| Background and ObjectiveThe Chinese medicine Jinquegen is the root or root bark of the Caragana sinica(buc,hoz)rehd of the leguminous plant Caragana.The original plant is born on the hillside,roadside and under the forest.From August to September every year,the roots are dug up and divided into individual root branches.Fine roots and tails are removed.The rough skin and the wooden heart are removed.Cut into short sections and dried to form a medicinal the root of Caragana sinica.The root of Caragana sinica has the effect of phlegm and blood circulation,through diuretic,and qi and kidney.Originally recorded in the“Compendium of Materia Medica”,there are few studies on the anti-inflammatory effect of modern the root of Caragana sinica.Especially the research on the effect of the root of Caragana sinica on rheumatoid arthritis is still in the aspect of total extract,and there is still broad space to explore the effect of broom root on rheumatoid arthritis.For further research on the topic,the root of Caragana sinica was identified first to ensure drug correctly.According to the polarity,the total extract was extracted successively to separate the four parts,and the in vitro anti-inflammatory activity screening of the effective parts and the composition analysis of the active parts were carried out.The efficacy evaluation of the active parts on adjuvant type arthritis and the anti-inflammatory mechanism were provided,which provided references for the subsequent development and clinical application of the root of Caragana sinica.Methods(1)Firstly,the pharmacognotic identification of the root of Caragana sinica was carried out.After the identification by professor Yu Nianjun,the characteristics of the original plant such as sight,measurement,smell and taste were identified,and the microscopic morphology and TLC identification of the roots of medicinal parts were carried out.(2)The root of Caragana sinica was extracted and separated,and the total extract was extracted with 80%ethanol,then dissolved in double distilled water,and extracted with petroleum ether,chloroform,ethyl acetate and n-butanol in this order.The extracted and isolated parts act on the LPS-stimulated macrophage-induced inflammation model in vitro.The CCK-8 method is used to screen the concentration of safe cells,and the optimal active sites are screened by inflammatory factors NO,PGE2,TNF-αand IL-6.The adjuvant arthritis model was used to evaluate the efficacy of anti-RA in the active site by weight of the rat,degree of swelling of the toes,determination of serum inflammatory factors and HE slices.(3)The Thermo Q Exactive plus quadrupole-electrostatic field orbitrap high-resolution mass spectrometry was used to accurately analyze the ethyl acetate fraction of the root of Caragana sinica,based on the first-and second-order high-resolution mass spectrometry data from the instrument combined with the Compound Discoverer software.The components of ChemSpider,mzCloud were compared and combined with literature reports to efficiently identify the components.(4)Western blot was used to determine the effect of active site of the root of Caragana sinica on the expression of COX-2,iNOS and NF-κB pathways in LPS-stimulated macrophages and the immunofluorescence assay,in order to further explore the anti-inflammatory mechanism of the active part of Caragana sinica.Results(1)The traits of the medicinal properties,the microscopic characteristics of the powder and the transverse section of the root were all obvious,and the results of thin layer chromatography were better.The results were determined to be the leguminous plant Caragana.(2)CCK-8 showed no effect on cell viability at various concentrations of 100,50,25μg·mL~-11 at each site,integrated results NO,PGE2,TNF-αand IL-6 inflammatory factor,the root of Caragana sinica ethyl acetate portion is an anti-inflammatory active site.In vivo experiments showed that the ethyl acetate site can improve the mental state of the rats and reduce the swelling of the toes.It reduces the level of inflammatory factors and inhibit the infiltration of inflammatory cells in the ankle joint cavity of rats.what’s more,it reduces the damage of cartilage and reduce the formation of vasospasm.(3)Using Compound Discoverer software,the mass spectrometry data was compared with the information of the online data ChemSpider and mzCloud,and the components were intelligently identified.A total of 11 components were identified,which basically clarified the chemical basis of the site.(4)The ethyl acetate fraction of the root of Caragana sinica can significantly inhibit the expression of iNOS and COX-2 proteins after LPS-treated macrophages.The expression of p-IκB and p-P65 protein was inhibited significantly.In the immunofluorescence experiment,the ethyl acetate site inhibited the entry of P65 into the nucleus and the expression of transcription factors in the nucleus.ConclusionsThe systematic biopharmaceutical identification of the root of Caragana sinica medicinal materials laid the foundation for further study of this medicinal material.The pharmacodynamic evaluation of in vitro and in vivo strongly demonstrated that the ethyl acetate fraction of the root of Caragana sinica is the active site of anti-rheumatoid arthritis.At the same time,the ethyl acetate site of the root of Caragana sinica can inhibit the activation of NF-κB inflammatory pathway,further inhibit the secretion of inflammatory mediators and pro-inflammatory cytokines such as NO,PGE2,IL-6,etc.c. |
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