Study On The Composition And Quality Of Medicinal Materials Of Shegan For The Treatment Of Exogenous Cough

Purpose:The rhizome of Belamcanda chinensis（L.）DC that belongs to Iridacea,has the effect of clearing heat,detoxifying,eliminating phlegm,and pharynx.In this study,six isoflavones were used as the research object,adopting the macrophage inflammatory model induced by LPS,study six composition of isoflavones inhibition of cytokine and inflammatory medium;To establish proper animal models close to clinical treatment of exogenous cough,the animal model was used to study the effects of monomer composition and compatibility on cough frequency,lung tissue morphology,and arachidonic acid metabolites in the guinea pig cough model infected with syncytial virus.Through correlation analysis and regression analysis,the optimal composition was determined,through correlation analysis and regression analysis,to determine the best compatibility proportion between components;Based on the optimal compatibility proportion,the quality evaluation of different radix dried medicinal materials was carried out to provide a basis for the research on the quality of traditional Chinese medicine and medicinal materials.Material and method:1 Material1.1 Experimental animals and cell lines:Guinea pigs,250³00 g（Beijing Experimental Animal Center）;Respiratory syncytial virus（Donated by Wuhan Institute of Virology,CAS）;Hep-2 cells（Jiangsu Qishi Biotechnology Co.,LTD.）;RAW264.7 mouse mononuclear macrophage leukemia cells（Institute of Biochemistry and Cell Biology,CAS）.1.2 Experimental samples:tectoridin,iridin,tectorigenin,irigenin,irisflorentin,and dichotomitin.（Jiangsu Yongjian Medicine Technology Co.,LTD.）The main products of the traditional Chinese medicine and medicinal materials are the main producing areas and the market of medicinal materials.1.3 Main reagents:LPS,DMSO（American Sigma Company）;MTT（Amresco）;DMEM,FBS（CORNING Company）;Penicillin/streptomycin liquid（Chinese Academy of Medical Sciences Institute of Biomedical Engineering）;TNF-α,IL-6,PGE₂ mouse Elisa kit,SP guinea pig Elisa kit.（Nanjing Jiancheng Bioengineering Institute）;NO,BCA kit,trypsin（Beyotime Biotechnology co.,LTD.）;Na₂HPO4,12H₂O,KH₂PO4,KCl and NaCl（Sinopharm Chemical Reagent co.,LTD.）;Citric acid（Shenyang Reagent Plant）;2 Method2.1 Effects of six isoflavones on the release of cytokines and inflammatory mediators from LPS-induced macrophagesLPS-induced RAW264.7 macrophages were used to establish an in vitro inflammation model.ELISA was used to determine the inhibitory effects of 6 components with different concentrations on inflammatory cytokines IL-6,TNF-αand inflammatory mediators NO and PGE2;Western Blot was used to detect iNOS.And COX-2 protein expression.2.2 Study on monomer composition and its compatibility based on exogenous cough model2.2.1 Establishment of respiratory syncytial virus model in guinea pigs:Establishment of a guinea pig RSV cough model by means of smoking plus RSV intranasal injection.Cigarette+sawdust caused airway hyperresponsiveness for 7 days.RSV nasal drops were given on the second and third days,respectively.2 times,RSV culture of lung tissue of guinea pigs in blank group and model group was performed,HE morphology of lung tissue was observed,and airway hypersensitivity（the number of cough）was measured.2.2.2 Anti-inflammatory effects and mechanisms of six isoflavones on the cough model of RSV infection after radiotrapping:The guinea pig RSV model of cough was used to inoculate six isoflavones.The number of coughs,lung tissue morphology,and lung tissue were measured in model animals.SP content,tissue immunohistochemistry SP,VR1,protein gene product NF-200,PGP-9.5 expression,SP and capsaicin receptor NK1,VR1 mRNA expression and arachidonic acid metabolism pathway in lung tissue,and related analysis.2.2.3 Based on post-infection cough model,the relationship between the activity of radioactivity and the compatibility of the cough model was studied:According to the uniform design scheme table,four studies were conducted to study the relationship between