The Mechanism Of Detoxification And Retention Effect Of Terminalia Chebula On Radix Aconiti Kusnezoffii Based On Pharmacokinetics

Objective Based on the pharmacokinetic process,the mechanism of reducing toxicity and preserving effect of Terminalia chebula?HZ?on Radix Aconiti Kusnezoffii?CW?was studied.Methods?1?Chemical binding of HZ and CW.In vitro:three aconitines were mixed with tannic acid?TA?in PBS solution to determine the content changes of three aconitines at different time points.In vivo:rats were randomly divided into two groups:CW group and HZ?CW?1?1?group.The rats were given 0.12g/kg of CW.After a single intragastric administration,carotid artery blood was taken at different time points to determine the concentration of three aconitines in blood.?2?Effects of HZ on absorption of CW.Caco-2 cell model was established.The components of three aconitines were detected by UPLC/Q Exactive MS.The bidirectional transportion of three aconitines in Caco-2 cell model was studied.The effects of P-gp inhibitor and tannin acid?TA?on the translocation of three aconitines were studied.?3?Effect of HZ on the distribution of CW.In vitro:the equilibrium dialysis method was used to simulate the binding process of three diester aconitines to plasma proteins in vivo.The binding rates of plasma proteins at different concentrations of three aconitines and the effects of TA on their binding rates were studied.Rats were randomly divided into different groups,in which the control group was given the same amount of 0.05%sodium CMC-Na solution,group H was administered with0.36 g/kg of HZ,and the another groups were given 0.12 g/kg of CW for 7consecutive days,and the heart,liver,spleen,lung,kidney,brain and fat tissues were taken 0.5 h after the last administration.?4?Effects of HZ on metabolism of CW.Metabolism of three aconitines was studied by in vitro incubation of rat liver microsomes.Samples were taken at different time points.The residual concentration of three aconitines at different time points was obtained.The metabolic rate was non-linear regression to the substrate concentration,that is,the curve of reaction speed to the substrate concentration was obtained.Finally,the metabolic parameters of three aconitines were obtained.At the same time,it was incubated together to determine whether HZ had any effect on the metabolism of CW.?5?Effect of HZ on excretion of CW.Collection of urine and fecal samples.Rats were randomly divided into different groups,in which the control group was given the same amount of0.05%sodium CMC-Na solution,group H was administered with 0.36 g/kg of HZ,and the another groups were given 0.12 g/kg of CW for 1 day.Urine and fecal samples were collected in metabolic cages at different time points.After uratan anesthesia,bile duct intubation was performed in rats.Bile samples were collected every 1 hours.Bile samples were collected within 12 hours.Results In vitro chemical binding experiments,we found that TA had no obvious chemical binding with aconitine?AC?,mesaconine?MA?and hypaaconitine?HA?,but the contents of three aconitines in blood were significantly different?P<0.05?between CW group and CW?HZ?1?1?group.It proved that the biological reaction played a more role in"detoxification and preservation effect".?2?In Caco-2 cell model,the cumulative transports of three aconitines were positively correlated with incubation time.Papp values of three aconitines had no statistical difference,and PDR values were close to1.5.The P_apppp values?AP-BL?of the three aconitines increased significantly after adding verapamil,a P-gp inhibitor,which further proved that AC,MA and HA were affected by the efflux protein P-gp in the absorption.When the ratio of three aconitines to tannic TA was 1:1 and 1:0.5,the absorption of aconitine was significantly inhibited?P<0.05?,but there was no significant effect on the conversion of effluent.?3?In vitro:the plasma protein binding rates of the three aconitine groups were all low plasma protein binding rates?about 30%?,with no significant difference between the groups and no significant dose dependence.When the ratio of three aconitine and TA was 1:0.1 and 1:0.075,the protein binding rate was significantly decrased?P?0.05?.When the ratio of three aconitines to tannic acid reached 1:0.025,the protein binding rate did not decrease significantly?P>0.05?.In vivo:After continuous intragastric administration for one week,the distribution of three aconitines was the highest in kidney,followed by liver,lung,spleen and heart.The distribution of three aconitines was also detected in the brain,but the content was too low to quantify.Aconitine,neoaconitine and hypaaconitine were not found in fat.Compared with group S,the distribution of tissues in Terminalia kusnezoffii group?Z Group?and different compatibility groups of CW-HZ decreased?P<0.05?.The contents of three aconitines from L-H were Z group<CW-HZ?1?1?group<CW-HZ?3?1?group<CW-HZ?1?3?group<CW group.the maximum reaction rates(V_max)of AC,MA and HA are 387.59ng·min^-1·mg protein^-1,337.83ng·min^-1·mg protein^-1and 450 ng·min^-1·mg protein^-1,the micron constants?K_m?are1124ng·L^-1,962.8ng·L^-1and 1278ng·L^-1,the internal removal rates(CL_int)are0.34?L·min^-1·mg protein^-1?0.35?L·min^-1·mg protein^-1?0.352?L·min^-1·mg protein^-1,respectively.?5?The three aconitines in Radix Aconiti are excreted mainly through the kidney.The order of cumulative excretion of AC in each group was CW group>CW-HZ?1?1?group>CW-HZ?1?3?group>CW-HZ?3?1?>Z Group.The order of cumulative excretion of MA and HA was opposite to that of AC.The order of excretion half-life of three aconitines wasCW-HZ?3?1?group>CW-HZ?1?1?group>CW-HZ?1?3?group>CW group>Z group.The excretion of the three aconitine were almost unchanged after 24 hours.Basically no excretion of bile and feces.Conclusion Terminalia chebula has an effect on the absorption,distribution and excretion of Radix Aconiti in the pharmacokinetic process,thus playing a role of"detoxification and preservation effect",while Terminalia chebula has no obvious inhibition or promotion effect on the metabolism of Radix Aconiti.