CN114250220A - Preparation and application of dopamine-coated sea urchin-shaped manganese dioxide hollow microspheres - Google Patents
Preparation and application of dopamine-coated sea urchin-shaped manganese dioxide hollow microspheres Download PDFInfo
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- CN114250220A CN114250220A CN202111598592.8A CN202111598592A CN114250220A CN 114250220 A CN114250220 A CN 114250220A CN 202111598592 A CN202111598592 A CN 202111598592A CN 114250220 A CN114250220 A CN 114250220A
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- acetylcholinesterase
- manganese dioxide
- dopamine
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- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 title claims abstract description 139
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 title claims abstract description 76
- 239000004005 microsphere Substances 0.000 title claims abstract description 61
- 229960003638 dopamine Drugs 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 108010022752 Acetylcholinesterase Proteins 0.000 claims abstract description 82
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- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 claims abstract description 9
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- 239000012286 potassium permanganate Substances 0.000 claims description 12
- ZRJBHWIHUMBLCN-SEQYCRGISA-N Huperzine A Natural products N1C(=O)C=CC2=C1C[C@H]1/C(=C/C)[C@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-SEQYCRGISA-N 0.000 claims description 11
- HXJUTPCZVOIRIF-UHFFFAOYSA-N sulfolane Chemical compound O=S1(=O)CCCC1 HXJUTPCZVOIRIF-UHFFFAOYSA-N 0.000 claims description 11
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- ZRJBHWIHUMBLCN-UHFFFAOYSA-N Shuangyiping Natural products N1C(=O)C=CC2=C1CC1C(=CC)C2(N)CC(C)=C1 ZRJBHWIHUMBLCN-UHFFFAOYSA-N 0.000 claims description 10
- ZRJBHWIHUMBLCN-YQEJDHNASA-N huperzine A Chemical compound N1C(=O)C=CC2=C1C[C@H]1\C(=C/C)[C@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-YQEJDHNASA-N 0.000 claims description 10
- ZRJBHWIHUMBLCN-BMIGLBTASA-N rac-huperzine A Natural products N1C(=O)C=CC2=C1C[C@@H]1C(=CC)[C@@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-BMIGLBTASA-N 0.000 claims description 10
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
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- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 claims description 6
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- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 claims description 5
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- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 claims description 2
- 210000000582 semen Anatomy 0.000 claims description 2
- GANZODCWZFAEGN-UHFFFAOYSA-N 5-mercapto-2-nitro-benzoic acid Chemical compound OC(=O)C1=CC(S)=CC=C1[N+]([O-])=O GANZODCWZFAEGN-UHFFFAOYSA-N 0.000 claims 2
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- MCHZKGNHFPNZDP-UHFFFAOYSA-N 2-aminoethane-1,1,1-triol;hydrochloride Chemical compound Cl.NCC(O)(O)O MCHZKGNHFPNZDP-UHFFFAOYSA-N 0.000 abstract 1
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- ASUTZQLVASHGKV-JDFRZJQESA-N galanthamine Chemical compound O1C(=C23)C(OC)=CC=C2CN(C)CC[C@]23[C@@H]1C[C@@H](O)C=C2 ASUTZQLVASHGKV-JDFRZJQESA-N 0.000 description 2
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- GFFIJCYHQYHUHB-UHFFFAOYSA-N 2-acetylsulfanylethyl(trimethyl)azanium Chemical class CC(=O)SCC[N+](C)(C)C GFFIJCYHQYHUHB-UHFFFAOYSA-N 0.000 description 1
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- XSVMFMHYUFZWBK-NSHDSACASA-N Rivastigmine Chemical compound CCN(C)C(=O)OC1=CC=CC([C@H](C)N(C)C)=C1 XSVMFMHYUFZWBK-NSHDSACASA-N 0.000 description 1
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- ASUTZQLVASHGKV-UHFFFAOYSA-N galanthamine hydrochloride Natural products O1C(=C23)C(OC)=CC=C2CN(C)CCC23C1CC(O)C=C2 ASUTZQLVASHGKV-UHFFFAOYSA-N 0.000 description 1
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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Abstract
The invention provides a preparation method of dopamine-coated sea urchin-shaped manganese dioxide hollow microspheres, which comprises the steps of completely dissolving manganese dioxide hollow microspheres in a trihydroxymethyl aminomethane hydrochloride solution, adding dopamine hydrochloride, carrying out ultrasonic treatment for 10-25 min, centrifuging, washing and drying to obtain dopamine-coated manganese dioxide hollow microspheres. The hollow microsphere has the advantages of thermal stability, easy synthesis, porous structure, low density, short diffusion path, high specific surface area, rich active sites and the like, and the formed aminated manganese dioxide hollow microsphere is favorable for immobilizing acetylcholinesterase, so that the hollow microsphere is used as a novel enzyme immobilization carrier, combines the capillary electrophoresis technology after the acetylcholinesterase is immobilized, has the characteristics of high separation efficiency, short analysis time, less sample consumption, low operation cost and the like when used for screening the acetylcholinesterase inhibitor from traditional Chinese medicines, and provides technical support for discovery of new medicines for treating Alzheimer's disease.
