CN112816592A - Preparation of immobilized acetylcholinesterase and application thereof in screening and identifying enzyme inhibitor - Google Patents
Preparation of immobilized acetylcholinesterase and application thereof in screening and identifying enzyme inhibitor Download PDFInfo
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Abstract
The invention provides a preparation method of immobilized acetylcholinesterase, which is characterized in that dopamine is adopted to modify cellulose filter paper, and then the acetylcholinesterase is covalently bonded on the cellulose filter paper through Michael addition/Schiff base reaction, so that the immobilized acetylcholinesterase is obtained. The invention also provides application of the acetylcholinesterase inhibitor in screening and identifying acetylcholinesterase inhibitors. The immobilized acetylcholinesterase disclosed by the invention has the advantages of good stability, wide pH resistance and temperature resistance ranges, strong enzyme activity, high utilization rate, easiness in control, low preparation cost, suitability for large-scale preparation, high-throughput screening of enzyme inhibitors and the like.
Description
Technical Field
The invention belongs to the technical field of new drug screening chemical processes, and particularly relates to preparation of immobilized acetylcholinesterase and application thereof in screening and identifying enzyme inhibitors.
Background
The enzyme is a biocatalyst, widely exists in each cell of a living body, participates in promoting the normal operation of metabolism of the living body, and maintains all life activities. At present, enzymes have become important research objects and targets of related subjects such as analytical chemistry, drug screening, clinical medicine and the like due to the characteristics of strong specificity, mild conditions, high catalytic efficiency and the like in catalytic reaction. Enzyme inhibitors are effective in reducing the activity of enzymes associated with diseases, thereby preventing and treating diseases, and many enzyme inhibitor drugs are used for treating the associated diseases. Although most of the enzyme inhibitor drugs can relieve or alleviate the symptoms of diseases clinically, the drugs (such as acetylcholinesterase inhibitor, donepezil, galantamine, rivastigmine and tacrine) have limited curative effect, high price and side effects, which severely restrict the application. The natural products (such as plants, traditional Chinese medicines, national medicines, foods and the like) have the characteristics of rich resources, safety, effectiveness, environmental friendliness, small toxic and side effects and the like, and are one of the important sources of natural enzyme inhibitors. Therefore, screening enzyme inhibitors from natural products by establishing different models has become an important means for finding new drugs.
Currently, screening methods for enzyme inhibitors include in vitro enzyme activity inhibition, bioaffinity chromatography, and enzyme-ligand affinity binding. Among them, the enzyme-ligand affinity binding method is a screening method based on the principle that a receptor (enzyme) and a ligand (active small molecule compound) are bound by affinity, and is considered to be a rapid, simple and effective method for screening active compounds from a complex matrix. Receptors related to the enzyme-ligand affinity binding method comprise free enzyme and immobilized enzyme, wherein the immobilized enzyme is more and more concerned due to the characteristics of good stability, wide pH and temperature resistant range, reusability, rapid separation from a reaction system and the like. The separation of the immobilized enzyme from the reaction system can be realized by a common immobilized enzyme carrier (such as a magnetic material) through centrifugation, filtration or special environment/equipment.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides preparation of immobilized acetylcholinesterase and application thereof in screening and identifying enzyme inhibitors. Aims to solve the problems of poor free enzyme stability, expensive and not easy-to-obtain enzyme carrier materials, complex enzyme immobilization process, difficult separation of immobilized enzyme and a reaction system, low enzyme utilization rate, large consumption of samples and solvents, high labor intensity, inapplicability to simultaneous screening of various components and the like. The cellulose filter paper is used as a novel enzyme immobilization carrier, and has the advantages of low price, no need of preparation, simple preparation process of enzyme immobilization, particularly immediate separation, and avoidance of complicated centrifugation or filtration operation in the separation process. The immobilized acetylcholinesterase established by the invention is combined with a high performance liquid phase-flight time mass spectrum model, has the characteristics of high speed, high efficiency, high sensitivity, low sample/enzyme consumption, high flux, high enzyme utilization rate and the like, can realize the screening of the acetylcholinesterase inhibitor from natural products, and has important scientific value and practical value in the aspect of screening the enzyme inhibitor.
The invention provides a preparation method of immobilized acetylcholinesterase, which is characterized in that dopamine is adopted to modify cellulose filter paper, and then the acetylcholinesterase is covalently bonded on the cellulose filter paper through Michael addition/Schiff base reaction, so that the immobilized acetylcholinesterase is obtained.
Preferably, the specific method for modifying the cellulose filter paper by using dopamine comprises the following steps: dissolving dopamine hydrochloride in a phosphate buffer solution to obtain a dopamine buffer solution with the concentration of 1-5 mg/mL, immersing cellulose filter paper in a container containing the dopamine buffer solution, sealing, placing in a constant-temperature oscillator at 25-50 ℃, taking out after reacting for 1-15 hours, washing with ultrapure water to be neutral, and drying.
Preferably, the specific method for covalently binding acetylcholinesterase on the cellulose filter paper comprises the following steps: and (3) placing the modified cellulose filter paper in a container containing acetylcholinesterase, sealing, placing in a constant-temperature oscillator at 25-50 ℃, taking out after reacting for 1.5-5.5 hours, washing by using a phosphate buffer solution, and drying.
The invention provides immobilized acetylcholinesterase, which is prepared by the preparation method.
The invention provides application of the immobilized acetylcholinesterase in screening and identifying acetylcholinesterase inhibitors.
The invention provides a method for screening and identifying enzyme inhibitors, which comprises the following steps:
(1) adding a positive control substance into a sample solution to be tested to prepare an enzyme inhibitor model solution, placing the immobilized acetylcholinesterase of claim 5 into the enzyme inhibitor model solution for incubation, and after the incubation is finished, cleaning the immobilized acetylcholinesterase by using a phosphate buffer solution and a methanol solution in sequence and collecting filtrate, namely screening to obtain an enzyme inhibitor;
(2) injecting the filtrate into an ultra-high performance liquid chromatography-time-of-flight mass spectrometer for chromatographic analysis and structural identification; and (3) the chromatographic peak of the positive control substance appears on the total ion flow diagram to indicate that the experiment is effective, the rest chromatographic peaks are active compounds, and the qualitative analysis of the active compounds is carried out according to MS and MS/MS data obtained by the time-of-flight mass spectrum.
