Screening method of beta-lactamase inhibitor

Abstract

The invention provides a screening method of a beta-lactamase inhibitor, and belongs to the technical field of drug screening. According to the method, a colorimetric sensing technology is adopted, cellulose membrane test paper is taken as a carrier, the cefoxitin is taken as a substrate, the cellulose membrane test paper is immersed into a solution containing the cefoxitin, and the cellulose membrane test paper loaded with the cefoxitin substrate is obtained. On the basis of the principle that beta-lactamase can hydrolyze amido bonds of cephalosporin to cause the color of a cephalosporin solution to be changed from yellow to red, a beta-lactamase solution is dropwise added to cellulose membrane test paper loaded with a cephalosporin substrate, and the color of the test paper is changed from yellow to orange red; when the beta-lactamase inhibitor exists, the color of the test paper is not changed. Therefore, the cellulose membrane test paper loaded with the cefoxitin substrate can be used for screening whether complex systems such as traditional Chinese medicines have beta-lactamase inhibitory activity or not. According to the method, a sample with beta-lactamase inhibitory activity can be efficiently and accurately screened out.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a method for screening beta-lactamase inhibitors. Background Art

[0002] Resistance of microorganisms, especially Gram-negative bacteria, to β-lactam antibiotics is often mediated by β-lactamases. β-lactamases are enzymes that catalyze the hydrolysis of the β-lactam ring, which degrades β-lactam antibiotics and renders them ineffective in their antibacterial activity, leading to bacterial resistance to these antibiotics. This resistance mechanism has become an important challenge in global public health, especially in hospital infections and critical care, where the spread of resistant bacteria has made treatment more difficult.

[0003] To address this problem, researchers have developed β-lactamase inhibitors that can slow down or prevent the activity of β-lactamases, thereby protecting β-lactam antibiotics from degradation and restoring the sensitivity of β-lactamase-producing bacteria to these antibiotics. However, some β-lactamase inhibitors currently on the market cannot effectively inhibit all types of β-lactamases, especially some β-lactamases that are widely present in drug-resistant bacteria. For example, KPC (β-lactamase produced by Klebsiella pneumoniae), C and D β-lactamases, etc., are all highly resistant to current inhibitors. This further limits the effectiveness of the application of β-lactam antibiotics in clinical treatment.

[0004] Therefore, it is urgent to explore new β-lactamase inhibitors, especially those that can effectively inhibit β-lactamases that cannot be cured or inhibited clinically, and to combine them with β-lactam antibiotics to treat drug-resistant infections. This is not only of great significance for combating antibiotic resistance, but also provides new strategies and hopes for future clinical treatment.

[0005] Therefore, we aimed to construct an innovative colorimetric sensing system for efficient screening of β-lactamase inhibitors in traditional Chinese medicines, thus contributing to protecting public health. Summary of the invention

[0006] In view of this, the present invention provides a method for screening β-lactamase inhibitors, which can efficiently and accurately screen β-lactamase inhibitors.

[0007] The technical solution adopted by the present invention to solve the technical problem is as follows: 1. A method for screening a β-lactamase inhibitor, comprising the following steps: (1) Immerse the cellulose membrane test paper in the nitrocefin solution for about 10 minutes to obtain a cellulose membrane test paper containing nitrocefin. (2) After preparing β-lactamase to an appropriate concentration, place it in an EP tube, add the sample solution to be screened, incubate at room temperature for 30 minutes, take an appropriate amount and drop it on the cellulose membrane test paper containing nitrocefin. After 1 minute, observe the color change. If the color of the test paper is yellow, it means that the sample has β-lactamase inhibitory activity; if the color of the test paper is orange-red, it means that the sample does not have β-lactamase inhibitory activity or the activity is weak. According to the reaction color of the above test paper, samples with β-lactamase inhibitory activity are screened out. (3) By measuring the half-inhibitory concentration of the screened samples on β-lactamase, the activity of the screened β-lactamase inhibitors is determined.

[0008] Preferably, the method for screening β-lactamase inhibitors in step (1) is characterized in that the concentration of the nitrocefin solution is 50 μg / mL.