Description
Technical Field
The invention relates to a preparation method of dopamine-coated manganese dioxide hollow microspheres, and also relates to a method for preparing immobilized acetylcholinesterase by using the dopamine-coated manganese dioxide hollow microspheres as immobilized enzyme carriers, and application of the immobilized acetylcholinesterase in screening acetylcholinesterase inhibitors in traditional Chinese medicines.
Background
Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases in the elderly that is associated with a reduction in the neurotransmitter acetylcholine (ACh) in the brain. The pathogenesis of AD has not yet been fully elucidated, and at present, the theory of abeta (Amyloid β -protein), the theory of cholinergic activity, the theory of abnormal phosphorylation of Tau (Microtubule-associated protein Tau) protein, the theory of oxidative stress, and the theory of inflammatory response are mainly used. Currently, the main drugs for the clinical treatment of AD are acetylcholinesterase inhibitors, including tacrine, donepezil, rivastigmine, galantamine and huperzine a. By inhibiting the activity of acetylcholinesterase, the cholinergic function of AD patients can be improved, and the level of neurotransmitter acetylcholine in brain can be increased, so that the purpose of treating AD can be achieved.
Because of relatively low toxicity and definite therapeutic effect, Chinese herbs become the hot point of research on the research and development of the drugs for improving learning and memory abilities. Therefore, the development of acetylcholinesterase inhibitors from traditional Chinese medicines, which have the advantages of suitability for long-term administration to patients, high selectivity, small toxic and side effects, wide action range and the like, has become an important means for finding new drugs for treating alzheimer's disease (a.p. Murray, m.b. Faraoni, m.j. Castro, n.p. Alza, v.cavallaro,Curr. Neuropharmacol.,2013(11)388-413). At present, methods for screening acetylcholinesterase inhibitors mainly include an Ellman's colorimetric method based on an ultraviolet spectrophotometer, a TLC thin-layer biological self-development method, a fluorescence analysis method, a liquid chromatography-mass spectrometry combined method and the like. An Ellman's colorimetric method based on an ultraviolet spectrophotometer is the earliest method for screening acetylcholinesterase inhibitors, and the screening principle is that acetylcholinesterase hydrolyzes a substrate, namely chlorinated acetylthiocholine into thiocholine, and the thiocholine reacts with a color-developing agent, namely 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) to generate a compound, namely 5-sulfur-2-nitrobenzoic acid (TNB) with characteristic absorption at the wavelength of 412 nm. The addition of the acetylcholinesterase inhibitor can obviously inhibit acetylcholineThe activity of the esterase can be measured by an ultraviolet spectrophotometer to increase or decrease the absorbance, and the inhibitory activity of a sample to be measured on the acetylcholinesterase can be indirectly measured. The Ellman's colorimetric method has the advantages of simple operation, low cost and the like, thereby being widely used. In the screening process of enzyme inhibitors, immobilized enzymes are favored by researchers due to the characteristics of wide range of pH value and temperature tolerance, good storage stability, repeated use, easy storage and the like (X.Liu, I.Azhar, H.Khan, Q.S. Qu, M.M. Tian, L. Yang,J. Chromatogr. A,2020 (1609) 460454)。
manganese dioxide has good thermal stability, easy synthesis, porous structure and abundant active sites. In addition, manganese dioxide hollow microspheres (h-MnO) with sea urchin shape2) Has the characteristics of low density, short diffusion path, high surface area and the like, and has wide application prospect in the aspect of enzyme immobilization carriers. However, the manganese dioxide hollow microspheres in the shape of sea urchins are used as enzyme immobilization carriers for immobilizing acetylcholinesterase, the stability of free enzymes is poor, and the enzyme immobilization efficiency is low, so that the manganese dioxide hollow microspheres are modified to be used as immobilized acetylcholinesterase carriers for screening acetylcholinesterase inhibitors.
