| The extraction, isolation&purification of two main compounds geniposidic acid(GPA) and pinoresinol diglucoside(PDG) from Eucommia Ulmoides Oliver were studied in this dissertation. Two kinds of analysis methods were established for simultaneous quantification of four active ingredients of Eucommia Ulmoides Oliver. The main research contents and results are as follows:1. The optimal extraction, isolation&purification of GPA from the extract of Eucommia Ulmoides Oliver leaves were obtained.(1) The solvent extraction method assisted by ultrasonic wave was adopted to extract GPA from the extract of Eucommia Ulmoides Oliver leaves. The optimal technology conditions were obtained as follows:60%(V/V) ethanol-water as extraction solvent, extraction temperature60℃, extraction times2, and extraction time60mins for each time. Under the optimal technology mentioned above, the extraction yield of GPA is up to93.42%.(2)The column chromatography method with macroporous adsorption resins was adopted to isolate and purify GPA from the extract of Eucommia Ulmoides Oliver leaves.The S-8macroporous adsorption resin was selected as the preferred isolation material by static adsorption and desorption experiments.The optimal desorption technology conditions of GPA from S-8macroporous adsorption resin were obtained by dynamical adsorption and desorption experiments as follows:water as the desorption solvent, desorption solvent dosage300mL, flow rate of desorption solvent3mL/min, the pH value of isolated sample1.0. The flow rate of desorption solvent is the most important influencing factor to the desorption process. Under the optimal technology mentioned above, the desorption rate of GPA is up to89.64%.2. The combined extraction, isolation&purification methods and technology of GPA and PDG from raw bark of Eucommia Ulmoides Oliver were obtained.(1) The water extraction method assisted by cellulose enzymeµwave was adopted to extract GPA and PDG from raw bark of Eucommia Ulmoides Oliver. The optimal technology conditions were obtained as follows:water as extraction solvent, the ratio of the material to water1:15(g/mL), the ratio of the material to cellulose enzyme solution(0.5%)1:1(g/mL), extraction temperatures60℃, microwave radiating times2and microwave radiating time30mins. Under the optimal technology mentioned above, the extraction yield of GPA and PDG is up to90.98%and92.43%respectively.(2) The column chromatography method with macroporous adsorption resins was adopted to isolate and purify GPA and PDG from the extract of raw bark of Eucommia Ulmoides Oliver raw skin.The S-8macroporous adsorption resin was selected as the preferred isolation material by static adsorption and desorption experiments.The optimal desorption technology conditions of GPA and PDG from S-8macroporous adsorption resin were obtained by dynamical adsorption and desorption experiments as follows:water as desorption solvent of GPA, desorption solvent dosage300mL, flow rate of desorption solvent3mL/min; and then40-50%(V/V) ethanol-water as desorption solvent of PDG, desorption solvent dosage250mL, flow rate of desorption solvent5mL/min. Under the optimal technology mentioned above, the desorption rate of GPA and PDG are up to88.56%%and89.34%respectively.Two kinds of purificated powdered product were obtained after concentrating and freeze-drying from the eluents mentioned above. The purity of CPA and PDG reached78.24%and76.8%respectively.3. Two kinds of analysis methods were established for simultaneous quantification of four active ingredients of Eucommia Ulmoides Oliver.(1) A kind of analysis methods by reverse high performance liquid chromatography(HPLC) was established for simultaneously quantifying of GPA,CA, GP and Rutin of the extract from Eucommia Ulmoides Oliver leaves.The optimal analysis condition by HPLC was obtained as follows:Shimadzu High Performance Liquid Chromatography Equipment, SPD-20A UV detector, ODS-C18column (250mm4.6mm,5μm), column temperature37℃, detection wavelength254nm, injection volumn5μL, mobile phase:mixture of methanol(A) and0.1%(V/V) phosphoric acid-water (B), flow rate was0.8mL/min, gradient elution program was0.01min-30.00mi:70→30(the percent of B in A+B),30.00min-30.01min:30→70,30.01min~40.00min:70。 GPA,CA,GP and Rutin were well isolated under the optimal analysis condition mentioned above. The calibration curve was linerar in the range of the injection volumn of0.3~2.4μg(GPA),0.26~1.28μg(CA),0.05-1.375μg(GP) and0.12~1.5μ.g(Rutin), respectly. The RSD of accuracy of the analysis method was1.838%(GPA),1.907%(CA),1.094%(GP) and1.349%(Rutin), respectly. The recovery rate was98.31%~101%(GPA).98.78%~101%(CA),99.23%~100.9%(GP) and97.60%~102%(Rutin), respectly. The analysis method can meet the analysis requirement because of its good accuracy.(2) A kind of analysis methods by reverse high performance liquid chromatography(HPLC) was established for simultaneously quantifying of GPA,CA, PDG and Rutin of the extract from Eucommia Ulmoides Oliver.The optimal analysis condition by HPLC was obtained as follows: Shimadzu High Performance Liquid Chromatography Equipment, SPD-20A UV detector, ODS-C18column (250mm4.6mm,5μm), column temperature37℃, detection wavelength was0.01min-7.00min:254nm,7.01min-12.50min:277nm,12.51min-40.00min:254nm, injection volumn5μL, mobile phase:mixture of methanol(A) and0.1%(V/V) phosphoric acid-water (B), flow rate was0.8mL/min, gradient elution program was0.01min~30.00min:70->30(the percent of B in A+B),30.00min-30.01min:30→70,30.01min~40.00min:70。GPA,CA,PDG and Rutin were well isolated under the optimal analysis condition mentioned above. The calibration curve was linerar in the range of the injection volumn of0.3-2.4μg(GPA),0.26~1.28μg(CA),0.05-1.375μg(GP) and0.12-1.5μg(Rutin), respectly. The RSD of accuracy of the analysis method was2.074%(GPA),0.973%(CA),2.019%(GP) and1.981%(Rutin), respectly. The average recovery rate was101.93%(GPA),97.44%(CA),96.93%(GP) and104.56%(Rutin), respectly. The analysis method can meet the analysis requirement with satifisfactory accuracy. |
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