Research On Preparation Process Optimization And Jaundice Abating Mechanism Of Canhuang Tablet

Objective: Canhuang tablet is a special clinical drug used in the Fifth Medical Center of PLA General Hospital for the treatment of chronic hepatitis accompanied by persistent jaundice-residual jaundice.It is a Chinese medicine formulated from Coptidis Rhizoma,Indigo Naturalis,Alumn and Curcumae Radix.Although the prescription has a definite effect on clearing jaundice,its mechanism is not clear so far.Besides,the proportion of Indigo Naturalis and Alumn in the prescription has a great disparity that effects the uniformity of powder and the quality of formed tablets in the preparation process.The purpose of this study was to resolve the existing problems in preparation process and clarify its jaundice abating mechanism to provide a basis for quality control of the preparation and further development.Method:（1）Optimizing the preparation process of Canhuang tablet: The optimum ultrafine grinding process of single components in the prescription was selected,and 3 kinds of ultrafine grinding process of Canhuang tablet were designed according to the results of selection and the preparation method of compound particles of single medicinal Materials.The particle size distribution,specific surface area,bulk density,vibration density and angle of repose of the ultrafine powders were inspected.The pattern of the powders was scanned and analyzed by electron microscope and the thermal stability of the powders was investigated by differential calorimetry.The characteristics of 3 ultrafine grind powders were compared.Based on above comparison,small-scale production was carried out with different ultrafine grinding processes and implemented high temperature,high humidity and strong light tests to investigate the appearance,tablet weight difference,disintegration time,fragility and berberine content of the Canhuang tablet and evaluated the contribution of different ultrafine grinding processes to the quality and stability of Canhuang tablet.（2）Exploration of the jaundice abating mechanism of Canhuang tablet based on molecular docking:The components of Coptidis Rhizoma,Indigo Naturalis,Alumn and Curcumae Radix were screened in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）under the conditions of: Oral absorptivity（≥30%）,relative molecular mass（≤500）,hydrogen bond donor amount（≤5）,hydrogen bond receptor amount（≤10）,lipid-water partition coefficient（≤5）.Each of the drug like component is used Discovery Studio 2016（DS 2016）to hydrogenate and given to CHARMm force field and defined as a molecular docking ligand.Jaundice-related genes were obtained from the CTD database（http://ctdbase.org/）with keywords "jaundice" and "cholestatic liver disease” and inputted into the Drug-Bank database（https://www.drugbank.ca/）to search for the targets and PDB IDs of listed drugs.The target protein related to jaundice was downloaded from PDB database（http://www.rcsb.org/pdb/home/home.do）by ID.DS 2016 was used to delete the original ligand and water molecule in target protein,removes polyconformation,supplements amino acid residues,hydrogenates,and defines as molecule docking acceptor.Use DS 2016 software Lib Dock module to set docking parameters,Max hits to save: 200,max number of hits: 1000,minimum Lib Dock score: 105,conformation method: FAST,the defined ligand components and receptors were docked one by one,and the evaluation results were given by Lib Dock score.Standardized the name of component-target Lib Dock score≥105.Imported the component-target data into Gephi 0.9.2 software,used Fruchterman-Reingold to layout and depict the component-target interaction network.Use Gephi 0.9.2 statistic module to analyze the he density,median and average path and evaluated the stability of the network.Identify the essential components of Canhuang tablet,which contributed significantly to the stability of the network.The mechanism of the abating the jaundice of Canhuang tablet was discussed respectively according to the function of targets.