Method for screening active ingredients for inhibiting acetylcholin esterase in traditional Chinese medicine based on colorimetric sensing combined with affinity chromatography technology
By combining colorimetric sensing and affinity chromatography, the problem of cumbersome and low efficiency of the screening method for acetylcholinesterase inhibitors in the prior art is solved, and the method of efficiently screening of active components of acetylcholinesterase from traditional Chinese medicine is achieved, which is simple and convenient Fast and accurate.
Patent Information
- Application Number
- CN202411585390.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-01-07
AI Technical Summary
The existing acetylcholinesterase inhibitor screening methods are cumbersome, time-consuming, and complex sample components and many interfering substances, making it difficult to efficiently and accurately screen out effective active ingredients that inhibit acetylcholinesterase.
Using a method based on colorimetric sensing combined with affinity chromatography technology, acetylcholinesterase active samples were detected through hydrogen peroxide test strips, and acetylcholinesterase affinity chromatography carrier was prepared by modifying amino silica using glutaraldehyde to prepare acetylcholinesterase affinity chromatography carrier, combining liquid Combination technology is used to screen and identify active ingredients in traditional Chinese medicines that inhibit acetylcholinesterase.
It realizes simple, fast, efficient and precise screening of active ingredients inhibiting acetylcholinesterase from traditional Chinese medicine, improves the sensitivity and efficiency of drug screening, and can quickly identify and identify active ingredients that inhibit the action of acetylcholinesterase.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug screening, and in particular relates to a method for screening components inhibiting acetylcholinesterase activity in traditional Chinese medicine based on colorimetric sensing combined with affinity chromatography technology. Background Art
[0002] Acetylcholinesterase (AChE) is a key enzyme in the central nervous system that can rapidly catalyze the hydrolysis of the neurotransmitter acetylcholine (ACh) into inactive choline. Inhibition of AChE can lead to the accumulation of ACh in the synaptic cleft, resulting in blocked nerve conduction. AChE has regained attention due to its association with Alzheimer's disease (AD) and other neurodegenerative diseases characterized by low ACh levels caused by AChE catabolism. Therefore, developing an effective strategy for detecting AChE activity with high sensitivity, fast response, and simple operation has become a top priority for drug screening and clinical diagnosis of Alzheimer's disease.
[0003] At present, the commonly used acetylcholinesterase inhibitors in clinical practice include donepezil and carbaladin. Donepezil belongs to the hexahydropyridine oxide class and is commonly used to treat mild or moderate Alzheimer's dementia symptoms. Taking the drug can achieve the purpose of alleviating the condition and is a commonly used drug for the treatment of Alzheimer's disease; carbaladin mainly increases the function of neurons releasing choline in the brain by inhibiting acetylcholinesterase. If used in combination with other types of cholinesterase inhibitors, cholinergic drugs and depolarizing muscle relaxants, it can enhance its effect and produce a synergistic effect, while its combination with anticholinergic drugs may interfere with its efficacy; it is a cholinesterase inhibitor, which has a selective inhibitory effect on acetylcholinesterase, has the effect of promoting memory retrieval, enhancing memory retention and strengthening muscle contraction strength, and is commonly used to treat benign memory disorders, myasthenia gravis, and memory disorders caused by dementia patients and organic brain lesions.
[0004] Some existing screening methods, such as capillary electrophoresis, electrochemical method and fluorescence technology screening, still have shortcomings due to the complex sample composition, more interfering substances, cumbersome operation and time-consuming.
[0005] Based on this, we carried out research on methods for screening active components of acetylcholinesterase. Summary of the invention
[0006] In view of this, the purpose of the present invention is to create a method for screening acetylcholinesterase inhibitors based on a colorimetric sensor combined with affinity chromatography technology, which can simply, quickly, efficiently and accurately screen out active ingredients that effectively inhibit acetylcholinesterase from a large number of traditional Chinese medicines.
