Study On Mechanism Of Study On The Preparation Process,Quality Standard,Stability And Network Pharmacology Of Wenshen Nuanpi Granule

Objective: Objective to optimize the preparation process of Wenshen Nuanpi granules(WSNP),establish the quality standard,conduct stability investigations,analysis of key components by UPLC-Q-TOF-MS/MS and preliminary exploration of effective ingredients and targets in prescription through network pharmacology.Methods: Preparation process: Through orthogonal experiments and comprehensive analysis,the best water extraction process was screened and validation test was conducted;Small test samples were prepared according to the optimized extraction process conditions,and the added excipients were investigated in the state of granules,and pilot production of Wenshen Nuanpi granules was carried out according to the small test formability process parameters.Research on quality standard of Wenshen Nuanpi granules: The relevant checks on Wenshen Nuanpi granules were carried out with reference to the general checks on granules in the Chinese Pharmacopoeia(2020 edition);thin layer chromatography identification study of Wenshen Nuanpi granules by TLC method;The content of psoralen and iso-psoralen in Wenshen Nuanpi granules was studied by HPLC,and the draft quality standard of Wenshen Nuanpi granules was initially established.Stability test: Long-term and accelerated stability studies were conducted on the pilot granules to determine the storage conditions and predict the expiration date of the product based on the results of check items,identification,content determination,etc.UPLC-Q-TOF-MS/MS analysis: Chemical components in granules were identified through high-resolution mass spectrometry data.Network pharmacology study: The active ingredients and associated targets of the eight herbs in the formula were collected using TCMSP,the specialized chemical database of the Shanghai Institute of Organic Chemistry,Chinese Academy of Sciences,relevant literature,Pub Chem,Swiss ADME,Uniprot database and Swiss Target Prediction;The disease gene was obtained by entering "Diabetic Diarrhea" in a database such as Gene Cards;Using Cytoscape to construct " Chinese herbal ingredients-targets-DD " network map and screen out key ingredients and core genes from it;Input of intersecting genes in Metascape to obtain GO and KEGG enrichment results,i.e.the biological processes and pathways of action involved;Using Cytoscape to create " Chinese herbal ingredients-DD-targets-pathways " map to obtain key ingredients and key targets;The structures of the components were then downloaded using Pub Chem,and the PDB database was queried and downloaded for the structures of the proteins,which were optimized and processed separately before applying Auto Dock for molecular docking to combine the ability to evaluate the stability of the potential core components of the Wenshen Nuanpi formula to bind to the targets.Results: Optimized preparation process conditions: decoction in water 3 times for boiling 1 hour each,with the amount of water added 5.5,4 and 4 times the weight of the herbs on three occasions.Based on the projection of the amount of raw drugs to be taken and the combination of the stable nature of granules,which can maintain the advantages of liquid formulations with fast onset of action,we chose to make granules.Purified water is used as a wetting agent and dextrin is used as an excipient for granulation.Quality standards:Wenshen Nuanpi granules were qualitatively identified by TLC for eight herbs,including Psoraleae Fructus and so on,and the spots of the thin layer maps of seven herbs in the formula,such as Psoraleae Fructus,Euodia rutaecarpa,and so on,showed clear color at the corresponding positions,and they were included in the standards.In the linearity experiments of psoralen and iso-psoralen,the linearity with peak area was better when the content of psoralen was between 0.02176μg and 0.21760μg and the iso-psoralen was between 0.02164μg and 0.21640μg.The content limit of this product,the psoralen contained in the index component psoralen and iso-psoralen,should not be less than 1.00mg/bag.Stability study: the storage condition of Wenshen Nuanpi granules is sealed and the validity period is tentatively 6 months.UPLC-Q-TOF-MS/MS analysis: 50 chemical components of Wenshen Nuanpi particles were quickly identified.Network pharmacology: The potentially relevant drug components and targets of WSNP were collected and screened through various databases and literature,and a total of 124 targets were intersected with DD-related genes.The main pharmacological components are quercetin,β-sitosterol,etc.while the main target proteins are PTGS2,ADRB2,etc.The main biological processes involved were lipopolysaccharide response,cellular response to nitrogen compounds,etc.The pathways of action were cancer pathway,AGE-RAGE signaling pathway in diabetic complications,PI3K-AKT signaling pathway,etc.After molecular docking,the five main key components screened bound well to the five key target proteins,among which ellagic acid bound most stably to ADRB2.Conclusion: The optimized preparation process of Wenshen Nuanpi granules was good,and the quality standard of Wenshen Nuanpi granules was initially established.A variety of chemical components in WSNP granules were identified by UPLC-Q-TOF-MS/MS analysis.The network pharmacological analysis predicts that WSNP exert their effects on the treatment of diabetic diarrhea through PTGS2,ADRB2 and other targets affecting cancer pathways,AGE-RAGE signaling pathways in diabetic complications,PI3K-AKT signaling pathway and other pathways,providing a basis for the clinical study of Wenshen Nuanpi granules.