Disclosure of Invention
The invention aims to provide a preparation method of a dopamine-coated sea urchin-shaped manganese dioxide hollow microsphere;
the invention also aims to provide the application of the dopamine-coated echinoid manganese dioxide hollow microspheres as a carrier in immobilizing acetylcholinesterase;
the invention further aims to provide application of the immobilized acetylcholinesterase based on the dopamine-coated sea urchin-shaped manganese dioxide carrier in screening of acetylcholinesterase inhibitors in traditional Chinese medicines.
Preparation of dopamine-coated sea urchin-shaped manganese dioxide hollow microspheres
(1) Preparing hollow manganese dioxide microspheres: dissolving potassium permanganate in ultrapure water, adding hydrochloric acid, and stirring at room temperature for 30-40 min; transferring the obtained mixed solution into a high-pressure reaction kettle of polytetrafluoroethylene, and reacting for 3-5 h at 120-140 ℃;cooling to room temperature after the reaction is finished, centrifuging, washing the obtained product with ethanol and ultrapure water for a plurality of times, and drying to be anhydrous to obtain the manganese dioxide hollow microspheres (h-MnO)2) (ii) a The molar ratio of potassium permanganate to hydrochloric acid is 1: 10-1: 29.
(2) Preparing dopamine-coated manganese dioxide hollow microspheres: completely dissolving the prepared manganese dioxide hollow microspheres in Tris (hydroxymethyl) aminomethane hydrochloride solution (Tris-HCl), adding dopamine hydrochloride, carrying out ultrasonic treatment for 10-25 min, centrifuging, washing the obtained product with ethanol and ultrapure water for several times, and drying to be anhydrous to obtain dopamine-coated manganese dioxide hollow microspheres (h-MnO)2@ PDA). The mass ratio of the hollow manganese dioxide microspheres to the dopamine hydrochloride is 1: 1-1: 4.
Fig. 1 is a transmission electron microscope image and a scanning electron microscope image of the hollow manganese dioxide microspheres and dopamine-coated hollow manganese dioxide microspheres of the present invention. Transmission electron microscope and scanning electron microscope tests show that the prepared manganese dioxide microspheres have obvious hollow structures (fig. 1a and 1 b), and dopamine is modified on the surfaces of the dopamine-coated manganese dioxide microspheres (fig. 1c and 1 d) besides the original hollow structures. Research shows that the specific surface area of the material can be increased by coating dopamine on the surface of the hollow manganese dioxide microsphere, and the formed aminated hollow manganese dioxide microsphere can realize immobilization of acetylcholinesterase through Schiff base reaction between glutaraldehyde and amino.
Secondly, preparation of immobilized acetylcholinesterase
Manganese dioxide hollow microsphere (h-MnO) coated with dopamine2@ PDA) is dispersed in phosphate buffer solution, Glutaraldehyde (GA) solution is added, and activation is carried out for 2-4 h under the condition of violent shaking; then adding an acetylcholinesterase solution into the suspension under the condition of violent shaking, shaking for 1-3 h at room temperature, centrifuging to collect a mixture, and washing by using a phosphate buffer solution to obtain immobilized acetylcholinesterase (h-MnO)2@ PDA immobilized AChE). The immobilized acetylcholinesterase obtained is dispersed in phosphate buffer and dissolved in water at 4oAnd (C) storing. A scheme for the preparation of immobilized acetylcholinesterase is shown in FIG. 2.
The pH value of the phosphate buffer solution is 5-11, and the mass percentage of the glutaraldehyde solution is 5-25%; the concentration of the acetylcholinesterase solution is 0.2-1 mg/mL. The mass ratio of h-MnO2@ PDA to glutaraldehyde is 1: 940-1: 1900; the mass ratio of h-MnO2@ PDA to acetylcholinesterase is 1: 0.04-1: 0.09.
