| Objective:To investigate the method of extracting Erianin from Dendrobium nobile,and optimize the extraction process by central composite design-response surface methodology;to prepare Erianin TPGS/F68 mixed micelles,evaluate the pharmaceutical characteristics,study the drug release and preliminary pharmacodynamics in vitro of mixed micelles.Methods:Erianin was extracted from Dendrobium nobile by reflux extraction method;on the basis of single factor experiment,the material-liquid ratio,methanol-ethanol ratio and reflux time were taken as independent variables,the extraction ratio of Erianin was taken as the dependent variable;central composite design-response surface methodology was used to select the optimum extraction process of Erianin through multiple linear regression and binomial fitting each level of independent variables;Erianin mixed micelles were prepared by a film-thin hydration method with D-α-tocopherol polyethyleneglycol 1000 succinate and poloxamer188 as the carriers;the preparation technology was optimized by orthogonal test with the entrapment efficiency as the index;the characterizations of the micelles were measured by critical micelle concentration,diameter of particles,etc;the drug release in vitro of the mixed micelles was evaluated;the stability test of the mixed micelles was evaluated;the preliminary pharmacodynamics in vitro of mixed micelles was evaluated by MTT assay.Results:The optimized extraction process of Erianin was as follow:material-liquid ratio 6.5:1,methanol-ethanol ratio 2.2:1,reflux temperature 70℃,the extraction times 2,eflux time 3.2h each time,and the optimized extraction ratio was（0.056±0.002）%.The content determination method of Erianin of Dendrobium extracting solution was established by HPLC method;the Erianin was separated by gradient elution,and the resolution was good.The linear relationship of Erianin was good between the concentration and peak area in the range of 14.88-297.6 mg·L-1,and the regression equation was A=25.25C+21.835（r=0.9991）;the RSD of low,medium,and high concentrations of were2.57%,1.28%,2.54%and 2.45%,3.04%,2.33%,in the investigation of intraday and day to day precision;the average recovery of this method was 99.56%,and RSD was 2.57%.The optimized preparation of the Erianin TPGS/F68 mixed micelles was as follow:Erianin 10 mg,D-α-tocopherol polyethyleneglycol 1000 succinate 30 mg,poloxamer 188 10mg,ethanol 10 ml,ultrapure water 10 ml,hydration temperature 35℃,stirring rate 60 rpm,stirring time 20 min,and the average entrapment efficiency was（84.97±1.52）%.The content determination method of Erianin of Erianin TPGS/F68 mixed micelles was established by HPLC method;the mobile phase was methanol-acetonitrile-water（30:30:40）;the supplementary materials used were not interfered with the determination of content,and the specificity was good.The linear relationship of Erianin was good between the concentration and peak area in the range of 10.37-207.4 mg·L-1,and the regression equation was A=29.923C+59.503（r=0.9996）;the RSD of low,medium,and high concentrations were2.90%、1.77%、1.90%and 2.90%、2.71%、1.95%,in the investigation of intraday and day to day precision;the average recovery of this method was 99.04%,and RSD was 1.46%.The average diameter of particles of micelles was（53.92?1.65）nm;the PDI was（0.289?0.030）;the Zeta potential was（-12.5?2.05）m V;the average drug loading efficiency was（16.52±0.88）%;the p H was（5.22?0.16）;and the critical micelle concentration was 33.92mg·L-1.The release regularity of Erianin TPGS/F68 mixed micelles in vitro was close to Weibull equation,and the regression equation was Ln[ln（1/1-Q）]=1.6991lnt-1.9930;Erianin TPGS/F68 mixed micelles could be stored at（6±2）℃for half a year.The MTT assay of human breast cancer MCF-7 cells and mouse breast cancer 4T1 cells showed as follow:when the concentration of Erianin was 0.23 mg·ml-1,the cell viability of MCF-7 cells and 4T1 cells in the Erianin group were 43.9%and 45.9%,while the cell viability in the Erianin TPGS/F68 mixed micelles group were 17.6%and 21.5%.When the concentration was over 0.23 mg·ml-1,Erianin TPGS/F68 mixed micelles group had a better inhibitory effect than Erianin group（p<0.05）in the same concentration.That showed Erianin prepared as mixed micelles can enhance the inhibitory effect of cells proliferation.MTT assay’s result showed that the blank TPGS/F68 mixed micelles had a little cytotoxicity.Conclusion:Erianin has been successfully extracted from Dendrobium nobile in this project,and the extraction process has been optimized by central composite design-response surface methodology;the Erianin TPGS/F68 mixed micelles has been prepared by the film-thin hydration method,and the appearance,diameter of particles,potential and CMC of Erianin mixed micelles’characterizations were up to the standard;the entrapment efficiency and drug loading efficiency were good;the rate of drug release of Erianin TPGS/F68 mixed micelles in vitro was gentler than Erianin solution;both MCF-7 cells and 4T1 cells can be inhibited by Erianin,and compared with Erianin,Erianin TPGS/F68 mixed micelles has better inhibitory effect. |
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