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Mesoporous biofilm chromatographic column based on target protein and application of mesoporous biofilm chromatographic column to screening of active components in natural products

A biofilm and chromatographic column technology, applied in the field of screening active ingredients in natural products, can solve the problems of poor stability and reproducibility, easy shedding of cell membrane proteins, loss of activity, etc., achieving good reproducibility, wide application range, and difficult shedding effect

Active Publication Date: 2020-04-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immobilization of receptor protein in receptor chromatography has the problems of activity loss and protein loss. The column life of cell membrane chromatography is short, and the cell membrane protein is easy to fall off or lose activity, and the stability and reproducibility are poor.
The lack of necessary receptors in the ordinary liposome membrane can neither comprehensively simulate the retention behavior of drugs on the membrane, nor can it specifically screen for lead compounds that bind to receptors
[0005] Among the existing chromatographic methods for screening α-glucosidase inhibitors, it is difficult to peel off the cell membrane by cell membrane chromatography, and the activity decays rapidly with time, so it cannot be reused
Liposome chromatography lacks receptors on the surface of liposomes, making it impossible to study the interaction between drugs, membranes and receptors. Research the interaction between drugs, membranes and receptors
Cell and animal level screening cycle is long and expensive

Method used

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  • Mesoporous biofilm chromatographic column based on target protein and application of mesoporous biofilm chromatographic column to screening of active components in natural products
  • Mesoporous biofilm chromatographic column based on target protein and application of mesoporous biofilm chromatographic column to screening of active components in natural products
  • Mesoporous biofilm chromatographic column based on target protein and application of mesoporous biofilm chromatographic column to screening of active components in natural products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] (1) Dissolve 4g P123 and 4.616g potassium chloride (KCl) in hydrochloric acid solution (120ml H2O and 20ml 37%wt concentrated HCl), and stir at room temperature until the solution becomes transparent. (2) Add 3ml of TMB under stirring, stir for 10h, then add 9.2ml of TEOS dropwise, and stir vigorously for 10min. (3) The mixture was kept at a constant temperature in a water bath at 35°C for 24h, transferred to an autoclave and heated at 100°C for 24h, cooled, washed, filtered, dried, and calcined in a muffle furnace at 540°C for 10h to obtain a mesoporous silica material. SEM picture as figure 1 shown. (4) Mix the mesoporous silica material with 1ml 100,000 U / ml α-glucosidase solution, 1ml 1mg / mL glutathione solution, 28ml 2.5mmol / L sodium acetate buffer solution with a pH of 4.0, and place in an ice bath Stir and adsorb for 6 hours, filter, and vacuum-dry at room temperature for 2 hours to obtain a mesoporous material loaded with α-glucosidase receptors. SEM picture ...

Embodiment 2

[0040] 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 The column alcohol solution and the acarbose solution were separated on a blank mesoporous chromatographic column and a membrane-coated enzyme protein mesoporous material chromatographic column, chromatographic conditions: mobile phase 0.01mMpH7.5Na 2 HPO 4 12H 2 O buffer, flow rate v=0.2ml / min, column temperature T=40°C, wavelength λ=210nm;

[0041] The retention time of miglitol on the blank mesoporous chromatographic column was 8.03min, and the retention time on the membrane-coated enzyme protein mesoporous material chromatographic column was 15.16min. The retention time of acarbose on the blank mesoporous chromatographic column was 7.57min, and the retention time on the membrane-coated enzyme protein mesoporous material chromatographic column was 13....

Embodiment 3

[0043] (1) Take 10g of Schisandra chinensis, add 150ml 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 10ml of pure water to obtain the alcoholic extract of Schisandra chinensis. (2) Take 5 μl of Schisandra chinensis alcohol extract, and use mesoporous biofilm chromatographic column to separate. T=40°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 = 250nm. (4) Perform mass spectrometry analysis on the collected Schisandra eluate.

[0044] Schisandra chinensis has 3 ...

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Abstract

The invention discloses a mesoporous biofilm chromatographic column based on target protein and an application of the mesoporous biofilm chromatographic column to screening of active components in natural products. A preparation method of the mesoporous biofilm chromatographic column based on the target protein comprises the following steps: mixing a mesoporous silica material with an alpha-glucosidase solution, a glutathione solution and a sodium acetate buffer solution, and carrying out a stirring reaction in an ice bath to obtain the mesoporous silica material loaded with an alpha-glucosidase receptor; fully dissolving soybean lecithin and cholesterol in chloroform to obtain a mixed solution; dipping the obtained alpha-glucosidase receptor loaded mesoporous silica material into the obtained mixed solution; and removing an organic solvent by reduced pressure distillation, forming a layer of phospholipid membrane on a surface of the alpha-glucosidase receptor-loaded mesoporous silicamaterial, then washing and drying to obtain a mesoporous stationary phase wet packing column covered with the phospholipid membrane, and obtaining the mesoporous biofilm chromatographic column based on the target protein. The receptor biofilm chromatographic column based on the target protein is applied to screening of the active components of the natural products.

Description

technical field [0001] The invention relates to a method for screening active components in natural products, in particular to a method for screening active components in natural products based on target protein mesoporous biofilm chromatography. Background technique [0002] Drug screening refers to the use of appropriate screening methods and techniques to establish a suitable screening model to obtain high-efficiency lead compounds from natural or synthetic compounds that may be used as drugs, and to detect their pharmacological and pharmacodynamic activities. Evaluation, the method of evaluating the medicinal prospect of a certain compound, is a key step in shortening time, reducing cost and reducing risk in the development of new drugs. [0003] Today's drug screening methods mainly include overall animal level screening, cellular level drug screening, and molecular level drug screening. However, due to the high price of animal level screening, the unknown pathological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/89
CPCG01N30/02G01N30/06G01N30/89
Inventor 单伟光楼晓艺郭倩侯晓蓉陈秋
Owner ZHEJIANG UNIV OF TECH
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