Fig. 3 is a graph showing the results of pH stability (a) and thermal stability (b) of dopamine-coated manganese dioxide hollow microsphere immobilized acetylcholinesterase. As can be seen, compared with the free enzyme, the immobilized acetylcholinesterase has the advantages of pH value (pH tolerance range is 5-11), stability and temperature tolerance (temperature tolerance range is 50-90)oC) Wide range, repeated use (5 times of repeated use, 63.78 percent of the initial activity is still kept), and the like. Therefore, the dopamine-coated sea urchin-shaped manganese dioxide hollow microspheres as a novel enzyme immobilization carrier have good chemical and physical stability, and can be used for screening acetylcholinesterase inhibitors. In addition, the prepared immobilized acetylcholinesterase has the advantages of wide range of pH value and temperature tolerance, good storage stability, repeated use, easy storage and the like.
Feasibility study of immobilized acetylcholinesterase for screening enzyme inhibitor
Representative acetylcholinesterase inhibitor huperzine A is used as a positive drug, and the positive drug is prepared into a solution. The established manganese dioxide microsphere immobilized acetylcholinesterase coated with dopamine is applied to the research of positive drug inhibition kinetics. The method comprises the following specific steps:
(1) mixing 50 μ L of immobilized acetylcholinesterase with 25 μ L of developer 5, 5-dithiobis- (2-nitrobenzoic acid) (DTNB), adding into 50 μ L of enzyme-catalyzed substrate acetylthiocholine chloride (AThCH), adding into the mixture at 37 μ LoC, incubating for 10min, centrifuging after the incubation is finished to terminate the reaction, collecting supernatant, and determining the peak area of the reaction product of the blank group by capillary electrophoresis;
(2) adding 25 μ L positive control drug huperzine A into 50 μ L immobilized acetylcholinesterase at 37%oC, incubating for 10 min; then adding enzyme reaction substrate acetylthiocholine chloride AThCH50 μ L and color-developing agent DTNB25 μ L into the above solutionIn a pre-incubation solution at 37oAnd C, incubating for 10min, collecting supernatant after the incubation is finished, and determining the peak area of the reaction product by capillary electrophoresis.
FIGS. 4a and b show the peak areas of the products of steps (1) and (2), respectively. As can be seen from FIG. 4, the peak area of the product obtained in step (2) is smaller than that of the product obtained in step (1), which indicates that the positive control drug huperzine A has an inhibitory effect on the activity of immobilized acetylcholinesterase.
Chromatographic conditions for the capillary electrophoresis assay described above: quartz capillary column: 50 μm i.d. x 33cm, limited length 24.5cm (Jian Feng chromatographic devices, Inc. in North and Hebei Yongnian); and (3) sample introduction mode: sampling at 50 mbar multiplied by 3 s; separation voltage: 25 kV; detection wavelength: 412 nm.
FIG. 5 is a graph showing reciprocal double plots of the concentrations of huperzine A in the immobilized acetylcholinesterase, respectively, 0. mu.M (a), 20. mu.M (b), 40. mu.M (c) and 70. mu.M (d). As can be seen from FIG. 4, the four curves intersect in the second quadrant and the Michaelis constant K increases with increasing huperzine A concentrationmThe maximum reaction rate v increases with increasing huperzine A concentrationmaxThe reduction shows that the huperzine A is a mixed inhibitor, and the feasibility of applying the prepared immobilized acetylcholinesterase to an enzyme inhibitor screening model in the medicine is demonstrated.
Fourthly, screening natural drug enzyme inhibitor by immobilized acetylcholinesterase
(1) Preparing a traditional Chinese medicine extracting solution: air-drying the natural medicine by using a pulverizer, pulverizing, ultrasonically extracting for 1-3 h by using ultrapure water, and centrifuging; the combined supernatants were evaporated to dryness on a rotary evaporator and dissolved in phosphate buffer (30 mM, pH 9.0) to give 250mg/mL of the herbal extract. Wherein the natural medicines comprise Inula salix, herba Inulae, semen Ziziphi Spinosae, rhizoma corydalis, Atractylodis rhizoma, fructus Zanthoxyli and fructus Schisandrae.
