Screening method of acetaldehyde dehydrogenase inhibitor

Abstract

The invention provides a screening method of an acetaldehyde dehydrogenase inhibitor, and belongs to the technical field of drug screening. The method comprises the following steps: 1, reacting acetaldehyde dehydrogenase (ALDH), acetaldehyde and nicotinamide adenine dinucleotide in a water bath at 37 DEG C to generate acetic acid and reduced nicotinamide adenine dinucleotide; and 2, heating the reactants in a water bath at 60 DEG C for a certain time, adding 1-methoxy-5-methylphenazine methyl sulfate salt and water-soluble tetrazole-8, and changing the reaction system from light purple to orange yellow. If the ALDH inhibitor is added in the first step, the final reaction system solution is kept in a light purple state. According to the principle, a sample with ALDH inhibitory activity is screened out, then the carrier material fixed with ALDH is used as a medium, and the screened sample with ALDH inhibitory activity is subjected to component separation and identification by adopting affinity chromatography and liquid chromatography-mass spectrometry, so that the acetaldehyde dehydrogenase inhibitor is efficiently and accurately screened out.

Description

Technical Field

[0001] The invention belongs to the technical field of drug screening, and in particular relates to a method for screening acetaldehyde dehydrogenase inhibitors. Background Art

[0002] As the quality of human life improves, scientific diet has received increasing attention from all walks of life, and people are increasingly concerned about alcohol addiction, a major health problem. When people drink alcohol, alcohol is first converted into acetaldehyde by alcohol dehydrogenase in the liver, which is a toxic intermediate in alcohol metabolism. Acetaldehyde dehydrogenase (ALDH) is an important enzyme mainly located in the liver, which can oxidize acetaldehyde into harmless acetic acid, thereby protecting the human body from the harmful effects of acetaldehyde. ALDH deficiency can lead to alcohol poisoning, which is mainly manifested as facial flushing, tachycardia, nausea and headache after drinking. Therefore, it helps prevent poisoning and alcoholism, which is crucial to protecting human health. In addition to playing a key role in alcohol metabolism, ALDH is also associated with a variety of health outcomes. Because of its ability to metabolize acetaldehyde, a potential carcinogen, it has anti-cancer properties. However, it is worth noting that some food ingredients may contain ALDH inhibitors, which can reduce the activity of ALDH, thereby affecting the alcohol metabolism process and increasing the risk of adverse health effects. In order to efficiently identify ALDH inhibitors in food, the development of highly sensitive and specific biosensing technologies is indispensable.

[0003] Currently, the most widely used enzyme inhibitor screening methods include UV-visible spectroscopy, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and electrochemical methods. However, there are few reports on the screening methods for ALDH inhibitors. The commonly used strategy is to develop enzyme activity sensors based on enzyme reaction products. Since sensor analysis can quickly identify enzyme inhibitory active components in complex samples, it has been widely used in determining enzyme activity and screening inhibitors.

[0004] Since carbon nanotubes (CNTs) were first discovered by Japanese electron microscopist Lijima in 1991 during the preparation of C60 by arc method, they have shown great application potential in the field of biosensing due to their unique structure, large specific surface area, excellent conductivity and mechanical strength, and good biocompatibility. The development of biosensors based on carbon nanotubes is due to the good biocompatibility of carbon nanotubes, the potential to be applied to specific engineered surfaces, and high-precision results.

[0005] Therefore, the present invention aims to construct an innovative colorimetric sensing system and explore carbon nanotubes as ALDH carrier materials for efficient screening of ALDH inhibitors in food. This study not only helps to gain a deeper understanding of the mechanism of action of ALDH, but also provides a powerful technical tool for the field of food safety testing, thereby contributing to protecting public health. Summary of the invention

