Exploration And Pharmacokinetics Study Of Main Active Ingredients Of Yuanhu Zhitong Formula In Rats

As natural drugs, Traditional Chinese Medicine (TCM) and its preparationscontain a great variety of components. This gives modern science a very difficulttask to find which components are the active components for the curative effectof TCM. Meanwhile, the quality control of TCM is also very difficult forindividual differences in natural products and difference pharmaceuticaltechnologies. The unknown active ingredients and the difficulty in qualitycontrol have been the bottleneck of the development of TCM.Interaction between drugs and human body is the only way in which alldrugs play their efficacy. After administration of TCM, the blood of human mustcontain the original components of TCM and many other components whichproduced by the metabolic activities and stress activities of the human body.These components are the potential active ingredients of TCM, which containthe curative components. For this reason, TCM plasma pharmacochemistry, anew strategy in the study of TCM, has played an important role in exploring the active ingredients of TCM. In addition, TCM is famous for its multicomponentand multitarget advantages. So pharmacokinetics studies play an important rolein studying the interaction of different drugs which are taken in the same time.In the present study, we choose Yuanhu Zhitong Formula (YZF) as theresearch object. Through TCM plasma pharmacochemistry andpharmacokinetics study of YZF, the present study has studied the activeingredients and its pharmacokinetic parameters and tissue distributioncharacteristics.1Objective1.1Establish the chemical analysis methods of total alkaloids and totalcoumarins of Corydalis yanhusuo (CYO) and Angelica Dahurica (ADA), whichare the formulating drugs of YZF, in vivo and in vitro of rats, and explore themain active ingredients of CYO and ADA.1.2Establish the analysis methods of tetrahydropalmatine (TDE) andimperatorin (IMP) in plasma and tissues of rats, and investigate theirpharmacokinetic parameters and tissue distribution characteristics.1.3Preliminary clarifying the effects which IMP has on the pharmacokineticparameters of TDE.2Methods2.1Explortion of main active ingredients of CYO2.1.1Extraction and chemical analysis of total alkaloids of CYO2.1.1.1Total alkaloids extraction methods The present research used thepreparation method of Yuanhu Zhitong soft capsule in the pharmacopoeia of thePeople’s Republic of China to produce the extraction of CYO. Due to thecharacteristics of alkaloids which could dissolve in acid water easily and could not dissolve in alkali water, total alkaloids were extracted from the extraction ofCYO.2.1.1.2Analysis methods for total alkaloids The present research adopted anHPLC method to analyse total alkaloids of CYO. Chromatographic condition:Column: Phenomenex Luna-C18column (250×4.6mm,5.0μm); The mobilephrase: acetonitrile (A) and0.1%phosphoric acid water (adjusted withtriethylamine to pH5.7, B); Gradien elution program:0~15min,20%A;15~30min,20~25%A;30~50min,25~80%A;50~60min,80%A; Columntemperature:30°C; Flow rate:1mL/min; Detective wave length:280nm;Injection sample volume:10μL.2.1.2Chemical analysis of plasma samples2.1.2.1Preparation of plasma samples After a single oral administration oftotal alkaloids of CYO, about1mL blood was collected from portal vein at0.5h.The blood was anticoagulated and then centrifuged at3500r/min for10min.The plasma samples were abtained.2.1.2.2Pretreatment methods of plasma samples500μL of plasma sampleand100μL of NaOH (1mol/L) were added in a glass tube. After mixing,3mLchloroform was added in the glass tube, and then vortex-mixed for5min.; theobtained mixture was centrifuged at3500r/min for10min (n=2). The twiceextract liquor was collected and dried under a stream of nitrogen at40°C, andthe analysis samples were obtained.2.1.2.3Analysis methods for plasma samples Chromatographic conditionwas the same as “analysis methods for total alkaloids” and the Injection samplevolume was20μL.2.2Exploration of main active ingredients of ADA2.2.1Extraction and chemical analysis of total coumarins of ADA2.2.1.1Total coumarins extraction methods The present research used the preparation method of Yuanhu Zhitong soft capsule in the pharmacopoeia of thePeople’s Republic of China to produce the extraction of ADA. Taking advantageof the characteristics of coumarins which could dissolve in ethyl acetate easily,total coumarins were extracted