[0007] The technical solution adopted by the present invention to solve the technical problem is as follows: A method for screening components inhibiting acetylcholinesterase activity in traditional Chinese medicine based on colorimetric sensing combined with affinity chromatography technology, characterized in that it comprises the steps of: (1) Screening of samples with the ability to inhibit acetylcholinesterase activity: Dissolve the Chinese herbal extract into an appropriate concentration and add it to the acetylcholinesterase solution. After reacting for 30 minutes, add the acetylcholine solution. After reacting for another 30 minutes, add the choline oxidase. After sampling, use a hydrogen peroxide test strip to perform a color reaction. When the color of the hydrogen peroxide test strip becomes significantly lighter, it is a Chinese herbal extract that inhibits acetylcholinesterase activity.
[0008] (2) Preparation of acetylcholinesterase affinity chromatography carrier material: glutaraldehyde and aminosilica are shaken and reacted at room temperature for a certain period of time to add aldehyde groups to the silica. The mixture is centrifuged and then washed and filtered. Acetylcholinesterase is added and reacted for a certain period of time to allow the aldehyde groups of aminosilica to covalently bind to the carboxyl groups of acetylcholinesterase. Lysine is then added and shaken for a certain period of time to block the excess aldehyde groups on the silica, thereby obtaining an acetylcholinesterase silica (AChE@SiO2) affinity chromatography carrier material.
[0009] (3) Screening and identification of active ingredients that inhibit acetylcholinesterase in traditional Chinese medicine: Affinity chromatography is used to separate traditional Chinese medicine ingredients that can bind to acetylcholinesterase. The traditional Chinese medicine extract with active ingredients that inhibit acetylcholinesterase screened out in step (1) is dissolved into an appropriate concentration and added to the AChE@SiO2 affinity chromatography carrier material obtained in step (2). After sufficient shaking and reaction for a certain period of time, the unadsorbed supernatant is removed by centrifugation. The solution that binds to AChE@SiO2 is obtained by elution with a suitable solvent, which is the eluent. The initial solution of the traditional Chinese medicine extract, the unadsorbed solution, and the eluent are respectively injected into a high-performance liquid chromatograph for detection. By analyzing the changes in the peak areas of the components in different solutions, the components that bind to AChE@SiO2 are screened out; the components that bind to AChE@SiO2 are identified by liquid chromatography-mass spectrometry; and the active ingredients that inhibit acetylcholinesterase are finally obtained by measuring the half-maximum inhibition concentration of the screened and identified binding components on acetylcholinesterase.
[0010] Preferably, the screening of samples having the ability to inhibit acetylcholinesterase activity in step (1) is characterized in that the concentration of the Chinese herbal medicine extract solution is 2.5 mg / mL.
[0011] Preferably, the screening of samples having the ability to inhibit acetylcholinesterase activity in step (1) is characterized in that the volume ratio of the traditional Chinese medicine extract, acetylcholinesterase, acetylcholine and choline oxidase is 2:2:4:1.
[0012] Preferably, the preparation of the acetylcholinesterase affinity chromatography carrier material in step (2) is characterized in that the detailed steps are as follows: dispersing aminosilica in PBS buffer, adding glutaraldehyde and shaking to activate for 2 hours, washing with water 3 times, adding acetylcholinesterase, placing the mixture in an oscillator and shaking for 4 hours to fix the acetylcholinesterase on the SiO2 surface, then adding lysine solution and shaking for 2 hours, centrifuging the composite mixture, washing with water 3 times to obtain the AChE@SiO2 carrier material, and dispersing and storing it in PBS buffer.
[0013] Preferably, the screening and identification of the active components inhibiting acetylcholinesterase in the traditional Chinese medicine in step (3) is characterized in that the affinity chromatography method is specifically: the chromatographic column is XBridge C18 (4.6 × 150 mm; 3.5 μm), the detection wavelength was 320 nm, the mobile phase A was 0.1% phosphoric acid, the mobile phase B was methanol, the gradient elution program was 0 min-98% A; 3 min-88%A; 12 min-85% A; 20 min-77% A; 35 min-67% A; 45 min-55% A; 48 min-98% A; 50 min-98%A, the column temperature was 35°C, and the flow rate was 0.8 mL / min.
