Neuraminidase inhibitor screening method
By combining fluorescence sensing and affinity chromatography, screening the active components of neuraminidase in traditional Chinese medicines has solved the problem of difficulty in effective screening in the existing technology, achieving efficient and accurate screening of NA inhibitors, and providing new antiviral treatment options.
Patent Information
- Application Number
- CN202510088647.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-04-08
AI Technical Summary
The prior art is difficult to effectively screen the active components of neuraminidase inhibitors in traditional Chinese medicines, and traditional synthetic NA inhibitors may lead to drug resistance and side effects.
Using a method based on fluorescence sensing binding affinity chromatography technology, samples with inhibitory NA activity were quickly identified through fluorescence sensors, and components that bind to NA were separated by affinity chromatography, and NA inhibitors were finally identified.
It has achieved the ability to screen out the active NA from traditional Chinese medicines for simple, quick, efficient and precise screening, overcoming the shortcomings of traditional methods, and providing new antiviral treatment options.
Smart Images
Figure CN119780056A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug screening, and specifically relates to a method for screening components inhibiting neuraminidase activity in traditional Chinese medicine based on fluorescence sensing combined with affinity chromatography technology. Background Art
[0002] Neuraminidase (NA) is a key enzyme for viral replication and is present on the surface of many viruses, especially influenza viruses (such as influenza A and B viruses). It plays a key role in the viral life cycle by removing sialic acid from the surface of host cells and promoting the release and spread of viruses. In recent years, significant progress has been made in antiviral drugs targeting NA, such as oseltamivir and zanamivir. However, long-term use of chemically synthesized NA inhibitors may lead to viral resistance and may cause a variety of side effects, such as indigestion, headache, nausea, etc., affecting patients' medication compliance. Natural medicines have rich structural diversity and can provide more options for the development of new NA inhibitors. Many natural medicines have been proven to be safe and effective in traditional applications, usually with fewer side effects and are more acceptable to patients. Therefore, screening NA inhibitors in natural medicines can not only overcome some of the shortcomings of current synthetic drugs, but also may bring new breakthroughs in antiviral treatment.
[0003] At present, sensor analysis is widely used to determine enzyme activity and screen inhibitors because it can quickly identify complex samples containing inhibitory active ingredients. At the same time, affinity chromatography is based on the interaction between specific molecules (such as antibodies and antigens, enzymes and substrates, receptors and ligands, etc.) to achieve the separation and purification of active ingredients. It has been proven to be effective in separating and identifying active ingredients from complex samples. There are no reports on combining sensor analysis with affinity chromatography for the screening of NA inhibitors.
[0004] Based on this, we constructed a method based on fluorescence sensing combined with affinity chromatography technology to quickly and accurately screen active ingredients in traditional Chinese medicine that inhibit neuraminidase, in order to provide new ideas for the screening of antiviral active ingredients in traditional Chinese medicine. Summary of the invention
[0005] The present invention provides a method for screening neuraminidase inhibitors, which can simply, quickly, efficiently and accurately screen out active ingredients capable of inhibiting neuraminidase from a large number of traditional Chinese medicines.
[0006] The technical solution adopted by the present invention to solve the technical problem is as follows: A method for screening active ingredients inhibiting neuraminidase in traditional Chinese medicine based on fluorescence sensing combined with affinity chromatography technology, characterized in that it comprises the steps of: (1) Screening of samples with neuraminidase inhibitory activity: After extraction, the Chinese herbal medicine was dissolved into an appropriate concentration and added to the neuraminidase (NA) solution. After 30 minutes of reaction, the sample was sampled and tested for fluorescence using a test strip loaded with 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid sodium salt (4-MUNANA). When the test strip has no fluorescence or the fluorescence intensity is weak, it has NA inhibitory activity. In addition, the Chinese herbal medicine extract was dissolved into an appropriate concentration and added to the NA solution. After 30 minutes of reaction, 4-MUNANA was added and the fluorescence intensity of the solution after the reaction was detected by a fluorescence detector. When there is no fluorescence or the fluorescence intensity is weak, it is a sample with NA inhibitory activity. (2) Separation and identification of components with neuraminidase inhibitory activity: Affinity chromatography was used to separate components that can bind to NA. First, NA was co-incubated with ferrous oxide (Fe3O4) magnetic beads to obtain NA@Fe3O4 immobilized enzyme carrier material. Then, the sample with NA inhibitory activity selected by (1) was added to the immobilized enzyme at a volume ratio of 1:1. After sufficient reaction for 30 minutes by oscillation, the unadsorbed supernatant was centrifuged and discarded. The NA@Fe3O4 binding solution was eluted with a suitable solvent. The eluate was injected into a liquid chromatograph and a liquid-mass spectrometer for detection, and the components that can bind to NA were obtained and identified. The NA inhibitor was finally obtained by measuring the half-inhibitory concentration of the selected binding components on NA.