（3）Experimental study on jaundice abating mechanism of Canhuang tablet:The normal group was given 10 m L·kg^-1·d^-1 normal saline for 7 consecutive days and 10 m L·kg^-1 soybean oil for intragastric administration.In model group,10 m L·kg^-1·d^-1 was administered with saline for 7 consecutive days,and 75 mg·kg^-1 was administered with alpha-naphthalene isothiocyanate（ANIT）.In the Canhuang tablet（CHP）group,0.315 g·kg^-1·d^-1 preparetion process optimiaed Canhuang tablet solution was administered for 7 consecutive days,and 75 mg·kg^-1 ANIT was administered intragastrically to establish the model.In the high dose Canhuang tablet（HCHP）group,0.710 g·kg^-1·d^-1 preparetion process optimiaed Canhuang tablet solution was administered for 7 consecutive days,and 75 mg·kg^-1 ANIT was administered intragastrically to establish the model.In the original Canhuang tablet（OCHP）group,0.315 g·kg^-1·d^-1 original preparetion Canhuang tablet solution was administered for 7 consecutive days,and 75 mg·kg^-1 ANIT was administered intragastrically to establish the model.Ursodeoxycholic acid（UDCA）group was given 0.135 g·kg^-1·d^-1 UDCA solution for 7 days,and 75 mg·kg^-1 ANIT was given intragastrically to establish the model.On the 5th day,2 hours after administration,except the normal group,all other group were given ANIT to duplicate jaundice model,while the normal group was given soybean oil to make model.On the 7th day,2 hours after the administration,intraperitoneal injection was processed with 20% urethane(20 m L·kg^-1)solution and blood collection from abdominal aorta,liver and ileum tissues were collected,serum TBIL,TBA,ALT,AST,ALP were detected,and liver pathological changes were observed by HE staining.RT-PCR was used to detect the expression of FXR,PXR,CAR,CYP7A1,UGT1A1,BSEP,MRP2,NTCP,OATP1A1 mRNA in the liver tissue and expression of FXR,ASBT,IBABP,MRP2 mRNA in ileum tissue.The expression levels of FXR,CYP7A1,UGT1A1,BSEP,MRP2,NTCP,OATP1A1 protein in the liver tissue and expression of FXR,IBABP,MRP2 proteins in ileum were detected by Western blot.Result:（1）The optimum ultrafine comminution time of the single ingredient of the CHP was determined by screening: Coptis Rhizoma 25 min,Indigo Naturalis 10 min,Alumn 5 min,Curcumae Radix 25 min.The optimum comminution condition of process 1 is that 4 kinds of components are mixed in proportion and then ultrafine comminuted for 20 minutes.The optimum comminution condition of process 2 is 25 min Coptis Rhizoma and Curcumae Radix and 10 min for Indigo Naturalis and Alumn,In process 3,first grinding Coptis Rhizoma and Curcumae Radix for 25 min,add Indigo Naturalis and grind 10 min,add Alumn and grind for 5 min.Comparing the characteristics of powder prepared by the three grinding processes,The process 3 is outstanding in the evaluation of particle size distribution and specific surface area,process 2 is the best in the evaluation of loose density,vibration density and angle of repose.Scanning particle pattern by electron microscope,no composite structure was found in the powder of process 1,and its dispersion was not uniform,the size of powder particles varied greatly.Process 2 powders have occasional composite particle structure,but the size of the powders varies greatly,and the particles are stratified and less uniformly dispersed.In process 3,the honeycomb-like composite particle structure with regular structure can be seen in the whole field of view,the smaller particles adhere to the larger particle surface,and the powder is uniformly dispersed.The specific heat capacity of process 1 is 262.1 J·g^-1,that of process 2 is 242.7 J·g^-1,that of process 3 is 295.9 J·g^-1,which is the best of the three processes.All three kinds of ultrafine grinding process improved the yield of intermediate and finished product.The result of high temperature,high humidity and strong light test shows that the quality of process 2 and 3 is improved and stabilized.（2）A total of 174 constituents were obtained by screening the drug like properties of the constituents of CHP,of which 25 were Coptis Rhizoma,their main structural types are isoquinoline alkaloids,organic acids and lignans.14 were from Indigo Naturalis,mainly indole alkaloids.135 from Curcumae Radix,mainly composed of sesquiterpenes and diphenylheptane（curcumin）.32 targets related to the treatment of jaundice were collected,14 potential targets and 37 potential active ingredients were screened by molecular docking.Component-target interaction network analysis showed that 10 components（moupinamide,canadine,quercetin,demethoxycurcumin,obacunone,curcumin,corchoroside A,berlambine,alnusone,naringenin）were associated with monoamine oxidase A,prostaglandin synthase 2,endothelial nitric oxide synthase,inducible nitric oxide synthase,cytochrome P450 2E1,GTP cyclization hydrolysis.These associations play an important role in the stability of componenttarget network.They may play a therapeutic role in jaundice by regulating bilirubin metabolism,bile acid synthesis and transport,inhibiting immune and inflammatory responses,and affecting liver collagen formation.