[0009] Preferably, the method for screening β-lactamase inhibitors in step (2) is characterized in that the concentration of the β-lactamase solution is 100 μg / mL-1000 μg / mL.

[0010] Preferably, the method for screening β-lactamase inhibitors in step (2) is characterized in that the concentration of the sample solution is 250 μg / mL.

[0011] 2. A β-lactamase inhibitor, characterized in that it comprises a Chinese medicine stir-fried spiny jujube seed extract.

[0012] It can be seen from the above technical scheme that the present invention provides a screening method for β-lactamase inhibitors, which belongs to the field of drug screening technology. This method adopts colorimetric sensing technology, takes cellulose membrane test paper as a carrier, takes nitrocefin as a substrate, and immerses the cellulose membrane test paper in a solution containing nitrocefin spores to obtain a cellulose membrane test paper carrying nitrocefin substrate. Based on the principle that β-lactamase can hydrolyze the amide bond of nitrocefin, causing the color of the nitrocefin solution to change from yellow to red, a β-lactamase solution is dripped onto the cellulose membrane test paper carrying the nitrocefin substrate, and the color of the test paper changes from yellow to red; when a β-lactamase inhibitor is present, the color of the test paper remains unchanged. Therefore, the cellulose membrane test paper carrying the nitrocefin substrate can be used to screen whether a complex system such as traditional Chinese medicine has the activity of inhibiting β-lactamase. This method can efficiently and accurately screen out samples with the activity of inhibiting β-lactamase. At the same time, the method was used to screen out the Chinese medicine stir-fried spiny jujube seeds, which has the effect of inhibiting the activity of β-lactamase and can be used as a β-lactamase inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0014] Figure 1 Schematic diagram of the β-lactamase inhibitor screening method of the present invention.

[0015] Figure 2 The color changes of different concentrations of β-lactamase on the test paper and its standard curve.

[0016] Figure 3 To verify positive and negative drugs using colorimetric sensing methods.

[0017] Figure 4 To screen traditional Chinese medicines with β-lactamase inhibitory activity using colorimetric sensing method. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The embodiment of the invention discloses a method for screening beta-lactamase inhibitors by using colorimetric sensing, and records the inhibitory activity of the screened traditional Chinese medicine stir-fried spiny jujube seeds on beta-lactamase. Example 1

[0020] β-lactamase qualitative test and standard curve test. Cut the cellulose membrane test strip into appropriate size (about 0.5 cm in length and width), and then immerse it in a centrifuge tube containing nitrocefin (50 μg / mL) solution for 10 minutes. Then, add appropriate amount of β-lactamase solution of different concentrations (100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL) to the test strip. After 1 minute, use a smartphone to record the color of the test strip. The results are shown in the table below. Figure 2As shown in A. The results show that within the concentration range of 100 μg / mL-1000 μg / mL, as the concentration of β-lactamase solution gradually increases, the color of the test strip changes from light yellow to orange-red. At the same time, a series of β-lactamase solutions with concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, and 125 μg / mL were prepared, 300 μL of each solution was taken, and 300 μL of 50 μg / mL nitrocefin solution was added, mixed, and the absorbance at 482 nm was measured by ultraviolet spectrophotometer to establish a β-lactamase standard curve. The results are shown in Figure 2 B. The results show that with the increase of β-LS concentration, the absorbance value gradually increases, that is, the concentration of β-lactamase solution is directly proportional to the absorbance value, and the linear equation is y=0.10487+0.00527x, R 2 =0.9919. Example 2