[0007] Preferably, the screening of samples having neuraminidase inhibitory activity in step (1) is characterized in that the concentration of the Chinese herbal medicine extract solution is 200 μg / mL.
[0008] Preferably, the screening of samples having neuraminidase inhibitory activity in step (1) is characterized in that the volume ratio of the traditional Chinese medicine extract, NA and 4-MUNANA is 1:1:1.
[0009] Preferably, the preparation of the neuraminidase affinity chromatography carrier material in step (2) is characterized in that the detailed steps are as follows: dispersing the ferroferric oxide magnetic beads in PBS buffer, adding glutaraldehyde and shaking to activate for 2 hours, washing with water 3 times, adding NA, placing the mixture in an oscillator and shaking for 4 hours to fix NA on the Fe3O4 surface, then adding lysine solution and shaking for 2 hours, centrifuging the composite mixture, washing with water 3 times to obtain the NA@Fe3O4 carrier material, and dispersing and storing it in PBS buffer.
[0010] Preferably, the preparation of the neuraminidase affinity chromatography carrier material in step (2) is characterized in that the affinity chromatography method is specifically as follows: the chromatographic column is C18 (4.6×250 mm; 5 μm), the detection wavelength is 270 nm, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is methanol, the gradient elution program is 0 min-75% A; 10 min-35% A; 55 min-10% A; the column temperature is 30°C, and the flow rate is 0.8 mL / min.
[0011] Preferably, the preparation of the neuraminidase affinity chromatography carrier material in step (2) is characterized in that the LC-MS method is specifically 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, the gradient elution program is 0 min-75% A; 10 min-35% A; 55 min-10% 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).
[0012] It can be seen from the above technical scheme that the present invention provides a method for screening components with neuraminidase inhibitory activity in traditional Chinese medicine based on fluorescence sensing and affinity chromatography technology, which belongs to the field of drug screening technology. The first step of this method is to use fluorescence sensor technology to quickly identify samples with NA inhibitory activity. The principle is that NA can catalyze the substrate 4-MUNANA to generate 4-MU, and 4-MU can make the test paper show blue fluorescence; when the screened sample has NA inhibitory activity, the test paper has no fluorescence or weak fluorescence intensity. The second step is to identify the components in the sample with NA inhibitory activity by affinity chromatography combined with liquid chromatography-mass spectrometry technology. The method is to fix NA on ferroferric oxide (Fe3O4) to form NA@Fe3O4 affinity chromatography carrier material; incubate the traditional Chinese medicine extract to be screened with the affinity chromatography carrier material for a certain period of time, separate the mixture with a magnet, remove the non-adsorbed liquid, and elute with a suitable solvent to obtain an eluate; HPLC is used to analyze and compare the changes in the peak areas of the initial liquid, non-adsorbed liquid, and eluate of the traditional Chinese medicine extract, so as to screen out the traditional Chinese medicine components that have adsorption effects on NA; and then use liquid chromatography-mass spectrometry technology to identify the components. This method can simply, quickly, efficiently and accurately screen out active ingredients that inhibit NA from traditional Chinese medicine. 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 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.
[0014] Figure 1 It is a schematic diagram of the screening method process of the present invention.
[0015] Figure 2 The fluorescence sensor test strip detects the neuraminidase activity results and the neuraminidase standard curve.