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

[0007] The technical solution adopted by the present invention to solve the technical problem is as follows: 1. A method for screening acetaldehyde dehydrogenase inhibitors, comprising the following steps: (1) Screening of samples with ALDH inhibitory activity: In the first step, ALDH, acetaldehyde and nicotinamide adenine dinucleotide (NAD+) solutions were mixed in a volume ratio of 6:1:5 and heated in a 37°C water bath for a certain period of time. Acetaldehyde was oxidized to acetic acid by ALDH and NAD+ was reduced to reduced nicotinamide adenine dinucleotide (NADH) by ALDH. In the second step, the above reaction system was heated in a 60°C water bath for a certain period of time to remove the remaining NAD+, and then 2 mM water-soluble tetrazolium-8 (WST-8) and 2 mM 1-methoxy-5-methylphenazine methylsulfate (1-mPMS) were added. In the presence of 1-mPMS, NADH can reduce WST-8 to orange-yellow formazan, causing the reaction system to change from light purple to orange-yellow. At this time, the formazan generated in the reaction system is proportional to the amount of NADH in the sample. If an ingredient with ALDH inhibitory activity is added in the first step, the final reaction system solution will remain light purple. According to the above principle, by adding the sample solution to be screened in the first step, the samples with ALDH inhibitory activity are screened according to the color of the final reaction system. (2) Separation and identification of components with ALDH inhibitory activity: Affinity chromatography is used to separate components that can bind to ALDH. First, ALDH is co-incubated with multi-walled carbon nanotubes (MWCNTs) to obtain an ALDH@MWCNTs immobilized enzyme carrier material. Then, the sample with ALDH inhibitory activity screened by (1) is added to the immobilized enzyme at a volume ratio of 1:1. After shaking for sufficient reaction for 30 minutes, the unadsorbed supernatant is centrifuged and discarded. The ALDH@MWCNTs binding solution is eluted with a suitable solvent. The eluate is injected into a liquid chromatograph and a liquid-mass spectrometer for detection, respectively, to obtain and identify the components that can bind to ALDH. By measuring the half-inhibitory concentration of the screened binding components on ALDH, the ALDH inhibitor is finally obtained.

[0008] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (1) is characterized in that the concentration of the acetaldehyde solution is 0.1%-1%.

[0009] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (1) is characterized in that the concentration of the ALDH enzyme solution is 0.5 mg / mL-1.5 mg / mL.

[0010] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (1) is characterized in that the concentration of the NAD+ solution is 5 mM-20 mM.

[0011] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (1) is characterized in that: the heating in a 37° C. water bath for a certain period of time is 20-50 minutes.

[0012] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (1) is characterized in that: the heating in a 60° C. water bath for a certain period of time is 20-50 minutes.

[0013] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (1) is characterized in that the concentration ratio of the two solutions of WST-8 and 1-mPMS is 3-4:2-4.

[0014] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors described in step (2) is characterized in that: the preparation steps of the ALDH@MWCNTs immobilized enzyme carrier material are as follows: the carboxyl functionalized MWCNTs are activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide solution for 2 hours, washed with water 3 times, and then ALDH is added, and the mixture is placed in an oscillator for 2 hours to fix ALDH on the surface of MWCNTs, and the composite mixture is separated by filtration, and washed with PBS buffer (pH=7.4) 3 times to obtain ALDH@MWCNTs immobilized enzyme.

[0015] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (2) is characterized in that: the affinity chromatography method is specifically: the chromatographic column is C 18 (4.6 × 250 mm), the detection wavelength was 254 nm, the mobile phase A was 0.1% formic acid, the mobile phase B was methanol, and the gradient elution program was 0 min-70% A; 4 min-70% A; 14 min-52% A; 24 min-52%A; 25 min-70% A; 30 min-70% A.

[0016] Preferably, the method for screening acetaldehyde dehydrogenase inhibitors in step (2) is characterized in that: the LC-MS method is specifically: the chromatographic column is C 18 (3.5 μm; 2.1 mm × 50 mm), mobile phase A was 0.1% formic acid, mobile phase B was methanol, and the gradient elution program was 0 min-70% A; 4 min-70% A; 14 min-52% A; 24 min-52% A; 25 min-70% A; 30 min-70% A. The nebulizer pressure was 35 psi, the drying gas flow rate was 8 L / min, the gas temperature was 320°C, the sheath gas temperature was 350°C, the gas flow rate was 11 L / min, and the mass range was selected as 50-3000 (m / z).

