Study On Water Extraction Process,Quality Control And Pharmcodynamic Comparison Of Ganle Granules

ObjectiveGanle Granule is an in-hospital preparation of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.It is mainly used for acute and chronic hepatitis,liver fibrosis,active stage of liver cirrhosis and hepatitis virus carriers.It has been used for more than ten years with definite curative effect.The aim of this project is to systematically and normatively study the preparation process optimization,quality standard improvement and pharmacodynamics comparison of Ganle granules,and lay an experimental foundation for its further development.Method1.Optimization of Water Extraction Process of Ganle GranulesAccording to the reference pre-experimental results and literature,the factors affecting various extractions were investigated,and the optimal extraction process was optimized by orthogonal test method;the chlorogenic acid content,paeoniflorin content,naringin content,neohesperidin content and dry extract yield were obtained.In order to evaluate the index,the effect of water addition,extraction time,extraction times and soaking time on the water extraction process of Ganle granules was investigated by L9（34）orthogonal test method,and the optimized process was verified.2.Qualitative and Quantitative Study and Content Comparison of Ganle GranulesTLC was used to identify eleven traditional Chinese medicines in the prescription,among which six components were identified,namely Yinchen,Paeonia lactiflora,Bupleurum,Astragalus membranaceus,Fructus Aurantii and Radix Isatidis.Three-dimensional TLC was obtained by three-dimensional scanning of TLC.The contents of chlorogenic acid,paeoniflorin,naringin and neohesperidin were determined by ultra-high performance liquid chromatography,and the contents of the two processes were compared,The contents of chlorogenic acid,paeoniflorin,naringin and neohesperidin were determined by ultra-high performance liquid chromatography,and the contents of the two processes were compared.The fingerprints of the two processes were investigated and the quality of the Ganle granules was evaluated.3.Comparative Study on the Efficacy of Two Processes of Ganle Granules in Rat Model of“Liver Injury”.Fifty rats were randomly divided into shame group,model group,Ganle granules current processing group（6.0g/kg）,Ganle granule original processing group（6.0g/kg）and positive drug colchicine group(0.1×10^-3g/kg).Except the normal group,the other groups used carbon tetrachloride-induced liver fibrosis model,50%carbon tetrachloride（CCl₄）0.1mL/100g,twice a week for 12 weeks.From the 7th week of modeling,the drug-administered group was given the corresponding test drugs by intragastric administration,and the normal group the shame group and the model group were intragastrically administered with the same volume of normal saline for 6 weeks.At the end of the experiment,blood was collected from the abdominal aorta,the contents of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）,total bilirubin（TBIL）,direct bilirubin（DBIL）,and total protein were determined by colorimetry.（TP）,albumin（ALB）,alkaline phosphatase（ALP）,glutamyl transpeptidase（GGT）,malondialdehyde（MDA）,superoxide dismutase（SOD）,total nitric oxide synthase（TNOS）and inducible nitric oxide synthase（iNOS）activity,malondialdehyde（MDA）,superoxide dismutase（SOD）,total nitric oxide synthase（TNOS）and inducible nitric oxide in liver homogenate Enzyme（iNOS）activity in serum were detected.At the same time,the liver tissue of the fixed site was taken,HE and Masson staining were used to observe the degree of hepatic histopathological damage,and the ultrastructural changes of hepatocytes such as intracellular organelles were observed by electron microscopy.Results1 Water extraction process optimization resultsThe best water extraction process of Ganle granules is to add 8 times of water to the medicinal materials,soaking time is 2h,water is taken 3 times,each time 1h.2 Thin layer identification results and content determination resultsThe thin layer identification of the eleven herbs in the compound of Ganle granules finally identified the six medicinal herbs of Yinchen,Baiji,Bupleurum,Radix Astragali,Radix,and Clam.The content of the two processes was determined to be better than the original process.The content of chlorogenic acid,paeoniflorin,naringin and neohesperidin in the current process were 0.322±0.009mg/g,6.170±0.029 mg/g,6.360±+0.030mg/g,4.88±0.023mg/g and the RSD（n=10）values were 1.67%,1.52%,1.52%and 1.49%,respectively.3 Comparative study on the efficacy of two processesCompared with the model group,the two groups of Ganle granules can significantly reduce the contents of ALT,AST,TBIL,DBIL,AKP,GGT,MDA,TNOS and iNOS in liver fibrosis rats（P<0.05,P<0.01）.The levels of TP,SOD and ALB were increased（P<0.05,P<0.01）,which alleviated the degree of liver fibrosis in rats.And the current process group of Ganle granules reduced or increased the level of each factor index slightly better than the original process group.Conclusion1 The preferred water extraction process is scientific,reasonable,stable and feasible,providing a more reliable experimental basis for the large-scale production of Ganle granules.2 TLC identification method is simple,negative and non-interference;the content of index components in the current process group is higher than that in the original process group;the quality control method is accurate,specific and reproducible.3 The two preparation processes of Ganle granules can effectively inhibit the liver fibrosis induced by CCl₄.However,compared with the original process group,the current process group of the Ganle granules can reduce the degree of liver fibrosis in rats better than the original process group.Or raise the level of each factor indicator is better than the original process group.