Preferably, step (1) is preceded by the following steps: taking a representative acetylcholinesterase inhibitor as a positive control substance, taking a control substance with little or no inhibition activity on the activity of acetylcholinesterase as a negative control substance, mixing the positive control substance and the negative control substance to prepare an artificial mixed model solution, placing the immobilized acetylcholinesterase of claim 5 in the artificial mixed model solution for incubation, washing the solution by using a phosphate buffer solution and a methanol solution after the incubation is finished, collecting filtrate, further verifying by using a high performance liquid chromatography-flight time mass spectrometry combined technology, and if the positive control substance is screened out from the filtrate, successfully preparing the immobilized acetylcholinesterase;
preferably, the positive control is huperzine A; the negative control substances are gallic acid and ferulic acid.
Preferably, in the step (1), the dosage of the immobilized acetylcholinesterase is 1-5 tablets, and the diameter is 1-12 mm; the dosage of the enzyme inhibitor model solution is 200-500 mu L.
Preferably, in step (1), the incubation conditions are as follows: incubating for 10-40 min at 25-50 ℃.
Preferably, when the sample to be detected is myrobalan, the setting conditions of all parameters in the high performance liquid chromatography-time-of-flight mass spectrometry are as follows:
chromatographic conditions are as follows: a chromatographic column: ZORBAX Eclipse Plus C18 column (3 mm. times.150 mm, 1.8 μm); column temperature: 35 ℃; mobile phase C: 0.1% formic acid-acetonitrile and mobile phase D: 0.1% formic acid-water; the flow rate is 0.250 mL/min; sample introduction amount: 2 mu L of the solution; gradient program:
changing the constant speed of the mobile phase C from 5% to 15% and the constant speed of the mobile phase D from 95% to 85% in 0-5 min;
5-30 min, changing the uniform speed of the mobile phase C from 15% to 35%, and changing the uniform speed of the mobile phase D from 85% to 65%;
30-32 min, changing the constant speed of the mobile phase C from 35% to 5%, and changing the constant speed of the mobile phase D from 65% to 95%;
mass spectrum conditions: the mass spectrum model is as follows: agilent Q-TOF 6545 time-of-flight mass spectrometry; an ion source: electrospray ion source (ESI); detection mode: a negative ion mode; scanning range: m/z is 50-1500; temperature of the drying gas: 350 ℃; flow rate of drying gas: 12.0L/min; the flow rate of the sheath gas: 10.0L/min; atomizing gas pressure: 40 psi; capillary voltage: 3500V; collision voltage: 135V; MS/MS optimized collision energy of parent ions: 10 to 60 eV.
The invention overcomes the defects of the prior art and provides a method for screening and identifying a natural enzyme inhibitor by combining immobilized acetylcholinesterase with an ultra-high performance liquid-flight time mass spectrum model. First, the present invention immobilizes acetylcholinesterase on cellulose filter paper. Secondly, respectively constructing an artificial mixed model of the immobilized enzyme screening enzyme inhibitor and a model of the immobilized enzyme screening natural enzyme inhibitor according to the principle of affinity binding reaction between a receptor (enzyme) and a ligand (active small molecular compound). And then carrying out chromatographic analysis and structure identification on the eluent obtained in the immobilized enzyme screening natural enzyme inhibitor model by establishing an ultra-high performance liquid chromatography-time-of-flight mass spectrometry technology, and finally identifying to obtain the natural enzyme inhibitor. Finally, investigating the inhibition activity of the screened compound on enzyme by an in-vitro acetylcholinesterase activity inhibition rate detection method; the affinity binding site and affinity binding force of the screened compound and acetylcholinesterase are predicted and calculated through molecular docking. And further verifying the reliability and effectiveness of the immobilized enzyme screening natural enzyme inhibitor model.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects:
(1) the cellulose filter paper is used as a novel enzyme immobilization carrier, the price is low, the preparation is not needed, the enzyme immobilization process is simple, and the most prominent characteristic is that the prepared immobilized enzyme does not need special equipment, and only tweezers are needed to instantly take out the immobilized enzyme filter paper from a reaction system, so that the separation from a reaction mixture can be instantly realized, and the enzymatic reaction or the reaction of the enzyme and an inhibitor is timely stopped.
(2) In the process of screening various enzyme inhibitors from a complex matrix, compared with free enzyme, the immobilized acetylcholinesterase has the advantages of good stability (the immobilized acetylcholinesterase is repeatedly used for 10 times and still keeps 60-70% of the initial activity of the enzyme), pH resistance (the pH tolerance range is 4.0-9.0), wide temperature resistance range (the temperature tolerance range is 25-60 ℃), strong enzyme activity (compared with the activity of the free enzyme, the Km value of the immobilized enzyme is slightly higher, which shows that the activity difference with the free enzyme is not large), high utilization rate (the immobilized enzyme can be repeatedly used and shows that the utilization rate is high), easiness in control, low preparation cost, suitability for large-scale preparation and the like.
(3) The method has the characteristics of rapidness, high efficiency, high sensitivity, low sample/enzyme consumption (in the screening process, the sample consumption is only 400ul, and the enzyme consumption is only 3, so that 26 natural acetylcholinesterase inhibitors can be screened out, the low sample/enzyme consumption is shown, and the good embodiment is still obtained in the experimental process), high flux and the like, and is suitable for simultaneously screening and identifying various acetylcholinesterase inhibitors from natural medicine extracts.
(4) Compared with the synthetic drugs, the acetylcholinesterase inhibitor obtained by screening by the method has the advantages of low price, environmental protection, safety, small toxic and side effects and the like.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a flow chart of the present invention for screening and identifying natural enzyme inhibitors by using immobilized acetylcholinesterase in combination with ultra high performance liquid-time-of-flight mass spectrometry (see example 1).