[0017] It can be seen from the above technical scheme that the present invention provides a method for screening acetaldehyde dehydrogenase inhibitors, which belongs to the field of drug screening technology. In the first step of this method, ALDH, acetaldehyde and NAD+ are placed in a 37°C water bath for reaction. ALDH can oxidize acetaldehyde to acetic acid, and NAD+ is reduced to NADH by ALDH. In the second step, the above reaction system is placed in a 60°C water bath and heated for a certain period of time to remove the remaining NAD+, and then WST-8 and 1-mPMS are added. NADH can reduce WST-8 to orange-yellow formazan in the presence of 1-mPMS, so that the reaction system changes from lavender to orange-yellow. At this time, the formazan generated in the reaction system is proportional to the amount of NADH in the sample. If an ingredient with ALDH inhibitory activity is added in the first step, the final reaction system solution remains lavender. According to the above principle, by adding the sample solution to be screened in the first step, the sample with ALDH inhibitory activity is screened out according to the color of the final reaction system. Then, using a carrier material immobilized with ALDH, affinity chromatography combined with mass spectrometry is used to separate and identify the components of the sample with ALDH inhibitory activity, thereby screening out acetaldehyde dehydrogenase inhibitors. This method can efficiently and accurately screen acetaldehyde dehydrogenase inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 It is a schematic diagram of the metabolic process of ethanol in the human body and the screening method of the acetaldehyde dehydrogenase inhibitor of the present invention.

[0020] Figure 2It shows the optimization results of ALDH concentration, NAD+ concentration, WST-8 to 1-mPMS ratio, different reaction times, and ALDH standard curve.

[0021] Figure 3 For positive and negative drug validation of colorimetric sensing methods, and screening of foods with ALDH enzyme inhibitory activity.

[0022] Figure 4 It is used for positive and negative drug validation of affinity chromatography methods, as well as for the separation and identification of active ingredients in foods with ALDH enzyme inhibitory activity.

[0023] Figure 5 To verify the inhibitory effect of the two screened inhibitors on ALDH enzyme activity and determine the half-inhibitory concentration IC 50 Determination of value. DETAILED DESCRIPTION

[0024] 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.

[0025] The embodiment of the invention discloses a method for screening acetaldehyde dehydrogenase inhibitors by using colorimetric sensing and affinity chromatography, and records the inhibitory activity of daidzein obtained by screening on acetaldehyde dehydrogenase. Example 1

[0026] Optimization experiment of acetaldehyde concentration. Prepare a series of acetaldehyde solutions with concentrations of 0.0001%, 0.001%, 0.01%, 0.1%, and 1%, take 5 μL of each, add 30 μL of 1 mg / mL ALDH and 25 μL of 10 mM NAD+ and mix well. After heating the above solutions in a 37°C water bath for 30 minutes, continue to heat in a 60°C water bath for 30 minutes, then add 5 μL of WST-8 and 1-mPMS with a concentration of 2 mM, respectively, record the color changes of the solutions, and detect the absorbance of each solution at a wavelength of 450 nm. Results are shown in Figure 2 As shown in A. The results show that as the concentration of acetaldehyde increases, the color of the solution gradually changes from light purple to orange-yellow, and the higher the absorbance value, when the acetaldehyde concentration reaches 0.1%, the color of the solution no longer changes, and the absorbance value reaches the highest. Therefore, the preferred acetaldehyde concentration is 0.1%-1%. Example 2

[0027] NAD+ concentration optimization experiment. Prepare a series of NAD+ solutions with concentrations of 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 20 mM, take 25 μL of each, add 30 μL of 1 mg / mL ALDH and 5 μL of 0.1% acetaldehyde and mix well. After heating the above solutions in a 37°C water bath for 30 minutes, continue to heat in a 60°C water bath for 30 minutes, then add 5 μL of WST-8 and 1-mPMS with a concentration of 2 mM, record the color changes of the solutions, and detect the absorbance of each solution at a wavelength of 450 nm. Results are shown in Figure 2 B. The results show that as the NAD+ concentration increases from 1.25 mM to 10 mM, the solution color gradually changes from light purple to orange-yellow; but as the NAD+ concentration continues to increase, the solution color changes from dark to light; that is, when the NAD+ concentration is 10 mM, the solution color is the darkest and the corresponding absorbance value is the highest. Considering the NAD+ concentrations of 5 mM and 20 mM, the preferred NAD+ concentration is 5-20 mM. Example 3

