A screening method for anti-Alzheimer's disease drugs based on zeolite adsorption

A screening method and technology for symptomatic drugs, which can be applied to measurement devices, instruments, scientific instruments, etc., can solve problems such as false positives, and achieve the effects of good reproducibility, reasonable process design, and improved screening efficiency.

Active Publication Date: 2018-06-26
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some compounds may adsorb on the separation membrane, leading to false positive results

Method used

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  • A screening method for anti-Alzheimer's disease drugs based on zeolite adsorption
  • A screening method for anti-Alzheimer's disease drugs based on zeolite adsorption
  • A screening method for anti-Alzheimer's disease drugs based on zeolite adsorption

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 The preparation method of adsorbing acetylcholinesterase zeolite is preferred

[0034] 1. Experimental method:

[0035] Dissolve 5ml of the prepared acetylcholinesterase solution (2mg / ml) in 200mM PBS buffer at pH 8.0, put 5mg of zeolite into the buffer, stir gently, take out 20μl of incubation solution at regular intervals, The amount of protein remaining in the incubation solution was determined by the Bradford method.

[0036] The three factors affecting the adsorption were investigated respectively: firstly, different types of zeolites (Y type, ZSM-5 type, Beta type) were incubated with a certain amount of acetylcholinesterase solution (5mL, 2mg / mL) to investigate the different types of zeolites. Effect of zeolites on adsorption. Secondly, the effect of PBS buffer with different pH values ​​(5.8, 6.2, 6.8, 7.2 and 8.0) on the adsorption was investigated. Thirdly, the influence of different incubation temperatures (25°C, 30°C, 37°C, 45°C) on the adsor...

Embodiment 2

[0042] Example 2 Research on various factors of the screening method for anti-Alzheimer's disease drugs based on zeolite adsorption

[0043] 1. Experimental method

[0044] 1.1 Dissolve 5ml of the prepared acetylcholinesterase solution (2mg / ml) in 200mM PBS buffer at pH 8.0, put 5mg of zeolite into the buffer, stir gently, incubate at 25°C for 20min, and suck off solution, wash the zeolite with PBS buffer solution to obtain the zeolite adsorbed acetylcholinesterase.

[0045] 1.2 In a centrifuge tube, add 20 μL of coptisine solution (1 mg / mL), add 0.025 mg of zeolite adsorbing acetylcholinesterase prepared in step 1.1, add 200 μL of PBS buffer, and adjust the solution to different pH values ​​(5.7, 6.2, 6.8, 7.4, 8.0) and different ionic strengths (10, 50, 100, 200, 500, 1000mM), and then incubated in different incubation temperatures (20, 25, 30, 37, 45°C) in different water baths time (5, 10, 20, 30, 40 min), respectively suck out the incubation solution, add 200 μL of PBS ...

Embodiment 3

[0055] Example 3 Screening of Anti-Alzheimer's Disease Components in Corydalis Corydalis Extract

[0056] 1. Experimental method

[0057] 1.1. Weigh 1g of Corydalis Corydalis powder, add 50ml of water, reflux for 1h, filter, centrifuge the filtrate at 13400rpm for 10min, absorb the supernatant for later use.

[0058] 1.2. Dissolve 5ml of the prepared acetylcholinesterase solution (2mg / ml) in 200mM PBS buffer at pH 8.0, put 5mg of zeolite into the buffer, stir gently, incubate at 25°C for 20min, and absorb The solution was removed, and the zeolite was washed with PBS buffer solution to obtain the zeolite adsorbing acetylcholinesterase.

[0059] 1.3. In a 1.5mL centrifuge tube, add 20 μL of Corydalis extract solution (10 mg / mL) obtained in step 1 and the zeolite adsorbing acetylcholinesterase obtained in step 2, and add 180 μL of PBS buffer solution (so that the ionic strength is 10mM, pH value 7.4), incubated at 37°C for 20 minutes, centrifuged and sucked out the incubation s...

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Abstract

The invention discloses a zeolite adsorption based screening method of a medicine resistant to an Alzheimer disease. According to the method, the screening is carried out based on the affinity selection-liquid mass spectrometry of zeolite; through adsorbing acetylcholin esterase on the zeolite, an acetylcholin esterase inhibitor in an officinal plant extract is screened. According to the zeolite adsorption based screening method of the medicine resistant to the Alzheimer disease, factors, such as factors of the type of the zeolite, the pH (potential of Hydrogen) value of a solution, adsorption time, an adsorption temperature and the like, which influence adsorption, are systematically optimized; moreover, factors, such as factors of the pH of an incubation solution, the ionic strength of the incubation solution, the temperature of the incubation solution, incubation time and the like, which influence a to-be-sieved medicine, are systematically optimized. A high-efficiency, correct, sensitive and high-reproducibility method for screening a component resistant to the Alzheimer disease is established; the zeolite adsorption based screening method of the medicine resistant to the Alzheimer disease has important application value.

Description

technical field [0001] The invention relates to a method for screening drugs, in particular to a method for screening anti-Alzheimer's disease drugs with high screening efficiency and based on zeolite adsorption. Background technique [0002] At present, the method based on affinity selection-mass spectrometry is used more and more in the screening of active ingredients of medicinal plants. This method finds target binders in the mixture through target affinity selection, and then identifies the binders, which can be greatly improved. Speed ​​up the screening process. These methods include affinity ultrafiltration, magnetic beads, eddy current chromatography, affinity cell membrane chromatography, etc. These methods can be divided into two categories according to the state in which the selected target exists: solubilized enzymes and immobilized enzymes. The method of immobilizing enzymes can simulate the real environment of membrane-bound enzymes or proteins, and can be qu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/34
CPCG01N30/02G01N30/34G01N2030/027
Inventor 陶益蒋妍慧李伟东蔡宝昌
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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