Process Optimization Of Yixin Badiranjibuya Granule Based On Fingerprint

Objective:(1)Establishment of the determination method and characteristic fingerprint of the active ingredients in Yixin Badiranjibuya granules.(2)Optimize the extraction process of Yixin Badiranjibuya granules.(3)Optimize the forming process of Yixin Badiranjibuya granules.(4)The content of active ingredients,characteristic fingerprints and preliminary in vitro efficacy of the optimized preparation and the commercially available preparations are compared.Methods:(1)Simultaneous determination of luteolin-7-O-β-D-glucuronide(LG),apigenin-7-O-glucuronide(APG),rosmarinic acid(RA),tilianin(TL)and diosmetin-7-O-β-D-glucuronide(DG)in Yixin Badiranjibuya granules by High performance liquid chromatography(HPLC)and establishment of their characteristic fingerprints.Ten batches of commercial preparations of Yixin Badiranjibuya granules are characterized and analyzed by the similarity evaluation of traditional Chinese medicine fingerprints and systematic cluster analysis.(2)Single factor and Box-Behnken design response surface methodology are adopted,with ethanol concentration,solvent times,and extraction time as independent variables.Taking the content of LG,APG,RA,DG,TL and the paste yield as the dependent variable.The response surface method(RSM)is used to analyze the influence of independent variables on dependent variables,and the optimal conditions are determined for verification experiments.(3)Single factor test is used to investigate the types and amounts of auxiliary materials and binders that affect the forming process by comprehensive scoring method of multiple indicators such as granules hygroscopicity,forming rate,and solubility,and the optimal forming process conditions are optimized.(4)Intervention is given to extracts from commercially available preparations and extracts from process-optimized preparations.Changes in the activities of lactate dehydrogenase(LDH),creatine kinase isoenzyme(CK-MB),superoxide dismutase(SOD)and malondialdehyde(MDA)in cardiomyocytes are measured.Comparing the effects of commercially available preparations and process-optimized preparations on H9c2 cardiomyocyte enzymes.Results:(1)A method for simultaneous determination of 5 representative components by HPLC fingerprint of Yixin Badiranjibuya granules was established.The contents of LG,APG,RA,DG and TL in ten batches of Yixin Badiranjibuya granules were 0.2795-0.4499,0.0824-0.1353,0.1848-0.4721,0.1490-0.3326,0.3112-0.6233 mg/g respectively.A total of 13 common peaks were calibrated in the fingerprint spectrum,and the chemical components of 5 main characteristic peaks were clearly identified,namely LG(Peak 2),APG(Peak 6),RA(Peak 7),DG(Peak 8),TL(Peak 11).The similarity values between the fingerprints of the ten batches of commercial preparations and the control fingerprints ranged from 0.598 to 1.000.Cluster analysis was divided into two categories.(2)The optimal extraction process is as follows: 55% ethanol,30 times the amount of solvent,1 extraction,6 h.And carry out the verification test,the RSD is 2.46%.(3)Best forming process: dry extract is obtained by the best extraction process,add the forming auxiliary lactose according to the material ratio(1:2),pass through an 80-mesh sieve and mix evenly,add an appropriate amount of 80% ethanol solution to make a soft material,14-mesh sieve Granulate,dry at 50°C,and granulate to obtain Yixin Badiranjibuya granules.The contents of LG,APG,RA,DG and TL in ten batches of laboratory-made Yixin Badiranjibuya granules were 1.2777-1.3536,0.4329-0.4534,3.0737-3.1890,1.1367-1.2224,6.1632-6.4995 mg/g,respectively.The chromatographic peak area and peak number of each batch of particles in the fingerprint spectrum were similar,and the difference between the spectra was small,the similarity is greater than 0.900,and the similarity was high.(4)In the in vitro efficacy test,compared with the control group,the activities of LDH,CK-MB and MDA in the model group were significantly increased(P<0.0001);compared with the model group,the activities of LDH,CK-MB and MDA in the process optimized preparation-ethanol extraction(EE)group and the commercial preparation-water extraction(WE)group were decreased(P<0.001 and P<0.05);the degree of reduction in the EE group was higher than that in the WE group(P<0.05 and P<0.01);compared with the control group,the SOD activity in the model group was significantly decreased(P<0.0001);compared with the model group,the SOD activity in the EE group and WE group was increased(P<0.001 and P<0.05),no significant difference.Conclusion:(1)The established HPLC method for content determination is fast and simple,and has a good linear relationship.(2)The extraction process is stable and feasible.(3)After optimization,the granules have high forming rate,low moisture absorption rate,high melting rate,and uniform granule appearance,which achieves the purpose of optimization.(4)The contents of LG,APG,RA,DG and TL in the commercial preparation and the process optimized preparation are quite different.Process optimization is reasonable and feasible.The established characteristic fingerprint can be used for the quality control of Yixin Badiranjibuya granules.According to the results of myocardial enzyme determination,the protective effect of the extract from the optimized preparation on myocardial ischemia-reperfusion injury is slightly better than that of the commercial preparation.