(2) Immobilizing acetylcholinesterase (h-MnO)2@ PDA immobilized AChE) and a color-developing agent 5, 5-dithiobis- (2-nitrobenzoic acid) (DTNB) are added into an enzyme catalysis substrate acetylthiocholine chloride (AThCH) for incubation, and after the incubation is finished, a supernatant is collected and a blank group is determined by capillary electrophoresisPeak area of the reaction product;
(3) adding a natural medicine extracting solution into immobilized acetylcholinesterase for pre-incubation, adding an enzyme reaction substrate, namely acetylthiocholine chloride and a color-developing agent DTNB into the pre-incubation solution for incubation, collecting supernatant after the incubation is finished, and determining the peak area of a reaction product through capillary electrophoresis;
in the steps (2) and (3), the dosage of the immobilized acetylcholinesterase is 50-200 mu L, the dosage of the enzyme catalysis substrate AThCH is 50-200 mu L, the dosage of the color development agent DTNB is 20-100 mu L, and the dosage of the huperzine A is 25-100 mu L; the incubation conditions were: 35 to 37oAnd C, incubating for 15-35 min.
The pre-incubation conditions in step (3) are as follows: 35 to 37oAnd C, incubating for 10-20 min.
Chromatographic conditions for capillary electrophoresis assay: quartz capillary column: 50 μm i.d. x 33cm, limited length 24.5cm (Jian Feng chromatographic devices, Inc. in North and Hebei Yongnian); and (3) sample introduction mode: sampling at 50 mbar multiplied by 3 s; separation voltage: 25 kV; detection wavelength: 412 nm.
(4) The calculation formula of the inhibition rate of the acetylcholine esterase is as follows:
inhibition ratio (%) = (1-Ab/Aa) × 100
In the formula, Aa represents the absorbance of the sample without the addition of the enzyme; ab represents the absorbance of the added enzyme and the test sample (drug extract).
FIG. 6 is a graph showing the results of screening acetylcholinesterase inhibitors from 7 herbal extracts under chromatographic conditions. Research shows that corydalis tuber, white atractylodes rhizome and inula sallina show good inhibition effect on acetylcholinesterase, and the inhibition rates are 85.0%, 80.1% and 76.1% respectively under the concentration of 100 mg/mL. In addition, the schisandra chinensis shows the strongest inhibitory activity, the inhibition rate is 96.7% when the initial concentration is 100mg/mL, and the schisandra chinensis still shows the strong inhibitory action when the concentration is 50mg/mL, and the inhibition rate is as high as 93.8%. Such excellent inhibition rate indicates that some acetylcholinesterase inhibitors may be present in these Chinese medicinal water extracts.
In conclusion, the dopamine-coated echinoid manganese dioxide hollow microsphere prepared by the invention has the advantages of thermal stability, easy synthesis, porous structure, low density, short diffusion path, high specific surface area, rich active sites and the like, and the formed aminated manganese dioxide hollow microsphere is favorable for immobilizing acetylcholinesterase, so that the immobilized acetylcholinesterase is used as a novel enzyme immobilization carrier, and the acetylcholinesterase is immobilized and then combined with a capillary electrophoresis technology, so that the method for screening the acetylcholinesterase inhibitor from the traditional Chinese medicine has the characteristics of high separation efficiency, short analysis time, low sample consumption, low operation cost and the like (in the screening process, the total sample dosage is only 145 mu L), and provides technical support for finding new medicines for treating Alzheimer's disease.
Drawings
Fig. 1 is a transmission electron microscope image and a scanning electron microscope image of the hollow manganese dioxide microspheres and dopamine-coated hollow manganese dioxide microspheres of the present invention.
FIG. 2 is a flow chart showing the preparation of immobilized acetylcholinesterase of the present invention.
Fig. 3 is a graph showing the results of pH stability (a) and thermal stability (b) of the immobilized acetylcholinesterase of dopamine-coated manganese dioxide hollow microspheres of the present invention.
FIG. 4 shows the absorbance of the blank product and the absorbance of the positive control drug huperzine A in the presence of the product (a).
FIG. 5 is a graph showing the reciprocal double plots of immobilized acetylcholinesterase.
FIG. 6 is a diagram showing the results of screening acetylcholinesterase inhibitors from 7 kinds of Chinese medicinal extracts.
Detailed Description
The following will further illustrate the preparation method of the dopamine-coated manganese dioxide hollow microsphere immobilized acetylcholinesterase and the application of the dopamine-coated manganese dioxide hollow microsphere immobilized acetylcholinesterase inhibitor in combination with the capillary electrophoresis technology.