[0028] Experiment on the optimal ratio of WST-8 and 1-mPMS. Take 5 EP tubes, add 30 μL 1 mg / mL ALDH, 5 μL 0.1% acetaldehyde and 25 μL 10 mM NAD+ respectively and mix them. After heating the above solutions in a 37°C water bath for 30 minutes, continue to heat in a 60°C water bath for 30 minutes, and then add 5 μL of WST-8 and 1-mPMS of different concentrations. The concentrations of the two reagents in each EP tube are: ①3 mM WST-8 and 2 mM 1-mPMS, ②3 mM WST-8 and 4 mM 1-mPMS, ③3 mM WST-8 and 3 mM 1-mPMS, ④4 mM WST-8 and 3 mM 1-mPMS, ⑤4 mM WST-8 and 2 mM 1-mPMS. Record the color changes of the above solutions, and detect the absorbance value of each solution at a wavelength of 450 nm. Results are shown in. Figure 2 C. The results show that WST-8 and 1-mPMS at different concentration ratios all show darker colors and higher absorbances. Therefore, the concentration ratio of WST-8 and 1-mPMS solutions can be 3-4:2-4. Example 4

[0029] Optimization experiment of the first heating time. Take 5 EP tubes, add 30 μL 1 mg / mL ALDH, 5 μL 0.1% acetaldehyde and 25 μL 10 mM NAD+ and mix them. Heat the above solutions in a 37°C water bath for 10 minutes, 20 minutes, 30 minutes, 40 minutes and 50 minutes respectively, and then continue to heat in a 60°C water bath for 30 minutes, then add 5 μL of WST-8 and 1-mPMS with a concentration of 2 mM, record the color change of the solution, and detect the absorbance value of each solution at a wavelength of 450 nm. The results are shown in Figure 2 D. The results show that as the reaction time increases, the color of the solution gradually changes from light orange to orange. When the reaction time is 30 minutes, the solution color is the darkest, and the corresponding absorbance value is the highest. Considering that the solution color is darker when the reaction time is 20-50 minutes, the first heating time is preferably 20-50 minutes. Example 5

[0030] Optimization experiment of the second heating time. Take 5 EP tubes, add 30 μL 1 mg / mL ALDH, 5 μL 0.1% acetaldehyde and 25 μL 10 mM NAD+ respectively and mix well. After heating the above solutions in a 37°C water bath for 30 minutes, continue to heat in a 60°C water bath for 10 minutes, 20 minutes, 30 minutes, 40 minutes and 50 minutes respectively, and then add 5 μL of WST-8 and 1-mPMS with a concentration of 2 mM respectively, record the color change of the solution, and detect the absorbance value of each solution at a wavelength of 450 nm. The results are shown in Figure 2 E. The results show that as the reaction time increases, the color of the solution gradually changes from light orange to orange. When the reaction time is 30 minutes, the solution color is the darkest, and the corresponding absorbance value is the highest. Considering that the solution color is darker when the reaction time is 20-50 minutes, the second heating time is preferably 20-50 minutes. Example 6

[0031] ALDH linear range and standard curve experiment. Prepare a series of ALDH solutions with concentrations of 0.5 mg / mL, 0.75 mg / mL, 1.0 mg / mL, 1.25 mg / mL, and 1.5 mg / mL, take 30 μL of each, add 5 μL of 0.1% acetaldehyde and 25 μL of 10 mM NAD+ and mix well. After heating the above solutions in a 37°C water bath for 30 minutes, continue to heat in a 60°C water bath for 30 minutes, then add 5 μL of WST-8 and 1-mPMS with a concentration of 2 mM, respectively, record the color changes of the solutions, and detect the absorbance of each solution at a wavelength of 450nm. Results are shown in Figure 2F. The results show that as the concentration of ALDH increases, the color of the solution gradually changes from light purple to orange-yellow, and the absorbance value gradually increases, that is, the concentration of ALDH and the absorbance value are in direct proportion, and the linear equation is y=0.6843x-0.1712, R 2 =0.9989. Therefore, the ALDH concentration can be 0.5 mg / mL-1.5 mg / mL. Example 7

