| ObjectsBerberine(BBR),the main active ingredient of Rhizoma coptidis and Phellodendron amurense,is widespread in clinical application.However,numerous pharmacokinetic studies show that the concentration of BBR in plasma is extremely low when administrated orally.Besides,it is widely distributed,slowly eliminated and rapidly metabolized in vivo.Limited concentration of BBR in blood cann’t explain the excellent efficacy,in addition,many metabolites of BBR show lower effects than itself on anti-inflammatory or multiple active targets in liver,so there may be other metabolites with stronger activity than BBR.BBR shows the similar pharmacokinetic characters with the drugs which bound to protein.It indicates that BBR probably combines with proteins in blood.Red blood cell(RBC),which possess both metabolic and targeting functions,is the most abundant blood cell in the blood.And the most abundant protein of RBC is hemoglobin(Hb).Therefore,the study on the metabolism and combination of BBR in RBC is helpful to illuminate the pharmacokinetic process of BBR as well as other protoberberine alkaloids.Methods1.Establishment of analysis method of concentration of BBR,OBB in blood(1)Pretreatment of free drug on blood sample250 pL of blank rat blood was mixed with 50 μL of BBR solution(10 μg/mL),the solution was incubated for 2 h in 37℃,then the 1 mL of acetonitrile solution was added to the mixture.Protein was fully precipitated,and then solution was centrifuged for 10 min at speed of 8000 r/min.Supernatant was removed,concentrated and finally redissolved with 200 μL acetonitrile.(2)Pretreatment of bonding drug on blood sample250 μL of blank rat blood was mixed with 50 μL of BBR solution(10 μg/mL).The solution was incubated for 2 h in 37℃ and added with five times amount of 15%HCL and subsequently heated in water bath at 60℃ for 6 h.After sample was centrifuged at 8000 r/min for 10 min,the supernatant was removed and enriched by SPE column.The eluent was concentrated and then redissolved at 200 μL of acetonitrile.(3)Discovery of oxyberberine(OBB),a new metabolite of BBRBlood samples of SD rats after intravenous injection of BBR(2.5mg/kg)were treated with "HCL hydrolysis method".The metabolite was identified by IT-TOF-MS,the fragment information was compared with the literatures,and then the metabolite was preliminarily confirmed.The retention time of metabolite was further compared with the purified metabolite,which obtained by synthesis,and finally determined.(4)Establishment of HPLC analysis method for blood samplesThe samples were determined by HPLC method:phenomenex Luna C18(250 mm x 4.6 mm,5 microns),mobile phase:0.05%formic acid solution(A),acetonitrile,(B).The samples were analyzed at 345 nm with a flow rate of 1 mL/min.(5)Test of HPLC analysis method for blood samplesBBR and OBB were respectively mixed with blank whole blood to obtain a series of biological samples.Piperine was added as the internal standard and the blood samples were treated according to the "HCL hydrolysis method".The concentration of sample was used as the abscissa,and the ratio of the peak area of sample to the internal standard was used as the ordinate to fit the standard curve.Blood samples with high,medium and low concentrations were made and measured for consecutive 3 days to obtain precision and accuracy of the intraday and inter-day.The extraction recovery rate was considered as the ratio of peak area of the drug in the blood sample to that in the acetonitrile at the same concentration,and the method recovery rate was calculated by the ratio of measured concentration to added concentration of drug.Blood samples were kept at the condition of room temperature and80℃ to determine the stability of samples at room temperature(48 h)and freeze-thaw(once or three times).2.Pharmacokinetic analysis of free BBR when administered orally and intravenouslyPharmacokinetic study of BBR was conducted in the ways of oral administration(400 mg/kg)and intravenous(2.5 mg/kg).The concentration of free BBR was determined by the way of "Pretreatment of free drug".The blood concentration-time profiles were made and then the pharmacokinetic parameters were calculated by the software "Drug and Statistics 2.0".3.The effect of different blood samples on the free concentration of BBR and OBB25 μL of BBR、OBB(500 μg/mL)was mixed with 250 μL of blank whole blood and plasma respectively,and then the solution were incubated in 37℃ for 2 h.The samples were prepared according to the "pretreatment of free drug",and analyzed by the "HPLC analysis method for blood samples".The concentration of drug in different blood samples were compared.4.Effects of BBR on blood cellsAtotal of 48 rats were randomly divided into six groups.They were intravenous injected with vehicle(normal saline),BBR(2.5 mg/kg),respectively.The whole blood sample(about 2 mL)was collected into a K2EDTA tube at 5 min,30 min and 120 min after administration.Blood routine examination was subsequently conducted to evaluate the effects of BBR on blood cells.5.Pharmacokinetic analysis of free and bonding BBR,OBB when intravenousA total of 8 rat were administrated with BBR(2.5 mg/kg)by intravenous.The blood sample was collected in different time points and divided into two equal aliquots.The