Liposome biofilm chromatographic column based on target protein and application of liposome biofilm chromatographic column to screening of active component in natural product
A technology of liposome and biomembrane, which is applied in material separation, material analysis, measuring devices, etc. It can solve the problems of poor stability and reproducibility, high price, long screening cycle, etc., and achieves simple preparation and low sample consumption , The effect of high screening accuracy
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Embodiment 1
[0031] ①The silica gel was refluxed in 20% hydrochloric acid (v / v) for 8 hours, washed with distilled water until neutral, dried in vacuum at 120°C for 24 hours, and set aside; ②Dissolve 0.21g soybean lecithin (PC) and 0.07g cholesterol in 30ml of chloroform; ③ Take 100μl of 700,000 u / ml α-glucosidase, dissolve it in 1ml of ionized water to make a 70,000 u / ml enzyme solution, add the same volume of 1mg / ml glutathione, and mix it with the solution in step ②, Ultrasonic vibration forms an emulsion; ④ Put the emulsion and activated silica gel in an eggplant-shaped bottle, and slowly evaporate the organic solvent under reduced pressure at 25°C to remove the organic solvent, and a layer of phospholipid film encapsulating the enzyme is formed on the surface of the silica gel; ⑤Wash the silica gel covered with phospholipid membrane with phosphate buffer to remove unimmobilized liposomes and enzymes, and dry in a vacuum oven at 40°C to remove water and remaining organic solvents to obt...
Embodiment 2
[0037]Put 0.1g of miglitol and acarbose standard products in 10ml volumetric flasks respectively, and dilute with pure water to obtain 10mg / ml miglitol solution and 10mg / ml acarbose solution, respectively take 20μl miglitol Column alcohol solution and acarbose solution are separated on LBC and RLBC chromatographic columns, chromatographic conditions: mobile phase 0.01mM pH7.5 Na 2 HPO 4 12H 2 O buffer, flow rate v=0.2ml / min, column temperature T=35°C, wavelength λ=210nm;
[0038] The retention time of miglitol on the LBC chromatographic column is 10.82min, and the retention time on the RLBC chromatographic column is 17.03min. The retention time of acarbose on the LBC chromatographic column is 10.06min, and the retention time on the RLBC chromatographic column is 15.37min. Components that demonstrate inhibition of α-glucosidase show prolonged retention on the RLBC column.
Embodiment 3
[0040] (1) Take 2 g of Schisandra chinensis, add 30 ml of methanol, reflux at 70°C for 2 hours, and then filter. The filtrate is placed in an eggplant-shaped bottle, and the methanol is removed by vacuum rotary drying at 55°C. The remaining part is dissolved in 40 ml of pure water to obtain the alcoholic extract of Schisandra chinensis. (2) Take 20 μl of Schisandra chinensis alcohol extract, and use RLBC to separate, the chromatographic conditions are as follows: mobile phase: 0.01mM pH 7.5Na 2 HPO 4 12H 2 O-citric acid buffer, flow rate v=0.2ml / min, column temperature T=35°C, wavelength λ=250nm. (3) The Schisandra alcohol extract, the collected Schisandra eluate, and the Schisandra alcohol A standard were used as samples for reverse-phase chromatographic separation. The chromatographic conditions were as follows, chromatographic column: XDB-C18 (4.6×250mm) reverse-phase Phase, methanol: water = 70:30 (v / v), flow rate v = 0.8ml / min, column temperature T = 25°C, wavelength λ ...
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