| Objective: To conduct a re-evaluation study on the process and quality of the listed variety "Kunning granules",mainly including a re-evaluation study on the basis of pharmacological substances,a re-evaluation study on extraction process,a re-evaluation study on quality standards,and a re-evaluation study on metabolomics.Methods: Ultra high performance liquid chromatography quadrupole time of flight tandem mass spectrometry(UHPLC-Q-TOF-MS/MS)was used to analyze the chemical components of Kunning granules.Interaction analysis was performed on the components obtained from the mass spectrometry through network pharmacology,and network topology analysis was used to screen the key components for the treatment of functional abnormal uterine bleeding;The anticoagulant activity of Kunning granules was determined using blood coagulation method and fibrinogen plate method.HPLC fingerprints of 10 batches of Kunning granules were established using high-performance liquid chromatography,and their spectral efficiency relationship was established.Common components in network pharmacology results and spectral efficiency analysis were selected as their main Q-markers,and anticoagulant activity was used as an indicator to verify and confirm their anticoagulant Q-markers,clarifying the pharmacological substance basis of Kunning granules;Based on the factors of water addition ratio,extraction time,and extraction frequency,the weight coefficients of Q-marker components were determined using the AHP method.The comprehensive scores of their content and extraction rate,as well as anticoagulant activity,were used as indicators to optimize and determine the extraction process and parameters of Kunning granules using the design space method;According to the provisions of the fourth part of the 2020 edition of the Chinese Pharmacopoeia,the content determination of improved Kunning granules was studied,and UV,HPLC and other methods were used to establish the quality standards for intermediate Kunning granules,improved Kunning granules,and original Kunning granules;Using metabolomics techniques,the serum pharmacology of improved Kunning granules and original Kunning granules on normal and Qi stagnation and blood stasis rats was studied.Results: 1.475 chemical components of Kunning Granule were obtained by mass spectrometry analysis,and 22 key components such as quercetin,genistein,salvianolic acid B,apigenin,triptolide,paeoniflorin,emodin,naringin,baicalein,neohesperidin,hesperidin,etc.were taken as the material basis of potential disease treatment by network pharmacology;Combined with the spectrum effect relationship,salvianolic acid B,paeoniflorin,naringin and neohesperidin were identified as the main Q-Markers of the blood activating effect of Kunning Granules.The efficacy verification results showed that these four components were stable and feasible as the main Q-Markers of the blood activating effect of Kunning Granules,and could be used as the material basis for its anticoagulant activity.2.Design the spatial method to obtain the extraction process range of Kunning granules,which includes extraction time(X1)of 30-65 minutes,extraction frequency(X2)of 2-3 times,solvent ratio(X3)of 12-13 times.The optimal extraction process is 3 times,extraction time of 30 minutes,and water addition of 12 times.The validation experimental results show that the process is stable and feasible.3.In the simultaneous determination of four Q-Marker components: the statistical analysis of the content of paeoniflorin,naringin,neohesperidin and salvianolic acid B in the intermediate of improved particles and the intermediate of Kunning particles with the original process showed that the P values were 0.70,0.58,0.35 and 0.03.There was no significant difference between the content of paeoniflorin,naringin and neohesperidin in the intermediate of Kunning particles with the improved process and the intermediate of Kunning particles with the original process,but there was a difference between salvianolic acid B.According to the determination results,the content of paeoniflorin,naringin,neohesperidin and salvianolic acid B in the intermediate of Kunning granules shall not be lower than 0.79%,2.27%,2.33% and 0.90%.The statistical analysis of the contents of paeoniflorin,naringin,neohesperidin and salvianolic acid B in the improved granules and the original granules showed that the P values were 0.64,0.60,0.20 and 0.01.There was no significant difference in the contents of paeoniflorin,naringin and neohesperidin in the improved and original Kunning granules,and there was a difference in salvianolic acid B.According to the determination results,the content of paeoniflorin,naringin,neohesperidin and salvianolic acid B in Kunning granules shall not be less than 0.31%,0.95%,0.97% and0.34%.In the study of UV visible fingerprint spectra,there was no significant difference(P=0.56)in the peak absorption of the control fingerprint spectra between the improved Kunning particle intermediate and the original process Kunning particle intermediate at 200-800 nm.According to the measurement results,the similarity range of Kunning granule intermediates is specified to be 0.77-1.00.There was no significant difference(P=0.82)in the peak absorption of the improved Kunning granules compared to the original process Kunning granules in the reference fingerprint spectrum at 200-800 nm after statistical analysis.According to the measurement results,the similarity range of Kunning granules is specified to be 0.78-1.00.In the study of HPLC fingerprint,there was no significant difference(P=0.66)in the similarity between the improved Kunning granule intermediate and the original process Kunning granule intermediate and the control fingerprint after statistical analysis.According to the measurement results,it is stipulated that the similarity of the HPLC fingerprint of the intermediate of Kunning granules should not be less than 0.80.Improved fingerprint similarity between Kunning granules and the original process Kunning granules.Conclusion: The study confirmed that salvianolic acid B,paeoniflorin,naringin and neohesperidin are stable and feasible Q-Markers for Kunning Granules Kun to play the role of anticoagulation.The optimized process is stable and feasible after verification.After the process improvement,there is no significant difference between the content of paeoniflorin,naringin components,UV fingerprint and HPLC fingerprint and the original process.The content of salvianolic acid B and neohesperidin is different from the original process,and there is no significant change in the quality of Kunning Granules after the improvement,It indicates that the improved Kunning granules are stable and feasible,and a relatively complete quality standard for the intermediate and finished products of Kunning granules has been established,providing a reference for improving the quality of Kunning granules;Before and after improvement,there was no significant change in the material basis of Kunning granules’ metabolic effects on normal rats;The mechanism of action of Kunning Granules in treating qi stagnation and blood stasis has been clarified.After reevaluation,the production cost of Kunning granules has been reduced,the quality standards have been improved,and its mechanism of action has been clarified.The research results can provide reference for its production changes and clinical use. |
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