one was treated with "pretreatment of free drug",and another one was treated with "pretreatment of bonding drug".The blood concentration-time profiles were made and the pharmacokinetic parameters were compared by the software "Drug and Statistics 2.0".6.Study on the interaction mechanism of BBR,OBB and HbBHb was mixed with BBR and OBB to obtain a series of solutions.And then the means of UV-vis absorption spectra,fluorescence spectra,synchronous fluorescence spectra,circular dichroism(CD)spectra and Raman spectra were utilized to measure the systems of BHb-BBR and BHb-OBB.Besides,molecular docking experiment was carried out to simulate the combination of BBR,OBB and BHb.The optimal conformation acquired by computer simulation was used to the analysis of binding mode.Result1.Investigation of determined method of drug concentration in bloodOBB,a new metabolite was found in the hydrolyzed blood samples of rats after intravenous of BBR.And calibration curves of BBR and OBB had good linearity(r>0.99),the precisions(RSD)and accuracy(RE)of two curves both within 15.0%,the extraction recovery and method recovery of them were more than 75%and 94.93%.So the analysis methods meet the requirements of pharmacokinetic experiments.2.Pharmacokinetic analysis of free BBR when administered orally and intravenouslyConcentration of BBR in blood rapidly decreased 5 min after intravenous and maintained at a low level for 24 h.1 h after oral administration of BBR,the Cmax was only 0.260 g/mL,and the drug was eliminated sharply.AUC0-t of BBR in two ways of administration are limited(0.681 μg/mL*h and 6.422μg/mL*h,respectively).3.Interaction between BBR,OBB and blood componentsThe concentration of BBR in whole blood was much lower than that in plasma when incubated for 2 h.The results of blood routine examination show that platelet count and white blood cell count in BBR group and OBB group failed to achieve statistically significant difference compared with the vehicle group(p>0.05).But treatment with BBR and OBB significantly reduced the RBC and Hb in rat(p<0.05 vs.vehicle group).4.Pharmacokinetic analysis of free drug and bonding drug when intravenousCompared to free BBR,the Co of bonding BBR was significantly improved,AUC0-t and AUC0-∞ were remarkably magnified,and t1/2z was significantly extended(p<0.01).It is noteworthy that a certain amount of bonding OBB was found in blood,but rarely seen free in the blood.5.Study on the interaction mechanism of BBR,OBB and HbThough the binding of BBR and OBB to BHb are both static process,the Stern-Volmer quenching constant(Ksv)between BBR and BHb exhibited larger than that of OBB as well as the quenching rate constant(kq).Electrostatic force is the main binding force in BHb-BBR system,while the BHb-OBB system may take place predominantly via the van der Waals and hydrogen bonds interactions.Both BBR and OBB can reduce the intensity of BHb.Besides,BBR can affect the molecular environment of Trp and Tyr,but OBB can only affect the Trp when interacting with BHb and the change is smaller than BBR.Both BBR and OBB can affect the secondary structure and tertiary structure of BHb.In the molecular docking,similarΔG values and location of BBR and OBB had been observed when interacting with BHb,and both BBR and OBB link the same amino acid residue(Arg-141),but OBB also connect with Lys-127 via hydrogen bond.Conclusion1.Concentration of free BBR is low in whole blood,but its new metabolite OBB has been found in protein-binding drugsAfter oral and intravenous administration of BBR and incubation in vitro,the concentration of free BBR was very low.But the contents of erythrocytes,neutrophils,monocytes and Hb in the blood were significantly decreased by intravenous of BBR.While OBB,the novel metabolite of BBR was found in the high concentration of protein-bonding drugs.Low concentration of free BBR and decreased blood routine indexes may be connected with the binding effect of BBR and blood components,and its metabolite,OBB may also interact with blood components.2.BBR mainly binds to Hb in vivo and is largely metabolized as bonding OBBAfter intravenous administration of BBR,the concentration of free BBR in the whole blood of rats was very low,while the concentration of bonding BBR was significantly increased,and the content of bonding OBB was relatively high,which may be the main metabolite of BBR.However,few free OBB was detected.It was speculated that BBR was mainly binds to Hb in vivo,and a large amount of prototype drug was metabolized as bonding OBB.3.The combination of BBR,OBB and BHb were demonstrated by experiments in vitroThe combinations of BBR,OBB and Hb were confirmed by the spectroscopy experiments in vitro.There is one binding site between BBR,OBB and BHb,respectively.And the bonding effect is strong.Predominant effect of BHb-BBR system is electrostatic force,while Van der Waals forces and hydrogen bonds play the main role in BHb-OBB system.The strong combination of BBR,OBB and BHb further affects the secondary and tertiary structures of BHb.Specifically,the content of a-helix is reduced,the stretching mode of Ca-Cm is changed,and the amino acid residues of BHb is affected(BBR:Trp and Tyr,OBB:Trp)... |
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