[0014] Preferably, the screening and identification of the active ingredients inhibiting acetylcholinesterase in the traditional Chinese medicine in step (3) is characterized in that the LC-MS method is specifically as follows: the chromatographic column is Symmetry C18 (2.1 mm × 50 mm; 3.5 μm), the mobile phase A is 0.1% formic acid, the mobile phase B is 0.1% formic acid acetonitrile, and the gradient elution program is 0 min-98% A; 2 min-98% A; 8 min-90% A; 15 min-88% A; 18 min-75% A; 28 min-55% A; 35 min-45% A; 50 min-10% A; 55 min-98% A; 60 min-98% A. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320°C, the sheath gas temperature is 350°C, the gas flow rate is 11 L / min, and the mass range is selected as 50-3000 (m / z).
[0015] It can be seen from the above technical scheme that the present invention provides a method for screening active ingredients that inhibit acetylcholinesterase in traditional Chinese medicine based on biosensor and affinity chromatography technology, which belongs to the field of drug screening technology. The first step of this method uses colorimetric sensor technology to quickly identify samples that inhibit acetylcholinesterase activity. The principle is that acetylcholinesterase can catalyze acetylcholine to produce choline, which can produce hydrogen peroxide under the action of choline oxidase, making the hydrogen peroxide test strip turn blue; when there are active ingredients that inhibit acetylcholinesterase in the screened sample, the color of the hydrogen peroxide test strip becomes significantly lighter. The second step is to identify the components in the sample that inhibit the activity of acetylcholinesterase by affinity chromatography combined with liquid chromatography-mass spectrometry technology. The method is to use glutaraldehyde to add aldehyde groups to aminosilica, then react with acetylcholinesterase, and then use lysine to block the excess aldehyde groups in aminosilica to form AChE@SiO2 affinity chromatography carrier material; react the Chinese herbal medicine extract to be screened with the affinity chromatography carrier material for a certain period of time, centrifuge to remove the non-adsorbed liquid, and elute with a suitable solvent to obtain the eluate; use high performance liquid phase analysis to compare the changes in the peak areas of the initial liquid, non-adsorbed liquid, and eluate of the Chinese herbal medicine extract, so as to screen out the Chinese herbal medicine components that have adsorption effects on acetylcholinesterase; and then use liquid chromatography-mass spectrometry technology to identify the components. This method can simply, quickly, efficiently and accurately screen out active components that inhibit acetylcholinesterase from Chinese herbal medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] 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 description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0017] Figure 1 It is a schematic diagram of the screening method process of the present invention.
[0018] Figure 2 The results of acetylcholinesterase activity and acetylcholinesterase standard curve detected by hydrogen peroxide test strips.
[0019] Figure 3 This is the result diagram of validating the screening model for the acetylcholinesterase positive inhibitor donepezil.
[0020] Figure 4 This is a diagram showing the results of the colorimetric sensor test paper of the present invention screening 10 kinds of traditional Chinese medicines.
[0021] Figure 5 This is the HPLC mass spectrum of wolfberry leaf extract and the screened compounds.
[0022] Figure 6The IC50 inhibitory concentration of chlorogenic acid on acetylcholinesterase was obtained by screening 50 Measurement result diagram. DETAILED DESCRIPTION
[0023] 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.
[0024] The embodiment of the present invention discloses a method for screening components inhibiting acetylcholinesterase activity in traditional Chinese medicine based on colorimetric sensing combined with affinity chromatography technology, comprising the following steps: Example 1
[0025] Preparation experiment of affinity chromatography carrier material AChE@SiO2: 50 mg of aminosilica was dispersed in 4 ml PBS buffer, glutaraldehyde was added and shaken for 2 hours for activation, washed with water three times, and then acetylcholinesterase was added. The mixture was placed in an oscillator and shaken for 4 hours to fix acetylcholinesterase on the SiO2 surface. 1 g of lysine was dissolved in 10 ml PBS buffer and added to the above product. The mixture was shaken for 2 hours. The composite mixture was separated by centrifugation. After washing with water three times, the AChE@SiO2 carrier material was obtained, and finally packaged and stored in 5 ml PBS buffer. Example 2
