Effects Of Atractylodes Macrocephala On Phase ? Metabolism Of Strychnos Nux Vomica In Liver Microsomes

Strychnos nux vomica is a commonly used medicine in TCM clinic.Its main active ingredients are brucine and strychnine,which are widely used in the treatment of rheumatoid arthritis and nervous system diseases.However,Strychnos nux vomica has strong toxicity,which limits its clinical application.Toxicological studies have shown that Atractylodes macrocephala could reduce the toxicity of Strychnos nux vomica,but the specific mechanism of attenuation is not fully clear.Previous studies have shown that Atractylodes macrocephala can reduce the toxicity of nux vomica by inhibiting intestinal absorption of active ingredients of nux vomica,but the toxicity of nux vomica has not been reported in vitro.Therefore,from the point of view of in vitro metabolism,this paper aims to study the difference of enzymatic kinetics of nux vomica in phase I metabolism,and to acquire the phenotype of enzymatic metabolism of active ingredients of nux vomica.Studying the effect of Atractylodes macrocephala on drug metabolic enzymes and explaining the mechanism of Atractylodes macrocephala in reducing toxicity of nux vomica in metabolism are of great significances for effectively evaluating one's safety level and clinical safe medication of it.The main works of this paper are as below: 1.Studying on the effect of Atractylodes macrocephala on the metabolism kinetics of phase I enzymes in the active constituents of nux vomica;2.Determining on the interaction of nux vomica with CYP450 enzymes;3.Investigating the interaction between extracts of Atractylodes macrocephala and CYP450 enzymes.1.Effect of Atractylodes macrocephala on Phase I Enzyme Metabolism Kinetics of Active Components of Strychnos nux vomicaIn this chapter,in vitro incubation technology was used to study the enzymatic kinetics of brucine and strychnine in rat liver microsomes after compatibility of Atractylodes macrocephala and brucine.The effects of Atractylodes macrocephala on phase I enzymatic metabolism of brucine and strychnine were obtained by comparing the kinetic parameters of brucine and strychnine.The results showed that the metabolic parameters Km,Vmax and Clint of brucine and strychnine in rat liver microsomes were 12.03±2.65 and 9.27±1.53 g/L,0.15±0.04 and 0.12±0.02 ?g/(min·mg·protein),0.012 4 ±0.000 7 and 0.012 8 ±0.000 3 min/mg,respectively.The metabolic rates of brucine and strychnine decreased with the increase of the concentration of Atractylodes macrocephala and strychnine in the co-incubation of Atractylodes macrocephala macrocephala.The metabolic rates of brucine and strychnine in low,medium and high concentration groups of brucine-atractylodes macrocephala were significantly higher than those in single brucine group,which were 69.0%±4.4% and 70.9%±4.0%,61.6%±2.5% and 64.6%±2.4%,57.1%±2.3% and 59.9%±1.9% in brucine-atractylodes macrocephala group and 37.5%±9.1% and 42.8%±7.9% in brucine-atractylodes macrocephala macrocephala group.The results showed that brucine and strychnine could be significantly metabolized,and the metabolic rate could reach more than 30%.It indicated that brucine had phase I metabolic characteristics and could be further studied on phase I metabolism of brucine.The combination of Atractylodes macrocephala and brucine could reduce the enzymatic kinetic parameters of the active components of brucine,which indicated that Atractylodes macrocephala could effectively slow the clearance of brucine in liver.It is speculated that the interaction between Atractylodes macrocephala and Semen Strychni may affect the metabolism of Semen Strychni,and the specific mechanism may be related to the level of phase I metabolic enzymes.2.The Interaction between Strychnos nux vomica and CYP450 EnzymeIn this chapter,the enzymatic pathways of brucine and strychnine were studied by chemical inhibitor method.The CYP450 phenotypes involved in brucine and strychnine metabolism were identified by using alpha-naphthalene flavone(CYP1A2),nocardione(CYP2C19),sulfobenzene