Fig. 2 is a total ion flow diagram of the filtrate of the artificial mixed model.
FIG. 3 is a comparison graph of total ion flow chromatograms of myrobalan extract and filtrate in a negative ion mode and a total ion flow graph of filtrate in a positive ion mode in example 1 of the present invention; wherein, the graph A is a total ion chromatogram of the myrobalan extract without being incubated and screened by the immobilized enzyme, the graph B is a total ion chromatogram of the myrobalan extract incubated and screened by the immobilized enzyme, and the graph C is a total ion chromatogram of the filtrate in the positive ion mode.
FIG. 4 is the second-level mass spectrum of the compounds 1,3, 6-O-galloyl-beta-D-glucose, punicalagin, myrobalan tannic acid and germacranin obtained by screening in example 1 of the present invention.
FIG. 5 shows the molecular docking prediction of binding sites and mechanism of action of agrimonine and acetylcholinesterase in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The preparation of the immobilized acetylcholinesterase and the application of the immobilized acetylcholinesterase in screening and identifying enzyme inhibitors by combining high performance liquid chromatography-flight time mass spectrometry are as follows:
1. preparation of immobilized acetylcholinesterase
Modifying cellulose filter paper with dopamine, and then carrying out Michael addition/Schiff base reaction to covalently bond acetylcholinesterase on the cellulose filter paper, thereby obtaining the immobilized acetylcholinesterase. The method specifically comprises the following steps:
firstly, accurately weighing 10-45 mg of dopamine hydrochloride, and fully dissolving the dopamine hydrochloride into a phosphate buffer solution with the concentration of 0.05-1M and the pH value of 2.0-10.0 to obtain a dopamine buffer solution with the concentration of 1-5 mg/mL. And immersing the cellulose filter paper in a culture dish containing dopamine buffer solution with the concentration of 1-5 mg/mL, sealing the culture dish by using a sealing film, placing the culture dish in a constant-temperature oscillator with the temperature of 25-50 ℃, taking out the culture dish after reacting for 1-15 hours, washing the cellulose filter paper by using ultrapure water until the residual liquid on the surface is neutral, drying the cellulose filter paper in an oven with the temperature of 25-50 ℃ after sucking the residual water on the surface by using dry and clean filter paper, and preparing a certain number of round small paper sheets with the diameter of 1-12 mm by using a paper puncher.
Secondly, placing a certain number of small paper sheets in a culture dish containing an acetylcholinesterase solution with the concentration of 2.0-7.0U/mL and the pH value of 3.0-9.0, sealing the culture dish, placing the culture dish in a constant-temperature oscillator with the temperature of 25-50 ℃, taking out after reacting for 1.5-6.5 hours, washing the culture dish by using a phosphate buffer solution with the concentration of 5-40 mmol/L, pH value of 2.0-10.0, washing the culture dish for a plurality of times, sucking off residual liquid on the surface by using dry and clean filter paper, and drying the paper sheets in an oven with the temperature of 25-50 ℃ to obtain the immobilized acetylcholinesterase.
2. Artificial mixed model for constructing immobilized acetylcholinesterase screening enzyme inhibitor
Representative acetylcholinesterase inhibitors were used as positive control (huperzine A), and control with little or no inhibitory activity against acetylcholinesterase activity was used as negative control (gallic acid and ferulic acid). Mixing the positive control substance and the negative control substance to prepare an artificial mixed model with the concentration of 0.5-5 mg/mL, placing 1-10 small pieces of immobilized acetylcholinesterase obtained in the step (1) in an artificial mixed model solution with the volume of 100-600 mu L and the concentration of 0.5-5 mg/mL, incubating at 20-70 ℃ for 5-50 min, after the incubation is finished, quickly taking out the immobilized enzyme paper sheet from the reaction system by using tweezers, washing the immobilized enzyme paper sheet for 1-5 times by using a phosphate buffer solution with the value of 100-600 mu L, pH being 9.0, discarding filtrate, eluting for 1-5 times by using 100-600 mu L of an organic solvent (50% methanol aqueous solution by volume percentage), collecting the filtrate, the method comprises the steps of screening an acetylcholinesterase inhibitor (positive control) from an artificial model solution, and carrying out high performance liquid chromatography-time of flight mass spectrometry on filtrate to verify the effectiveness and reliability of screening the enzyme inhibitor by an immobilized acetylcholinesterase model.
In the experiment, huperzine A (acetylcholinesterase inhibitor) is used as a positive control substance, gallic acid and ferulic acid are used as negative control substances, and 3 natural standard substances are used for constructing an artificial mixed model, so that the aim is to verify that the acetylcholinesterase inhibitor (huperzine A) can be successfully screened out by the immobilized enzyme in a complex matrix (an environment in which the acetylcholinesterase inhibitor and the non/weak acetylcholinesterase inhibitor coexist), and the non/weak acetylcholinesterase inhibitor (gallic acid or ferulic acid) cannot be screened out. The purpose of adding the negative control is to form an artificial mixed model on one hand, so that the target solution contains both acetylcholinesterase inhibitor and non/weak acetylcholinesterase inhibitor, and simulate the complex matrix environment of the actual natural medicine extracting solution. On the other hand, in order to eliminate the possibility of false positive, if the immobilized enzyme can screen out one or both of the negative controls (gallic acid or ferulic acid), then the method has the possibility of false positive in the actual sample screening process.
The setting conditions of all parameters in the high performance liquid chromatography-time-of-flight mass spectrometry technology are as follows:
chromatographic conditions are as follows: a chromatographic column: ZORBAX Eclipse Plus C18 column (3 mm. times.150 mm, 1.8 μm); column temperature: 35 ℃; mobile phase: 0.1% formic acid-acetonitrile (a) and 5mM ammonium acetate-water (B); flow rate: 0.250 mL/min; sample introduction amount: 2 mu L of the solution; gradient program:
changing the constant speed of the mobile phase A from 5% to 70% and the constant speed of the mobile phase B from 95% to 30% in 0-2 min;
changing the uniform speed of the mobile phase A from 70% to 30% and the uniform speed of the mobile phase B from 30% to 70% for 2-9 min;
and (3) 9-10 min, uniformly changing the mobile phase A from 30% to 95%, and uniformly changing the mobile phase B from 70% to 5%.
