Intra-herb Interactions: Primary Metabolites In Coptidis Rhizoma Extract Improved The Pharmacokinetics Of Oral Berberine Hydrochloride In Mice

Aim:The crude extract of a traditional Chinese medicine（TCM）is a mixture of secondary metabolites and primary metabolites of the original plant.Some primary metabolites may have pharmacokinetic synergistic effects on the active constituents,usually secondary metabolites,in the extract.Rhizoma Coptidis is a representative heat-cleaning and one of the most commonly used TCMs in clinical practice.Berberine hydrochloride（BBR）is the most important and representative active constituent and pharmacokinetic marker of Rhizoma Coptidis.However,the poor solubility and significant first-pass effect resulted in the oral bioavailability of BBR as low as 0.36%.The previous research of our group found that there are pharmacokinetic synergistic constituents of BBR in the extract of Rhizoma Coptidis.For this reason,this thesis intended to determine the existence form of the main small molecule primary metabolites contained in Rhizoma Coptidis under the actual administration situation,and to study their effects on the pharmaceutical and pharmacokinetic properties of BBR and related mechanism,in order to explain the interactions between the constituents of Rhizoma Coptidis.This research would help to reveal the scientific connotation of the co-acting effects of multiple constituents of Rhizoma Coptidis.Methods:In the first part of this thesis,according to the content ratio of four major small molecular primary metabolites in Rhizoma Coptidis extract,i.e.,malic acid,sucrose,glucose,and choline,stable NADES were prepared by using the heating method,and the preparation process was optimized.Nuclear magnetic resonance（NMR）analysis showed that there was still a significant hydrogen bond interaction between nades even after 1000 fold dilution.The safety of the NADES and its dilutions was evaluated based on the acute toxicity experiment in mice and cytotoxicity in MDCK-MDR1 cells.The effects of NADES dilutions on the pharmacokinetics of oral BBR were investigated in mice.The effects of NADES and its dilutions on the water solubility of BBR were studied.The effects of NADES dilutions on the trans-membrane transport of BBR in intestinal gut sacs and across MDCK-MDR1 monolayer cells were determined in vitro.Whether NADES dilutions can open tight junctions reversibly was also evaluated.The effect of NADES dilutions on the metabolism of BBR in intestinal S9 was also investigated.In the second part of this thesis,the above methods were used to study the acute toxicity and cytotoxicity of malic acid alone in mice,and to determine the effect of malic acid on the pharmacokinetics of oral BBR in mice and related mechanisms of action such as influences on solubility and intestinal absorption.Results:Firstly,a viscous and transparent NADES with a density of about 1.32mg/m L and good stability within 48 hours was prepared.NMR analysis showed that even after 1000-fold dilution,the NADES still retains significant hydrogen bond interactions between molecules.The NADES had significant acute toxicity in mice.The volume of administration of 0.2 m L/kg could lead to the death of all mice within6 hours.But its 10%dilution had no acute toxicity in mice.In addition,the 1%dilution of NADES had no significant cytotoxicity.Pharmacokinetic studies showed that the C_maxof berberine in the portal vein of mice that were orally administered with1%and 10%dilutions of NADES dissolved BBR（200 mg/kg）were 3.04 and 4.57times of those in the control group,which received water dissolved BBR.In addition,the AUC_{0-12 h}values were 2.45 and 3.22 times of those in the control group,respectively.In the livers,the values of C_maxwere 1.75 and 2.08 times,and the values of AUC_{0-12 h}were 1.12 and 1.65 times of those in the control group,respectively.The solubility of BBR in pure water was about 2.5 mg/m L,but the solubility in the prepared NADES was at least 20 mg/m L（p<0.01）.Compared with the NADES,the solubility of BBR in its 10%dilution was decreased sharply but was still significantly better than that in pure water（p<0.01）.Unexpectedly,the 10%dilution of NADES significantly promoted the efflux of BBR in the isolated intestinal gut sacs（p<0.01）.When the concentration of NADES dilution was greater than 1%,it significantly increased the absorption of FD4 in the mouse gut sacs in a concentration-dependent manner,indicating that NADES dilutions