[0021] β-lactamase positive and negative drug verification experiment. Prepare a series of potassium clavulanate (β-lactamase positive drug, Potassium clavulanate) solutions with concentrations of 0 ng / mL, 10 ng / mL, 100 ng / mL, and 1000 ng / mL, take 300 μL of each, add 300 μL of 1 mg / mL β-lactamase solution, mix well, and react at room temperature. After 30 minutes, add appropriate amounts to the test paper, and record the color change of the test paper after 1 minute. The results are shown in Figure 3 As shown in A. The results showed that as the concentration of β-lactamase positive drug clavulanate potassium increased, the color of the test paper gradually changed from orange-red to yellow, indicating that clavulanate potassium inhibited the activity of β-lactamase. Under the same conditions, 50 μg / mL of argatroban, allopurinol and zanamivir were used as negative controls. 300 μL of each was added to 300 μL of 1 mg / mL β-lactamase solution. After mixing, the mixture was reacted at room temperature. After 30 minutes, an appropriate amount was added to the test paper. After 1 minute, the color change of the solution was recorded. Results are shown in Figure 3 B. The results show that the color of the negative control test paper is consistent with the blank, both orange-red, indicating that the negative drug has no inhibitory effect on β-lactamase. The above results fully demonstrate that the established colorimetric sensing method can be used for the screening of β-lactamase inhibitors. Example 3

[0022] An experiment using colorimetric sensing technology to screen traditional Chinese medicines with β-lactamase inhibitory activity. Take 7 EP tubes, first add 300 μL of 1 mg / mL β-lactamase, then add 300 μL of 250 μg / mL traditional Chinese medicine extracts, namely, Cyperus rotundus, Paeonia lactiflora, Lycium barbarum, Lycium barbarum, Saposhnikovia divaricata, Phellodendron chinense and stir-fried Semen Ziziphi spinulosae. After mixing the above solutions, react at room temperature for 30 minutes, then drop appropriate amounts onto the test strips, and record the color change of the solution after 1 minute. Results are shown in Figure 4 The results showed that the color of the test paper after adding the stir-fried spiny jujube seed extract turned yellow significantly, indicating that the activity of β-lactamase decreased after reacting with the stir-fried spiny jujube seed extract, that is, the stir-fried spiny jujube seed extract had the effect of inhibiting the activity of β-lactamase. Example 4

[0023] The half inhibitory concentration IC of the extract of stir-fried spiny jujube seeds on β-lactamase 50 Determination experiment. Prepare a series of stir-fried spiny jujube kernel extract solutions with concentrations of 800 μg / mL, 600 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL, and 2.5 μg / mL, take 300 μL of each, add 300 μL of 1 mg / mL β-lactamase solution, mix well, react at room temperature for 30 minutes, use an ultraviolet spectrophotometer to detect the absorbance of each solution at a wavelength of 482 nm, and calculate the IC value of stir-fried spiny jujube kernel against β-lactamase 50 The results showed that the IC 50 The value was 143.53 μM, indicating that the stir-fried spiny jujube seed extract had a good inhibitory effect on β-lactamase activity.

[0024] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0025] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for screening β-lactamase inhibitors, characterized in that: The main steps include: (1) Immerse the cellulose membrane test paper in the nitrocefin solution for about 10 minutes to obtain a cellulose membrane test paper containing nitrocefin. (2) After preparing β-lactamase to an appropriate concentration, place it in an EP tube, add the sample solution to be screened, incubate at room temperature for 30 minutes, take an appropriate amount and drop it on the cellulose membrane test paper containing nitrocefin. After 1 minute, observe the color change. If the color of the test paper is yellow, it means that the sample has β-lactamase inhibitory activity; if the color of the test paper is orange-red, it means that the sample does not have β-lactamase inhibitory activity or the activity is weak. According to the reaction color of the above test paper, samples with β-lactamase inhibitory activity are screened out. (3) By measuring the half-inhibitory concentration of the screened samples on β-lactamase, the activity of the screened β-lactamase inhibitors is determined.

2. The method for screening β-lactamase inhibitors according to claim 1, characterized in that: The concentration of the β-lactamase solution is 100 μg / mL-1000 μg / mL.

3. The method for screening β-lactamase inhibitors according to claim 1, characterized in that: The concentration of the nitrocefin solution is 50 μg / mL.

4. The method for screening β-lactamase inhibitors according to claim 1, characterized in that: The concentration of the sample solution was 250 μg / mL.

5. A β-lactamase inhibitor, characterized in that: Including Chinese medicine stir-fried Chinese jujube seed extract.

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