Mass spectrum conditions: the mass spectrum model is as follows: agilent Q-TOF 6545 time-of-flight mass spectrometry; an ion source: electrospray ion source (ESI); detection mode: positive/negative ion mode; scanning range: m/z is 50-500; temperature of the drying gas: 350 ℃; flow rate of drying gas: 12.0L/min; the flow rate of the sheath gas: 10.0L/min; atomizing gas pressure: 40 psi; capillary voltage: 4000V/3500V; collision voltage: 135V.
3. And (3) constructing a model for screening natural enzyme inhibitors by immobilized acetylcholinesterase.
(1) Preparing a sample solution to be tested. And ultrasonically extracting 50-600 g of sample for 1-5 times by using a proper solvent with the volume of 100-1000 mL, combining extracting solutions and concentrating to obtain an extract. Dissolving the extract with 50-300 mL of distilled water, transferring to macroporous adsorption resin column chromatography for elution at each part, performing gradient elution with distilled water and ethanol solutions (10-100%) with different concentration gradients, collecting eluates according to different polarities, and concentrating to obtain dry extract samples.
(2) And (4) selecting a sample solution to be tested. And (3) inspecting the inhibition rate of each dry paste sample solution on the enzyme activity by adopting an in-vitro enzyme activity inhibition rate detection method, and selecting the dry paste sample with the strongest inhibition activity as the sample solution to be detected.
(3) And (3) screening and identifying a natural enzyme inhibitor by using the immobilized acetylcholinesterase. And (3) adding the representative acetylcholinesterase inhibitor huperzine A in the step (2) serving as a positive control into a sample solution to be detected to prepare a natural enzyme inhibitor model solution with a certain concentration (the concentration of the huperzine A is 2mg/mL, and the concentration of the sample solution to be detected is 10 mg/mL). Incubating 100-600 mu L of natural enzyme inhibitor model solution and 1-5 pieces of immobilized acetylcholinesterase small paper sheets at 20-70 ℃ for 10-60 min. After incubation is finished, quickly taking out the immobilized enzyme small paper sheet from the reaction system by using tweezers, washing the enzyme sheet for 1-5 times by using 100-600 mu L of phosphate buffer solution, and discarding filtrate; washing the enzyme tablet 1-5 times with 100-600 μ L organic solvent (30-70% methanol (v: v)) and collecting the filtrate, i.e. screening the acetylcholinesterase inhibitor (active small molecular compound) from the complex matrix at high flux.
4. Chromatographic analysis and structure identification of ultra-high performance liquid chromatography-time-of-flight mass spectrometer
Concentrating the filtrate, redissolving, filtering with 0.22 μm microporous membrane, and injecting into ultra high performance liquid chromatography-time of flight mass spectrometer for chromatographic analysis and structure identification. The chromatographic peak of huperzine A appearing on the total ion flow chart shows that the experiment is effective, the rest chromatographic peaks are active compounds, and qualitative analysis of each compound is carried out according to MS and MS/MS data obtained by time-of-flight mass spectrometry. It is intended that these active compounds are very likely to be potential acetylcholinesterase inhibitors.
Example 1
In this example, an acetylcholinesterase inhibitor is screened and identified from a natural product myrobalan extract by combining immobilized acetylcholinesterase with an ultra-high performance liquid-time-of-flight mass spectrometry model, and the screening process is shown in fig. 1.
1. Preparation of immobilized acetylcholinesterase
Modifying cellulose filter paper with dopamine, and then carrying out Michael addition/Schiff base reaction to covalently bond acetylcholinesterase on the cellulose filter paper, thereby obtaining the immobilized acetylcholinesterase. The method specifically comprises the following steps:
firstly, 32mg of dopamine hydrochloride is accurately weighed and fully dissolved in a phosphate buffer solution with the concentration of 0.1M and the pH value of 8.5 to obtain a dopamine buffer solution with the concentration of 2 mg/mL. And then immersing the cellulose filter paper in a culture dish containing dopamine buffer solution with the concentration of 2mg/mL, sealing the culture dish by using a sealing film, placing the culture dish in a constant-temperature oscillator at the temperature of 30 ℃, taking out the culture dish after reacting for 5 hours, washing the cellulose filter paper by using ultrapure water until the residual liquid on the surface is neutral, drying the cellulose filter paper in a drying oven at the temperature of 35 ℃ after sucking the residual water on the surface by using dry and clean filter paper, and preparing a certain number of round small paper sheets with the diameter of 6mm by using a paper puncher.
Secondly, placing a certain number of small paper sheets in a culture dish containing acetylcholinesterase solution with the concentration of 4.0U/mL and the pH value of 6.0, sealing the culture dish, placing the culture dish in a constant-temperature oscillator at 30 ℃, taking out after reacting for 3.5 hours, washing the culture dish by using phosphate buffer solution with the concentration of 20mmol/L, pH value of 6.0, washing the culture dish for a plurality of times, sucking residual liquid on the surface by using dry and clean filter paper, and drying the culture dish in a drying oven at 35 ℃ to obtain the immobilized acetylcholinesterase.
