Comparative Study On Antitussive Expectorant Effects Of Endophytic Microorganisms And Different Extracts From Glycyrrhizae Radix Et Rhizoma

OBJECTIVE By studying and comparing with antitussive and epectorant pharmacodynamics between metabolites of endophytic microorganisms from Glycyrrhizae radix et rhizoma and water decoction,total Flavonioids and total Saponins of its host plant,to make clear differences between metabolites of endophytic microorganisms from Glycyrrhizae radix et rhizoma and water decoction,total Flavonioids and total Saponins of its host plant.To explore whether there is the same efficacy between metabolites of endophytic microorganisms and its host,so that we can provide a new idea for expanding medicinal resources of Glycyrrhizae.METHODS1.An ultrasonic extraction method was applied to extract total flavone and total saponins from wild and cultivated Glycyrrhizae radix et rhizoma,ultraviolet spectrophotometer was used to measure their content.An extraction method was applied to separate crude total flavone and crude total saponins from wild and cultivated Glycyrrhizae,and ultraviolet spectrophotometer was used to measure their purity.2.By measuring the tracheal excretion of phenol red to evaluate the volume of sputum excretion of metabolites of endophytic microorganisms from Glycyrrhizae radix et rhizoma and water decoction,total Flavonioids and total Saponins of its host plant on mice.By inducing sputum model of turbid phlegm obstructing lung to observe general situations of rats and detect lung function,as well as pathological changes of lung tissue,the content of inflammatory factor such as TNF-?,COX-2,IL-17,and IL-23,so that discussed the difference of intervention and eliminating phlegm on rats with turbid phlegm obstructing lung between metabolites of endophytic microorganisms from Glycyrrhizae radix et rhizoma and water decoction,total Flavonioids and total Saponins of its host plant.3.Cough-leading methods with Ammonium hydroxide in mice,and cough-leading method with mechanical simulation in rat were applied.Half effect time(ET50),R for mice and times of cough in 90 minutes for rats were used as indicators to observe anti-tussive action of metabolites of effective endophytic microorganisms from Glycyrrhizae radix et rhizoma and water decoction,total Flavonioids and total Saponins of its host plant.4.BLAST homology comparison and phylogenetic tree construction was applied to identify endophytic microorganisms from Glycyrrhizae radix et rhizoma with the role of relieving a cough or eliminating phlegm.RESULTS1.The results shows that 5.23mg·g-1of total saponins and 16.28mg·g-1of total flavone in wild glycyrrhiza uralensis,3.20mg·g-1 of total saponins and 6.81mg·g-1 of total flavone in the cultivated glycyrrhiza uralensis were detected.434.64mg·g-1of crude total saponins was separated from wild glycyrrhiza uralensis with the purity of 43.36%.750.48mg·g-1 of crude total flavone was separated from wild glycyrrhiza uralensis with the purity of 75.05%.445.88mg·g-1 of crude total saponins was separated from the cultivated glycyrrhiza uralensis with the purity of 45.59%.718.45mg·g-1of crude total flavone was separated from the cultivated glycyrrhiza uralensis with the purity of 71.85%.2.JTYB029,JTZB005,JTZB006,JTZB058,JTZB060 and JTZB063 can increase weasand excretion of phenol red for normal mice(P<0.05 or 0.01).The role of eliminating phlegm is similar to water decoction,total Flavonioids and total Saponins of its host plant.The experimental results of turbid phlegm obstructing lung showed that compared with the model group,various indicators of function test in JTYB029,JTZB005,JTZB006,JTZB058 and JTZB063 groups were slightly increased.The content of TNF-?,COX-2,IL-17 and IL-23 were reduced(P<0.05 or P<0.01).Moreover,the intervention effect on rats with turbid phlegm obstructing lung is equal to water decoction,total Flavonioids and total Saponins of its host plant.The lung tissue of JTYB029,JTZB005,JTZB006,JTZB058,JTZB063 groups shows bronchial epithelium was more complete than the model group,the cilia were increased and the bronchial wall was thickened slightly,infiltration of submucosal inflammatory cells was better than that of model group,mild alveolar enlargement,pulmonary interstitial inflammatory cell infiltration was alleviated.3.The cough test shows that JTYB029,JTZB005,JTZB006,JTZB043,JTZB060,JTZB063 could extend cough incubation,reduce restrain times of cough for rats with mechanical stimulation within ninety minutes.It has the role of relieving a cough(P<0.01 or P<0.05),which is similar to water decoction,total Flavonioids and total Saponins of its host plant.In addition,each effective strain group slightly extended ET50 of ammonium hydroxide cough-leading in mice.JTZB005,JTZB006,JTZB014,JTZB020,JTZB043,JTZB060,and JTZB063 have the obvious role of relieving a cough(R>150%).JTYB006 and JTZB058 have the role of relieving a cough(R>130%).4.JTYB029,JTZB005,JTZB006,JTZB043,JYZB058,JTZB060,and JTZB063 are Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus.CONCLUSION1.Endophytic microorganisms from Glycyrrhizae radix et rhizoma as JTYB029,JTZB005,JTZB006,JTZB058,and JTZB063 have the have the same eliminating phlegm activity with its host plant.It also have the improvement of lung function and lung tissue in rats with phlegm turbidity obstructing lung.2.Endophytic microorganisms from Glycyrrhizae radix et rhizoma as JTYB029,JTZB005,JTZB006,JTZB043,JTZB060,and JTZB063 have the same antitussive activity with its host plant.And antitussive effect is better than or equal to water decoction or total Flavonioids or total Saponins of its host plant.3.According to this experiment,endophytic microorganisms from Glycyrrhizae radix et rhizoma as JTYB029,JTZB005,JTZB006,and JTZB063 have the same eliminating phlegm and antitussive activity with its host plant.Hint: JTYB029 can replace wild Glycyrrhizae radix et rhizoma,JTZB005,JTZB006 and JTZB063 can replace cultivated Glycyrrhizae radix et rhizoma.4.JTYB029,JTZB005,JTZB006,JTZB043,JTZB058,JTZB060,and JTZB063 belong to bacillus.