from the extraction of ADA.2.2.1.2Analysis methods for total coumarins The present research adoptedan HPLC method to analyse total alkaloids of CYO. Chromatographic condition:Column: Phenomenex Luna-C18column (250×4.6mm,5.0μm); The mobilephrase: acetonitrile (A) and water0.1%phosphoric acid (adjusted withtriethylamine to pH6.0, B); Gradient elution program:0～5min,4%A;5～30min,4～28%A;30～55min,28～31%A;55～80min,31～70%A;80～90min,70～80A;90～95min,80A; Column temperature:40°C; Flow rate:1mL/min;Detective wave length:300nm; Injection sample volume:10μL.2.2.2Chemical analysis of plasma samples2.2.2.1Preparation of plasma samples After a single oral administration oftotal coumarins of ADA, about1mL blood was collected from portal vein at2.5h. The blood was anticoagulated and then centrifuged at3500r/min for10min.The plasma samples were obtained.2.2.2.2Pretreatment methods of plasma samples500μL of plasma sampleand3mL ethyl acetate were added in a glass tube, and then vortex-mixed for5min; the obtain mixture was centrifuged at3500r/min for10min (n=2). Thetwice extract liquor was collected and dried under a stream of nitrogen at40°C,and the analysis samples were obtained.2.2.2.3Analysis methods for plasma samples Chromatographic conditionwas the same as “analysis methods for total coumarins” and the Injection samplevolume was20μL.2.3Plasma pharmacokinetics study of TDE in rats2.3.1Preparation of plasma samples After a single oral administration of TDE, about0.4mL blood was collected from retinal vein plexus at0,0.083,0.17,0.25,0.5,1,2,3,4,6,8,10,12h. The blood is anticoagulated and thencentrifuged at3500r/min for10min. The plasma samples were obtained.2.3.2Pretreatment methods of plasma samples200μL of plasma sample,10μL of internal standard solution (corydaline; CDE) and50μL of NaOH (1mol/L) were added in a glass tube. After mixing,1.5mL n-hexane-2-propanol(95:5) was added in the glass tube, and then vortex-mixed for5min; theobtained mixture was centrifuged at3500r/min for10min. The extract liquorwas collect and dried under a stream of nitrogen at40°C, and the analysissamples were obtained.2.3.3Analysis methods for plasma samples The present research adopted aninternal standard method for the detection of TDE in rat plasma. Column:Hypersil BDS C18column (250mm×4.6mm i.d.,5μm); The mobile phrase:acetonitrile-0.1%phosphoric acid water (adjusted with triethylamine to pH6.2)(60:40); Column temperature:30°C; Flow rate:1mL/min; Injection samplevolume:20μL; Detective wave length:280nm.2.4Plasma pharmacokinetics study of IMP in rats2.4.1Preparation of plasma samples After a single oral administration ofIMP, about0.4mL blood was collected from retinal vein plexus at0,0.25,0.5,1,1.5,2,2.5,3,4,5,6,8,10h. The blood is anticoagulated and then centrifuged at3500r/min for10min. The plasma samples were obtained.2.4.2Pretreatment methods of plasma samples200μL of plasma sampleand10μL of internal standard solution (isoimperatorin; ISO) were added in aglass tube. After mixing,1.5mL ethyl acetate-light petroleum (1:1) was addedin the glass tube, and then vortex-mixed for5min; the obtained mixture wascentrifuged at3500r/min for10min. The extract liquor was collect and driedunder a stream of nitrogen at40°C, and the analysis samples were obtained. 2.4.3Analysis methods for plasma samples The present research adopted aninternal standard method for the detection of IMP in rat plasma. Column:Hypersil BDS C18column (250mm×4.6mm i.d.,5μm); The mobile phrase:acetonitrile-water (60:40); Column temperature:25°C; Flow rate:1mL/min;Injection sample volume:20μL; Detective wave length:300nm.2.5Tissue distribution study of IMP in rats2.5.1Preparation of tissue samples After a single oral administration of IMP,brain, heart, liver, spleen, lung, kidney, uterus, ovary were immediately collectedfrom rats at0.5,1.5,2.5,4,6,8h, respectively. Then they were put into thephysiological saline rapidly to exclude the remaining bloodstain. After dryingthe moisture with filter paper, heart, liver, spleen, lung, kidney, uterus, ovarywere stored at-80°C until analysis. Cortex, cerebellum, brain stem,diencephalon, striatum and hippocampus were separated from brain tissuesrapidly on ice, and collected into tubes separately. They were also stored at-80°C until analysis.2.5.2Pretreatment methods of tissue samples Cortex, cerebellum, brainstem, diencephalon, striatum, hippocampus, heart, liver, spleen, lung, kidney,uterus, ovary were weighted for wet weight and homogenized in physiologicalsaline