CN115920793A - Preparation and application of angiotensin-converting enzyme ACE (angiotensin-converting enzyme) functionalized magnetic nano-microspheres - Google Patents
Preparation and application of angiotensin-converting enzyme ACE (angiotensin-converting enzyme) functionalized magnetic nano-microspheres Download PDFInfo
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Abstract
The invention discloses preparation and application of angiotensin converting enzyme ACE (angiotensin converting enzyme) functionalized magnetic nano microspheres, wherein the functionalized magnetic nano microspheres are prepared from magnetic graphene oxide, ethyl orthosilicate, aminopropyl triethoxysilane and angiotensin converting enzyme ACE; the method positions Angiotensin Converting Enzyme (ACE) targets, captures ACE ligands in fructus Choerospondiatis extract by means of ACE functional modified nano magnetic microspheres, characterizes the synthesized magnetic microspheres by means of Fourier transform infrared spectroscopy, a vibration sample magnetometer, a scanning electron microscope and a transmission electron microscope, and identifies the angiotensin converting enzyme ligands captured from the extract by means of UPLC-Q-active orbitrap-MS/MS. The method can quickly and efficiently screen 5 ACE inhibitory active ingredients from the fructus Choerospondiatis extract, and the synthesized ACE functionalized magnetic nano-microsphere can be screened specifically, and has good precision and high efficiency.
Description
Technical Field
The invention belongs to the technical field of functional materials and analysis, and particularly relates to preparation and application of angiotensin converting enzyme ACE (angiotensin converting enzyme) functionalized magnetic nano microspheres.
Background
Coronary Heart Disease (CHD) is one of the most harmful cardiovascular diseases in the world, is easy to cause myocardial ischemia, arrhythmia and the like, and seriously harms the physical and mental health of people. The fructus choerospondiatis is a traditional Chinese medicine used by Mongolian medicine and Tibetan medicine, has the effects of relieving heyi, calming heart, regulating qi and blood, promoting qi and blood circulation, relieving pain and the like, and has great value in the aspect of treating cardiovascular diseases. However, how to screen out target active ingredients with high specific activity from fructus choerospondiatis and clarify the drug effect material basis of fructus choerospondiatis for preventing and treating coronary heart disease has become a technical bottleneck for further research and development.
Modern pharmacological research shows that a large amount of active ingredients in the fructus choerospondiatis have various physiological activities and are used for resisting cardiovascular diseases such as arrhythmia, cardiac ischemia and the like in a cardiovascular system through various action mechanisms. Angiotensin Converting Enzyme (ACE) has the effects of resisting myocardial ischemia, resisting heart failure and the like, can increase blood supply of coronary arteries to expand epicardial coronary arteries, improve collateral circulation, prevent coronary artery spasm and increase coronary artery blood flow, is one of important action mechanisms for treating coronary heart disease, can develop and research active ingredients capable of being combined with ACE in fructus choerospondiatis, and is expected to find effective ingredients for treating coronary heart disease.
Fishing techniques based on receptor-ligand interactions have the unique advantage of identifying biomolecular interactions, allowing the search for the corresponding ligand for the receptor from complex mixtures; magnetic Nanoparticles (MNPs) have superparamagnetism, are easily modified and have high coupling capacity, and can play a role in bioanalysis/separation by virtue of the characteristics. Qualitative analysis of small molecule active ingredients based on magnetic ligand fishing combined with mass spectrometry, liquid phase technology or liquid mass spectrometry has become a powerful tool for interaction between biomacromolecules and active small molecules. Compared with separation methods such as centrifugation, ultrafiltration, dialysis, precipitation, electrophoresis and the like, the functionalized magnetic microspheres can quickly and specifically capture corresponding ligands, and are particularly suitable for screening active compounds from complex natural products. The method can be used for quickly screening certain enzyme inhibitors in the complex mixture, and can provide a new solution for extracting and identifying active pharmaceutical ingredients such as traditional Chinese medicines.