pyrazole(CYP2C9),quinidine(CYP2D6)and ketoconazole(CYP3A4)as inhibitors.At the same time,different concentrations of brucine extract and CYP450 enzyme probe drug were incubated,and the metabolic rate of each enzyme probe drug was taken as an index to investigate its effect on the activity of phase I metabolic enzyme.The results showed that with the increase of inhibitor concentration,the metabolism of brucine and strychnine was inhibited most obviously by ketoconazole,the inhibition rate was more than 90%,followed by sulfamethoxazole,the inhibition rate was about 40%,the remaining three inhibitors only partially inhibited,the inhibition rate was between 20% and 40%.It indicated that brucine was mainly metabolized by CYP3A4 enzyme,secondly by CYP2C9 enzyme,and a little by CYP1 A CYP2C9 enzyme.2.CYP2C19 and CYP2D6 metabolism.The effect of brucine seed extract on CYP450 enzyme activity showed that brucine seed had strong inhibitory effect on CYP1A2 and CYP2D6,with IC50 values of 1.48 and 0.31 mg/mL,respectively,but weak inhibitory effect on other enzymes,with IC50 values greater than 10 mg/mL.The above results provide a basis for the possible interaction between nux vomica through enzymatic metabolic pathway and affecting drug metabolic enzyme activity,and have important theoretical guiding significance for clinical drug design.At the same time,it can be seen that the active ingredients of nux vomica are mainly metabolized by CYP3A4 enzyme,but they have no significant effect on CYP3A4 enzyme activity themselves.It can be concluded that Atractylodes macrocephala may play a role in regulating CYP3 activity.The activity of A4 enzymes can reduce the metabolism of active components in nux vomica,which can be used as the mechanism of reducing the toxicity of nux vomica.3.The Interaction of Atractylodes macrocephala Extract and atractylenolide II on CYP450 EnzymeIn this chapter,the effects of extracts of Atractylodes macrocephala on CYP450 enzyme activity were investigated.The effects of different concentrations of Atractylodes macrocephala and probe drugs on CYP450 enzyme activity were observed.Then,the interaction of atractylenolide II on CYP450 enzyme was further studied with atractylenolide II as the active component of Atractylodes macrocephala macrocephala The results showed that Atractylodes macrocephala has potential induction effect on CYP3A4 enzyme,the induction rate is about 30%.Atractylodes macrocephala has strong inhibitory effect on CYP1A2 and CYP2D6.The IC50 values are 5.12 and 4.43 mg/mL,respectively.It has weak inhibitory effect on other enzymes,and the IC50 value is more than 100 mg/mL.In the study of interaction between atractylenolide II and CYP450 enzyme,it was found that atractylenolide II was mainly metabolized by CYP3A4 enzyme,partly by CYP1A2 and CYP2C19 enzyme,while other enzymes were hardly involved in metabolism.In the study of CYP450 enzyme activity,atractylenolide II was found to have non-competitive inhibition on CYP1A2 and CYP3A4.Ki was 154.51 and 139.82 ?M,respectively,and CYP2C19,CYP2C9 and CYP2D6 were competitive inhibition.Ki values were 200,66.79 and 138.63 ?M,respectively.In conclusion,atractylenolide II was not easy to interact with other drugs and competed with brucine for CYP3A4 enzyme metabolism.The interaction of atractylodes macrocephala extract and atractylenolide II on CYP450 enzymes can confirm that atractylodes macrocephala can inhibit CYP1A2 and CYP2D6 enzymes activities,thereby weakening the undesirable enlargement of pharmacodynamics induced by brucine and strychnine,while atractylodes macrocephala can partly induce CYP3A4 enzymes activity.In the process of metabolic competition between atractylodes macrocephala macrocephala and nux vomica,since the ability of the former is stronger than that of the latter,thus the metabolism of CYP3A4 enzyme to atractylodes macrocephala increased.Meanwhile,the metabolic rate of brucine and strychnine decreased,which may be one of the important mechanisms that atractylodes macrocephala.reduced the toxicity of nux vomica.