[0016] Figure 3 This is the result diagram of the validation screening model for the neuraminidase positive inhibitor zanamivir.
[0017] Figure 4 This is a result diagram of the validation screening model for neuraminidase negative inhibitors.
[0018] Figure 5 This is a diagram showing the results of the fluorescent sensor test paper of the present invention screening 20 kinds of traditional Chinese medicines.
[0019] Figure 6 This is the high performance liquid chromatogram of the Chinese medicine Bupleurum extract and the mass spectrum of the screened compounds.
[0020] Figure 7 The IC50 inhibition concentrations of saikosaponin a, saikosaponin d and baicalin on neuraminidase were screened. 50 Measurement result diagram. DETAILED DESCRIPTION
[0021] 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.
[0022] The embodiment of the present invention discloses a method for screening components with neuraminidase inhibitory activity in traditional Chinese medicine based on fluorescence sensing combined with affinity chromatography technology, comprising the following steps: Example 1
[0023] Preparation experiment of affinity chromatography carrier material NA@Fe3O4: Weigh 10 mg of Fe3O4 magnetic nanoparticles, coat SiO2 and -NH2 groups to activate them, then incubate with 1 mL PBS containing 0.5 mL glutaraldehyde for 2 h, wash with water 3 times, add NA, and place the mixture in an oscillator for 4 h to fix NA on the SiO2 surface. In addition, weigh 500 mg of lysine and dissolve it in 5 mL PBS, vortex for 10 min, and react with the above system for 2 h. The mixture was separated by magnetic separation, washed with water 3 times, and the NA@Fe3O4 carrier material was obtained, which was stored in 5 mL PBS buffer. Example 2
[0024] Neuraminidase fluorescence sensor test paper sensitivity experiment: First, PBS was used as a solvent to prepare a 4-MUNANA stock solution with a concentration of 50 μg / mL. Subsequently, the polyester film test paper was cut into a size of about 0.5 cm×0.5 cm, immersed in the 4-MUNANA stock solution for about 30 s and then taken out. Then, it was immersed in NA solutions with concentrations of 100μ g / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL and 1μg / mL for 30 min, taken out, and the fluorescence intensity was observed at 365 nm, and the image was recorded using a smartphone. The results showed that the fluorescence intensity of the test paper was positively correlated with the concentration of the NA solution, that is, the higher the concentration of the NA solution, the stronger the NA fluorescence ( Figure 2 A). Example 3
[0025] Neuraminidase standard curve experiment: 100 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL and 1 μg / mL NA were added to 50 μg / mL 4-MUNANA solution and reacted for 30 min. ex =365 nm / em =445 nm. The standard curve was established with the concentration of NA solution as the horizontal axis and the fluorescence intensity of the reaction solution as the vertical axis. The results showed that there was a linear relationship between the concentration of NA solution and the corresponding fluorescence intensity in the range of 1~100 μg / mL ( Figure 2 B). Example 4
[0026] The neuraminidase positive inhibitor zanamivir was used to verify the fluorescence sensing part of this method: first, the test paper was immersed in a 4-MUNANA solution with a concentration of 50 μg / mL for about 30 s and then taken out for use. At the same time, 100 μL of NA with a concentration of 100 μg / mL was taken and reacted with zanamivir (neuraminidase inhibitor) with concentrations of 0 μg / mL, 50 μg / mL, 100 μg / mL and 150 μg / mL for 30 min. Then, the prepared test paper coated with 4-MUNANA was added to the above reaction systems and incubated for 30 s before being taken out. The fluorescence intensity of the test paper was recorded at 365 nm. The results are shown as follows: Figure 3 A. Take 100 μL of NA with a concentration of 100 μg / mL and react with 0 μg / mL, 50 μg / mL, 100 μg / mL and 150 μg / mL of zanamivir for 30 min, then add 4-MUNANA with a concentration of 50 μg / mL and react for another 30 min. ex =365 nm / em =445 nm to measure the fluorescence intensity of the above reaction solution. Figure 3 B. The results showed that as the concentration of zanamivir increased ( Figure 3 abcd in A and 3B correspond to zanamivir at concentrations of 0 μg / mL, 50 μg / mL, 100 μg / mL, and 150 μg / mL, respectively). The fluorescence of the test strip was significantly weakened, and the fluorescence intensity of the solution was also significantly reduced. The above shows that NA activity is inhibited by zanamivir. This result suggests that the fluorescence sensing model established by this method can be used for the rapid screening of components that inhibit NA activity. Example 5