Disclosure of Invention
The invention aims to provide preparation and application of angiotensin converting enzyme ACE functionalized magnetic nano microspheres, and to capture ACE ligand components in fructus choerospondiatis extract based on a magnetic ligand fishing technology. The method has good specificity and high sensitivity, and the synthesized magnetic nano-microspheres can be recycled and have low cost. Compared with other methods, the method can obviously improve the experimental efficiency of screening the active ingredients in the traditional Chinese medicine and the natural products, combines the liquid phase, mass spectrum and liquid mass spectrometry technologies to carry out qualitative analysis on the active ingredients, has high specificity, good precision and low cost, and provides convenient conditions for screening a large amount of specific bioactive compounds.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the functionalized magnetic nano-microspheres are prepared from magnetic graphene oxide, tetraethoxysilane, aminopropyl triethoxysilane and angiotensin converting enzyme ACE.
A method for preparing angiotensin converting enzyme ACE functionalized magnetic nanospheres, which comprises the following steps:
(1) Synthesizing magnetic graphene oxide: by Fe 3+ And Fe 2+ Preparing Magnetic Graphene Oxide (MGO) nanoparticles by a chemical coprecipitation method; graphene oxide GO and FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 O is dispersed in distilled water, and the reaction condition is that nitrogen protects and isolates air, and the mixture is heated and stirred; adjusting pH of the mixed solution to alkaline condition, stirring, filtering suspension after reaction, separating magnetic material with the help of external magnetic field, distilling respectivelyWashing with water and ethanol, drying and grinding to obtain magnetic graphene oxide MGO nano particles;
(2) Synthesis of SiO 2 Coated magnetic graphene oxide: measuring a certain amount of tetraethoxysilane, adding distilled water, and ultrasonically stirring; adding the obtained milky white solution and the magnetic graphene oxide into absolute ethyl alcohol, mechanically stirring, gradually dropwise adding a glutaraldehyde solution, and continuing to react; finally, separating with magnet, synthesized GO @ Fe 3 O 4 @SiO 2 And (SMGO) nano particles are washed by 2% nitric acid, distilled water and absolute ethyl alcohol in sequence and dried to obtain the product.
(3) Synthesizing ACE functionalized magnetic nano microspheres: dispersing the SMGO nano-particles into an ethanol-water solution, and adjusting the pH to be acidic by 0.1mol/L hydrochloric acid; then slowly dropwise adding aminopropyl triethoxysilane APTES, stirring and continuously reacting, separating the synthesized product by using a magnet, washing by using ethanol and drying; dispersing the dried solid in 5% glutaraldehyde solution, and separating with magnet to recover SMGO-NH 2 Washing the nanoparticles with Tris-HCl buffer solution to remove redundant glutaraldehyde; mixing with buffer solution containing ACE, and shaking to synthesize ACE functional magnetic microsphere (SMGO-ACE).
Graphene oxide GO and FeCl in step (1) 3 ·6H 2 O and FeCl 2 ·4H 2 The mass ratio of O is as follows: 0.5:2.16:0.8, the reaction temperature rose to 70 ℃.
The volume ratio of the ethyl orthosilicate, the distilled water and the absolute ethyl alcohol in the step (2) is 7:21:65.
the volume ratio of aminopropyltriethoxysilane APTES to ethanol to glutaraldehyde is 1:50:5.
the saturation magnetization of the prepared magnetic nano-microsphere ACE modified silicon dioxide coated magnetic nano-microsphere is 13.20 emu 8729g -1 。
An application of angiotensin converting enzyme ACE functionalized magnetic nano-microspheres is applied to capturing active ingredients in fructus Choerospondiatis extract and rapidly screening out potential ACE enzyme inhibitors.