the activity and the cough model of guinea pigs infected with syncytial virus.The number of coughs,lung morphology,and SP were measured.Substance content,SP receptor NK1,capsaicin receptor VR1 protein expression,receptor NK1,VR1 mRNA expression,and arachidonic acid metabolites,will be selected for compatibility analysis and data for regression analysis.2.3 Study on the quality of Rhizoma Belamcandae based on the best combinationBased on the ratios of tectorigenin,irigenin,irisflorentin and dichtotmitin,the content of four components in the 14 batches of herbs in the main producing area and the distribution place of stem borer was determined and the relationship was analyzed.Determine the best medicine materials for the treatment of cough.Result:1 Effects of six isoflavones on cytokines and inflammatory mediators released by LPS-induced macrophages1.1 Effect on the activity of RAW264.7 cells:The effect of each component on the viability of macrophages was examined by MTT assay.The results showed that the content of tectoridin was 0-250μg/ml,the content of iridin was 0-205.5μg/ml,the content of tectorigenin was 0-64μg/ml,the content of irigenin was 0-176μg/ml,and the content of irisflorentin was 0-14.7μg/ml,the cell survival rate was greater than 95%and dichotomitin0-45.1μg/ml.Compared with the control group（0μg/ml）,there was no significant difference in the dose of each group.1.2 Effects on NO,PGE₂,IL-6 and TNF-αreleased from RAW264.7 cells:The contents of NO,PGE₂,IL-6 and TNF-αin the cell supernatant were determined by ELISA.Compared with the blank group,LPS significantly increased NO,PGE₂,IL-6,and TNF-αlevels in the model group.After adding different concentrations of the drug,the levels in the drug-administered groups had different degrees compared with the model group.Decreased（P<0.05 or P<0.01）.The results showed that the high concentration of tectorigenin,irigenin,and irisflorentin could significantly inhibit the secretion of NO;tectoridin,iridin,tectorigenin,irigenin,irisflorentin,and dichotomitin in the high-concentration group,the secretion of PGE2 could be inhibited;the high concentrations of tectorigenin and irigenin could significantly inhibit the secretion of IL-6;the high concentration of iridin,tectorigenin,irigenin,and dichotomitin inhibition of TNF-αsecretion,and this effect is dose-dependent.1.3 Effect of tectorigenin and irigenin on iNOS and COX-2 protein expression:After LPS stimulation of macrophages,the expression of iNOS and COX-2 in the model group was increased compared with the blank group,and the high concentrations of tectorigenin and irigenin were administered.The group can significantly inhibit the expression of iNOS and COX-2.2 Guinea pig RSV infection cough model replication2.1 RSV virus detection results in lung tissue:Compared with the blank group,lung tissue homogenates in the model group were cultured on Hep-2 cells until day 2 when the cells began to swell and round,and the cell gaps increased,forming a fusion on the 4th day.Cells that have RSV lesions.2.2 Pathological results of lung:The alveolar septum structure was clear in the blank group,and there was no inflammatory cell infiltrate in the lung interstitium.In the model group,pulmonary bronchiolitis and alveolar inflammation were gradually aggravated,the degree of inflammatory cell infiltration gradually increased,and the alveolar structure was gradually destroyed.Increased,but no significant pulmonary interstitial inflammation and alveolar fusion.2.3 Number of guinea pigs cough:The number of coughs in the model group was significantly higher than the blank group within 5 minutes,and the results were statistically significant.3 Effects of six isoflavones in the Rhizoma Belamcandae on airway inflammation in guinea pig cough model infected with RSV3.1 Effect of citrate-induced cough:In the administration group,iridin,irisflorentin,dichotomitin,and Chinese medicine positive control drug had a significant inhibitory effect on the number of coughs in model animals,tectoridin,tectorigenin,and irigenin were no significant effect,and all