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A screening method for antithrombotic drugs based on magnetic bead separation

A technology of antithrombotic drugs and screening methods, which is applied in the field of drug screening, can solve the problems of membrane materials, proteins and compounds affecting the accuracy of experiments, poor compatibility between elution solvents and mass spectrometry, and retention of unbound compounds. Short screening time, simple and economical equipment, good reproducible results

Active Publication Date: 2018-08-21
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method will cause many unbound compounds to remain in the sample, and the non-specific binding of membrane materials to proteins and compounds will also affect the accuracy of the experiment
Another method is to immobilize the protein on the column as a stationary phase, and use the elution solvent to elute the compounds in sequence according to the binding strength to the protein, but the required elution solvent is not compatible with mass spectrometry
[0004] At present, no one has reported the method of selecting thrombin as a drug target and establishing a screening method for thrombin inhibitors based on magnetic bead separation by applying magnetic bead bonding technology.

Method used

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  • A screening method for antithrombotic drugs based on magnetic bead separation

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Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Preparation of thrombin-bonded magnetic beads

[0027] Take a certain volume of magnetic beads, use an equal volume of 25mM MES solution (pH 6) to wash twice, twice for 10min, add an equal volume of freshly prepared EDC solution and NHS solution (50mg / ml) to the washed magnetic beads , mix well, and incubate at room temperature for 30 min with slow shaking. After incubation, the tube was placed on the magnet for 4 minutes, the supernatant was removed, and finally washed twice with 25 mM MES solution (pH 6) to obtain activated magnetic beads for later use.

[0028] Take 25, 50, 75, and 100 μL of activated magnetic beads respectively, add 200 μL of thrombin (1 mg / ml) solution, shake and mix well, and incubate overnight at room temperature. After incubation, place the tube on the magnetic beads for 4 minutes, remove the supernatant remaining from the binding, and use the Bradford method to determine the protein content in it. Add 100 μL of 0.5% BSA solution to ...

Embodiment 2

[0035] Example 2 Research on various factors of antithrombotic drug screening method based on magnetic bead separation

[0036] (1) Using scutellarin as a model drug, optimize the denaturation and elution solvent

[0037] Take 10 μL of thrombin-bonded magnetic beads prepared in Example 1 above, add 20 μL of scutellarin reference solution (0.1 mg / ml) and 170 μL of PBS buffer (pH 7.4) and mix, and incubate for 30 min. After the incubation, use 200 μL PBS buffer was washed 4 times, and the eluate was analyzed by liquid phase. At last, the volume concentration was 10%, 30%, 50%, 70% and 90% acetonitrile-water solution (v / v) and volume concentration 10%, 30% respectively. %, 50%, 70% and 90% methanol-water solution (v / v) for denaturation elution, and the denaturation eluents were respectively taken for liquid phase analysis.

[0038] (2) Using scutellarin as a model drug, optimize the incubation time

[0039] Take 10 μL of thrombin-bonded magnetic beads prepared in Example 1 above,...

Embodiment 3

[0053] Example 3 Screening of Antithrombotic Components of Erigeron breviscapine

[0054] (1) Take a certain volume of magnetic beads, wash them twice with an equal volume of 25mM MES buffer solution, each time for 10min, after washing, add an equal volume of 50mg / ml EDC solution and 50mg / ml NHS solution, mix well, and store at room temperature Incubate with slow shaking, after incubation, place on the magnet for 4min, remove the supernatant, and then wash with 25mM MES buffer solution to obtain activated magnetic beads, set aside;

[0055] (2) Take the activated magnetic beads obtained in step (1), add thrombin solution, shake and mix evenly, incubate at room temperature, after incubation, place on the magnetic beads for 4 minutes, remove the remaining supernatant from bonding, and obtain coagulation Enzyme-bonded magnetic beads;

[0056] (3) Weigh 1g of Erigeron breviscapine powder, add 50ml of methanol, reflux for 2h, filter, centrifuge the filtrate at 13400rpm for 10min, ...

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Abstract

The invention discloses a method for screening an antithrombotic drug based on magnetic bead separation. According to the method, magnetic separation and chemical information collection (liquid chromatography-mass spectrometry) techniques are adopted; a target protein bonder is analyzed; the method is used for screening and evaluating the antithrombotic action of a drug. According to the method for screening the antithrombotic drug based on the magnetic bead separation, an optimal incubation temperature, an incubation time, a pH (potential of Hydrogen) value of a solution during incubation, ionic strength and a denaturizing cleaning solution are screened out through lots of experiments. A screening and evaluating method provided by the invention can be used for screening a large-scale natural product library or a combinatorial chemical library; in comparison with a conventional ultraviolet screening method, the screening efficiency can be obviously improved; a screening time is shortened; moreover, a synergistic effect between compounds can be discovered; the method for screening the antithrombotic drug based on the magnetic bead separation has the advantages of strong operability, quickness, high efficiency, accuracy, good repeatability and the like.

Description

technical field [0001] The invention relates to a drug screening method, in particular to an antithrombotic drug screening method based on magnetic bead separation with high screening efficiency, and belongs to the field of antithrombotic drug screening and evaluation methods. Background technique [0002] A thrombus is a blood clot that forms in a blood vessel and impedes or blocks blood flow in the circulatory system. At present, antithrombotic drugs can be divided into three categories: anticoagulant drugs, antiplatelet aggregation drugs and thrombolytic drugs. After the vascular endothelial cells are damaged, the production of thromboplastin increases, which promotes the formation of thrombin, and the thrombin A2 also increases. At the same time, the production of anticoagulant prostacyclin decreases, which easily induces thrombosis. Therefore, inhibiting thrombin can prevent thrombus formation, and people have discovered many natural thrombin inhibitors from natural pl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06C07K1/14
CPCC07K1/14G01N30/02G01N30/06G01N2030/027G01N2030/065
Inventor 陶益蒋妍慧李伟东蔡宝昌
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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