[0032] ALDH positive inhibitor verification experiment. Prepare a series of furazolidone (ALDH positive inhibitor, Furazolidone) solutions with concentrations of 0 μM, 0.5 μM, 1.0 μM, and 10 μM, respectively. Take 50 μL of each solution, add 30 μL of 1.5 mg / mL ALDH solution, mix well, and react at 37°C for 30 minutes. Then add 5 μL of 0.1% acetaldehyde solution and 25 μL of 10 mM NAD+ solution, mix well, and react in a 37°C water bath for 30 minutes. Subsequently, heat the reaction system in a 60°C water bath for 30 minutes, and then add 5 μL of 2 mM WST-8 and 2 mM 1-mPMS. Record the color change of the solution, and detect the absorbance value of each solution at a wavelength of 450 nm. Results are shown in. Figure 3 As shown in A. The results show that as the concentration of furazolidone, an ALDH positive inhibitor, increases, the color of the solution gradually changes from orange to light, and the absorbance value gradually decreases. The above results indicate that furazolidone inhibits ALDH activity, causing the solution color to become lighter or return to light purple. That is, the established colorimetric sensing method can be used for the screening of ALDH inhibitors. Example 8

[0033] ALDH negative inhibitor verification experiment. Take 50 μL each of PBS, allopurinol, dabigatran etexilate, and zanamivir, add 30 μL of 1.5 mg / mL ALDH solution, mix well, and react at 37°C for 30 minutes. Then add 5 μL of 0.1% acetaldehyde solution and 25 μL of 10 mM NAD+ solution, mix well, and react in a 37°C water bath for 30 minutes. Subsequently, heat the reaction system in a 60°C water bath for 30 minutes, and then add 5 μL of 2 mM WST-8 and 2 mM 1-mPMS. Record the color change of the solution and detect the absorbance of each solution at a wavelength of 450 nm. Results are shown in Figure 3As shown in B. The results show that the addition of ALDH negative inhibitors such as PBS, allopurinol, dabigatran and zanamivir does not significantly change the solution color and absorbance. The above results show that allopurinol, dabigatran and zanamivir do not have ALDH inhibitory activity, resulting in no change in solution color. The results of negative inhibitors once again prove that the established colorimetric sensing method can be used for the screening of ALDH inhibitors. Example 9

[0034] The ALDH inhibitor screening experiment in food was carried out using colorimetric sensing. Take 11 EP tubes, add 30 μL 1.5 mg / mL ALDH to each tube, and then add 50 μL food extract to each tube, which are watermelon [ Citrullus lanatus (Thunb.) Matsum. et Nakai], cucumber ( Cucumis sativus L.), tomato ( Solanum lycopersicum L.), Chrysanthemum( Dendranthema morifolium Tzvel.), green tea[ Camellia sinensis (L.) O. Ktze.], soybean[ Glycine max (L.) merr.], cashew ( Anacardium occidentale L.), red dates ( Ziziphus jujuba Mill.), wolfberry ( Lycium barbarum L . ), Rose( Rosa rugosa Thunb.), purple grapes ( Vitis romanetii Roman. du Caill. ex Planch . ), the above solutions were mixed and reacted at 37°C for 30 minutes. Then 5 μL of 0.1% acetaldehyde solution and 25 μL of 10 mM NAD+ solution were added, mixed and placed in a 37°C water bath for 30 minutes. Subsequently, the reaction system was heated in a 60°C water bath for 30 minutes, and 5 μL of 2 mM WST-8 and 2 mM 1-PMS were added. The color change of the solution was recorded, and the absorbance value of each solution was detected at a wavelength of 450 nm. The results are shown in Figure 3 C. The results show that the absorbance value of the system solution added with soybeans is significantly reduced, indicating that the activity of ALDH is reduced after reacting with soybean extract, that is, soybean extract has the effect of inhibiting ALDH activity, and there are components in soybean extract that can inhibit ALDH activity. Example 10