groups had no significant effect on incubation period.3.2 Pathological effects on lung tissue of RSV-infected guinea pigs:The model group showed typical airway inflammation in the acute phase,congestive edema of the alveolar wall,massive infiltration of inflammatory cells,enlargement of some alveolar cavities,and destruction of the alveolar structure.There was a different effect of alleviating the symptoms of infection in each of the administration groups.3.3 Influence of SP content in lung tissue of guinea pigs:The content of SP substance in the model group was significantly higher than that in the blank group.In the administration group,tectoridin,iridin,tectorigenin,irigenin,irisflorentin,dichotomitin,and Chinese medicine positive control medicine were significantly reduced the SP effect in lung tissue,with the strongest effect of iridin and irigenin.3.4 Effect on immunohistochemical parameters:The staining results showed that compared with the blank group,dark yellow positive reactants were seen in the lung tissue of the guinea pigs in the model group,and the color of each drug reaction substance was lighter than that of the model group;semi-quantitative results showed that the model The expressions of SP,VR1,PGP-9.5 and NF-200 in the group were significantly higher than those in the blank group.The concentrations of tectorigenin,irigenin,irislorentin,dichotomitin and Chinese medicine positive drug group were all significantly lower SP,VR1 expression than those of the control group.Tectoridin,tectorigenin,irigenin,and irislorentin can significantly reduce the expression of PGP-9.5.Tectoridin,iridin,tectorigenin,irigenin,irisflorentin,dichotomitin,Chinese medicine positive drug,and western drug positive drug group were significantly reduced NF-200 expression.3.5 Effects on the three metabolic pathways of arachidonic acid:In the four metabolites of the COX metabolic pathway,the content of PGE₂ in the model group was not significantly different from that in the blank group,and the contents of PGD₂,TXB₂,and 11 HETE were significantly higher than those in the blank group.The content of PGD₂,TXB₂,and 11HETE in the drug was differently reduced.Tectoridin,irigenin,and western medicine positive drug groups could significantly reduce the PGE₂ content.Tectoridin,iridin,Chinese medicine positive drug group,and western drug positive drug group were significant reduced the content of TXB₂ in the model.In the AA metabolic pathway,the AA content in the model group was not significantly different from that in the blank group,and there was no effect of reducing the AA content in each administration group.The content of 16HETE in the metabolite of the model group was significantly higher than that in the model group.The changes of 8.9-DHET,11.12-DHET,and 14.15-DHET were not significant in the groups.No significant decrease in the level of 16HETE was observed in each drug group.Tectoridin,iridin,tectorigenin,irisflorentin,chinese medicine positive control medicine and western medicine positive drug groups significantly reduced the effect of 14.15-DHET in the model.In the LOX metabolites,the content of 5HETE and 15HETE in the model group was significantly higher than that in the blank group,and the administration group was decreased to varying degrees effect.3.6 Correlation analysis results:There was a negative correlation between tectorigenin and NF-200 protein expression,AA content,metabolites 8.9-DHET,11.12-DHET,14.15-DHET,and 16HETE in P450 pathway.Irigenin and VR1,NF-200 Protein expression was negatively correlated with the contents of metabolites 8.9-DHET,11.12-DHET,and 14.15-DHET in the P450 pathway.Irisflorentin and SP substance,VR1,PGP-9.5 protein expression,COX pathway metabolites TXB2,11HETE,LOX The content of 5HETE was negatively correlated with the metabolites of the pathway.Dichotomitin was negatively correlated with the number of coughs and SP substances,and negatively correlated with the expression of NF-200 protein and the metabolites of 8.9-DHET,11.12-DHET,and 14.15-DHET.4 The effect of the combination on the airway inflammation