| ObjectiveUsing modern analytical chemistry to optimize extraction and purification process of annua acid in artemisia annua dregs,exploring the potential efficacy of Artemisia annua acid by vitro experiments,providing foundation and scientific basis for antitumor activity of artemisinic acid,to make full ues of the dregs and enhance the utilization value of Artemisia annua.MethodsThe qualitative analysis of artemisinic acid was determined by TLC,content of artemisinic acid by HPLC.Optimize the extraction of Artemisia annua acid from the dregs of artemisia annua by response surface design;later,the product was preliminary purified by solvent extraction and then enriched by column chromatography at the last.Methyl thiazolyl tetrazolium(MTT)assay was used to observe the growth of SMMC-7721 cells.Apoptosis of the cells was analyzed by Hoechst-33258 staining.Annexin V-FITC/P staining was used to quantify the percentages of apoptic cells in the total cell population.Result1 Establish analysis methods of artemisinic acidSetting up a determination method of Artemisia acid by HPLC,in the range of 0.209?2.09?g,the regression equation is Y=1029500X+6056.35904(R=0.9999),the recovery rate is 96.63%,RSD=1.26%(n=6).2 Extraction ProcessThe best extraction process parameters of artemisinic acid was 91%ethanol as the extract,the liquid ratio was 8.5,extraction time was 42min,under which the acid extraction of Artemisia annua was 0.458mg/g,the regression model was highly significant(R2 =0.9859),and good fit.3 preliminary purification processPurified by solvent extraction.Crude extract was suspended in an aqueous solution,adjusting pH11,adding the same volume of petroleum ether and washed twice,to remove some impurities.Then the pH was adjusted to 5,a 1:1 ratio of liquid material is added and extracted twice with petroleum ether,the extract was dried,and finally the resultant product content of 5.724%.4 silica gel column chromatographySilica gel column chromatography was used separate annua acid further.Petroleum ether yielded to colorless,the petroleum ether-ethyl acetate(9:1)to give the fractions,then,ethyl acetate and petroleum ether was concentrated to precipitate impurities,Artemisia annua acid was in the mother liquor.The purity of Artemisia annua acid was 76.38%.5 artemisinic acid in vitro anti-tumor activityAfter artemisinic acid effecting on human hepatoma cell SMMC-7721 24h,48 h,72 h,the half inhibitory concentration(IC50)were 71.067?g/mL,35.800 ?g/mL,13.308?g/mL,this could significantly indicated that artemisinic acid can inhibit SMMC-7721 cells.Hoechst-33258 staining results presented obvious apoptotic morphology of SMMC-7721 cells treated with arteannuic acid at various concentrations for 48h.The result of Annexin V-FITC/P double-staining indicated that the apoptotic rate increased significantly with arteannuic acid in dose-dependent manner.Discussion1 Establish a artemisinic acid analysis methodsOptimizating the mobile phase and wavelength of HPLC,the determination of the method was accurate,simple,good stability,and suitable for measuring content of Artemisia annua acid.2 Extraction ProcessObtaining the optimum extraction conditions of artemisinic acid by response surface designs.The liquid ratio was 8.5:1 and 91%ethanol as the extraction,extracting three times for 42min.The extraction process was stable,laying the foundation for the study of post-production process.3 Purification TechnologyAfter the extraction solvent extraction,the content of artemisinic acid of extract was 5.724%;and ten enriched by Silica gel column chromatography,the purity was 76.38%.After purification,the artemisinic acid content of the original extract of about 0.4%to 76.38%,4 artemisinic acid in vitro anti-tumor activityAfter artemisinic acid effecting on human hepatoma cell SMMC-7721 24h,48 h,72 h,the half inhibitory concentration(IC50)were 71.067?g/mL,35.800 ?g/mL,13.308?g/mL,this could significantly indicated that artemisinic acid can inhibit SMMC-7721 cells.Hoechst-33258 staining results presented obvious apoptotic morphology of SMMC-7721 cells treated with arteannuic acid at various concentrations for 48h.The result of Annexin V-FITC/P double-staining indicated that the apoptotic rate increased significantly with arteannuic acid in dose-dependent manner. |
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