Study On Ultrasonic Extraction And Separation Of Ursolic Acid From Paulownia Leaves

Paulownia, as a widely distributed species, the leaves contain a variety of bioactivematters, among which ursolic acid has shown a series of important pharmacological functions,such as hepatoprotective, anti-inflammatory, antibacterial, anti-tumor, enhancing immune,hypoglycemic and hypolipidemic, and has been widely employed in medicine and healthproducts, household chemicals, functional foods and many other aspects. In this paper, theultrasonic extraction process and separation process of ursolic acid from Paulownia leaves wasstudied. Meanwhile, a HPLC analysis method has been developed for the simultaneousquantification of two isomers, ursolic acid and oleanolic acid in Paulownia leaves. Thematerial selection of Paulownia leaves was studied in terms of Paulownia species, harvest timeand storage method.On the basis of investigating effects of HPLC mobile phase and column on the separationand determination of ursolic acid and oleanolic acid, the analysis time and the resolution ofursolic acid and oleanolic acid as response values, the main influencing factors of HPLC wereoptimized by central composite design. An effective and fast method for the separation anddetermination of ursolic acid and oleanolic acid by HPLC was established. The results showedthat oleanolic acid and ursolic acid were effectively separated within23min with the resolutionof1.739, when the chromatographic separation was performed on a Shim-pack ODS-CLC (M)column kept at21℃, using the methanol and aqueous phase containing0.05ï¼…phosphoric acidwith the volume ratio of91.7:8.3as the mobile phase at a flow rate of0.6mL/min, and thedetection wavelength was set at210nm. In the optimized chromatographic conditions，standard curve equations of oleanolic acid and ursolic acid were: y=649520x–178493(r=0.9991) and y=502791x–294016(r=0.9993), in the range of0.44~8.75μg and0.92~18.37μg, respectively. The results of precision, stability, repeatability and recovery test show that the HPLC method can be used to analyze and determinate oleanolic acid and ursolic acidin Paulownia leaves.In order to select the appropriate Paulownia leaf material, and make full and effective useof resources, the content differences of ursolic acid and oleanolic acid in Paulownia leaveswere determinated in different Paulownia species, different raw material storage methods anddifferent harvest time. The results showed that the leaves of Paulownia fortunei (Seem.) Hemsl.had the highest contents of ursolic acid and oleanolic acid, followed by the leaves ofPaulownia elongata S. Y. Hu, while Paulownia tomentosa (Thunb.) Steud. leaves weresignificantly lower. Air-dried Paulownia leaves showed higher contents of ursolic acid andoleanolic acid rather than fresh leaves and refrigerated leaves. From may, the ursolic acid andoleanolic acid contents in Paulownia leaves monthly increased， reaching the highest inOctober.In order to investigate ultrasonic extraction technology of ursolic acid from Paulowniaelongate leaves, central composite design coupled with response surface analysis was adoptedto study the effects of influencing factors including ethanol concentration (X1), ratio of liquidto solid (X2) and ultrasonic time (X3) on the extraction yield of ursolic acid. The results showedthat the effects of ultrasonic factors on the extraction yield of ursolic acid from Paulowniaelongate leaves were in the following order: ethanol concentration (X1)> ultrasonic time (X3)>ratio of liquid to solid (X2), and the optimal extraction conditions were as follows:89%ethanol solution as extraction solution,31:1(mL/g) as ratio of liquid to solid,37min asultrasonic time. Under the optimal conditions, the extraction yield of ursolic acid reached14.80mg/g, which was close to the predicted response value. Therefore, the quadratic regressionmodel can be used to predict the ultrasonic extraction conditions of ursolic acid fromPaulownia elongate leaves.A tracking study on the effectiveness and ursolic acid loss of each separation method wereperformed by HPLC, and a purification process route "n-hexane scrubbingâ†'ethanoldissolvingâ†'acid-precipitation" was established. Acoording to the purification process, thepurity of the crude ursolic acid separated from Paulownia leaves was39.1%. The crude ursolic acid were further purified by silica gel column chromatography, with n-hexane-ethyl acetate-acetic acid as the eluent. The column was packed with wet method, and the sample was addedwith dry method. Using gradient elution with the eluent proportions of8:1:0.08,6:1:0.08,4:1:0.04, ursolic acid in Paulownia leaves got better separation, and the purity of ursolic acidreached87.4%, containing9.8%oleanolic acid, also. When the ursolic acid separated bycolumn chromatography was recrystallized with ethanol three times, the purity of ursolic acidwas increased to93.6%, and the content of oleanolic acid was reduced to4.9%.The study on ultrasonic extraction and separation of ursolic acid from Paulownia leaveslaid a theoretical and practical foundation for the comprehensive development and utilizationof Paulownia resources, and expanded broader plant sources for ursolic acid.