in a cough model of guinea pigs infected with RSV4.1 The effects of different compatibility ratios on different pharmacological parameters in model animals were different:Compatibility 1^#6#were all involved in COX metabolism in lung tissue,in which compatibility 1^#inhibited the number of coughs and decreased the content of SP in lung tissue and the expression of 4 related proteins.Participate in LOX metabolism in lung tissue.Compatible with 2^#to suppress the number of cough;Reduce the expression of VR1 and NF200 protein in lung tissue.Compatible with 3^#to reduce the expression of VR1 and NF-200 protein in lung tissue and participate in the metabolism of P450 in lung tissue.Compatible with 4^#to reduce the NF-200 protein expression,involved in lung tissue P450 and LOX metabolism.Compatibility 5^#reduce the expression of NF-200 protein in lung tissue,involved in the lung tissue LOX metabolism.Compatibility 6^#reduce the expression of NF-200 protein in lung tissue.4.2 Regression analysis results:The content was correlated with the corresponding pharmacological indices and regression analysis was performed.The regression equations for cough and different pharmacological indexes were obtained.After the test,the regression equations of the 4 indicators were significant and are summarized as follows:tectorigenin,irigenin,irisflorentin and dichotomitin had the effect of inhibiting cough.The most effective ratio was X1=36 mg,X2=54 mg,X3=36 mg,and X4=12 mg.The effects of VR1 protein were significantly reduced by tectorigenin,irigenin,irisflorentin and dichotomitin,and the most effective ratio was X1=36 mg,X2=54 mg,X3=36 mg,and X4=12 mg.Irigenin and irisflorentin both decreased PGD2 in lung tissue.the best combination ratio was X1=3.6 mg,X2=54 mg,X3=36 mg,X4=1.2 mg.Tectorigenin and dichotomitin have a role in reducing lung tissue 11,12 DHET,the best ratio of X1=36 mg,X3=3.6 mg,X4=12 mg.5 Study on the quality of radix dried medicinal materials based on the external cough medicine.To tectorigenin,irigenin,irisflorentin and dichotomitin the best compatibility proportion for inspection basis,by means of 14 batches of producers and distribution of content determination of shoot dry herbs,iris flavin found four group of organs in tectorigenin,irigenin,irisflorentin and dichotomitin,content with the best compatibility proportion.Conclusion:1.The six isoflavones in the stem can inhibit the production of inflammatory mediators NO and PGE₂ in LPS-induced macrophages,reduce the production of inflammatory cytokines IL-6 and TNF-α,and have good anti-inflammatory activity in vitro.The effects of tectorigenin and irigenin were the best.Tectoridin,iridin,irisflorentin and dichotomitin were the most effective.The mechanism of action may be through inhibiting the expression of iNOS and COX-2 protein and inhibiting the release of NO and PGE₂.2.Expression of SP,VR1,protein gene products NF-200 and PGP-9.5 in cough frequency,lung tissue morphology,SP content in lung tissue,tissue immunohistochemical indices of six isoflavones in guinea pigs infected with syncytial virus cough model The expression of NK1,VR1 mRNA and the AA metabolites in lung,SP and capsaicin receptors all had different degrees of inhibition.The mechanisms,mechanisms and mechanisms of inflammation caused by cough after antiviral infection by tectoridin,iridin,tectorigenin,irigenin,irisflorentin and dichotomitin were studied at the global,tissue,and molecular levels.3.After isobaric isoflavones are allocated to each other,synergistic effects are achieved through multiple targets to enhance the efficacy of cough-induced inflammation after antiviral infection.Compared with other compatibility ratios,tectorigenin,irigenin,irisflorentin and dichotomitin（6:9:6:2）can reduce the number of coughs in animals,decrease the expression of receptor protein in tissues,and reduce AA.Metabolites are produced.4.The proportion of isoflavones in 4 batches of medicine materials is close to the optimal ratio,which can be used as the next research medicinal material for coughing after viral infection.