The application method comprises the following steps:
(1) Magnetic nano-microsphere capturing active ingredient
Extracting a certain amount of fructus Choerospondiatis powder with ethanol for three times, mixing extractive solutions, filtering, lyophilizing, and adding methanol to dissolve; dispersing the ACE functionalized silicon dioxide coated magnetic nano microspheres in the fructus Choerospondiatis extract, capturing ligands combined with angiotensin converting enzyme, washing with Tris-HCl buffer solution to remove non-specific combined ligands, and incubating with methanol to elute the extracted ligands;
(2) Active ingredients of UPLC-Q-active orbitrap-MS/MS qualitative analysis
And filtering the eluent by a 0.22-micron membrane, injecting an UPLC-Q-active orbitrap-MS/MS system to qualitatively analyze active ingredients captured by the magnetic nano-microspheres, and further verifying the specificity of the synthesized magnetic microsphere fishing ligand.
Preparing a fructus choerospondiatis extracting solution: the fructus Choerospondiatis powder is extracted with 75% (v/v) ethanol at 85 deg.C in water bath, and the concentration of the extractive solution is 2 mg/mL after dissolving in methanol.
The mass-volume ratio of the functionalized magnetic microspheres to the extracting solution used in the application is 5: and 4, stirring at 200 rpm for 30min, eluting the magnetic microspheres for 3 times by using Tris-HCl buffer solution with the pH of 7.0, removing non-specific binding ligands, incubating for 1h with 1mL of methanol, and dissociating the extracted ligands.
Compared with the prior art, the technical scheme of the invention can obtain the following beneficial effects:
(1) The ACE functionalized magnetic nano-microsphere is synthesized, and a rapid, simple and convenient method can be established for detecting and screening the active components interacting with ACE from fructus Choerospondiatis.
(2) The magnetic nano-microsphere with the ACE function has the advantages of good specificity, good precision, low cost and capability of realizing high-throughput screening.
(3) The established method can be used for fixing macromolecular targets and provides convenient conditions for screening a large number of specific bioactive compounds from complex natural products.
Drawings
FIG. 1 shows SMGO-NH 2 And infrared spectroscopy of SMGO-ACE;
FIG. 2 is a magnetization curve for SMGO, SMGO @ ACE;
FIG. 3 is a scanning electron microscope image of SMGO and SMGO @ ACE;
FIG. 4 is a TEM image of SMGO and SMGO @ ACE.
Detailed Description
The invention is further described with reference to the accompanying drawings:
the functionalized magnetic nano-microspheres are prepared from magnetic graphene oxide, tetraethoxysilane, aminopropyl triethoxysilane and angiotensin converting enzyme ACE.
A preparation method of angiotensin converting enzyme ACE functionalized magnetic nano microspheres comprises the following steps:
(1) Synthesizing magnetic graphene oxide: by means of Fe 3+ And Fe 2+ Magnetic Graphene Oxide (MGO) nanoparticles were prepared by a chemical coprecipitation method. Mixing Graphene Oxide (GO) and FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 O is dispersed in distilled water, and the reaction conditions are that nitrogen is protected from air and heated and stirred. And adjusting the pH value of the mixed solution to be alkaline, continuously stirring, filtering the suspension after the reaction is finished, separating the magnetic material by means of an external magnetic field, washing with distilled water and ethanol respectively, drying and grinding to obtain the Magnetic Graphene Oxide (MGO) nanoparticles.
(2) Synthesis of SiO 2 Coated magnetic graphene oxide: measuring a certain amount of tetraethoxysilane, adding distilled water, and ultrasonically stirring; and adding the obtained milky white solution and the magnetic graphene oxide into absolute ethyl alcohol, mechanically stirring, and gradually dropwise adding a glutaraldehyde solution for continuous reaction. Finally, separating with magnet, synthesized GO @ Fe 3 O 4 @SiO 2 And (SMGO) nano particles are washed by 2% nitric acid, distilled water and absolute ethyl alcohol in sequence and dried to obtain the product.