Example one
1. Accurately weighing 0.4g of potassium permanganate, and fully dissolving the potassium permanganate in 20mL of ultrapure water; dropwise adding 2mL of concentrated hydrochloric acid, and continuously stirring at room temperature for 30 min; transferring the obtained mixed solution to high-pressure reaction of polytetrafluoroethyleneReacting for 4 hours at 120 ℃ in a kettle; cooling the high-pressure reaction kettle to room temperature, centrifuging to collect a product, washing the product for a plurality of times by using ethanol and ultrapure water, and drying to obtain the manganese dioxide hollow microsphere h-MnO2。
2. Taking 10mgh-MnO2Completely dissolving in 10mL Tris-HCl buffer solution (pH 8.5), and performing ultrasonic treatment for 15 min; adding 10mg of dopamine hydrochloride, carrying out ultrasonic treatment for 5min, centrifuging to collect a product, washing with ethanol and ultrapure water for several times, and freeze-drying to obtain dopamine-coated manganese dioxide hollow microspheres h-MnO2@PDA。
3. Taking 4mgh-MnO2@ PDA, dispersing in 4mL phosphate buffer solution (pH value is 10), carrying out ultrasonic treatment for 5min, adding 4mL15% glutaraldehyde solution, and activating for 4 h under the condition of violent shaking to obtain suspension; subsequently, 0.4mL of a 0.4mg/mL acetylcholinesterase solution was added to the suspension under vigorous shaking, and the mixture was shaken at room temperature for 1 hour, followed by centrifugation to collect the product, which was washed with a phosphate buffer solution having a pH of 10 to obtain immobilized acetylcholinesterase (h-MnO)2@ PDA immobilized AChE); finally, the immobilized acetylcholinesterase was dispersed in phosphate buffer and stored at 4 ℃.
4. Application of immobilized acetylcholinesterase in screening natural drug enzyme inhibitor
(1) Preparing a traditional Chinese medicine extracting solution: respectively pulverizing the air-dried 7 traditional Chinese medicines including Inula salicifolia, inula japonica, spina date seed, corydalis tuber, bighead atractylodes rhizome, pepper and schisandra chinensis by using a pulverizer, accurately weighing 1g of traditional Chinese medicine powder respectively, placing the powder into a conical flask, adding 25mL of ultrapure water, and carrying out ultrasonic extraction for 1 h. The extract was poured into a centrifuge tube and centrifuged at 3000rpm for 10 min. Extracting the residue with the above method for 1 time, centrifuging the extractive solution, mixing the supernatants, evaporating to dryness with rotary evaporator, dissolving the residue with 4mL phosphate buffer (30 mM, pH 9.0), and diluting to desired volume to obtain 250mg/mL Chinese medicinal extractive solution;
(2) the application of the immobilized acetylcholinesterase in screening acetylcholinesterase inhibitors in the traditional Chinese medicine comprises the following steps: mixing 50 μ L of immobilized acetylcholinesterase solution (concentration 0.4 mg/mL) with 100mg/mL of 25 μ L of Chinese medicinal extractive solution, and adding into the mixture at 35%oPreincubation for 10min at C, then 25. mu.L DTNB and 50. mu.L Acetylthiocholine chloride were added at 35oIncubate for 25min under C. After the incubation is finished, centrifuging to terminate the reaction, and allowing the collected supernatant to enter capillary electrophoresis for separation and detection;
(3) the traditional Chinese medicine extracting solution obtained in the step (1) is an inhibitor, and a contrast is set: adding enzyme, and obtaining blank control without adding test sample, wherein the specific process is as follows: 50 μ L of the immobilized acetylcholinesterase solution (concentration 0.4 mg/ml) was added to 25 μ L of phosphate buffer solution at 35%oPreincubation for 10min at C, then 25. mu.L DTNB and 50. mu.L Acetylthiocholine chloride were added at 35oIncubate for 25min under C. After the incubation is finished, the reaction is stopped by centrifugation, and the collected supernatant enters capillary electrophoresis for separation and detection. The inhibition rate of each traditional Chinese medicine extracting solution on the enzyme activity can be calculated through the steps (2) and (3).
FIG. 6 is a graph showing the results of screening acetylcholinesterase inhibitors from 7 herbal extracts under chromatographic conditions. Research shows that corydalis tuber, white atractylodes rhizome and inula sallina show good inhibition effect on acetylcholinesterase, and the inhibition rates are 85.0%, 80.1% and 76.1% respectively under the concentration of 100 mg/mL. Furthermore, schisandra chinensis shows the strongest inhibitory activity, and the inhibition rate is 96.7% at the initial concentration of 100 mg/mL.