[0035] Affinity chromatography support material - preparation and characterization of immobilized enzyme ALDH@MWCNTs. 20 mg of carboxyl functionalized MWCNTs were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide solution for 2 hours, washed three times with PBS buffer (pH=7.4), and then 3 mg of ALDH was added. The mixture was placed in an oscillator for 2 hours to immobilize ALDH on the surface of MWCNTs. The composite mixture was separated by filtration and washed three times with PBS buffer (pH=7.4) to obtain ALDH@MWCNTs immobilized enzyme. The properties of ALDH, MWCNTs and ALDH@MWCNTs were studied through a series of characterization experiments. The surface morphology of ALDH@MWCNTs was characterized by transmission electron microscopy (TEM) (Talos F200S); the elemental composition of MWCNTs and ALDH@MWCNTs was analyzed by Thermo Fisher K-ALPHA X-ray photoelectron spectrometer (XPS); the surface structure and properties of MWCNTs and ALDH@MWCNTs were studied by Bruker Dimension EDGE atomic force microscopy (AFM). The results showed that TEM results showed that ALDH free enzyme and immobilized enzyme had the same basic morphology without significant difference; XPS analysis results showed that the proportion of oxygen and nitrogen elements in the protein on the surface of MWCNTs increased, further proving the fixation of ALDH on the surface of MWCNTs. At the same time, AFM images showed obvious differences between ALDH and ALDH@MWCNTs. The above results show that ALDH was successfully immobilized on the surface of MWCNTs, and the ALDH@MWCNTs immobilized enzyme affinity chromatography carrier material was prepared. Embodiment 11

[0036] Affinity chromatography positive and negative adsorption verification experiment. To the 4 EP tubes containing ALDH@MWCNTs, add 200 μL of each of 50 μg / mL furazolidone (ALDH positive inhibitor, Furazolidone), zanamivir (ALDH negative inhibitor, Zanamivir), allopurinol (ALDH negative inhibitor, Allopurinol), and donepezil (ALDH negative inhibitor, Donepezil), mix well and place in an oscillator for 30 minutes, let stand, and centrifuge to obtain the supernatant, which is the unadsorbed liquid. Then add 200 μL of methanol to the remaining solid, mix well and place in an oscillator at room temperature for 30 minutes, centrifuge to obtain the supernatant, which is the eluent. The above drug initial solution, unadsorbed solution, and eluent were analyzed by high performance liquid chromatography. Results are shown in Figure 4A (adsorption rate), 4B (a: furazolidone initial solution; b: furazolidone non-adsorbed solution; c: furazolidone elution solution), 4C (a: zanamivir initial solution; b: zanamivir non-adsorbed solution), 4D (a: allopurinol initial solution; b: allopurinol non-adsorbed solution), 4E (a: donepezil initial solution; b: donepezil non-adsorbed solution). The results show that the ALDH positive inhibitor furazolidone has a good adsorption effect on ALDH@MWCNTs, while the three ALDH negative inhibitors have poor adsorption effect on ALDH@MWCNTs. That is, ALDH@MWCNTs can be used for affinity chromatography screening of ALDH inhibitors. Example 12

[0037] Affinity chromatography was used to screen the components in soybean extract that can bind to ALDH. The soybean extract with ALDH inhibitory activity obtained in Example 9 was used as the research object. 200 μL of soybean extract was added to the EP tube containing ALDH@MWCNTs, mixed and placed in an oscillator for 30 minutes, centrifuged to obtain the supernatant (non-adsorbed liquid), and then 200 μL of methanol was added to the remaining solid. After mixing, it was placed in an oscillator at room temperature for 30 minutes, centrifuged to obtain the supernatant (eluent), and the soybean extract initial liquid, non-adsorbed liquid, and eluent were respectively analyzed by high performance liquid chromatography. The results are shown in Figure 4 F (a: soybean extract initial solution; b: soybean unadsorbed solution; c: soybean eluate). The results show that in the HPLC of the soybean eluate, two chromatographic peaks can be clearly seen, that is, ALDH@MWCNTs successfully captured two components in soybean that can bind to ALDH. Example 13

[0038] Liquid chromatography-mass spectrometry was used to identify the components in soybeans that can bind to ALDH. 200 μL of soybean extract was added to the EP tube containing ALDH@MWCNTs, mixed and placed in a room temperature oscillator for 30 minutes, centrifuged to obtain the supernatant (unadsorbed liquid), and then 200 μL of methanol was added to the remaining solid, mixed and placed in a oscillator for 30 minutes, centrifuged to obtain the supernatant (eluent), and the soybean eluent was analyzed by liquid chromatography-mass spectrometry. The results are shown in Figure 4 G (a: mass spectrum of daidzein; b: mass spectrum of genistin), it was preliminarily determined that the components in soybean that can bind to ALDH are daidzein and genistin. Embodiment 14