2. Artificial mixed model for constructing immobilized acetylcholinesterase screening enzyme inhibitor
Representative acetylcholinesterase inhibitors were used as positive control (huperzine A), and control with little or no inhibitory activity against acetylcholinesterase activity was used as negative control (gallic acid and ferulic acid). Mixing the positive control substance and the negative control substance to prepare an artificial mixed model with the concentration of 1mg/mL, putting 3 small sheets of the immobilized acetylcholinesterase obtained in the step 1 into an artificial mixed model solution with the volume of 400 mu L and the concentration of 1mg/mL, incubating at 45 deg.C for 30min, after incubation, quickly taking out the immobilized enzyme paper from the reaction system with tweezers, washing the immobilized enzyme paper with 400 μ L, pH value 9.0 phosphate buffer solution for 3 times, discarding filtrate, eluting with 400 μ L organic solvent (50% methanol water solution by volume) for 3 times, collecting filtrate, the method comprises the steps of screening an acetylcholinesterase inhibitor (positive control) from an artificial model solution, and carrying out high performance liquid chromatography-time of flight mass spectrometry on filtrate to verify the effectiveness and reliability of screening the enzyme inhibitor by an immobilized acetylcholinesterase model. For example, fig. 2 is a total ion flow diagram of the filtrate, in which only the chromatographic peak of huperzine a appears and the chromatographic peaks of the negative control gallic acid and ferulic acid do not appear, which indicates that the immobilized enzyme and enzyme screening model is successfully prepared and is effective.
The setting conditions of all parameters in the high performance liquid chromatography-time-of-flight mass spectrometry technology are as follows:
chromatographic conditions are as follows: a chromatographic column: ZORBAX Eclipse Plus C18 column (3 mm. times.150 mm, 1.8 μm); column temperature: 35 ℃; mobile phase: 0.1% formic acid-acetonitrile (a) and 5mM ammonium acetate-water (B); flow rate: 0.250 mL/min; sample introduction amount: 2 mu L of the solution; gradient program:
changing the constant speed of the mobile phase A from 5% to 70% and the constant speed of the mobile phase B from 95% to 30% in 0-2 min;
changing the uniform speed of the mobile phase A from 70% to 30% and the uniform speed of the mobile phase B from 30% to 70% for 2-9 min;
and (3) 9-10 min, uniformly changing the mobile phase A from 30% to 95%, and uniformly changing the mobile phase B from 70% to 5%.
Mass spectrum conditions: the mass spectrum model is as follows: agilent Q-TOF 6545 time-of-flight mass spectrometry; an ion source: electrospray ion source (ESI); detection mode: positive/negative ion mode; scanning range: m/z is 50-500; temperature of the drying gas: 350 ℃; flow rate of drying gas: 12.0L/min; the flow rate of the sheath gas: 10.0L/min; atomizing gas pressure: 40 psi; capillary voltage: 4000V/3500V; collision voltage: 135V.
3. And (3) constructing a model for screening natural enzyme inhibitors by immobilized acetylcholinesterase.
(1) And preparing a myrobalan sample. Extracting fructus Chebulae 100g with distilled water 600mL for 2 times, mixing extractive solutions, and concentrating to obtain extract. Dissolving the extract with 150mL of distilled water, transferring to macroporous adsorption resin column chromatography for elution at each part, performing gradient elution with distilled water and ethanol solutions with different concentration gradients (volume percentage of 30%, 50%, 70% and 95%), collecting 5 parts of eluents according to different polarities, and concentrating to obtain 5 parts of dry paste samples.
(2) And selecting a test article. The method for detecting the inhibition rate of acetylcholinesterase activity in vitro (see the literature: Zhao HQ, Zhou SD, Zhang MM, et al. an in vitro AChE inhibition associated with UF-HPLC-ESI-QTOF/MS approach for screening and characterization of AChE inhibition from protein fragments [ J ]. Journal of pharmaceutical and biological Analysis,2016,120:235-240 ″, and the literature: Ning ZW, hai Zi, Huang T, et al. identification of alpha-glucose inhibition from protein fragments [ J ]. J. the inventor of acetylcholine inhibitors from proteins isolates UF-UPLC-Q/MS/TOF J. (see the literature: Acetylcholinesterase, pH 1902, pH 3, pH; the dry paste sample solution obtained in the step (1) is used as an inhibitor, and the following four controls are arranged at the same time: simultaneously adding enzyme and a test sample into the dry paste sample solution obtained by the steps; adding only enzyme, not adding test sample, and obtaining dry paste sample solution according to the above steps; dry paste-like sample solution obtained by the above steps without adding an enzyme and a test sample; the test sample was added only to the dry paste-like sample solution obtained in the above procedure without adding the enzyme. The inhibition rate of each dry extract-like sample solution on the enzyme activity was examined. Wherein, the calculation formula of the in vitro acetylcholinesterase activity inhibition rate is as follows:
inhibition ratio (%) ═ 1- (A)s-Abs)/(Ac-Abc)
In the formula, AcIndicates the absorbance of the enzyme with no test sample added; a. thebcRepresenting the absorbance of the test sample without the addition of enzyme; a. thesRepresents the absorbance of the added test sample and enzyme; a. thebsIndicates the absorbance of the test sample without enzyme.
Through the detection of the acetylcholinesterase inhibitory activity of 5 parts of dry paste sample solution, the second part of sample solution (i.e. the sample solution obtained by eluting with 30% by volume of ethanol) was found to have a strong inhibitory activity against acetylcholinesterase, its IC50The value was 0.34. + -. 0.02mg/mL, indicating that the second sample may be enriched for potential acetylcholinesterase inhibitors and was therefore selected as the test solution.
(3) And (3) screening and identifying a natural enzyme inhibitor by using the immobilized acetylcholinesterase. Adding the representative acetylcholinesterase inhibitor huperzine A of 2 as a positive control into the myrobalan extract (the second dry extract sample solution) of 3(2) to prepare a natural enzyme inhibitor model solution with a certain concentration (the concentration of huperzine A is 2mg/mL, and the concentration of the second dry extract sample solution of the myrobalan extract is 10 mg/mL). A volume of 400. mu.L of the native enzyme inhibitor model solution was incubated with 3 small sheets of immobilized acetylcholinesterase paper at 45 ℃ for 30 min. After the incubation is finished, quickly taking out the immobilized enzyme small paper sheet from the reaction system by using tweezers, washing the enzyme sheet for 3 times by using 400 mu L of phosphate buffer solution, and removing filtrate; washing enzyme tablet with 400 μ L organic solvent (50% methanol water solution by volume percentage) for 3 times, and collecting filtrate, i.e. screening natural acetylcholinesterase inhibitor (active small molecule compound) from complex matrix (second dry extract sample solution of fructus Chebulae extract) at high flux.