could open tight junctions between intestinal epithelial cells.Importantly,the absorption of FD4 in the gut sacs of mice pretreated with 10%dilution of NADES for 4 h did not increase significantly,which indicated that the open of the tight junctions between intestinal epithelial cells was reversible.The 0.3%dilution of NADES significantly reduced the efflux rate of BBR in the monolayer membrane of MDCK-MDR1 cells（p<0.01）.However,it should be noted that NADES dilutions not only increased the apparent permeability from AP to BL but also increased the apparent permeability from BL to AP,and the latter was more significant,indicating that the role of the NADES dilutions were mainly to open the tight junctions between intestinal epithelial cells.The results of in vitro metabolism experiments showed that when the concentration of NADES was greater than 0.3%,the production of berberine metabolite demethylberberine was significantly inhibited（p<0.05）;when the concentration of NADES was greater than 1%,it completely inhibited the production of demethylberberine.The results of the second part of this thesis firstly showed that the dissolving ability of NADES to BBR was mainly derived from malic acid.When the concentration of malic acid was higher than 40 mg/m L,the solubility of BBR was significantly increased（p<0.01）.The effect was not related to the acidity of malic acid.When the malic acid solution with a concentration of 490.5 mg/m L（consistent with the malic acid concentration in the NADES stock solution）was administered intragastrically with 0.2 m L/10 g body weight,the mice all died within 6 hours.However,no animals in the 49.0 mg/m L group showed signs of toxicity or death.After incubation with 4.9mg/m L malic acid for 4 h and 24 h,cell viability was completely lost（p<0.01）;but incubation with 1.47 and 0.49 mg/m L malic acid for 4 and 24 h showed no significant cytotoxicity（p>0.05）.The pharmacokinetic study showed that after oral administration of BBR（200 mg/kg）in 4.9 and 49.0 mg/ml malic acid solution preparation groups,the C_maxof BBR in portal vein was respectively 3.03 and 3.91times of that in pure water preparation group,and AUC_{0-12 h}was respectively 2.00 and2.74 times of that in pure water preparation group;in addition,the C_maxof berberine in liver was respectively 1.78 and 2.28 times of that in water preparation group,and AUC_{0-12 h}was respectively 1.09 and 1.35 times of that in water preparation group.Malic acid significantly promoted the intestinal absorption of BBR in the range of 4.9to 49.0 mg/m L in a concentration-dependent manner（p<0.01）.Malic acid also significantly promoted（p<0.05）the efflux of BBR when its concentration reached14.7 mg/m L or more.Malic acid solution significantly increased the absorption of FD4 in the ileum of mice in a concentration dependent and reversible manner.Conclusion:In this paper,a quaternary combination NADES with no integer stoichiometric ratio between the components was prepared.There are strong interactions between the components of the NADES and its dilutions.The NADES improved the solubility of BBR.The NADES showed significant acute toxicity in mice and cytotoxicity MDCK-MDR1 cells,but disappeared after 10 or 100 fold dilution,respectively.Both the 1%and 10%dilutions of the NADES significantly promoted the intestinal absorption of oral BBR and increased its exposure level in the liver.The mechanism was not only due to improving solubility,but also due to inhibiting intestinal metabolism of BBR,inhibiting P-glycoprotein-mediated efflux,and reversible opening the tight junctions between intestinal epithelial cells.Malic acid alone had similar effects to NADES dilutions containing the same concentration of malic acid.Malic acid had significant acute toxicity in mice and cytotoxicity in MDCK-MDR1 cells.Malic acid also promoted the intestinal absorption of BBR and increased its exposure level in the portal vein and lives of mice.The mechanism was similar to that of NADES,such as increasing the solubility of BBR and reversibly opening tight junctions between intestinal epithelial cells.The comprehensive analysis of the above results indicated that malic acid was the main component of the NADES that played a role in the pharmacokinetic and pharmacokinetic improvement of oral BBR,but the hydrogen bond interaction between components of the NADES was helpful to reduce the toxicity of malic acid and enhanced its dissloving capability and pharmacokinetic synergy for BBR.