conclusion: 1.According to identification and classification of metabolites in vivo,it was demonstrated that the main biotransformation reactions for polyacetylenes were oxidation and glucuronide conjugation.The excretion of polyacetylenes was mainly followed with feces.2.The result about the reduction ratio of substrates,it was preliminarily speculated that there were no significance influence of the activity of CYP450 enzyme for polyacetylenes.3.The metabolic properties of polyacetylenes were excellent,which can be used as a candidate for the development of antidepressant drugs.