[0027] The neuraminidase positive inhibitor zanamivir was used to validate the affinity chromatography part of this method: 200 μL of a zanamivir solution with a concentration of 100 μg / mL was taken and mixed with the same volume of NA@Fe3O4 affinity chromatography carrier material with a concentration of 400 μg / mL, placed in an oscillator for 30 min, and then the mixture was separated by a magnet, and the supernatant was collected as the non-adsorbed liquid. Subsequently, 1 mL of water was added to the remaining solid, mixed evenly, and placed in an oscillator for 30 min, and the mixture was separated by a magnet, and the supernatant was collected as the eluent. The above zanamivir solution, non-adsorbed liquid, and eluent were analyzed by high performance liquid chromatography injection. The chromatographic conditions are as follows: the chromatographic column is C18 (4.6 × 150 mm; 3.5 μm), the detection wavelength is 320 nm, and the elution is isocratic with 0.1% phosphoric acid aqueous solution-methanol (volume ratio of 70:30), the column temperature is 35°C, and the flow rate is 0.8 mL / min. The results are as follows Figure 3C (a-zanamivir initial solution, b-unadsorbed solution, c-eluted solution). The results show that zanamivir can bind to NA. This result suggests that the affinity chromatography model established by this method can be used to separate and identify NA binding components. Example 6
[0028] Neuraminidase negative inhibitors were used to verify the fluorescence sensing part of this method: first, the test paper was immersed in a 50 μg / mL 4-MUNANA solution for about 30 s and then taken out for use. At the same time, 100 μL of NA with a concentration of 100 μg / mL was taken and reacted with 100 μL of potassium clavulanate, norepinephrine and allopurinol with a concentration of 100 μg / mL for 30 min. Then, the prepared test paper coated with 4-MUNANA was added to the above reaction systems and incubated for 30 s before being taken out. The fluorescence intensity of the test paper was recorded at 365 nm. Another 100 μL of NA with a concentration of 100 μg / mL was taken and reacted with 100 μL of potassium clavulanate, norepinephrine and allopurinol with a concentration of 100 μg / mL for 30 min. Then, 50 μg / mL 4-MUNANA was added and reacted for another 30 min. ex =365 nm / em =445 nm to measure the fluorescence intensity of the above reaction solution. Figure 4 A (a-blank, b-clavulanate potassium, c-norepinephrine, d-allopurinol, e-zanamivir). The results showed that clavulanate potassium, norepinephrine and allopurinol did not cause significant changes in the fluorescence test paper and fluorescence intensity. The above results indicate that clavulanate potassium, norepinephrine and allopurinol do not have NA inhibitory activity, resulting in no change in fluorescence. The results of negative inhibitors once again confirmed that the established method can be used for the screening of NA inhibitors. Example 7
[0029] Neuraminidase negative inhibitors were used to validate the affinity chromatography part of this method: 200 μL of each of 100 μg / mL solutions of potassium clavulanate, norepinephrine, and allopurinol were taken and mixed with the same volume of 400 μg / mL NA@Fe3O4 affinity chromatography carrier material, placed in an oscillator for 30 min, and then separated the mixture with a magnet, and the supernatant was collected as the non-adsorbed liquid. Subsequently, 1 mL of water was added to the remaining solid, mixed evenly, and placed in an oscillator for 30 min. The mixture was separated with a magnet, and the supernatant was collected as the eluent. The above solutions of potassium clavulanate, norepinephrine, and allopurinol, the non-adsorbed liquid, and the eluent were analyzed by high performance liquid chromatography injection, respectively. The results are shown in the figure. Figure 4B (adsorption rate), 4C (a-potassium clavulanate initial solution, b-potassium clavulanate unadsorbed solution), 4D (a-norepinephrine initial solution, b-norepinephrine unadsorbed solution) and 4E (a-allopurinol initial solution, b-allopurinol unadsorbed solution). The results show that the adsorption rates of potassium clavulanate, norepinephrine and allopurinol are significantly lower than those of zanamivir, that is, NA@Fe3O4 can be used for affinity chromatography screening of NA inhibitors. Example 8