(3) Synthesizing ACE functionalized magnetic nano microspheres: the SMGO nanoparticles were dispersed in an ethanol-water solution and the pH was adjusted to acidic with 0.1mol/L hydrochloric acid. Then, APTES is slowly dropped into the mixture, the mixture is stirred and continuously reacted, and the synthesized product is separated by a magnet, washed by ethanol and dried. Dispersing the dried solid in 5% glutaraldehyde solutionIn the liquid, SMGO-NH is separated and recovered by a magnet 2 The nanoparticles were washed with Tris-HCl buffer to remove excess glutaraldehyde. Mixing with buffer solution containing ACE, and shaking to synthesize ACE functional magnetic microsphere (SMGO-ACE).
Wherein the mass ratio of Graphene Oxide (GO), ferric chloride hexahydrate and ferrous chloride tetrahydrate in the step (1) is as follows: 0.5:2.16:0.8, the reaction temperature is raised to 70 DEG C
The volume ratio of the ethyl orthosilicate, the distilled water and the absolute ethyl alcohol in the step (2) is 7:21:65.
the volume ratio of Aminopropyltriethoxysilane (APTES), ethanol-water and glutaraldehyde in the step (3) is 1:50:5.
the saturation magnetization of the magnetic nano-microsphere coated by silicon dioxide modified by the magnetic nano-microsphere ACE prepared by the invention is 13.20 emu 8729g -1 。
Example 1
Preparing ACE functional magnetic nano microspheres:
(1) Synthesizing magnetic graphene oxide: first, 0.50 g of graphene oxide was dispersed in 100 mL of distilled water, and then stirred to 70 ℃ with heat. 2.16 g FeCl 3 •6H 2 O and 0.80 gFeCl 2 •4H 2 O was dissolved in 40.0 mL of distilled water, and the solution was added to the above dispersion suspension at 70 ℃ and 300 rpm. After adjusting the pH to 10 with glutaraldehyde (25%, w/v), the dispersion was kept at 70 ℃ for a further 6 h. The whole reaction process is carried out under pure nitrogen flow. After the reaction was completed, the suspension was filtered, washed 3 times with distilled water and ethanol, respectively, and then dried in a vacuum oven at 60 ℃ for 6 hours. The product was ground and MGO nanoparticles were isolated with a magnet.
(2) Synthesizing silicon dioxide modified magnetic graphene oxide: 6.3 mL of distilled water and 2.1 mL of TEOS were stirred for 3 min and sonicated for 1 min to obtain a milky white solution. Adding the milky white solution into 49.5 mL of absolute ethyl alcohol, wherein the absolute ethyl alcohol comprises 0.50 g of magnetic graphene oxide GO @ Fe 3 O 4 And (3) nanoparticles. After stirring at 300 rpm for 10 min at 0 ℃, 2.0 mL of glutaraldehyde was added dropwise to the dispersion solution. Then at 0 ℃ for 10 h. Finally, using a magnet to divideIon, GO @ Fe 3 O 4 @SiO 2 (SMGO) nanoparticles were washed sequentially with 2% nitric acid, distilled water and absolute ethanol and dried in vacuo at 60 ℃ for 6 h.
(3) Preparing ACE functionalized magnetic microspheres: 0.10 g of SMGO nanoparticles were suspended in 50 mL of ethanol-water solution (1,v/v) and the pH was adjusted to 3-4 with 0.1mol/L hydrochloric acid. 1.0 mL of APTES was then added dropwise and stirring was continued at 50 ℃ for 5 h. The product was separated under the action of a permanent magnet, washed 4 times with ethanol and dried at 40 ℃ for 3 h. After suspending in 5.0 mL of 5% glutaraldehyde solution for 1h, 5.0 mg of SMGO-NH was extracted by magnetic separation 2 The nanoparticles were washed 3 times with Tris-HCl buffer (100mM, pH 7.0) to remove excess glutaraldehyde. Mixing with 2.0 mL buffer solution containing 10 mu LACE, shaking at 200 rpm at 4 ℃ for 16 h, and synthesizing the ACE functionalized magnetic microsphere (SMGO-ACE).