（3）The serum levels of TBIL,TBA,ALT,AST and ALP in model group were significantly higher than those in normal group（P < 0.01）,while the serum levels of TBIL,TBA,ALT,AST and ALP in CHP group were significantly lower（P < 0.01）.HE staining showed that the Liver tissues of normal rats were centered around interlobular veins,arranged regularly,cells are closely connected,with clear and complete cell structure,large and round cell nucleus,and no necrosis or inflammatory cell infiltration.In the model group,the pathological changes of liver tissue were obvious,the arrangement of hepatocytes was loose and disordered,most of the cell nucleus of hepatocytes atrophied and vacuoles were produced,and the junction between cells was destroyed.In CHP group,hepatic lesions were not obvious,hepatocytes arranged regularly,junctions were less destroyed,most nucleus were large and round,and vacuolar degeneration was rare.In the liver,compared with the model group,CHP could activate the expression of FXR mRNA and protein in rat liver tissue,significantly reduce the expression of CYP7A1 mRNA and protein（P <0.01）,promote the mRNA transcription of PXR and UGT1A1,promote the expression of UGT1A1（P <0.01）.CHP could significantly increase the expression of mRNA and protein of BSEP and MRP2 in liver tissue（P <0.01）.Compared with the model group,CHP could increase the expression of NTCP mRNA and protein,and significantly inhibit the expression of OATP1A1（P <0.01）.In ileum,compared with model group,both CHP and UDCA significantly increased mRNA transcription of FXR and protein expression（P<0.01）;CHP could significantly inhibit the transcription of mRNA of ASBT and increase the expression of MRP2 mRNA and protein（P<0.01）.Compared with the model group,CHP also promoted the expression of mRNA of IBABP and protein to normal level.Conclusion:（1）The ultrafine grinding process of CHP was optimized by using the method of composite particle preparation,which made the powder form composite particles with regular structure,uniform dispersion and good thermal stability.The problem of uniform mixing of Indigo Naturalis and Alumn was solved,the yield of intermediate was improved,and the quality stability of CHP was improved.（2）Molecular docking screened 37 potential active ingredients from CHP,and 14 therapeutic targets of jaundice.The jaundice abating mechanism may be related to the regulation of bilirubin metabolism,bile acid synthesis and transport,inhibition of immune and inflammatory response,and influence of liver collagen formation.（3）CHP can regulate the hepato-intestinal circulation of bile.The levels of transcription and protein expression of bile generation and transport related proteins in liver and ileum were detected.The result shows CHP increased the expression of FXR in the liver of jaundice model rats,inhibited the expression of CYP7A1 and OATP1A1,reduced the production of bile acid and reduced the uptake of bilirubin in the liver,and abated intrahepatic cholestasis.At the same time,CHP also increased the expression of BSEP and MRP2 in liver,promoted the excretion of bile acid and bilirubin in liver,abated cholestasis and reducing the damage of bile components to hepatocytes and bile duct cells.CHP can significantly increase the expression of UGT1A1 and MRP2 by activating the transcription of FXR and PXR genes in liver,and promote hydrophilic detoxification of unconjugated bilirubin and excretion from the liver.In conclusion,this research improved the quality stability of CHP,and clarified the mechanism of jaundice abating effect related to regulating the liver-intestinal circulation of bile,which provided a basis for ensuring the stability of curative effect and further development of this product.