| Objective:Huanbei Zhike recipe is a clinical experience prescription of Shanxi Provincial Hospital of traditional Chinese Medicine,which is effective in the treatment of cough variant asthma in children.In order to clarify the material basis of its efficacy,this study used spectral effect correlation analysis and serum pharmacochemistry to study the material basis of the antitussive effect of Huanbei Zhike recipe,which laid a foundation for the research and development of new drugs and quality control of the prescription.Methods: 1.Based on the spectral effect correlation technique,the material basis of antitussive effect of Huanbei Zhike recipe was studied.The cough latency,cough times and the contents of IL-4,IFN-γ,IL-5 and IgE in lung lavage fluid of mice were used as indexes to study the antitussive effects of water extract,ethyl acetate layer,n-butanol layer and water layer of Huanbei Zhike recipe.HPLC fingerprints of different polar parts of Huanbei Zhike recipe were established by high performance liquid chromatography(HPLC).The maps of different polar parts were compared,and the water extract chromatogram was used as reference.The common peaks and corresponding peak areas of each part of fingerprint were obtained.Bivariate correlation analysis was used to correlate the pharmacodynamic experimental data with the common peak area,and the chromatographic peaks with high correlation degree were assigned and designated based on the HPLC spectra of single drug and reference substance.2.Based on the technique of serum pharmacochemistry,the material basis of antitussive effect of Huanbei Zhike recipe was studied.The total ion flow diagram of Huanbei Zhike recipe in vitro was established by ultra-high performance liquid chromatography-four-stage rod time-of-flight mass spectrometryin electrospray positive and negative ion mode.The excimer ion peak,fragment ion and other information are used to speculate and identify the isolated compounds by comparing the database and the existing literature,and the total ion flow diagrams of the drug-containing serum and the blank serum of Huanbei Zhike recipe are established under the same conditions.The difference between the two spectra was comparedwith the total ion flow diagram of the sample solution in vitro,and the blood components of Huanbei Zhike recipe were speculated and characterized.Results: 1.Study on the correlation of Spectral efficiency(1)Study on antitussive activity of different polar partsCompared with the blank group,the contents of IL-4,IFN-γ,IL-5 and IgE in the lung lavage fluid of the model group were significantly higher than those of the blank group(P<0.05),which indicated that the model was successful.Compared with the model group,water extract,ethyl acetate layer,n-butanol layer and water layer had a downward trend on the times of cough and the contents of IL-4,IFN-γ,IL-5 and IgE in lung lavage fluid of mice,and all of them could prolong the latent period of cough in mice.It is suggested that the water extract of Huanbei Zhike recipe,ethyl acetate layer,n-butanol layer and water layer have certain therapeutic effects on cough in mice.(2)Establishment of fingerprint of different Polar partsBy optimizing the preparation conditions and chromatographic conditions of the sample,the HPLC characteristic map of the water extract of Huanbei Zhike recipe was established,and the methodological investigation was carried out.The results showed that the method was stable and feasible.Under the same conditions,the fingerprint of different polar parts was established.Taking the water extract as reference and processed by chromatographic workstation,the common peaks and corresponding peak areas of each part of the fingerprint were obtained.Among them,there were 46 common peaks of water extract,45 common peaks in ethyl acetate layer,43 common peaks in n-butanol layer and 3 common peaks in water layer.(3)Spectral correlation Analysis of antitussive effect of different Polar partsBivariate correlation analysis showed that there was a significant positive correlation between the content of 7 chromatographic peaks and cough latency,which were peaks 12,13,29,32,33,35 and 39,respectively,among which peak 32 had very significant significance.The content changes of the 4 chromatographic peaks were negatively correlated with the times of cough,which were 12,13,32,33,respectively,among which the 13 th and 32 nd peaks had very significant significance.It is indicated that these peaks have a great contribution to the antitussive effect of Huanbei Zhikerecipe.In addition,there were 15 chromatographic peaks with strong correlation with pharmacodynamic test results,which were 1,5,6,8,9,10,18,19,20,21,23,36,38,40,41,respectively,but there was no significant difference.It is suggested that these components also contribute to the antitussive effect of the prescription.(4)Identification and Analysis of active components based on single Drug and reference substanceBy comparing with the HPLC spectra of each single drug in the prescription,the common peaks were assigned and further identified by the reference substance.It was proved that peaks 5,9,12,14,17,24,25,26,27,33,44 and 46 were new chlorogenic acid,amygdalin,chlorogenic acid,caffeic acid,glycyrrhizin,naringin,hesperidin,neo-hesperidin and rosemary acid,baicalin,baicalein and wogonoside.Among them,new chlorogenic acid,amygdalin,chlorogenic acid and baicalin were the peaks with high correlation with antitussive effect,and chlorogenic acid and baicalin had significant correlation2.Study on Serum Pharmacochemistry(1)Qualitative Analysis of Chemical constituents in VitroThrough database comparison and literature review,a total of 68 compounds were isolated from the solution of Huanbei Zhike recipe in vitro,and 64 compounds were speculated and identified,including 38 flavonoids,10 organic acids and 6terpenoids,10 nitrogen-containing compounds,10 of which were confirmed by control.(2)Blood component analysisBy comparing the atlas of blank serum and drug-containing serum,the difference map showed 28 kinds of blood components,and 9 of them were prototype components by comparing with the total ion fluid of Huanbei Zhike recipe in vitro,19 species were metabolites.Combined with the existing literature reports,a total of 24 components were speculated and identified,most of which were prototypes and metabolites of flavonoids and organic acids.Conclusion: By using the two different methods of spectral effect correlation study and serum pharmacochemistry,it is clarified from different angles that the antitussive effect of Huabei Zhike recipe is based on flavonoids and organic acids.Ithas laid a foundation for new drug research and development,quality control and so on. |
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