Study On The Antitussive And Antiasthmatic Effects And The Mechanism Of The Effective Parts Of Botrychium Tematum

Objective The purpose of this thesis is to study the antitussive and antiasthmatic effects of active fraction of Botrychium using different animal models of asthma Furthermore,we observed its influence on the mRNA expression of leukotriene receptor to explore the mechanism of anti-asthma effective parts of Botrychium.Our study will provide a theoretical basis for resource use and new drug development of BotrychiumMethod The Botrychium ethanol extract was obtained by heating reflux extraction with 70%ethanol.The total extract was extracted successively with petroleum ether,chloroform,ethyl acetate and n-butanol,respectively.Petroleum ether layer,chloroform layer,the ethyl acetate layer and n-butanol layer were obtained.Ammonia-induced cough method,histamine-induced asthma method,OVA sensitization and atomization method were adopted to screen effective extraction parts of Botrychium.RT-PCR method and agarose gel electrophoresis were used to investigate the mRNA expression of CysLT1,2,moreover,2-△△Ct method was used to explore the expression levels of CysLT1 receptor of different parts of Botrychium.Results 1.In antitussive experiment,compared with the control group,the chloroform and ethyl acetate layer of Botrychium can significantly decrease cough times of mice within 3 min(P<0.01).2.In histamine-induced asthma experiment,compared with the control group,each extracts of Botrychium could prolong the incubation period of acute asthma in guinea pigs.The petroleum ether layer and chloroform layer can significantly prolong the incubation period(P<0.01);Ethyl acetate layer had significant statistical difference(P<0.05)3.In OVA sensitization and atomization asthma experiment,compared with the model group,each extract of Botrychium could reduce airway constriction index in asthmatic mice(Penh),the effect of petroleum ether layer、chloroform and n-butanol layer compared to all other parts were more pronounced(P<0.01);Botrychium chloroform layer and ethyl acetate layer significantly increased tidal volume(TV)(P<0.01);Compared with the model group,Botrychium chloroform and ethyl acetate layer can decreased significantly the percentage of eosinophils,lymphocytes and neutrophils in the BALF(P<0.01);Compared with the model group,the ethyl acetate layer of Botrychium can significantly reduce the IL-4/IFN-y ratio(P<0.05).4.The effect of Botrychium extract on mRNA expression of leukotriene receptor was examined,data showed that the mRNA level of CysLT1 was relatively stable and could be quantitated by 2-△△Ct method.The mRNA level of CysLT2 receptor was decreased and couldn’t be quantitatively analyzed with a lot of primer dimer products.Compared with the model group,all extracts of Botrychium could significantly reduce CysLT1 receptor mRNA expression.The chloroform layer and ethyl acetate layer were significantly difference(P<0.01),petroleum ether layer had significant statistical difference(P<0.05);CysLT2 receptor mRNA expression wasn’t obvious.Conclusion The study on anti tussive and antiasthmatic effects showed that chloroform layer and ethyl acetate layer play predominate roles in different animal models of asthma,such as ammonia-induced cough method,histamine-induced asthma method and OVA-induced asthma method.In addition,the effect of Botrychium on asthma was associated with the inhibition of mRNA expression of CysLT1 receptor in asthma mice model.