| ObjectiveTo investigate the optimum extraction condition of Flexible Xiangru Decoction Extract and study its anti-influenza effects,aiming at providing a basis for its clinical application and preparation.Methods1.Extraction process optimization of Flexible Xiangru Decoction Extract:Extracting volatile oil from Mosla chinensis Maxim:the swimming ability,soak time and decoct extraction time were the three factors in the experiment,with content of volatile oil as the index.L9(34)orthogonal table was selected to extracting the volatile oil from Mosla chinensis Maxim.Coating volatile oil by ?-CD:the ?-CD:oil,heating temperature and heating time were the three factors in the experiment,with the yield of oil content and ratio of inclusion complex as the lndexs.L9(34)orthogonal table was selected to coating volatile oil by ?-CD.Extracting Coptis chinensis:the ethanol concentration,solid-liquid ratio,reflux time and extraction times were the three factors in the experiment,with the content of Berberine hydrochloride and dry extract rate as the indexs.L9(34)orthogonal table was selected to extracting Coptis chinensis.Extracting Lonicera japonica Thunb.,Scutellaria baicalensis Georgi and Forsythia suspensa(Thunb.)Vahl.:the solvent dilution,ethanol concentration,reflux time and extraction times were the three factors in the experiment,with the content of chlorogenic acid,baicalin and phillyrin as the indexs.L9(34)orthogonal table was selected extracting Lonicera japonica Thunb.,Scutellaria baicalensis Georgi and Forsythia suspensa(Thunb.)Vahl.According to the orthogonal test results analyzed by SPSS 19.0,mathematical regression models of indicators were established as the sub-objective.By using SGAIAB beta 5008,a plug-in toolbox of Matlab 2009a software,the optimization of single-objective genetic algorithm and multi-objective genetic algorithm was searched.Extracting the Flexible Xiangru Decoction:the extraction of optimized Flexible Xiangru decoction was the mixture of aboved extractums and inclusion complex in recipe quantity.2.Study on anti-influenza effect of Flexible Xiangru Decoction Extract in chick embryo:different concentrations of OFE and TFE were mixed with equivalent virus H3N2(20EID50).Penicillin-Streptomycin Solution(10?L/mL)were respectively added to the above mixtures.These mixed solutions were put into the 37?incubator,then injected into the allantoic cavity of chick embryo immediately after neutralizing 1h.After 48 h incubation and being placed over night at 4?,the allantoic fluids were collected for the hemagglutination assay,to evaluate the anti-influenza effect of drugs in chick embryo.3.Study on anti-influenza effect of Flexible Xiangru Decoction Extract on influenza virus pneumonia in mice:mice were inoculated intranasally with mouse lung-adapted influenza virus strain A/PR/8/34.Viral pneumonia model in mice were established and were then treated with OFE of hige,middle and low dose.Tamiflu was used as positive control drug.The model control group and the normal control group were set as well.(1)The medication was given two days in advance.Mice were sacrificed at 5th day after inoculation,with lunges removed and weighted.The lung index and inhibiton ratio of lung index were analyzed,then lungs were fixed in formalin,embedded in paraffin,routinely sectioned.(2)The medication was given two days in advance.The incidence and deaths were observed in 14 days since the inoculation,with death-protection rate and prolong survival period calculated.Results1.Extraction process optimization of Flexible Xiangru Decoction Extract:the hightest yield rate of volatile oil from Mosla chinensis Maxim(1.66%)was gained at 1:11.7 solvent dilution,2 hours immersion,5.8 hours distillation.Following application of MOGA for extraction of Mosla chinensis Maxim,the yield of oil content and ratio of inclusion complex were 82.83%,80.69%.Extraction with ?-CD:oil of 8:1 for 57?,2.4 hours treatment cycles was proved to be the optimal condition.The optimum extraction of Coptis chinensis was as follows:ethanol concentration of 74%,ratio of solid to liquid of 1:10,reflux time of 1.2 hours and extraction times of 3 times,the content of Berberine hydrochloride and dry extract rate were 8.80%,25.03%.Preferably got optimum extraction condition of Lonicera japonica Thunb.,Scutellaria baicalensis Georgi and Forsythia suspensa(Thunb.)Vahl:1:7 solvent dilution,60%ethanol concentration,decocting for 3 times,once a hour.The content of chlorogenic acid,baicalin and phillyrin were 2.64%,8.12%,1.75%,respectively.2.Study on anti-influenza effect of Flexible Xiangru Decoction Extract in chick embryo:allantoic fluid was significantly decreased after the chick embryos were treated with the OFE(high dosage,middle dosage and low dosage)and TFE(high dosage and middle dosage)(P<0.01)by the allantoic cavity injection,compared with the virus control group.Allantoic fluid hemagglutinin was significantly decreased after the chick embryos were treated with the low dosage TFE(P<0.05).OFE and TFE can significantly and dose-dependently decrease allantoic fluid hemagglutinin of chick embryos.And OFE had better anti-influenza effects than TFE.3.Study on anti-influenza effect of Flexible Xiangru Decoction Extract on influenza virus pneumonia in mice:OFE had a significant effect on decreasing lung index,increasing inhibition rate of lung index and death protection rate,prolonging survival time of mice,which eased pneumonia lesions in mice induced by influenza virus.ConclusionI.The study identified through testing the optimum extraction condition ofFlexible Xiangru Decoction Extract was reasonably practicable,providing a feasible basis for its clinical application and preparation.2.OFE and TFE both had good effects on anti-influenza virusin chick embryo.However,the effects on anti-influenza virus of OFE were better than TFE.3.The in vivo study showed that OFE was of significance to anti-influenza virus on viral pneumonia model in mice. |
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