Concentrating the filtrate, redissolving, filtering with 0.22 μm microporous membrane, and injecting into ultra high performance liquid chromatography-time of flight mass spectrometer for chromatographic analysis and structure identification.
The setting conditions of all parameters in the high performance liquid chromatography-time-of-flight mass spectrometry technology are as follows:
chromatographic conditions 1: a chromatographic column: ZORBAX Eclipse Plus C18 column (3 mm. times.150 mm, 1.8 μm); column temperature: 35 ℃; mobile phase: 0.1% formic acid-acetonitrile (C) and 0.1% formic acid-water (D); the flow rate is 0.250 mL/min; sample introduction amount: 2 mu L of the solution; gradient program:
changing the constant speed of the mobile phase C from 5% to 15% and the constant speed of the mobile phase D from 95% to 85% in 0-5 min;
5-30 min, changing the uniform speed of the mobile phase C from 15% to 35%, and changing the uniform speed of the mobile phase D from 85% to 65%;
and (3) 30-32 min, uniformly changing the mobile phase C from 35% to 5%, and uniformly changing the mobile phase D from 65% to 95%.
Chromatographic conditions 2 (huperzine a): a chromatographic column: ZORBAX Eclipse Plus C18 column (3 mm. times.150 mm, 1.8 μm); column temperature: 35 ℃; mobile phase: 0.1% formic acid-acetonitrile (a) and 5mM ammonium acetate-water (B); flow rate: 0.250 mL/min; sample introduction amount: 2 mu L of the solution; gradient program:
changing the constant speed of the mobile phase A from 5% to 70% and the constant speed of the mobile phase B from 95% to 30% in 0-2 min;
changing the uniform speed of the mobile phase A from 70% to 30% and the uniform speed of the mobile phase B from 30% to 70% for 2-9 min;
and (3) 9-10 min, uniformly changing the mobile phase A from 30% to 95%, and uniformly changing the mobile phase B from 70% to 5%.
Mass spectrum conditions: the mass spectrum model is as follows: agilent Q-TOF 6545 time-of-flight mass spectrometry; an ion source: electrospray ion source (ESI); detection mode: positive (huperzine a)/negative ion mode (filtrate); scanning range: m/z is 50-1500; temperature of the drying gas: 350 ℃; flow rate of drying gas: 12.0L/min; the flow rate of the sheath gas: 10.0L/min; atomizing gas pressure: 40 psi; capillary voltage: 4000V (positive ions)/3500V (negative ions); collision voltage: 135V; MS/MS optimized collision energy of parent ions: 10 to 60 eV.
FIG. 3 is a comparison graph of total ion flow chromatograms of myrobalan extract and filtrate in a negative ion mode and a total ion flow graph of filtrate in a positive ion mode in example 1 of the present invention; wherein, the graph A is a total ion chromatogram of the myrobalan extract without being incubated and screened by the immobilized enzyme, the graph B is a total ion chromatogram of the myrobalan extract incubated and screened by the immobilized enzyme, and the graph C is a total ion chromatogram of the filtrate in the positive ion mode.
The applicant finds that the myrobalan extracting solution has higher correspondence in the negative ion mode, so that the myrobalan extracting solution is detected by selecting chromatographic conditions 1 and mass spectrometry in the negative ion mode. However, when huperzine a was selected in the same manner as myrobalan extract (chromatography condition 1 and mass spectrum in negative ion mode), no peak was detected. Then, the huperzine A can detect the chromatographic peak only under the chromatographic condition 2 and the positive ion mode through groping. Thus the same filtrate in negative ion mode and under chromatographic conditions 1 gave figure 3B (screened natural inhibitor) and in positive ion mode and chromatographic conditions 2 gave figure 3C (cationic control huperzine a). Because the peak of huperzine A is not in the same mode and chromatographic condition, the peak of huperzine A cannot appear in FIG. 3B, but the detection method is different, the same filtrate is detected, and the chromatographic peak of huperzine A appears in FIG. 3C, so that the obtained filtrate is completely proved to contain the positive control huperzine A, and the experiment is proved to be effective.
As shown in fig. 3, fig. 3C is a total ion flow diagram of the filtrate under the positive ion mode and the chromatographic condition 2, and a chromatographic peak of the positive control huperzine a appears in fig. 3C, which illustrates the effectiveness of the screening model; fig. 3B is a total ion flow graph of the filtrate under negative ion mode and chromatographic condition 1, and fig. 3B has 26 chromatographic peaks in total, which illustrates that the 26 active compounds are co-screened from the myrobalan extract (second dry paste sample solution), which means that the 26 active compounds are very likely to be potential acetylcholinesterase inhibitors in the second dry paste sample solution of the myrobalan extract.