[0039] Liquid chromatography combined with reference substance verification was used to further determine the components in soybeans that can bind to ALDH. 200 μL of soybean extract was added to the EP tube containing ALDH@MWCNTs, mixed and placed in an oscillator at room temperature for 30 minutes, centrifuged to obtain the supernatant (unadsorbed liquid), and then 200 μL of methanol was added to the remaining solid, mixed and placed in an oscillator for 30 minutes, and centrifuged to obtain the supernatant (eluent). In addition, appropriate concentrations of daidzein and genistin reference substance solutions were prepared, and the eluent, daidzein reference substance solution, and genistin reference substance solution were respectively analyzed by high performance liquid chromatography. Results are shown in Figure 4 H (a: genistin reference solution; b: daidzein reference solution; c: soybean eluate). The results showed that the two chromatographic peaks in the eluate were identified as daidzein and genistin. The above results confirmed that the components in soybean that can bind to ALDH are daidzein and genistin. Embodiment 15

[0040] Experiment on the inhibition of ALDH activity by daidzein. Prepare a series of daidzein reference solutions with concentrations of 10 μg / mL, 100 μg / mL, and 500 μg / mL, respectively. Take 50 μL of each solution, add 30 μL of 1.5 mg / mL ALDH solution, mix well, and react at 37°C for 30 minutes. Then add 5 μL of 0.1% acetaldehyde solution and 25 μL of 10 mM NAD+ solution, mix well, and react in a 37°C water bath for 30 minutes. Subsequently, heat the reaction system in a 60°C water bath for 30 minutes, and then add 5 μL of 2 mM WST-8 and 2 mM 1-mPMS. Record the color change of the solution, and detect the absorbance of each solution at a wavelength of 450 nm. Results are shown in. Figure 5 As shown in A. The results show that as the concentration of daidzein increases, the color of the solution gradually fades from orange-yellow, and the absorbance value gradually decreases. The above results indicate that daidzein has ALDH inhibitory activity, causing the color of the solution to change, and showing a concentration-dependent relationship. This result shows that daidzein has an inhibitory effect on ALDH activity. Example 16

[0041] Genistin inhibition experiment on ALDH activity. Prepare a series of genistin reference solutions with concentrations of 10 μg / mL, 100 μg / mL, and 500 μg / mL, respectively. Take 50 μL of each solution, add 30 μL of 1.5 mg / mL ALDH solution, mix well, and react at 37°C for 30 minutes. Then add 5 μL of 0.1% acetaldehyde solution and 25 μL of 10 mM NAD+ solution, mix well, and react in a 37°C water bath for 30 minutes. Subsequently, heat the reaction system in a 60°C water bath for 30 minutes, and then add 5 μL of 2 mM WST-8 and 2 mM 1-mPMS. Record the color change of the solution, and detect the absorbance of each solution at a wavelength of 450 nm. Results are shown in. Figure 5 B. The results show that with the increase of genistin concentration, the solution color and absorbance value do not change. The above results indicate that genistin does not have ALDH inhibitory activity, resulting in no change in solution color. This result indicates that genistin has no inhibitory effect on ALDH activity. Embodiment 17

[0042] IC50 of ALDH inhibitor daidzein 50 Determination experiment. Prepare a series of daidzein reference solutions with concentrations of 1400 μg / mL, 700 μg / mL, 500 μg / mL, 300 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL, take 50 μL of each, add 30 μL of 1.5 mg / mL ALDH solution, mix well, and react at 37°C for 30 minutes. Then add 5 μL of 0.1% acetaldehyde solution and 25 μL of 10 mM NAD+ solution, mix well, and react in a 37°C water bath for 30 minutes. Subsequently, the reaction system was heated in a 60°C water bath for 30 minutes, and 5 μL of 2 mM WST-8 and 2 mM 1-mPMS were added, and the absorbance of each solution was detected at a wavelength of 450 nm to calculate the IC value of daidzein for ALDH. 50 Value, see the result Figure 5 C. The results showed that the IC 50 The value was 194.09 μM. The above results further confirmed that daidzein has a good inhibitory effect on ALDH activity.

