| Objective:The Huanbeizhike Recipe comed from clinical experience preparations and was mainly used for the treatment of cough variant asthma,asthmatic bronchitis and other lung hyperactivity cough.Its clinical symptoms was chronic cough.In order to clarify its efficacy and mechanism of action,the study were carried out pharmacodynamic experiments such as antitussive,expectorant,antiasthmatic,antibacterial and antiallergic activities under the premise of the extraction process determined.A comprehensive and systematic study was conducted to initially clarify the mechanism of action in the treatment of cough variant asthma and provide a basis for the clinical using and new drug research and development.Methods:Through the literature research,the chemical constituents and pharmacological actions of various herbs in Huanbeizhike Recipe were summarized.Two extraction processes were protocolied,and the extraction process parameters were optimized by orthogonal test.In order to study efficacies of cough,expectorant,asthma for Huanbeizhike Recipe,this paper was adopted ammonia cough in mice test,acid cough in guinea pig citric test,phenolphthalein expectorant in rats test,capillary phlegm in rat test,mixed histamine asthma in guinea pig test.It was used instillation of streptococcus pneumoniae in nasal to establish model of lung heat in rat,and observed the thymus,spleen index,inflammatory cells and Inflammatory factors of the rat model's lung lavage fluid intervened by Huanbeizhike Recipe.By using CVA model in guinea pigs and detecting cough reaction caused,inflammatory cytokines and related-immune changes in indicators on the whole level,the mechanism of CVA in the treatment of Huanbeizhike Recipe has been defined preliminarily.Finally,based on the ~1H-NMR metabonomics techniques,right middle lobe lungs of guinea pigs with CVA were detected.According to the corresponding S-plot and its VIP value and t-test results,it were seeked endogenous differences metabolites related to CVA model in guinea pig.According to KEGG database,the relevant previous animal experiments and related literature,to expose its biological significance,and identify potential biomarkers and relation metabolic pathways,the mechanism of CVA in the treatment of Huanbeizhike Recipe has been probed.Results:1.Research on extraction processBy the extraction rate,content of total flavonoids,content of chlorogenic acid,cough and asthma efficacy as indexs,the two extraction processes were screened.And it was finally determined the whole water decoction.This paper has used orthogonal test for its parameters and ultimately determined the extraction process:the amount of water 10 times,boiling 3 times,each 1.5h.2.System pharmacodynamics(1)Effect of cough,expectorant,asthma:There were significant antitussive effects in different dose groups for Huanbeizhike Recipe(P<0.05),but there were significant differences between the Huanbeizhike Recipe group and the positive western medicine group(P<0.05).And the positive western medicine could better extend the curative effect of cough latency in guinea pigs than Huanbeizhike Recipe group.Different dose groups of Huanbeizhike Recipe had could significantly increase tracheal sputum secretion in mice and rats(P<0.05),and expectorant effecting had been similar to that of the positive drugs.Compared with the model group,the aminophylline group,CLHC group and Huanbeizhike Recipe groups could significantly prolong the latent period of asthma induction in guinea pigs,with significant difference(P<0.05).Different doses of Huanbeizhike Recipe groups,CLHC group and aminophylline group could prolong the latent period of asthma in guinea pigs,but with statistical significance(P<0.05).The result suggested that relieving asthma of Huanbeizhike Recipe was not as good as positive drug groups.(2)Effect of anti-streptococcus pneumoniae:Compared with the blank group,the edema fluid in the alveolar cavity of the model group rats was found in the model group.And it was the thickened of the vascular wall in the alveolar wall,the infiltred several small inflammatory cells in the pleura and formated the small abscesses.The body temperature of the model group was significantly increased,and prompted modeling successful.Compared with the model group,the leukocyte,neutrophil number and Th/Ts ratio in the whole blood of different doses groups of Huanbeizhike Recipe were significantly decreased(P<0.05).And the content IL-1?,IL-6,IL-4 and TNF-?were significantly decreased(P<0.05).(3)The role of treatment of CVA model in guinea pig:Compared with the blank group,the bronchial mucosa of the model group formed folds.And a large number of inflammatory cells(infiltrated by eosinophils)were infiltrated in mesenchyme.The result indicated that the CVA model was successfully replicated.After capsaicin cough,compared with the model group,the different doses groups of Huanbeizhike Recipe of the latent period and the number of cough within 5min were significantly different(P<0.01).And Compared with the Huanbeizhike Recipe groups and positive drug groups(CLHC and DEX),the latent period and the number of cough were no significantly different(P>0.05).Thymus index and immune index also had the upward trend in varying degree.Through testing on inflammatory cytokines and inflammatory cells in BALF in guinea pig,it was found that Huanbeizhike Recipe groups could regulate relatived amount of IL-5,IL-4,IL-10,IFN-?,LTE4,NGF and TNF-?(P<0.05).The effect was similar to that of DEX,and the effect on IFN-?and NGF were better than that of CLHC.The leukocytes,neutrophils,eosinophils and other inflammatory cells in BALF of guinea pigs had been regulated in varying degree.3.The mechanism of Huanbeizhike Recipe in the treatment of CVA model in guinea pig:There were 22 biomarkers,including amino acids(alanine,valine,leucine),organicacids(lacticacid),amines(glutamine,acetic acid),lipids(phosphatidylcholine,sphingolipid)and others(glucose)and so on.These metabolites had been mainly involved in the body's energy metabolism(pyruvate metabolism and fatty acid metabolism),amino acid metabolism(valine,leucine and isoleucine metabolism,glutamate and glutamine metabolism,arginine and proline metabolism).Huanbeizhike Recipe in the treatment of CVA mechanism of action was of great significance.Conclusion:The determined water extraction process could retain the main active ingredients in Huanbeizhike Recipe,and its process parameters were reasonable and feasible.The main pharmacodynamics studies had shown that Huanbeizhike Recipe had significant antitussive and expectorant effects,as well as antibacterial,anti-inflammatory and immunomodulatory effects.Further,based on1H-NMR metabonomics technology to explore the possible mechanism of the intervented by Huanbeizhike Recipe of CVA model in guinea pigs might be through energy metabolism and amino acid metabolism.The above studies provided experimental basis for the clinical application of Huanbeizhike Recipe and lay a foundation for further research and development of the prescription. |
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