solution (15-fold tissue weight for cortex, cerebellum, brain stem,diencephalon, heart, liver, spleen, lung and kidney or30-fold tissue weight forstriatum, hippocampus, uterus and ovary) separately by a homogenizer at0°C.Then the obtained region homogenates were centrifugated separately at12000r/min for10min and supernatant of each tissue was stored at-80°C untilanalysis. To each tissue sample,500μL supernatant and20μL of internalstandard solution (ISO) were added and vortex-mixed for30s. Then3mL aceticether-light petroleum (1:1) was added and the resulting solution wasvortex-mixed for5min. After centrifugation for10min at3500r/min, the upper organic layer was transferred to a clean glass centrifuge tube, and evaporatedunder a stream of nitrogen at40°C, and the analysis samples were obtained.2.5.3Analysis methods for tissue samples The methods were the same as2.4.3.2.6The effects which IMP has on the pharmacokinetic parameters of TDEThe procedure was the same as2.3.3Results3.1Exploration main active ingredients of total alkaloids of CYO Thepresent research adopted a validated HPLC method for the detection of totalalkaloids of CYO and total alkaloids in plasma of rat treated with total alkaloidsof CYO. The method could simultaneous quantitatively analyse10alkaloids(PTP, AYE, CTE, GUE, PME, BRE, DDE, TDE, THE, CDE) in alkaloids ofCYO and qualitative analyse6alkaloids (PTP, AYE, GUE, PME, TDE, CDE) inplasma sample. DDE, whose concentration was the highest in total alkaloids ofCYO, was not detected in plasma samples. In addition, TDE’s analgesic effect isbetter than CDE. Therefore, the present research chose TDE as the activeingredient of CYO.3.2Exploration main active ingredients of total coumarins of ADA Thepresent research also adopted a validated HPLC method for the detection ofcoumarins of ADA and total coumarins in plasma of rat treated with totalcoumarins of ADA. The method could simultaneous quantitatively analyse6coumarins (XTT, XTX, BAT, IPN, IMP, ISO) in total coumarins of ADA and allof them were detected in plasma sample. Therefore, the present research choseIMP as the active ingredient of ADA.3.3Pharmacokinetics study of TDE in rats Pharmacokinetic models of TDEwas two-compartment model. The main pharmacokinetic parameters of TDE: AUC(0-t):6.44±1.69mg/L/h; AUC(0-∞):8.62±3.03mg/L/h; T1/2z:6.92±4.35h;Tmax:0.63±0.23h; Cmax:2.29±0.82mg/L.3.4Pharmacokinetics study of IMP in rats Pharmacokinetic models of IMPwas two-compartment model. The main pharmacokinetic parameters of IMP:AUC(0-t):6.52±2.25mg/L/h; AUC(0-∞):6.79±2.03mg/L/h; T1/2z:1.75±1.39h;Tmax:2.5h; Cmax:1.45±0.49mg/L.3.5Tissue distribution of IMP IMP could easily traverse blood-brain barrier.The concentration of IMP was higher in striatum than the other areas of the ratbrain (P＜0.05). IMP was also detected in heart, liver, spleen, lung, kidney,uterus and ovary. The concentration of IMP in liver was the highest (P＜0.01).3.6The effects which IMP has on the pharmacokinetic parameters of TDEAfter oral administration of TDE and IMP, the main pharmacokinetic parametersof TDE was: AUC(0-t):15.55±3.22mg/L/h; AUC(0-∞):18.31±4.40mg/L/h; T1/2z:4.41±1.17h; Tmax:0.5h; Cmax:3.51±0.88mg/L. Compared with simpleadministration of TDE, AUC(0-t), AUC(0-∞)and Cmaxwere all enhanced (P＜0.01).Conclusion1The validated HPLC methods are suitable for the analysis of alkaloids both intotal alkaloids of CYO and the plasma of rat administered total alkaloids ofCYO, and the analysis of coumarins both in total coumarins of ADA and theplasma of rat administered total coumarins of ADA. Our research preliminarydemonstrates that TDE and IMP are the active ingredients of CYO and ADA.The present researches lay the foundation for the further active ingredients studyof YZF.2The validated HPLC methods are suitable for the pharmacokinetics study ofTDE and IMP. The results demonstrate that pharmacokinetic models of TDE and IMP are two-compartment model and IMP can enhance the concentration ofTDE in rat plasma. The concentrations in striatum of TDE and IMP are both thehighest in rat brain. The results indicate that two compounds maight playsynergistic effects in striatum. The validated methods lay the foundation for thefurther pharmacokinetics study of active ingredients of YZF.3For the first time, the present research demonstrates that TDE is the keycomponent of CYO and IMP is the key component of ADA. Furthermore, ourresearches preliminarily explain the mechanism of formala principle of YZF inmolecular level.