Example two
1. Accurately weighing 0.4g of potassium permanganate, and fully dissolving the potassium permanganate in 25mL of ultrapure water; dropwise adding 4mL of concentrated hydrochloric acid, and continuously stirring at room temperature for 40 min; transferring the obtained mixed solution into a polytetrafluoroethylene high-pressure reaction kettle, and reacting for 3 hours at 140 ℃; cooling the high-pressure reaction kettle to room temperature, centrifuging to collect a product, washing the product for a plurality of times by using ethanol and ultrapure water, and drying to obtain the manganese dioxide hollow microsphere h-MnO2；
2. Weighing 10mgh-MnO2Completely dissolving in 20mL Tris-HCl buffer solution (pH value is 9.5), and performing ultrasonic treatment for 25 min; adding 20mg of dopamine hydrochloride, carrying out ultrasonic treatment for 15min, centrifuging to collect a product, washing the product for several times by using ethanol and ultrapure water, and drying to obtain dopamine-coated manganese dioxide hollow microspheres h-MnO2@PDA；
3. Weighing 4mgh-MnO2@ PDA, dispersing in 8mL phosphate buffer solution (pH value is 9), adding 5mL25% glutaraldehyde solution after ultrasonic treatment for 15min, and activating for 4 h under the condition of vigorous shaking; then adding 0.5mL acetylcholinesterase solution with concentration of 0.6mg/mL into the suspension under the condition of vigorous shaking, shaking at room temperature for 2 h, centrifuging to collect the product, and washing with phosphate buffer solution with pH value of 9 to obtain immobilized acetylcholinesterase (h-MnO)2@ PDA immobilized AChE). Finally dispersing the mixture in phosphate buffer solution for storage;
4. the application of immobilized acetylcholinesterase in screening natural medicine enzyme inhibitor. The specific procedure is the same as in example 1. The screening results show that under the concentration of 100mg/mL, the inhibition rates of 7 traditional Chinese medicine extracting solutions on acetylcholinesterase are 73.5%, 57.9%, 14.9%, 86.8%, 80.7%, 74.5% and 96.2%, respectively.
EXAMPLE III
1. Accurately weighing 0.4g of potassium permanganate, and fully dissolving the potassium permanganate in 30mL of ultrapure water; dropwise adding 5mL of concentrated hydrochloric acid, and continuously stirring at room temperature for 35 min; transferring the obtained mixed solution into a high-pressure reaction kettle of polytetrafluoroethylene, and reacting for 4 hours at 130 ℃. Cooling the high-pressure reaction kettle to room temperature, centrifuging to collect a product, washing the product for a plurality of times by using ethanol and ultrapure water, and drying to obtain the manganese dioxide hollow microsphere h-MnO2；
2. Taking 10mgh-MnO2Completely dissolving in 30mL Tris-HCl buffer solution (pH value is 8), and carrying out ultrasonic treatment for 15 min; adding 30mg of dopamine hydrochloride, carrying out ultrasonic treatment for 25min, centrifuging to collect a product, washing the product for several times by using ethanol and ultrapure water, and drying to obtain dopamine-coated manganese dioxide hollow microspheres h-MnO2@PDA；
3. Taking 4mgh-MnO2@ PDA, dispersing in 10mL phosphate buffer (pH value 11), adding 6mL15% glutaraldehyde solution after 10min of ultrasonic treatment, and activating for 5 h under the condition of vigorous shaking to obtain suspension. Subsequently, 0.6mL of a 0.6mg/mL acetylcholinesterase solution was added to the suspension under vigorous shaking, the mixture was shaken at room temperature for 3 hours, centrifuged to collect the product, and washed with a pH 11 phosphate buffer solution to obtain a fixed complexChemical acetylcholinesterase h-MnO2@ PDA immobilized AChE. Dispersing in phosphate buffer solution, and storing at 4 deg.C;
4. the application of immobilized acetylcholinesterase in screening natural medicine enzyme inhibitor. The specific procedure is the same as in example 1. The screening results show that under the concentration of 100mg/mL, the inhibition rates of the 7 traditional Chinese medicine extracting solutions on acetylcholinesterase are 76.5%, 62.3%, 11.9%, 83.9%, 80.0%, 65.9% and 96.4% respectively.