[0030] Fluorescence sensor test paper was used to screen samples with neuraminidase inhibitory activity: first, the test paper was immersed in a 50 μg / mL 4-MUNANA solution for about 30 s and then taken out for use. A 100 μg / mL NA solution was prepared, 200 μL of each solution was taken, and 200 μL of different Chinese herbal medicine extract solutions (perilla, scutellaria, notopterygium, raw rehmannia, honeysuckle, schizonepeta, reed root, licorice, chuanxiong, bran-fried atractylodes, platycodon, astragalus, ephedra, saposhnikovia, cyperus, jujube seed, Xanthium sibiricum, Pseudostellariae, Poria, and Bupleurum) were added. After 30 min of reaction, the prepared test paper coated with 4-MUNANA was added to the above reaction systems and incubated for 30 s before being taken out. The fluorescence intensity of the test paper was recorded at 365 nm. The results are as follows: Figure 5 The results showed that the fluorescence of the test strip with Bupleurum extract was significantly weaker than that of other Chinese herbal medicine extracts (a-blank, b-Perilla, c-Scutellaria, d-Notopterygium, e-Rehmannia, f-Lonicera, g-Schizonepeta, h-Phragmites australis, i-Glycyrrhiza, j-Ligusticum chuanxiong, k-Atractylodes macrocephala, l-Planthoceras, m-Astragalus, n-Ephedra, o-Saposhnikovia, p-Cyperus rotundus, q-Semen Ziziphi spinulosus, r-Xanthium sibiricum, s-Pseudostellariae, t-Poria, u-Bupleurum), that is, Bupleurum extract has inhibitory NA activity. Example 9
[0031] The experiment of screening the components in Bupleurum that can bind to the NA@Fe3O4 affinity chromatography carrier material by affinity chromatography: Take 1 portion of NA@Fe3O4 carrier material, separate the mixture with a magnet, remove the PBS buffer, add 300 μL of Bupleurum extract solution, mix and place it in an oscillator for 30 min, separate the mixture with a magnet, and take the supernatant as the non-adsorbed liquid. Then add 200 μL of 50% acetonitrile to the remaining solid, mix well and place it in an oscillator for 30 min, separate the mixture with a magnet, and take the supernatant as the eluent. The initial solution of Bupleurum 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 × 250 mm; 5 μm), the detection wavelength was 270 nm, the mobile phase A was 0.1% phosphoric acid, the mobile phase B was methanol, the gradient elution program was 0 min-75% A; 10 min-35% A; 55 min-10% A; the column temperature was 30 °C, and the flow rate was 0.8 mL / min. 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; 3.5 μm), the mobile phase A was 0.1% formic acid, the mobile phase B was 0.1% formic acid acetonitrile, the gradient elution program was 0 min-75% A; 10 min-35% A; 55 min-10% 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 as follows Figure 6 The results show that in the chromatogram of the Bupleurum extract eluate ( Figure 6 A, a-initial solution of Bupleurum extract, b-unadsorbed solution, c-elution solution), multiple chromatographic peaks can be clearly seen, that is, the NA@Fe3O4 affinity chromatography carrier material successfully adsorbed multiple components in Bupleurum that can bind to NA. In the mass spectrum of the eluate of Bupleurum extract, the adsorbed components were identified as saikosaponin a and saikosaponin d ( Figure 6 B, saikosaponin a and saikosaponin d are isomers, corresponding to the same mass spectra) and baicalin (6C). At the same time, the three components identified by HPLC were verified by reference substances, and the results were consistent ( Figure 6 D, a- bupleurum extract eluate, b- bupleurosaponin a reference solution, c- bupleurosaponin d reference solution, d- baicalin reference solution), that is, the three components in bupleurum that can bind to NA are bupleurosaponin a, bupleurosaponin d and baicalin. Example 10
