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Method for simultaneously screening superoxide anion remover and xanthine oxidase inhibitor

A technology of xanthine oxidase and superoxide anion, which is applied in material separation, instruments, measuring devices, etc., can solve the problems of influence of result accuracy, poor parallelism and reproducibility, poor selectivity, etc., to avoid false positives and false positives. Negative result, good stability, good separation effect

Inactive Publication Date: 2015-07-22
CHINA PHARM UNIV
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Problems solved by technology

In actual drug screening, the candidate drugs are generally screened using independent xanthine oxidase inhibitors and superoxide anion scavengers, and finally the two test results are compared comprehensively. The demand is large, and the experimental workload is large, and because it is two independent experiments, the parallelism and reproducibility between the experimental results and data are poor
Disclosed in CN103364519A is a screening method for xanthine oxidase inhibitors and / or superoxide anion scavengers, which is a drug screening method that combines xanthine oxidase-thin-layer chromatography bioautographic technology with bioactivity assays, but This method cannot quantify the activity of the drug, and there are a large number of other interfering substances in the method, which is very prone to false positive and false negative results
[0008] However, the method of using HE as a probe and using a microplate reader to detect the fluorescence intensity of the compound after incubation with the O2- generation system has great limitations: 1) It is impossible to accurately determine whether the compound can directly scavenge O2- free radicals The effect still inhibits the activity of xanthine oxidase to reduce the O2- free radicals produced
2) A large number of literature studies have pointed out that fluorescent probes have poor selectivity in the actual use process, and the detection process is easily affected by other components such as intracellular pH, antioxidants, and cytochrome C, and is prone to problems such as self-oxidation and increased background fluorescence intensity. This brings difficulties to the common determination of fluorescently labeled superoxide anion and other components. Taking HE as an example, the E+ generated when HE reacts with other substances will interfere with the detection and affect the accuracy of the results.
Therefore, in the prior art, it is difficult to successfully use fluorescent probes coupled with other methods to simultaneously evaluate two or more activities of compounds.

Method used

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  • Method for simultaneously screening superoxide anion remover and xanthine oxidase inhibitor
  • Method for simultaneously screening superoxide anion remover and xanthine oxidase inhibitor
  • Method for simultaneously screening superoxide anion remover and xanthine oxidase inhibitor

Examples

Experimental program
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Embodiment 1

[0058] In order to examine the reliability of the method in this embodiment, Vc is used as O 2 - · As the positive drug of scavenger, febuxostat is used as the positive drug of XOD inhibitor, and the established method is verified.

[0059] (1) Buffer preparation: Precisely weigh KH 2 PO 4 0.0956g, K 2 HPO 4 ·3H 2 O 0.6946g, EDTA 1.862mg, and dilute to 50mL with ultrapure water to obtain a phosphate buffer (PB) containing 75mmol / L phosphate ions and having a pH value of 7.4.

[0060] (2) Preparation of substrate: Dissolving and preparing 40 mmol / L xanthine standard solution with 0.1 mol / L NaOH solution. Dilute with PB before use to obtain a 400 μmol / L substrate solution (the substrate solution needs to be freshly prepared every day).

[0061] (3) Enzyme preparation: 5 U / mL xanthine oxidase stock solution was prepared with phosphate buffered saline and stored in a -80°C refrigerator. Take it out before use, prepare 0.08U / mL working enzyme solution with phosphate-buffere...

Embodiment 2

[0087] The xanthine oxidase inhibitor described in this example is salvianolic acid C.

[0088] The sample is as follows: the total reaction volume is 200 μL, the salvianolic acid C with a final concentration of 100 μmol / L and the xanthine oxidase with a final concentration of 0.02 U / mL are co-incubated at 37°C for 2 minutes, and the final concentration of 100 μmol / L is added Xanthine and a fluorescent probe solution with a final concentration of 50 μmol / L were used to start the reaction. After incubation at 37°C for 5 minutes, the reaction was terminated by adding acetonitrile, and the internal standard galantamine with a final concentration of 6 μmol / L was added. After vortexing, centrifuge at 13,000 g After 10 minutes, the supernatant was taken as a sample for determination by ultra-high performance liquid chromatography-mass spectrometry.

[0089] The rest are the same as in Example 1; detect and calculate the 100 μmol / L salvianolic acid C standard substance to xanthine ox...

Embodiment 3

[0091] The xanthine oxidase inhibitor described in this embodiment is quercetin.

[0092] The sample is as follows: the total reaction volume is 200 μL, quercetin with a final concentration of 100 μmol / L is co-incubated with xanthine oxidase with a final concentration of 0.02 U / mL at 37°C for 2 minutes, and a substrate with a final concentration of 100 μmol / L is added Xanthine and fluorescent probe solution with a final concentration of 50 μmol / L start the reaction, incubate at 37°C for 5 minutes, add acetonitrile to terminate the reaction, add internal standard galantamine with a final concentration of 6 μmol / L, vortex and centrifuge at 13,000 g for 10 minutes , take the supernatant as a sample for ultra-high performance liquid chromatography-mass spectrometry determination.

[0093] The rest are the same as in Example 1; detect and calculate the 100 μmol / L quercetin standard substance to xanthine oxidase inhibitory rate 67.72% according to the detection steps of Example 1, t...

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Abstract

The invention relates to the field of drug screening, particularly a method for simultaneously screening a superoxide anion remover and a xanthine oxidase inhibitor. The method comprises the following steps: adding a fluorescent probe into a reaction system composed of a compound to be detected, a substrate and a target protein, reacting the probe with the superoxide anion for some time to generate a specific product 2-OH-E+, terminating the reaction by a proper process, carrying out quantitative analysis on uric acid and the 2-OH-E+ generated in the reaction system by using a high-performance liquid phase-mass spectrometer coupling technique, and identifying the structure of the compound. The changes of the contents of the uric acid and 2-OH-E+ before and after the reaction between the xanthine oxidase or superoxide anion are detected to analyze the inhibition rate of the natural product extract or monomeric compound xanthine oxidase or the clearance rate of the superoxide anion.

Description

technical field [0001] The invention relates to the field of drug screening, in particular to a new method for quickly and simultaneously screening xanthine oxidase inhibitors and superoxide anion scavengers. technical background [0002] Xanthine oxidase is a very important enzyme in the human body, which has been extensively studied in recent decades due to its important biological functions. Xanthine oxidase plays an extremely important role in the process of purine metabolism. It can catalyze the oxidation of hypoxanthine to xanthine, and further catalyze the oxidation of xanthine to uric acid, and this process is accompanied by the generation of superoxide anion. Excessive uric acid can be deposited in the joints to form an inflammatory reaction, causing gout. And superoxide anion not only has important biological functions, but also has close relationship with various diseases such as inflammation, cancer, diabetes, senile dementia, atherosclerosis and aging (see: Har...

Claims

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Application Information

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IPC IPC(8): G01N30/88
Inventor 陈君张文付钰李萍莫华燕周萍董馨
Owner CHINA PHARM UNIV
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