ACE functionalized magnetic nanospheres were characterized by fourier transform infrared spectroscopy (FTIR), scanning Electron Microscopy (SEM), vibrating Sample Magnetometer (VSM) and Transmission Electron Microscopy (TEM).
SMGO-NH 2 And the infrared spectrum of SMGO-ACE is shown in figure 1. SMGO-NH 2 In which 2N-H bonds are present and 1600cm -1 The absorption at (a) is due to the symmetric vibration of N-H, thus exhibiting a double peak; and 1600cm in SMGO-ACE -1 The absorption shows a clear single sharp peak, indicating that one of the N-H bonds is replaced and the newly formed C-N bond is at 1100cm -1 There was moderate absorption. Therefore, the result of FT-IR analysis provides a basis for the formation of SMGO-ACE nanometer materials.
The magnetic properties of the materials were studied with VSM. According to the curve of FIG. 2, the saturation magnetization of SMGO and SMGO-ACE are 98.42 emu 8729g -1 And 13.20 emu 8729g -1 Sufficient to separate the nanomaterial from the sample solution under an external magnetic field. Compared with SMGO, the magnetization intensity of SMGO-ACE is reduced by 85.22 emu 8729g -1 This is due to the enzyme grafted on the silica surface.
The morphological study of smgo @ ace nanocomposites is shown in figure 3. In previous studies, it was shown that graphene oxide isA single foil layer. Mixing Fe 3 O 4 After the particles are combined, the surface becomes rough with spherical or ellipsoidal matter. After coating with silica, the agglomeration phenomenon is reduced. After modification and ACE connection, a thin layer of graphene oxide is hardly visible, gaps generated by silicon dioxide aggregation are prevented from being filled, and the whole surface becomes rougher and fuller. The structural morphology of the SMGO @ ACE nanocomposite is shown in FIG. 4. Black dots (SMGO @ ACE) on the cross section are evenly anchored on the surface of the nanometer material, and a shell-shaped layered structure can be seen in the material. The above proves the successful synthesis of the ACE functionalized magnetic nano-microsphere.
Example 2 screening of active ingredients in the extract of choerospondias axillaris by using magnetic microspheres:
(1) Preparing an extracting solution: extracting fructus Choerospondiatis powder with 75% (v/v) ethanol for three times, and incubating in water bath at 85 deg.C. After filtration through three layers of gauze with a pore diameter of 4.5mm, the 3 extraction solutions were combined, freeze-dried and dissolved to 2 mg/mL with a methanol solution.
(2) The fishing process of the magnetic ligand comprises the following steps: suspending the ACE functional magnetic microspheres in an extracting solution, incubating at 37 ℃, stirring at 200 rpm, and magnetically separating ACE ligands. Then, the non-specifically bound ligand was removed by washing 3 times with Tris-HCl buffer (pH 7.0), and the extracted ligand was dissociated by incubating with a certain amount of methanol for 1 hour.
(3) UPLC-Q-active orbitrap-MS/MS analysis of active ingredients:
the chromatographic conditions were as follows:
a chromatographic column: a Shim-pack XR-ODS II column (75 mm. Times.3.0 mm,1.7 μm); mobile phase: a is 0.4% acetic acid-water, B: acetonitrile; the gradient elution procedure was: 0-2 mim:5-15% b: 15-25% by weight of B;3-7 min:25-45% of B;7-9 min:45-60% of B;9-10 min:60-75% by weight B: 75-5% by mass B;10.01-12 min:5% B, flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 10.μ L.
