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Method for screening active ingredients of traditional Chinese medicine by nano mesoporous material immobilized target protein

A technology of nano-mesoporous materials and active ingredients, which is applied in the field of nano-mesoporous materials to immobilize target proteins to screen active ingredients of traditional Chinese medicines. It can solve the problems of easy inactivation of target proteins, low reliability of screening results, and inability to judge the type of inhibition. Low column pressure, high reliability, and adjustable pore size

Active Publication Date: 2013-02-20
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a method for immobilizing the target protein with nanometer mesoporous material to screen the active ingredients of traditional Chinese medicine, which overcomes the method in the prior art of mixing the target protein with the substrate / Chinese medicine aqueous solution and incubating it for a period of time, and then using ultraviolet spectrophotometry Measure the absorbance value and screen the inhibitors. This method is easy to inactivate the target protein, and the screening results are not reliable, and it is impossible to judge the defects of the inhibitory type.

Method used

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  • Method for screening active ingredients of traditional Chinese medicine by nano mesoporous material immobilized target protein
  • Method for screening active ingredients of traditional Chinese medicine by nano mesoporous material immobilized target protein
  • Method for screening active ingredients of traditional Chinese medicine by nano mesoporous material immobilized target protein

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Embodiment 1

[0037] Taking diabetes target enzyme α-glucosidase as an example, its substrate is 4-nitrophenyl-α-D-pyranoside, and the typical inhibitor is acarbose.

[0038] The specific process and steps of this embodiment are as follows:

[0039] 1. Preparation of mesoporous materials: Use P123 as template, TMB as pore expander, BTESE as precursor, and CETES as -COOH coupling agent. By determining the ratio of P123 / TMB=4 / 3, the pore diameter is 13.38nm. Carboxylated organic mesoporous silica materials (-COOH-PMOs);

[0040] 2. Immobilization of α-glucosidase: using physical adsorption method, dissolve α-glucosidase in acetate buffer at pH 4.5, add the above-COOH-PMOs material, and incubate with stirring for 7 hours in an ice bath;

[0041] 3. Preparation of immobilized α-glucosidase inhibitor screening model: use water as the mobile phase, use a column packing machine to pack the above-mentioned immobilized α-glucosidase into a chromatographic column with a specification of 4.6*50mm, an...

Embodiment 2

[0062] Taking aldose reductase, the target enzyme of diabetes complications as an example, its coenzyme is NADPH, its substrate is DL-glyceraldehyde, and its typical inhibitor is epalrestat.

[0063] The specific process and steps of this embodiment are as follows:

[0064] 1. Preparation of mesoporous materials: use P123 as template, TMB as pore expander, BTESE as precursor, add -NH 2 As a coupling agent, by determining the ratio of P123 / TMB=4 / 2, an aminated organic mesoporous silica material (-NH 2 -PMOs);

[0065] 2. Immobilization of aldose reductase: using physical adsorption method, dissolve aldose reductase in pH 6.2 phosphate buffer, add the above-NH 2 -PMOs material, stirring and incubating in ice bath for 7h;

[0066] 3. Prepare the screening model for immobilized aldose reductase inhibitors: use water as the mobile phase, use a column packing machine to pack the above-mentioned immobilized aldose reductase into a chromatographic column with a specification of 4.6...

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Abstract

The invention relates to a method for screening active ingredients of traditional Chinese medicine by nano mesoporous material immobilized target protein. The method includes filling mesoporous material immobilized protein into a chromatographic column, preparing an immobilized target protein inhibitor screening model, dissolving inhibitor having representative inhibiting effect on the immobilized protein into buffer solution to serve as a mobile phase, introducing samples of substrates with saturation concentration every fixed time, collecting peak appearance fraction, replacing the mobile phase with buffer solution free of inhibitor after fixed times, introducing samples of the substrates with saturation concentration every fixed time, respectively freezing and drying the fraction, dissolving the fraction into water, separating the substrates from a product through the analytical type chromatographic column, recording peak area of the product, drawing an inhibition curve of the immobilized target protein inhibitor, checking the model and adopting the prepared model to dissolve aqueous extract of traditional Chinese medicine to be screened into the buffer solution to serve as the mobile phase. Whether the traditional Chinese medicine has inhibition effect or not and the process and the type of the traditional Chinese medicine can be judged according to the peak area of the product. The method has the advantages of high reliability, online performance and the like.

Description

technical field [0001] The invention relates to a method for screening active components of traditional Chinese medicine, in particular to a method for screening active components of traditional Chinese medicine by immobilizing target proteins with nanometer mesoporous materials. Background technique [0002] Drug screening is a step in the process of modern drug development to test and obtain specific physiologically active compounds, and it is the process of selecting compounds with higher activity on a specific target from a large number of compounds or new compounds through standardized experimental methods. Drug targets include genetic sites, receptors, enzymes, ion channels, nucleic acids and other biological macromolecules. Recent studies have shown that enzymes have many advantages as drug targets, and many important research results have been obtained. [0003] However, traditional Chinese medicine screening using the target protein as the target generally adopts th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 戴荣继王慧亨顾昊胡英慧安静邓玉林孟薇薇孟世英王丰潮
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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