Extraction And Separation Of Chemical Components Of Forsythia Fructus By Supercritical CO₂ Extraction And Molecular Distillation

PURPOSE: To explore the feasibility of extraction and separation active principles of traditional Chinese medicine by supercritical CO2 extraction and molecular distillation. To study the selection of the best operation condition of supercritical CO2 extraction and molecular distillation. To analyze the components in separate groups of Forsythia Fructus and compare the pharmacological activity of separate groups.METHODS:1. Orthogonal experiment of supercritical CO2 extraction of ForsythiaFructus(1) Select five factors and three levels: extraction pressure(20, 25, 30MPa), extraction temperature(35, 40, 45℃), separation pressure(6. 3, 6.7, 7. IMPa), separation temperature(30, 40, 50℃), degrees of crush(no crush, 12mu, 30mu), according to the table L,8 (37) to design the orthogonal experiment.(2) Select the best extraction condition of supercritical CO2 extraction by orthogonal experiment. Repeat experiment of the best extraction condition.(3) Study the influence of smuggle chemical preparation.(4) Separate and identify the extractive by GC-MS.2. Separate the extractive by molecular distillation(1) Separate the extractive twice according to condition of the heating temperature 100 ℃ ,vacuum degree 100Pa and heating temperature 200 ℃,vacuum degree 5Pa by molecular distillation. Obtain the distillate 1 and distillate2.(2) Separate and identify the distillate 1 and distillate2 by GC-MS;3. Extraction and separation of water-soluble components(1) Residue of Forsythia Fructus after supercritical CO2 extraction is boiled 3 times with 30000ml water, and merge the supernate and condense the supernate to 3000ml. Remove the precipitation by high-speed centrifugation. Filtered by filter paper, then obtain the water-extract solution.(2) Add the water-extract solution to the pretreated foramen magnum resin, elute it by 1000ml of water, 30%ethanol, 50%ethanol, 70%ethanol and 90%ethanol, respectively. Collect the eluent and condense to 100ml.(3) Identify by MS and HPLC-MS. 4. Pharmacodynamics experiment(1) Antipyretic experiment: 70 male SD rats are measured normal body temperature with mercury clinical thermometer, twice everyday and continuous for 3 days. On assay day, measure the normal body temperature of rats 1 time/hour, 3 times continuously, then select 60 rats that body temperature varring lower than 0. 3℃ and divide into 6 groups randomly and equally. Distilled water 10ml/kg, extractive 16g/kg, distillate 16g/kg, water-extract solution 16g/kg, aspirin 0.1 g/kg and water-extract solution 8g/kg is given intragastricly respectively. 1 hour later, subcutaneous inject 2, 4-dinitrophenol 0. 015g/kg on the back of rat, then measure the body temperature 1 time/20 minutes, continuous for 3 hour.(2) Analgetic experiment: Divide 100 mice into 5 groups randomly, 20-mice/ group. Normal saline (10ml/kg) and pethidine hydrochloride (0. 05g/kg) are injected intraperitoneally. 16g/kg extractive, distillate and water-extract solution is given intragastricly respectively. 30 minutes later, inject the 0. 5% acetic acid0. 2ml/mouse intraperitoneally. Observe and record whether mice appear twist body reaction (belly concaved, hind limb extended and buttocks raised), the time when mice appears twist body reaction, the number of times which mice appear twist body reaction and the number of mice that appears twist body reaction in 10 minutes, then calculate the analgetic percentage of each group.RESULT:1. Orthogonal experiment of supercritical CO2 extraction of Forsythia Fructus(1) The best extraction condition is: extraction pressure 30MPa, extraction temperature 35℃, separation pressure 6. 7MPa, separate temperature 50℃, degrees of crush 30mu.(2) The influence to experiment result of each factor is: extraction temperature> degrees of crush > extraction pressure > separation pressure > separation temperature. Extraction temperature, degrees of crush and extraction pressure can influence...