[0032] IC50% inhibition concentration of saikosaponin a, saikosaponin d and baicalin on neuraminidase 50Determination experiment: Accurately weigh appropriate amounts of saikosaponin a, saikosaponin d, and baicalin, and prepare about 15 series of concentrations, ranging from about 2 to 900 μg / mL (slightly different between different compounds). Take 200 μL of each of the above compounds with different concentrations and incubate with 200 μL of 100 μg / mL NA for 30 min, then react with 200 μL of 50 μg / mL 4-MUNANA for 30 min, and then ex =365 nm / em =445 nm to measure the fluorescence intensity of each reaction solution, and use Origin software to analyze the data and draw the curve of each compound concentration and inhibition rate. The equation is automatically fitted, and the inhibition rate is set to 50%. The IC 50 Value, see the result Figure 7 (A-saikosaponin a IC 50 , B-saikosaponin d IC 50 , C-baicalin IC 50 The results showed that the IC values of saikosaponin a, saikosaponin d and baicalin 50 The values were 177.32 μg / mL, 262.89 μg / mL, and 241.41 μg / mL, respectively. The above results show that saikosaponin a, saikosaponin d, and baicalin have good inhibitory effects on NA 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 with neuraminidase inhibitory activity in traditional Chinese medicine based on fluorescence sensing combined with affinity chromatography technology, comprising the following steps: (1) Screening of samples with neuraminidase inhibitory activity: After extraction, the Chinese herbal medicine was dissolved into an appropriate concentration and added to the neuraminidase (NA) solution. After 30 minutes of reaction, the sample was sampled and tested for fluorescence using a test strip loaded with 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid sodium salt (4-MUNANA). When the test strip has no fluorescence or the fluorescence intensity is weak, it has NA inhibitory activity. In addition, the Chinese herbal medicine extract was dissolved into an appropriate concentration and added to the NA solution. After 30 minutes of reaction, 4-MUNANA was added and the fluorescence intensity of the solution after the reaction was detected by a fluorescence detector. When there is no fluorescence or the fluorescence intensity is weak, it is a sample with NA inhibitory activity. (2) Separation and identification of components with neuraminidase inhibitory activity: Affinity chromatography was used to separate components that can bind to NA. First, NA was co-incubated with ferrous oxide (Fe3O4) magnetic beads to obtain NA@Fe3O4 immobilized enzyme carrier material. Then, the sample with NA inhibitory activity selected by (1) was added to the immobilized enzyme at a volume ratio of 1:
1. After sufficient oscillation for 30 minutes, the unadsorbed supernatant was centrifuged and discarded, and a suitable solvent was used to elute to obtain a solution bound to NA@Fe3O4. The eluate was injected into a liquid chromatograph and a liquid-mass spectrometer for detection, and the components that can bind to NA were obtained and identified. By measuring the half-inhibitory concentration of the screened binding components on NA, the NA inhibitor was finally obtained.
2. The method for screening an active ingredient for inhibiting neuraminidase according to claim 1, characterized in that: The concentration of the Chinese herbal medicine extract solution is 200 μg / mL.
3. The method for screening an active ingredient for inhibiting neuraminidase according to claim 1, characterized in that: The volume ratio of the traditional Chinese medicine extract, NA and 4-MUNANA is 1:1:
1.
4. The method for screening an active ingredient that inhibits neuraminidase according to claim 1, characterized in that: The affinity chromatography method is specifically as follows: the chromatographic column is C18 (4.6 × 250 mm; 5 μm), the detection wavelength is 270 nm, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is methanol, the gradient elution program is 0 min-75% A; 10 min-35% A; 55 min-10% A; the column temperature is 30°C, and the flow rate is 0.8 mL / min.
5. The method for screening an active ingredient for inhibiting neuraminidase according to claim 1, characterized in that: The LC-MS method is specifically 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, the gradient elution program is 0 min-75% A; 10 min-35% A; 55 min-10% 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).