[0026] Sensitivity experiment of acetylcholinesterase colorimetric sensor paper: Take 6 EP tubes, add 400 μL of 1mg / mL acetylcholine solution to each tube, then add 400 μL of acetylcholinesterase with concentrations of 2000, 1000, 500, 250, 125, and 62.5 μg / mL, respectively. After reacting for 30 minutes, add 200 μL of 50 μg / mL choline oxidase to each tube, continue to react for 30 minutes, add hydrogen peroxide test strips, and observe the color change. The results are as follows: Figure 2 As shown in A. The results show that as the concentration of acetylcholinesterase increases, the hydrogen peroxide test strip changes from colorless to blue. The higher the concentration of acetylcholinesterase, the darker the blue color of the hydrogen peroxide test strip. Example 3
[0027] Acetylcholinesterase standard curve experiment: Take 6 EP tubes, add 400 μL of 1 mg / mL acetylcholine solution to each tube, then add 400 μL of acetylcholinesterase with concentrations of 2000, 1000, 500, 250, 125, and 62.5 μg / mL, respectively. After reacting for 30 minutes, add 200 μL of 50 μg / mL choline oxidase, 200 μL of 5 μg / mL horseradish peroxidase, and 400 μL of 1 mg / mL 3,3',5,5'-tetramethylbenzidine. After reacting for 30 minutes, add 200 μL of 3,3',5,5'-tetramethylbenzidine colorimetric stop solution, measure the absorbance at 450 nm, and draw the acetylcholinesterase standard curve with concentration as the horizontal axis and absorbance as the vertical axis. The results are shown in the figure. Figure 2 B. The results showed that the absorbance value of acetylcholinesterase was linearly related to the concentration in the range of 62.5 to 2000 μg / mL, and the linear equation was y = 0.0004 x +0.0458, R² = 0.9994. Example 4
[0028] The acetylcholinesterase-positive drug donepezil was used to verify the colorimetric sensing part of this method: 200 μL of 1 mg / mL acetylcholinesterase solution was taken, and 200 μL of donepezil solution of different concentrations (1000, 200, 50, 0 μg / mL) was added. After reacting for 30 minutes, 400 μL of 500 μg / mL acetylcholine solution was added, and then reacted for another 30 minutes. 100 μL of 50 μg / mL choline oxidase was added, and after continuing to react for 30 minutes, a hydrogen peroxide test strip was added to observe the color change. The experiment was repeated three times. The results are as follows Figure 3 B. The results showed that compared with the blank, the blue color of the hydrogen peroxide test strip became significantly lighter in the presence of donepezil at different concentrations, indicating that the activity of acetylcholinesterase was inhibited by donepezil. This result suggests that the colorimetric sensing model established by this method can be used for the rapid screening of components that inhibit the activity of acetylcholinesterase. Example 5
[0029] The acetylcholinesterase-positive drug donepezil was used to verify the affinity chromatography part of this method: a 50 μg / mL donepezil solution was prepared, 200 μL was mixed with 500 μL of AChE@SiO2 affinity chromatography carrier material at a concentration of 400 μg / mL, placed in an oscillator for 30 minutes, centrifuged, and the supernatant was taken as the non-adsorbed liquid. Subsequently, 200 μL of methanol, acetonitrile, 50% methanol, and 50% acetonitrile were added to the remaining solid, mixed evenly, placed in an oscillator for 30 minutes, centrifuged, and the supernatant was taken as the eluent. The above donepezil solution, non-adsorbed liquid, and eluent were analyzed by high-performance liquid chromatography injection. The chromatographic conditions were as follows: the chromatographic column was C18 (4.6 × 150 mm; 3.5 μm), the detection wavelength was 320 nm, the mobile phase A was 0.1% phosphoric acid, the mobile phase B was methanol, and the gradient elution program was 0 min-98% A; 3 min-88% A; 12 min-85% A; 20 min-77% A; 35 min-67% A; 45 min-55% A; 48 min-98% A; 50 min-98% A. The column temperature was 35°C and the flow rate was 0.8 mL / min. The results were as follows: Figure 3 As shown in A (a: donepezil solution, b: unadsorbed solution, c: acetonitrile eluent, d: methanol eluent, e: 50% acetonitrile eluent, f: 50% methanol eluent). The results showed that donepezil could bind to acetylcholinesterase, and methanol, acetonitrile, 50% methanol and 50% acetonitrile could all be used as eluents. Example 6
