| Objectives: Using single-factor experiment, orthogonal test and Response Surface Method to optimize the extracting conditions of artemisiae argyi folium flavonoids and separating the chemical compositions by preparative HSCCC. In this study, an effieient new technological process for extraction by ulrtasonie-assisted,prepurification by solvent extraction, purification by HSCCC, and identification by UV、HPLC、LCMS-IT-TOF、1H-NMR was constructed. this technological process will not only provide technical support for its further pharmacological and clinical studies,but also supply the technical basis for the separation and purification of natural products and other traditional chinese herbal medicines.Methods:(1)Establish a standard curve with rutin as standard,the extraction rate of the total flavonoids in artemisiae argyi folium were determinated via the Na NO2-Al(NO3)3-Na OH coloration system using UV spectrophotometer.(2)The best solvent extracting the total flavonoids from artemisiae argyi folium was selected by extraction with methanol, ethanol, ethyl acetate and acetone according to the extraction rate.(3)The best method extracting the total flavonoids from artemisiae argyi folium was selected by extraction, reflux extraction and ultrasonic extraction method according to the extraction rate.(4)Time, temperature, solid-liquid ratio and volume fraction were selected to carry out single factor experiments at each level, and then select the best extraction conditions.(5)Four factors and three levels were selected to optimize the extraction conditions of total flavonoids from artemisia leaves by orthogonal method, and then select the best extraction conditions.(6)Four factors and three levels were selected to optimize the extraction conditions of total flavonoids from artemisiae argyi folium by RSM, and then select the best extraction conditions.(7)To establish a method for separating the chemical components of artemisiae argyi folium by HPLC. The optimal solvent system of HSCCC was determined by HPLC to calculate K value of the chemical components in each solvent system.(8)HSCCC was used to isolate and purify the chemical constituents in artemisiae argyi folium to obtain high purity compounds.(9)Structures of compounds were identified by UV, HPLC, LCMS-IT-TOF and 1H-NMR and the yeild were calculated.Results:(1)A method for the determination of total flavonoids in artemisiae argyi folium by UV was established and the methodology investigation results were good.(2)The extraction rate of total flavonoids with methanol, ethanol, ethyl acetate, acetone were 4.3%、4.08%、2.42%、1.79% respectively, so this study using methanol as the best extraction solvent of total flavonoids.(3)The extraction rate of total flavonoids by extraction, reflux extraction and ultrasonic extraction method were 1.12%、4.01%、4.63% respectively, so this study using ultrasonic extraction method as the best method of total flavonoids.(4)In single factor experiment, the optimal extraction conditions were extraction timeof 30 min, extraction temperature of 70 degrees, liquid ratio of 1:40, methanol volume fraction of 60%, the total flavonoids extraction rate was 4.29%.(5)The optimum extraction conditions by orthogonal test for the extraction time of 40 min, extraction temperature of 80 degrees, solid-liquid ratio of 1:30, volume fraction of 70%, the extraction rate of total flavonoids was 4.85%.(6)The optimum extraction conditions by response surface method for the extraction time of 46 min, extraction temperature of 74 degrees, liquid ratio of 1:42, methanol volume fraction of 66%, the total flavonoids extraction rate was 6.85%.(7)Conditions of HPLC method for the separation: column temperature is 25 degrees; velocity is 1.0 m L/min, flow phase A(acetonitrile), B(0.4% acetic acid): 0 min, 10%A; 10 min, 25%A; 20 min, 35%A; 30 min, 60%A, 35 min. 80%A; 37 min, 80%A; 40 min, 10%A; 43 min, 10%A. The detection wavelength is 280 nm.(8)By measuring the partition coefficient, the best two-phase solvent system is hexane- ethyl acetate- ethanol- water(3:7:4:6, v/v/v/v),the upper phase acted as stationary phase and the lower phase served as mobile phase. The separation process was performed at a flow rate of 2 m L/min, the rotation rate of the apparatus was set at 900 r/min, and the effluents were detected at 280 nm, 5 target compound was obtained in one step separation, the purities were 98.47%, 95.65%, 95.48%, 93.06%, 98.17% detected by HPLC.(9)The yield of 5 compounds were11.0%、3.6%、4.1%、7.5%、10.3% respectively, and five compounds were identified by Isochlorogenic acid A, luteolin, Cyanidin chloride, Jaceosidin and Eupatilin.Conclusions :(1)UV spectrophotometry method for the measurement of total flavonoids from artemisiae argyi folium was successfully established, the method is simple, rapid and accurate.(2)The optimum extraction solvent of total flavonoids is methanol, the best extraction method is the ultrasonic extraction, the flavonoids in artemisiae argyi folium could be extracted quickly and efficiently by taking these two methods,(3)Comparing single factor test, orthogonal method and RSM, the best extraction method is the RSM, we could extract the total flavonoids economically and efficiently by using this method.(4)HPLC method was established for the separation of the chemical components of artemisiae argyi folium, the method is fast and accurate.(5)The study established a metoed of separating and purifying 5 compounds in one step by HSCCC, the method is convenient and of high separation efficiency, and great importance for the applications in the food and medical fields.(6)An effieient new technological process for extraction by ulrtasonie-assisted, prepurification by solvent extraction, purification by HSCCC, and identification by UV、HPLC、LCMS-IT-TOF、1H-NMR was constructed. This technological process will supply the technical basis for the separation and purification of natural products and other traditional chinese herbal medicines. |
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