The mass spectrometry conditions were as follows:
an ion source: an electrospray ionization source (ESI source); electrospray voltage: and (3) positive electrode: 3.0 kV, negative electrode: -3.0 kV; scanning mode: positive and negative ion monitoring modes; flow rate of sheath gas: 35 Arb; auxiliary airflow: 10 Arb; capillary tubeThe temperature is 350 ℃; ion lens voltage frequency: 55; temperature of heat source of auxiliary gas: 350 ℃; automatic Gain Control (AGC): 1X 10 6 An ion;
maximum ion Implantation Time (IT) 50 ms, isolation window: 2.0 m/z; collision energy: 20-40 eV; scanning range: m/z 50-1500
5 ACE ligands which are flavonoids and organic acid compounds are detected successfully by UPLC-Q-active orbitrap-MS/MS analysis.
Performing inhibition experiment investigation on a lisinopril reference substance and an extracting solution by respectively fishing using SMGO, SMGO-NH2 and SMGO-ACE, and analyzing a chromatographic result to show that the peak area of lisinopril in an eluent is obviously increased after the SMGO-active ACE microspheres are captured; and MGO, SMGO-inactive ACE-fishing extract is difficult to detect or neglects lisinopril peak area, which confirms the specificity of the synthesized ACE functionalized magnetic microsphere for screening ACE ligands. The magnetic microspheres and the extracting solution are subjected to fishing-elution circulation for 5 times under the same condition, the affinity among SMGO, ACE and ACE ligands is stable, and the results show that the binding efficiency is still up to 90% of the first period by comparing the lisinopril peak area; and the binding rate of lisinopril was not significantly lost when stored at 4 ℃ for 5 days (RSD <8%, n = 5), and 3 batches of smgo @ ace showed similar binding efficiency (RSD < 10%).
The method positions Angiotensin Converting Enzyme (ACE) targets, captures ACE ligands in fructus Choerospondiatis extract by means of ACE functionalized nano magnetic microspheres, characterizes the synthesized magnetic microspheres by means of Fourier transform infrared spectroscopy, a vibration sample magnetometer, a scanning electron microscope and a transmission electron microscope, and identifies the angiotensin converting enzyme ligands captured from the extract by means of UPLC-Q-active orbitrap-MS/MS. The method can quickly and efficiently screen 5 ACE inhibitory active ingredients from the fructus Choerospondiatis extract, and the synthesized ACE functionalized magnetic nano-microsphere can be screened specifically, and has good precision and high efficiency. Compared with the ACE inhibitor lisinopril, the experiment has strong binding activity of the magnetic microspheres and the active ingredients in the fructus choerospondiatis, can provide ideas for extracting and separating the active ingredients from other natural medicines, traditional Chinese medicines and the like, and can quickly screen out target ligands which play pharmacological actions in complex components.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. An angiotensin converting enzyme ACE functionalized magnetic nano microsphere is characterized in that: the functionalized magnetic nano-microsphere is prepared from magnetic graphene oxide, ethyl orthosilicate, aminopropyl triethoxysilane and angiotensin converting enzyme ACE.
