Study On Synergic Anti-pruritus Mechanisms Of Action For The Radix Sophorae Flavescentis And Fructus Cnidii Herbal Pair Based On Network Pharmacology

Objective:This study aimed to explore the synergic anti-pruritus mechanisms of action for the Radix Sophorae Flavescentis(KS)and Fructus Cnidii(SCZ)herbal pair with the network pharmacology methods and to further verify through experiments.It provides new ideas for the study of the mechanism of traditional Chinese medicine formulas.Method:1.Prediction of the potential synergic anti-pruritus mechanism of KS and SCZ based on the network pharmacology methods.(1)The chemical compound data derived from RSF and FC were collected from the Traditional Chinese Medicine Systems Pharmacology Database(TCMSP).(2)We analyzed the molecular scaffolds from the active components of of KS and SCZ.The putative targets of the molecular scaffolds from RSF and FC were identified by the Annotated Scaffold Database(ASDB)and Pharmaceutical Target Seeker(PTS).(3)Anti-pruritus targets were derived from the Therapeutic Targets Database(TTD)and literatures.(4)Protein-protein interaction(PPI)analysis of predict targets and anti-pruritus targets were carried out.The important predict targets which have direct or indirect interaction with anti-pruritus targets could be obtained from the PPI analysis.To perform the pathway enrichment analysis could obtain the potential anti-pruritus pathway mechanism of KS and SCZ.2.Extraction and isolation of the main effective part of KS and SCZ.(1)To obtain the crude extract of KS and SCZ through heating reflux extraction method and ultrasonic extraction method respectively.(2)Purified the KS flavonoid fraction and SCZ coumarins fraction by solvent processing method;derived the alkaloids fraction from KS through macroporous resin purification method;Essential oil of SCZ was isolated by steam distillation method.(3)The high performance liquid chromatography(HPLC)method was used to determine the matrine and oxymatrine quantitative of KS alkaloids fraction,the trifolirhizin and sophoraflavanone G quantitative of KS flavonoid fraction and the osthole and imperatorin quantitative of SCZ coumarins fraction.It could evaluate the extract quality of difference fractions,and also could provide basic research data to anti-inflammatory studies in vitro.3.In vitro anti-inflammatory activity of the fractions and compounds of KS and SCZ.(1)The effect of each fractions or compounds on the cell viability of RAW264.7 cells was first examined.(2)We established an inflammatory cell model of lipopolysaccharide(LPS)-induced macrophage RAW264.7 inflammatory response.The content of NO inflammatory factors was used as index.And the griess method was used to determine the effect of fractions and compounds on anti-inflammatory.(4)The method of real-time PCR was used to detect the the effect of fractions and compounds on on mRNA expression of iNOS,IL-6 and COX2.Results:1.Network pharmacology analysisA total of 113 compounds of KS were obtained.After analyzing the chemical structure of the compounds,36 scaffolds were obtained and 406 targets were predicted.The components of SCZ were 114,39 scaffold were identified by chemical structure analysis and416 targets were predicted.81 anti-pruritus targets were obtained..30 predicted targets were same as anti-pruritus targets overlapping targets(including IL-2,COX2,ACHE,TRPV1 and so on).It indicated that these 30 targets are directly related to pruritus.After PPI network analysis,65 predicted targets which have important indirect relationship with anti-pruritus were obtained.We merged the direct and indirect predict targets as 95 key targets which were used for pathway enrichment analysis.The result of pathway enrichment analysis showed that these candidate targets are mainly enriched in 46 KEGG pathways and the first20 pathways with the smallest P-value were found.There are 4 immune-related pathways and 2 nerve-related pathways,which are:(1)Toll-like receptor signaling pathway;(2)Chemokine signaling pathway;(3)T cell receptor signaling pathway;(4)Fc epsilon RI signaling pathway;(5)Neurotrophin signaling