Of the 26 active compounds screened from the second dry paste sample of the above myrobalan extract (FIG. 3B), the qualitative resolution of the compound was performed based on the MS and MS/MS data obtained from the time-of-flight mass spectrometry, using peaks 4, 19, 21 and 26 as examplesAnd (6) analyzing. Peak 4 had a retention time of 9.94min, and its excimer ion peak was M/z1083.0634[ M-H ] as shown in FIG. 4A]-The molecular formula is presumed to be C48H28O30. The ion further obtains fragment ion M/z 781.0561[ M-H-302Da ]]-(loss of hexahydrodibenzoyl HHDP), M/z 600.9915[ M-H-482Da]-(loss of HHDP-glucosyl group), M/z 450.9959[ M-H-634Da]-(loss of galloyl-HHDP-glucosyl group) and the characteristic ion m/z300.9981[ ellagic acid-H]-. Therefore, the compound was identified as punicalagin. Peak 19 retention time 14.13min, shown in FIG. 4B, with an excimer peak of M/z 635.0915[ M-H [ ]]－The molecular formula is presumed to be C27H24O18. The ion further provides fragment ion M/z 483.0803[ M-H-galloyl [ ]]-M/z 465.0694[ M-H-gallic acid group ]]-M/z 313.05[ M-H-gallic acid group-galloyl group ]]-Characteristic example m/z 169.0146[ Gallic acid-H ]]-And m/z 125.0245[ gallic acid-H-CO ]2]-. Thus, the compound was identified as 1,3, 6-O-galloyl- β -D-glucose. Peak 21 retention time 15.26min, shown in FIG. 4C, with an excimer peak of M/z 951.0775[ M-H [ ]]－The molecular formula is presumed to be C41H28O27. The ion further obtains fragment ion M/z 933.0660[ M-H-18Da ]]-(the molecule loses one molecule of H2O, oxidation of the hexahydroxydibenzoyl radical (HHDP) in the molecule to dehydrohexahydroxydibenzoyl radical (DHHDP)) and the characteristic fragment ion m/z 300.9999[ ellagic acid-H ]]--. Thus, the compound was identified as germacylin. Peak 26 retention time 18.66min, shown in FIG. 4D, with an excimer peak of M/z 955.1096[ M-H [ ]]-, the presumed formula is C41H32O27. The ion further provides fragment ion M/z 785.0898[ M-H-gallic acid group [)]-、617.0798[M-H-chebuloyl]- (chebulayl is the oxidized form of HHDP, molecular weight 337Da), M/z 465.0704[ M-H-galloyl-chebulayl-H]-And 169.0146[ Gallic acid-H ]]-. In addition, there is a fragment ion m/z 337.0199[ chebulayl-H ]]-、m/z 319.0104[chebuloyl-H-H2O]And 275.0199[ chebulayl-H [ ]2O-CO2]-. Thus, the compound was identified as myrobalan tannic acid.
4. Further verifying the reliability and effectiveness of the immobilized acetylcholinesterase model in screening natural enzyme inhibitors
(1) Detecting that an active small molecular compound obtained by screening from a second dry paste sample solution of a myrobalan extracting solution has stronger inhibitory activity on acetylcholinesterase by an in-vitro acetylcholinesterase activity inhibition detection method, wherein the screened compounds 1,3, 6-O-galloyl-beta-D-glucose, punicalagin, myrobalan tannic acid and gerbera agrimonine have IC (integrated Circuit) on acetylcholinesterase50The values were 0.29. + -. 0.02, 0.47. + -. 0.03, 0.48. + -. 0.03 and 0.49. + -. 0.03mg/mL, respectively. IC from the 4 screened Compounds50The value shows that the immobilized acetylcholinesterase has reliability and effectiveness in screening the natural enzyme inhibitor from the second dry paste sample solution of the myrobalan extracting solution.
(2) The molecular docking is carried out by adopting AutoDock Vina software, and the affinity binding site and affinity binding force of the screened small molecular compound germacine and acetylcholinesterase in the second dry paste sample solution of the myrobalan extracting solution are predicted and calculated, as shown in figure 5, the screened compound germacine can well enter the active pocket of the acetylcholinesterase, can form hydrogen bond binding with amino acids Ser 347, Arg 24, Lys 51 and Gly 342 of the acetylcholinesterase, and the affinity binding force of the screened small molecular compound germacine and the acetylcholinesterase is-8.0 kcal/mol. The molecular docking result shows that the acetylcholinesterase inhibitor screened from the second dry paste sample solution of the myrobalan extracting solution by applying the immobilized acetylcholinesterase is reliable and effective.
Example 2
The preparation method of the immobilized acetylcholinesterase of the invention is as follows:
modifying cellulose filter paper with dopamine, and then carrying out Michael addition/Schiff base reaction to covalently bond acetylcholinesterase on the cellulose filter paper, thereby obtaining the immobilized acetylcholinesterase. The method specifically comprises the following steps:
firstly, 45mg of dopamine hydrochloride is accurately weighed and fully dissolved in a phosphate buffer solution with the concentration of 0.05M and the pH value of 10.0 to obtain a dopamine buffer solution with the concentration of 5 mg/mL. And then immersing the cellulose filter paper in a culture dish containing dopamine buffer solution with the concentration of 5mg/mL, sealing the culture dish by using a sealing film, placing the culture dish in a constant-temperature oscillator at 50 ℃, taking out the cellulose filter paper after reacting for 1 hour, washing the cellulose filter paper by using ultrapure water until the residual liquid on the surface is neutral, sucking the residual water on the surface by using dry and clean filter paper, drying the cellulose filter paper in an oven at 25 ℃, and preparing a certain number of round small paper sheets with the diameter of 12mm by using a paper puncher.
Secondly, placing a certain number of small paper sheets in a culture dish containing acetylcholinesterase solution with the concentration of 2.0U/mL and the pH value of 9.0, sealing the culture dish, placing the culture dish in a constant-temperature oscillator at 25 ℃, taking out after reacting for 1.5 hours, washing the culture dish by using phosphate buffer solution with the concentration of 40mmol/L, pH value of 10.0, washing the culture dish for a plurality of times, sucking residual liquid on the surface by using dry and clean filter paper, and drying the culture dish in a 50 ℃ drying oven to obtain the immobilized acetylcholinesterase.
Example 3
The preparation method of the immobilized acetylcholinesterase of the invention is as follows:
modifying cellulose filter paper with dopamine, and then carrying out Michael addition/Schiff base reaction to covalently bond acetylcholinesterase on the cellulose filter paper, thereby obtaining the immobilized acetylcholinesterase. The method specifically comprises the following steps:
firstly, 10mg of dopamine hydrochloride is accurately weighed and fully dissolved in a phosphate buffer solution with the concentration of 1M and the pH value of 2.0 to obtain a dopamine buffer solution with the concentration of 1 mg/mL. And then immersing the cellulose filter paper in a culture dish containing dopamine buffer solution with the concentration of 1mg/mL, sealing the culture dish by using a sealing film, placing the culture dish in a constant-temperature oscillator at 25 ℃, taking out the cellulose filter paper after reacting for 15 hours, washing the cellulose filter paper by using ultrapure water until the residual liquid on the surface is neutral, sucking the residual water on the surface by using dry and clean filter paper, drying the cellulose filter paper in an oven at 50 ℃, and preparing a certain number of round small paper sheets with the diameter of 1mm by using a paper puncher.