[0030] A colorimetric sensor was used to screen samples with acetylcholinesterase inhibitory activity. Take 200 μL of 1 mg / mL acetylcholinesterase, add 200 μL of different Chinese herbal extracts (wolfberry leaves, bitter beans, costus root, stir-fried chicken gizzard lining, stir-fried spiny jujube seeds, albizzia bark, radix schizonepetae, stir-fried white peony root, and scutellaria baicalensis), react for 30 minutes, add 400 μL of 500 μg / mL acetylcholine solution, react for another 30 minutes, add 100 μL of 50 μg / mL choline oxidase, continue to react for 30 minutes, add hydrogen peroxide test strips, and observe color changes. The experiment was repeated three times, and the results are as follows: Figure 4 As shown (a: blank, b: wolfberry leaf, c: bitter bean, d: costus root, e: stir-fried chicken gizzard lining, f: stir-fried spiny jujube seed, g: julibrissin bark, h: dipsacus root, i: stir-fried white peony root, j: cyanopteris chinensis). The results showed that the color of the hydrogen peroxide test strip with wolfberry leaf extract was significantly lighter than that of other Chinese herbal extracts, that is, wolfberry leaf extract has active ingredients that inhibit acetylcholinesterase. Example 7
[0031] Affinity chromatography was used to screen the components in wolfberry leaves that can bind to the AChE@SiO2 affinity chromatography carrier material. Take 1 portion of the AChE@SiO2 carrier material, centrifuge to remove the PBS buffer, add 500 μL of wolfberry leaf extract solution, mix and place in an oscillator to react for 30 minutes, centrifuge, and take the supernatant as the non-adsorbed liquid. Then add 200 μL of 50% acetonitrile to the remaining solid, mix well and place in an oscillator to shake for 30 minutes, centrifuge, and take the supernatant as the eluent. The initial solution of wolfberry leaf extract, the non-adsorbed solution, and the eluent were analyzed by high performance liquid chromatography. The chromatographic conditions were as follows: the chromatographic column was C18 (4.6× 150 mm; 3.5 μm), the detection wavelength was 320 nm, the mobile phase A was 0.1% phosphoric acid, the mobile phase B was methanol, and the gradient elution program was 0 min-98% A; 3 min-88% A; 12 min-85% A; 20 min-77% A; 35 min-67% A; 45 min-55% A; 48 min-98% A; 50 min-98% A. The column temperature was 35°C and the flow rate was 0.8 mL / min. The results are shown in Figure 5 A. Another portion of the eluate was taken for LC-MS analysis. The specific LC-MS method was as follows: the chromatographic column was C18 (2.1 mm × 50 mm; 3.5μm), the mobile phase A was 0.1% formic acid, the mobile phase B was 0.1% formic acid acetonitrile, and the gradient elution program was 0 min-98% A; 2 min-98% A; 8 min-90% A; 15 min-88% A; 18 min-75% A; 28 min-55% A; 35 min-45% A; 50 min-10% A; 55 min-98% A; 60 min-98% 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). The results are shown in Figure 5 B and 5C. The results show that in the chromatogram of the wolfberry leaf extract eluate ( Figure 5 A), multiple chromatographic peaks can be clearly seen, that is, the AChE@SiO2 affinity chromatography carrier material successfully adsorbed multiple components in wolfberry leaves that can bind to acetylcholinesterase. In the mass spectrum of the wolfberry leaf extract eluate, identification Figure 5 The compound marked by arrow A is chlorogenic acid, that is, one of the components in wolfberry leaves that can bind to acetylcholinesterase is chlorogenic acid. Example 8
[0032] IC50 of chlorogenic acid, an active component in Lycium barbarum leaves that inhibits acetylcholinesterase 50Determination experiment. Prepare a series of chlorogenic acid reference solutions with concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 100 μg / mL, and 200 μg / mL, take 200 μL of each, add 400 μL of 1 mg / mL acetylcholinesterase solution, mix well, and react at 37°C for 30 minutes. Then add 400 μL of 500 μg / mL acetylcholine solution, mix well, and react for 30 minutes. Subsequently, 200 μL of 50 μg / mL choline oxidase, 400 μL of 1 mg / mL 3,3',5,5'-tetramethylbenzidine, and 200 μL of 5 μg / mL horseradish peroxidase were added, and 200 μL of 3,3',5,5'-tetramethylbenzidine color stop solution was added after 30 minutes of reaction. Finally, the absorbance of each solution was detected at a wavelength of 450 nm to calculate the IC value of chlorogenic acid in inhibiting acetylcholinesterase. 50 Value, see the result Figure 6 The results showed that the IC 50 The value was 19.7 μg / mL. The above showed that chlorogenic acid has a good effect of inhibiting acetylcholinesterase activity.