pathway;(6)Neuroactive ligand-receptor interaction.This study focuses on the immune-related pathways.Immunity is closely related to inflammation.Many immune factors can stimulate,induce and modulate inflammatory responses.This part of the results laid the foundation for the next study of cell activity.2.Determination of compound contentA good linear relationship between the peak area integral value and concentration was found in the 10.56~84.48μg/mL(R~2=0.9999)and 10.76~86.08μg/mL(R~2=0.9999)concentration range for trifolirhizin and sophoraflavanone G,respectively.The content was determined by external standard peak area method.The content of trifolirhizin in the flavonoid part of KS was 2.68%,RSD=1.70%,and the content of sophoraflavanone G was6.71%,RSD=1.59%.Oxymatrine and matrine have a good linear relationship between the peak area integral value and concentration in the concentration range of 27.00~216.00μg/mL(R~2=0.9992)and 14.48~115.84μg/mL(R~2=0.9998),respectively.The content of oxymatrine and matrine in the alkaloid part of KS were 12.48%(RSD=1.32%)and 2.94%(RSD=0.51%),respectively.A good linear relationship between the peak area integral value and concentration was found in the 10.28~82.24μg/mL(R~2=0.9999)and 9.36~74.88μg/mL(R~2=0.9999)concentration range for osthole and imperatorin,respectively.The content of osthole and imperatorin in the coumarin part of SCZ were 43.12%(RSD=0.96%)and 19.83%(RSD=1.34%),respectively.4.In vitro anti-inflammatory activity of the fractions and compounds of KS and SCZThe results of cell viability test showed that the survived rate of RAW264.7 cells were more than 90%when the concentrations of KS and SCZ extract parts were 12.5μg/mL,6.25μg/mL and 3.125μg/mL.So the three concentrations were determined as the administration concentration in the next step.The results of the NO content test on KS and SCZ extract parts showed that the flavonoids fraction of KS has the best in inhibition in anti-inflammation and the average NO inhibition rates at the concentration of 12.5μg/mL was 49.78%.The coumarin fraction of SCZ had better inhibition to NO content,and the average NO inhibition rates at the concentration of 12.5μg/mL was 26.51%.The average NO inhibition rates reach to 84.86%(at the concentration of 12.5μg/mL)when combined the flavonoids fraction with coumarin fraction.The PCR results showed that:the iNOS,IL-6 and COX2 gene mRNA relative expression can be significantly reduced when combined the flavonoids fraction with coumarin fraction(at the concentration of 6.25μg/mL).According to the results of cell viability test,the three concentrations of 10μM,5μM and 2.5μM were selected as the compounds administration concentration in the next step.The results of anti-inflammation test on the compounds showed that sophoraflavanone G in the flavonoid fraction of KS had the best anti-inflammatory activity and the inhibitory rate of NO was 51.63%(the concentration of sophoraflavanone G was 10μM).Imperatorin at the coumarin fraction of SCZ had a relatively good inhibitory effect on NO,and the average inhibition rate of NO was 30.33%at 10μM.The combination of sophoraflavanone G and Imperatorin had better anti-inflammation activity.The average inhibition rate of NO was78.45%at 10μM.This inhibition rate was significantly higher than the positive drug(10μM quercetin,with average inhibition rate of NO was 51.13%).The real-time PCR results showed that COX2 and IL-6 mRNA relative expression could be significant down-regulate when sophoraflavanone G was combined with imperatorin.Conclusions:1.The network pharmacology analysis indicated that KS and SCZ may mainly have effect on immune-related pathways and neural-related pathways to treat pruritus.2.The flavonoids fraction of KS and the coumarins fraction of SCZ played a leading role in treating pruritus,and the combination of the two fraction had the best anti-inflammatory effects.3.The compound combination of sophoraflavanone G and imperatorin have better anti-inflammatory effects than any monomer compound.4.KS and SCZ could reducing the expression of IL-6 and COX2 related inflammatory genes to anti-pruritus.