[0043] 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.

[0044] 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 an acetaldehyde dehydrogenase inhibitor, comprising the following steps: (1) Screening of samples with acetaldehyde dehydrogenase (ALDH) inhibitory activity: In the first step, ALDH, acetaldehyde and nicotinamide adenine dinucleotide (NAD+) were mixed in a volume ratio of 6:1:5, and then heated in a 37°C water bath for a certain period of time. Acetaldehyde was oxidized to acetic acid by ALDH, and NAD+ was reduced to reduced nicotinamide adenine dinucleotide (NADH) by ALDH. In the second step, the above reaction system was further heated in a 60°C water bath for a certain period of time to remove the remaining NAD+, and then 2mM water-soluble tetrazolium-8 (WST-8) and 2mM 1-methoxy-5-methylphenazine methylsulfate (1-mPMS) were added. In the presence of 1-mPMS, NADH reduced WST-8 to orange-yellow formazan, causing the reaction system to change from light purple to orange-yellow. At this time, the formazan generated in the reaction system was proportional to the amount of NADH in the sample. If an ingredient with ALDH inhibitory activity was added in the first step, the final reaction system solution remained light purple. According to the above principle, by adding the sample solution to be screened in the first step, the samples with ALDH inhibitory activity are screened according to the color of the final reaction system. (2) Separation and identification of components with ALDH inhibitory activity: Affinity chromatography is used to separate components that can bind to ALDH. First, ALDH is co-incubated with multi-walled carbon nanotubes (MWCNTs) to obtain ALDH@MWCNTs immobilized enzyme carrier material, and then the sample with ALDH inhibitory activity screened by (1) is added to the immobilized enzyme at a volume ratio of 1:

1. After shaking for sufficient reaction for 30 minutes, the unadsorbed supernatant is centrifuged and discarded, and a suitable solvent is used to elute to obtain a solution bound to ALDH@MWCNTs. The eluate is injected into a liquid chromatograph and a liquid-mass spectrometer for detection, and the components that can bind to ALDH are obtained and identified. By measuring the half-inhibitory concentration of the screened binding components on ALDH, the ALDH inhibitor was finally obtained.

2. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The concentration of the acetaldehyde solution is 0.1%-1%.

3. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The concentration of the ALDH enzyme solution is 0.5 mg / mL-1.5 mg / mL.

4. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The concentration of the NAD+ solution is 5 mM-20 mM.

5. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The heating time in the 37° C. water bath is 20-50 minutes.

6. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The 60° C. water bath heating time is 20-50 minutes.

7. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The concentration ratio of the WST-8 and 1-mPMS solutions is 3-4:2-4.

8. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: In step (2), the preparation steps of the ALDH@MWCNTs immobilized enzyme carrier material are as follows: the carboxyl functionalized MWCNTs are activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide solution for 2 hours, washed with water 3 times, and then ALDH is added, and the mixture is placed in an oscillator for 2 hours to fix ALDH on the surface of MWCNTs, and the composite mixture is separated by filtration, and washed with PBS buffer (pH=7.4) 3 times to obtain ALDH@MWCNTs immobilized enzyme.

9. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The affinity chromatography method is specifically as follows: the chromatographic column is C 18 (4.6 × 250 mm), the detection wavelength was 254 nm, the mobile phase A was 0.1% formic acid, the mobile phase B was methanol, and the gradient elution program was 0 min-70% A; 4 min-70% A; 14 min-52% A; 24 min-52% A; 25min-70% A; 30 min-70% A.

10. The method for screening acetaldehyde dehydrogenase inhibitors according to claim 1, characterized in that: The LC-MS method is specifically as follows: the chromatographic column is C 18 (3.5 μm; 2.1 mm × 50 mm), mobile phase A was 0.1% formic acid, mobile phase B was methanol, and the gradient elution program was 0 min-70% A; 4 min-70% A; 14 min-52% A; 24 min-52% A; 25 min-70% A; 30 min-70% A. The nebulizer pressure was 35 psi, the drying gas flow rate was 8 L / min, the gas temperature was 320°C, the sheath gas temperature was 350°C, the gas flow rate was 11 L / min, and the mass range was selected as 50-3000 (m / z).

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