Secondly, placing a certain number of small paper sheets in a culture dish containing acetylcholinesterase solution with the concentration of 7.0U/mL and the pH value of 3.0, sealing the culture dish, placing the culture dish in a constant-temperature oscillator at 50 ℃, taking out after reacting for 6.5 hours, washing the culture dish by using phosphate buffer solution with the concentration of 5mmol/L, pH value of 2.0, washing the culture dish for a plurality of times, sucking residual liquid on the surface by using dry and clean filter paper, and drying the culture dish in a drying oven at 25 ℃ to obtain the immobilized acetylcholinesterase.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the immobilized acetylcholinesterase is characterized by comprising the following steps: modifying cellulose filter paper with dopamine, and then carrying out Michael addition/Schiff base reaction to covalently bond acetylcholinesterase on the cellulose filter paper, thereby obtaining the immobilized acetylcholinesterase.
2. The process for producing an immobilized acetylcholinesterase according to claim 1, wherein: the specific method for modifying the cellulose filter paper by using the dopamine comprises the following steps: dissolving dopamine hydrochloride in a phosphate buffer solution to obtain a dopamine buffer solution with the concentration of 1-5 mg/mL, immersing cellulose filter paper in a container containing the dopamine buffer solution, sealing, placing in a constant-temperature oscillator at 25-50 ℃, taking out after reacting for 1-15 hours, washing with ultrapure water to be neutral, and drying.
3. The process for the preparation of immobilized acetylcholinesterase according to claims 1 and 2, wherein: the specific method for covalently bonding acetylcholinesterase on the cellulose filter paper comprises the following steps: and (3) placing the modified cellulose filter paper in a container containing acetylcholinesterase, sealing, placing in a constant-temperature oscillator at 25-50 ℃, taking out after reacting for 1.5-5.5 hours, washing by using a phosphate buffer solution, and drying.
4. An immobilized acetylcholinesterase prepared by the method of any one of claims 1-3.
5. Use of the immobilized acetylcholinesterase of claim 4 in screening and identifying acetylcholinesterase inhibitors.
6. A method of screening and identifying enzyme inhibitors, characterized by: the method comprises the following steps:
(1) adding a positive control substance into a sample solution to be tested to prepare an enzyme inhibitor model solution, placing the immobilized acetylcholinesterase of claim 5 into the enzyme inhibitor model solution for incubation, and after the incubation is finished, cleaning the immobilized acetylcholinesterase by using a phosphate buffer solution and a methanol solution in sequence and collecting filtrate, namely screening to obtain an enzyme inhibitor;
(2) injecting the filtrate into an ultra-high performance liquid chromatography-time-of-flight mass spectrometer for chromatographic analysis and structural identification; and (3) the chromatographic peak of the positive control substance appears on the total ion flow diagram to indicate that the experiment is effective, the rest chromatographic peaks are active compounds, and the qualitative analysis of the active compounds is carried out according to MS and MS/MS data obtained by the time-of-flight mass spectrum.
7. The method of claim 6, wherein the screening and identification of enzyme inhibitors is performed by: the method also comprises the following steps before the step (1): taking a representative acetylcholinesterase inhibitor as a positive control substance, taking a control substance with little or no inhibition activity on the activity of acetylcholinesterase as a negative control substance, mixing the positive control substance and the negative control substance to prepare an artificial mixed model solution, placing the immobilized acetylcholinesterase of claim 5 in the artificial mixed model solution for incubation, washing the solution by using a phosphate buffer solution and a methanol solution after the incubation is finished, collecting filtrate, further verifying by using a high performance liquid chromatography-flight time mass spectrometry combined technology, and if the positive control substance is screened out from the filtrate, successfully preparing the immobilized acetylcholinesterase;
preferably, the positive control is huperzine A; the negative control substances are gallic acid and ferulic acid.
8. The method of claim 6, wherein the screening and identification of enzyme inhibitors is performed by: in the step (1), the dosage of the immobilized acetylcholinesterase is 1-5 tablets, and the diameter is 1-12 mm; the dosage of the enzyme inhibitor model solution is 200-500 mu L.
9. A method of screening and identifying enzyme inhibitors according to any one of claims 6 to 8 wherein: in the step (1), the incubation conditions are as follows: incubating for 10-40 min at 25-50 ℃.
10. The method of screening and identifying enzyme inhibitors according to claims 6-9, wherein: when the sample to be detected is myrobalan, the setting conditions of all parameters in the high performance liquid chromatography-flight time mass spectrometry are as follows:
chromatographic conditions are as follows: a chromatographic column: ZORBAX Eclipse Plus C18 column (3 mm. times.150 mm, 1.8 μm); column temperature: 35 ℃; mobile phase C: 0.1% formic acid-acetonitrile and mobile phase D: 0.1% formic acid-water; the flow rate is 0.250 mL/min; sample introduction amount: 2 mu L of the solution; gradient program:
changing the constant speed of the mobile phase C from 5% to 15% and the constant speed of the mobile phase D from 95% to 85% in 0-5 min;
5-30 min, changing the uniform speed of the mobile phase C from 15% to 35%, and changing the uniform speed of the mobile phase D from 85% to 65%;
30-32 min, changing the constant speed of the mobile phase C from 35% to 5%, and changing the constant speed of the mobile phase D from 65% to 95%;
mass spectrum conditions: the mass spectrum model is as follows: agilent Q-TOF 6545 time-of-flight mass spectrometry; an ion source: electrospray ion source ESI; detection mode: a negative ion mode; scanning range: m/z is 50-1500; temperature of the drying gas: 350 ℃; flow rate of drying gas: 12.0L/min; the flow rate of the sheath gas: 10.0L/min; atomizing gas pressure: 40 psi; capillary voltage: 3500V; collision voltage: 135V; MS/MS optimized collision energy of parent ions: 10 to 60 eV.
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