[0033] 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.
[0034] 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 components inhibiting acetylcholinesterase activity in traditional Chinese medicine based on colorimetric sensing combined with affinity chromatography technology, comprising the following steps: (1) Screening of samples with inhibitory effects on acetylcholinesterase activity: Dissolve the Chinese herbal extract into an appropriate concentration and add it to an acetylcholinesterase (AChE) solution. After reacting for 30 minutes, add the acetylcholine solution. After reacting for another 30 minutes, add the choline oxidase. After sampling, use a hydrogen peroxide test strip for color development. When the color of the hydrogen peroxide test strip becomes significantly lighter, it is a Chinese herbal extract that has the ability to inhibit acetylcholinesterase activity. (2) Preparation of acetylcholinesterase affinity chromatography carrier material: Glutaraldehyde and aminosilica are shaken at room temperature for a certain time to add aldehyde groups to the silica, centrifuged and filtered, acetylcholinesterase is added for a certain time to make the aldehyde groups of aminosilica covalently bond with the carboxyl groups of acetylcholinesterase, and then lysine is added and shaken for a certain time to block the excess aldehyde groups on the silica, thereby obtaining acetylcholinesterase silica (AChE@SiO2) affinity chromatography carrier material. (3) Screening and identification of components that inhibit acetylcholinesterase activity in traditional Chinese medicine: Affinity chromatography is used to separate traditional Chinese medicine components that can bind to acetylcholinesterase. The Chinese herbal medicine extract with active ingredients that inhibit acetylcholinesterase screened out in step (1) is dissolved into a suitable concentration and added to the AChE@SiO2 affinity chromatography carrier material obtained in step (2). After sufficient shaking and reaction for a certain period of time, the unadsorbed supernatant is centrifuged and removed. A solution that binds to AChE@SiO2 is obtained by elution with a suitable solvent, which is the eluent. The initial solution of the Chinese herbal medicine extract, the unadsorbed solution, and the eluent are respectively injected into a high-performance liquid chromatograph for detection. By analyzing the changes in the peak areas of the components in different solutions, the components that bind to the Chinese herbal medicine and AChE@SiO2 are screened out; the components that bind to AChE@SiO2 are identified by liquid chromatography-mass spectrometry; and the active components that inhibit acetylcholinesterase are finally obtained by measuring the half-maximum inhibition concentration of the screened and identified binding components on acetylcholinesterase.
2. The method for screening an active ingredient that inhibits acetylcholinesterase according to claim 1, characterized in that: The concentration of the Chinese herbal medicine extract solution is 2.5 mg / mL.
3. The method for screening an active component inhibiting acetylcholinesterase according to claim 1, characterized in that: The volume ratio of the traditional Chinese medicine extract, acetylcholinesterase, acetylcholine and choline oxidase is 2:2:4:
1.
4. The method for screening an active component inhibiting acetylcholinesterase according to claim 1, characterized in that: The affinity chromatography method is specifically as follows: the chromatographic column is C18 (4.6 × 150 mm; 3.5 μm), the detection wavelength is 320 nm, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is methanol, and the gradient elution program is 0 min-98% A; 3 min-88% A; 12 min-85% A; 20 min-77% A; 35 min-67% A; 45 min-55% A; 48 min-98% A; 50 min-98% A. The column temperature is 35°C and the flow rate is 0.8 mL / min.
5. The method for screening an active component inhibiting acetylcholinesterase according to claim 1, characterized in that: The LC-MS method is as follows: the chromatographic column is C18 (2.1 mm × 50 mm; 3.5 μm), the mobile phase A is 0.1% formic acid, the mobile phase B is 0.1% formic acid acetonitrile, and the gradient elution program is 0 min-98% A; 2 min-98% A; 8 min-90% A; 15 min-88% A; 18 min-75% A; 28 min-55% A; 35 min-45% A; 50 min-10% A; 55 min-98% A; 60 min-98% A. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320°C, the sheath gas temperature is 350°C, the gas flow rate is 11 L / min, and the mass range is selected as 50-3000 (m / z).