Study On Bioactive Components Of San-ao Decoction For Treating Bronchial Asthama

San-ao decoction,consists of Herba Ephedrae,Semen Armeniacae Amarum and Radix Glycyrrhizae,is a famous Chinese traditional prescription and has been used clinical to treat asthma for thousand of years.In clinic it is used to treat patients with symptoms of feeling cold,dizzy,cough,having a stuffy nose.Pharmacological research showed that San-ao decoction and its analogical complex prescription has signifcant functions of cold,bronchitis, bronchial asthma,dry pharynx and dry cough which caused by cough variant asthma and chronic pharyngistis.In order to clarify the chemical substancial fundation of San-ao decoctions and explore the chemical methords of studying Traditional Chinese Medicine(TCM),we systematically studied the chemical constituents of San-ao decoction and its analogical complex prescription,and evaluate the pharmacological activity.These work could offer gist for quality control and clinical application of this preparations.We used GC-MS Hyphenated Techniques and computer retrieval to study the volatile oils extracted with steam distillation from San-ao decoction and its analogical complex prescription.The volatile oil of San-ao decoction was identified up to 50 compounds;the volatile oil of San-ao decoction with Radix Asari was identified up to 86 compounds;the volatile oil of Wu-ao decoction was identified up to 105 compounds;the volatile oil of Qi-ao decoction was identified up to 84 compounds.And the volatile componets of the complex prescription are derived mainly from their major constituting herbs.But some componets could not detect in the volatile of the sigle herbs.GC-MS method is useful in analyzing the bioactive components of TCM.To investigate the volatile oil of analogical complex prescriptions of San-ao decoction in the development of RSV-induced,OVA-sensitized airway inflammation and the neutralization of Th1/Th2 subgroup,compared the effect of each prescription,screened out the best prescription whose therapeutic action aimed directly at asthma induced by virus.The differences in the increase value of R_L which compared with contribution in OVA/RSV group were significant when compared with those in PBS control group when stimulated by Ach(45,135,405μg/mL)(P<0.05,P<0.01).The amounts of total cellular score, lymphocytes,eosinophils,neutrophils in BALF of OVA/RSV group were increasing significantly(respectively P<0.01).The levels of IFN-γand IL-4,IL-5 of OVA/RSV group all heightened(P<0.01),especially,the levels of IL-4,IL-5 heightened much more significantly(respectively P<0.01).The degree of the changes of lung histopathology of OVA/RSV group were much more serious.The parallel prescription of San-ao decoction clearly inhibited RSV-induced increasing airway responsiveness to Ach,the number of TCS, macrophages,lymphocytes,eosinophils,especially the neutrophils:The IFN-γIL-4 and IL-5 levels in the supernatants of BALF all decreased obviously,compared with those in OVA/RSV group respectively(P<0.05,P<0.01).And the parallel prescription of San-ao decoction also could improve the pathological changes of peribronchitis.Among the total parallel prescription,Qi-ao decoction had a better inhibitory action to all type of indexes which could prove that the illness was asthma.The volatile oil of San-ao decoction and it's analogical complex prescription had some therapeutical effect on asthma model about virus. Among the total parallel prescription,Qi-ao decoction had a better therapeutic efficacy.And its partial mechanism of action mabe was that it could inhibite the amount of neutrophils, consequently,had a therapeutic efficacy which aimed directly at virus-induced asthma. Further more,it could confirm that neutrophil was one of the most important cells of virus-induced asthma.Twelve compounds were isolated from aqueous extract of San-ao decoction.Through chemical and spectral analysis the structures of twelve compounds were elucidated, including two glycosides,amygdaloside and glycyrrhizic acid;three flavonones, formononetin,liquiritigenin and isoliquiritigenin;one sterol,β-sitosterol;three organic acid, benzoic acid,triacontanoic acid and succinic acid;two alkaloid,l-ephedrin and d-pseudephedrine;and a hydrolysate of amygdaloside,named mandelonitrile.All of the compounds were first isolated from San-ao decoction.The study on the activity of Peroxisome proliferator-activated receptorγ(PPARγ) showed that the ETOH and BuOH extractions of San-ao decoction had pretty good action,and formononetin,liquiritigenin and isoliquiritigenin had significant action.The chemical constituents of the active fraction of San-ao decoction and its analogical complex prescriptions were analyzed by high performance liquid chromatography/ electrospray ionization mass spectrometry(HPLC/ESI-MS~n).34 major components were idetified by the ESI-MS,ESI-MS~n and ultraviolet(UV) spectra.Among them,4 active compounds such as benzoic acid,formononetin,liquiritigenin and isoliquiritigenin were identified by comparing with the reference substances.Furthermore,an HPLC-DAD method was established to simultaneously determime 5 constituents,including mandelonitrile, benzoic acid,formononetin,liquiritigenin and isoliquiritigenin in the active fraction of San-ao decoction and its analogical complex prescriptions.The linear ranges for mandelonitrile,benzoic acid,formononetin,liquiritigenin and isoliquiritigenin were 5.3～ 106,1.1～22,2.1～41.2,0.60～11.9,0.16～3.28μg/mL,all calibration curves showed good linear regression(r≥0.9998).The results suggest that developed method is available for screening active components from complex mixtures,such as the extracts of TCM.The main features and innovations in this dissertation are presented:1.This issue revealed the implementation of the process of the formation from the medcine-fomula-analogical complex prescriptions to recognize the basic ideas and to explore the scientific content of the TCM formula.2.The GC-MS was used to analyze the volatile components of San-ao decoction and its analogical complex prescriptions at the first time.And compare the effect of San-ao decoction and its analogical complex prescriptions on the asthma model about virus to reveal the relationship of the major constituting herbs and the formula and screen the best prescription.3.Study the active on PPARγ,found the formononetin,liquiritigenin and isoliquiritigenin in the EtOH fraction of San-ao decoction which had the best activation at the first time.Use of advanced instrumental analysis:avarieth of modern chromatographic techniques,especially HPLC/ESI-MS and fully automated HPLC analysis system provide a reliable technical support for the qulitative and quantitative analysis of complex chemical constituents.