| Objective: Through the characterization and quantification of Paeoniae Radix Rubra and the determination of the content of internal components,the grading standards of red peony medicinal materials and decoction pieces are established,and combined with network pharmacology and in vitro experiments,the biological evaluation and verification of anti-inflammatory and anticoagulation of different grades of Paeoniae Radix Rubra are carried out.The quality evaluation and clinical application of Paeoniae Radix Rubra provide a theoretical basis.Methods: Use vernier calipers to measure the diameter and length of Paeoniae Radix Rubra,use NH-300 color difference meter to measure the color value of Paeoniae Radix Rubra epidermis,section and powder;measure moisture,ash,extract and other items,and analyze Paeoniae Radix Rubra from different origins.The quality difference of peony.HPLC method was used for the determination of paeoniflorin and a method was established for the simultaneous determination of gallic acid,catechin,oxypaeoniflorin and paeoniflorin content in Paeoniae Radix Rubra;the HPLC method was used to establish a fingerprint determination method for Paeoniae Radix Rubra.Fingerprint consistency evaluation of medicinal materials and decoction pieces;correlation analysis and cluster analysis of the appearance and internal content of Paeoniae Radix Rubra using SPSS 21.0 and SIMCA 14.1 software,and the classification of Paeoniae Radix Rubra;Network pharmacology screening red Anti-inflammatory and anticoagulant active ingredients,related targets and pathways of Paeoniae Radix Rubra;use automatic coagulation analyzer to determine in vitro anticoagulation indexes PT,TT,FIB,APTT of different grades of decoction pieces;culture macrophages RAW264.7,The CCK-8 method was used to determine the cell viability values at different concentrations,and the ELISA method was used to determine the in vitro anti-inflammatory factors NO,IL-1β,IL-6,VEGFA and TNF-α of different grades of Paeoniae Radix Rubra decoction pieces.Results: Data quantification of the appearance characteristics of Paeoniae Radix Rubra,such as diameter,length and chroma.A method was established for the simultaneous determination of gallic acid,catechin,oxypaeoniflorin and paeoniflorin content in Paeoniae Radix Rubra.The method has good precision,repeatability and stability,and the sample recovery rate is 95%~105%.Established a Paeoniae Radix Rubra fingerprint measurement method,which has good precision,repeatability,and stability,and used this method to evaluate the consistency of the fingerprints of the medicinal materials and decoctions of Paeoniae Radix Rubra,and the results showed that the similarity was above 0.9.It shows that the Paeoniae Radix Rubra and Paeoniae Radix Rubra decoction pieces have good similarity.Using SIMCA 14.1 software to analyze the fingerprint peak area information,it can be concluded that peak 9 paeoniflorin is the first principal component,indicating that paeoniflorin is the main chemical component in Paeoniae Radix Rubra;Combining the appearance properties and internal content of Paeoniae Radix Rubra,a classification standard for Paeoniae Radix Rubra was established.The correlation analysis was carried out using SPSS21.0 software.The results showed that the classification of Paeoniae Radix Rubra was related to color.The reddish the color,the better the quality,SPSS21.0 software and SIMCA 14.1 software divide red peony medicinal materials into two categories,decoction pieces are divided into three categories,and the two analysis methods are mutually verified to ensure the accuracy of the classification results;network pharmacology studies find out Paeoniae Radix Rubra by constructing a component-target network 11 related active ingredients in Paeoniae Radix Rubra;by matching drug targets and disease targets,30 targets for the treatment of inflammation and 27 targets for the treatment of blood coagulation are marked,and the two with the highest correlation are found through the protein interaction network Targets IL-6 and VEGFA;the active ingredients directly related to these two targets can be obtained from the network diagram as paeoniflorin and ellagic acid.Paeoniflorin is an indicator component of Paeoniae Radix Rubra in the 2015 edition of the Chinese Pharmacopoeia,and has the highest content in Paeoniae Radix Rubra.It is the main pharmacologically active component of Paeoniae Radix Rubra.The enrichment analysis of GO and KEGG can conclude that Paeoniae Radix Rubra regulates cell proliferation and apoptosis,inhibits inflammation,protects vascular endothelium,and regulates the secretion of hormones through multi-protein and multi-signal pathways for anti-inflammatory and anti-coagulation,etc.Therapeutic effect.The results of four in vitro coagulation experiments show that Paeoniae Radix Rubra can significantly prolong the time of TT,PT,APTT,and FIB,and the effect results show a trend of type one> type two> type three,which can explain the anticoagulation of different grades of Paeoniae Radix Rubra.The degree is also different;the results of in vitro anti-inflammatory experiments show that treatment of RAW264.7 cells induced by LPS with different concentrations of Paeoniae Radix Rubra water extract can significantly inhibit the release of NO,TNF-α,IL-1β,IL-6,and VEGFA factors,And different grades of Paeoniae Radix Rubra showed a trend of one category> category two> category three,which was consistent with the results of the grade classification and mutually verified.In vitro biological evaluation experiments have verified the rationality of the classification.Conclusion: This experiment conducted a systematic study on the classification standards of Paeoniae Radix Rubra.The experiment verified that different grades of Paeoniae Radix Rubra have different effects on in vitro anti-inflammatory and anticoagulant effects.The higher the grade,the better the effect.It can be a standardized Paeoniae Radix Rubra.Shao quality standards provide theoretical basis and experimental basis. |
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