2. A method of preparing angiotensin converting enzyme ACE functionalized magnetic nanospheres according to claim 1, characterized in that: the method comprises the following steps:
(1) Synthesizing magnetic graphene oxide: by Fe 3+ And Fe 2+ Preparing Magnetic Graphene Oxide (MGO) nanoparticles by a chemical coprecipitation method; graphene oxide GO and FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 O is dispersed in distilled water, and the reaction condition is that nitrogen protects and isolates air, and the mixture is heated and stirred; adjusting the pH value of the mixed solution to be alkaline, continuously stirring, filtering the suspension after the reaction is finished, separating the magnetic material by means of an external magnetic field, washing with distilled water and ethanol respectively, drying and grinding to obtain Magnetic Graphene Oxide (MGO) nanoparticles;
(2) Synthesis of SiO 2 Coated magnetic graphene oxide: measuring a certain amount of tetraethoxysilane, adding distilled water, and ultrasonically stirring; adding the obtained milky white solution and the magnetic graphene oxide into absolute ethyl alcohol, mechanically stirring, gradually dropwise adding a glutaraldehyde solution, and continuing to react; finally, separating with magnet, synthesized GO @ Fe 3 O 4 @SiO 2 (SMGO) nanoparticlesWashing with 2% nitric acid, distilled water and absolute ethyl alcohol in sequence, and drying to obtain a product;
(3) Synthesizing ACE functionalized magnetic nano microspheres: dispersing the SMGO nano-particles into an ethanol-water solution, and adjusting the pH to be acidic by 0.1mol/L hydrochloric acid; then slowly dripping aminopropyltriethoxysilane APTES, stirring and continuously reacting, separating the synthesized product by using a magnet, washing by using ethanol and drying; dispersing the dried solid in 5% glutaraldehyde solution, and separating with magnet to recover SMGO-NH 2 Washing the nanoparticles with Tris-HCl buffer solution to remove redundant glutaraldehyde; mixing with buffer solution containing ACE, and shaking to synthesize ACE functionalized magnetic microsphere (SMGO-ACE).
3. The preparation method of the angiotensin-converting enzyme ACE functionalized magnetic nanosphere according to claim 2, wherein the preparation method comprises the following steps: graphene oxide GO and FeCl in step (1) 3 ·6H 2 O and FeCl 2 ·4H 2 The mass ratio of O is as follows: 0.5:2.16:0.8, the reaction temperature rose to 70 ℃.
4. The preparation method of the angiotensin-converting enzyme ACE functionalized magnetic nanosphere according to claim 2, wherein the preparation method comprises the following steps: the volume ratio of the ethyl orthosilicate, the distilled water and the absolute ethyl alcohol in the step (2) is 7:21:65.
5. the preparation method of the angiotensin-converting enzyme ACE functionalized magnetic nanosphere according to claim 1, wherein the preparation method comprises the following steps: the volume ratio of aminopropyltriethoxysilane APTES to ethanol to glutaraldehyde is 1:50:5.
6. an application of angiotensin converting enzyme ACE functionalized magnetic nano-microspheres is characterized in that: the method is applied to capture active ingredients in the fructus choerospondiatis extracting solution and quickly screen out potential ACE enzyme inhibitors.
7. The use of the angiotensin-converting enzyme ACE functionalized magnetic nanospheres according to claim 6, wherein the magnetic nanospheres comprise: the application method comprises the following steps:
(1) Magnetic nano-microsphere capturing active ingredient
Extracting a certain amount of fructus Choerospondiatis powder with ethanol for three times, mixing extractive solutions, filtering, freeze drying, and dissolving with methanol; dispersing the magnetic nano-microspheres coated with the ACE functionalized silicon dioxide in an fructus Choerospondiatis extracting solution, capturing a ligand combined with angiotensin converting enzyme, washing with a Tris-HCl buffer solution to remove a non-specific combined ligand, and incubating with methanol to elute the extracted ligand;
(2) UPLC-Q-active inhibition-MS/MS qualitative analysis active ingredient eluent is filtered by a 0.22 mu m membrane, injected into an UPLC-Q-active inhibition-MS/MS system to qualitatively analyze active ingredients captured by the magnetic nano microspheres, and further verifies the specificity of the synthesized magnetic microsphere fishing ligand.
8. The use of the angiotensin-converting enzyme ACE-functionalized magnetic nanospheres according to claim 7, wherein the angiotensin-converting enzyme ACE-functionalized magnetic nanospheres are characterized in that: the mass-volume ratio of the functionalized magnetic microspheres to the extracting solution used in the application is 5: and 4, stirring at 200 rpm for 30min, eluting the magnetic microspheres for 3 times by using Tris-HCl buffer solution with the pH of 7.0, removing non-specific binding ligands, incubating for 1h with 1mL of methanol, and dissociating the extracted ligands.
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Application publication date: 20230407 |