Example four
1. Accurately weighing 0.4g of potassium permanganate, fully dissolving the potassium permanganate in 40mL of ultrapure water, dropwise adding 6mL of concentrated hydrochloric acid, and continuously stirring at room temperature for 40 min; transferring the obtained mixed solution into a high-pressure reaction kettle of polytetrafluoroethylene, and reacting for 5 hours at 130 ℃; cooling the high-pressure reaction kettle to room temperature, centrifugally collecting a product, washing the product with ethanol and ultrapure water for a plurality of times, and drying to obtain the manganese dioxide hollow microspheres h-MnO2；
2. Taking 10mgh-MnO2Completely dissolving in 40mL Tris-HCl buffer solution (pH value is 10), and performing ultrasonic treatment for 25 min; adding 40mg of dopamine hydrochloride, carrying out ultrasonic treatment for 15min, centrifuging to collect a product, washing the product with ethanol and ultrapure water for several times, and drying to obtain dopamine-coated manganese dioxide hollow microspheres h-MnO2@PDA；
3. Taking 4mg of h-MnO2@ PDA, dispersing in 15mL phosphate buffer solution (pH value is 8), performing ultrasonic treatment for 10min, adding 8mL10% glutaraldehyde solution, and activating for 3h under the condition of violent shaking to obtain suspension; then adding 0.8mL acetylcholinesterase solution with concentration of 0.4mg/mL into the suspension under vigorous shaking, shaking at room temperature for 4 h, centrifuging to collect the product, washing with phosphate buffer solution with pH of 8 to obtain immobilized acetylcholinesterase (h-MnO)2@ PDA immobilized AChE), which was dispersed in phosphate buffer and stored at 4 ℃;
4. the application of immobilized acetylcholinesterase in screening natural medicine enzyme inhibitor. The specific procedure is the same as in example 1. The screening results show that under the concentration of 100mg/mL, the inhibition rates of 7 traditional Chinese medicine extracting solutions on acetylcholinesterase are 78.3%, 56.5%, 11.6%, 84.3%, 79.5%, 64.9% and 97.5%, respectively.
Claims (10)
1. A preparation method of a dopamine-coated sea urchin-shaped manganese dioxide hollow microsphere comprises the following steps:
(1) preparing hollow manganese dioxide microspheres: dissolving potassium permanganate in ultrapure water, adding hydrochloric acid, and stirring at room temperature for 30-40 min; transferring the obtained mixed solution into a high-pressure reaction kettle of polytetrafluoroethylene, and reacting for 3-5 h at 120-140 ℃; cooling to room temperature after the reaction is finished, centrifuging, washing the obtained product with ethanol and ultrapure water for a plurality of times, and drying to be anhydrous to obtain the manganese dioxide hollow microspheres (h-MnO)2) (ii) a The molar ratio of potassium permanganate to hydrochloric acid is 1: 10-1: 29;
(2) preparing dopamine-coated manganese dioxide hollow microspheres: completely dissolving the prepared manganese dioxide hollow microspheres in Tris (hydroxymethyl) aminomethane hydrochloride solution (Tris-HCl), adding dopamine hydrochloride, carrying out ultrasonic treatment for 10-25 min, centrifuging, washing the obtained product with ethanol and ultrapure water for several times, and drying to be anhydrous to obtain dopamine-coated manganese dioxide hollow microspheres (h-MnO)2@PDA)。
2. The method for preparing the dopamine-coated echinoid manganese dioxide hollow microspheres of claim 1, wherein the dopamine-coated echinoid manganese dioxide hollow microspheres are prepared by the following steps: the mass ratio of the hollow manganese dioxide microspheres to the dopamine hydrochloride is 1: 1-1: 4.
3. The dopamine-coated echinoid manganese dioxide hollow microsphere prepared by the method of claim 1 is used as a carrier for preparing immobilized acetylcholinesterase, and is characterized in that: dispersing dopamine-coated manganese dioxide hollow microspheres in a phosphate buffer solution, adding a glutaraldehyde solution, and activating for 2-4 hours under the condition of violent shaking; and then adding an acetylcholinesterase solution into the suspension under the condition of violent shaking, shaking for 1-3 h at room temperature, centrifuging, collecting the mixture, and washing with a phosphate buffer solution to obtain the immobilized acetylcholinesterase.
4. The dopamine-coated echinoid manganese dioxide hollow microsphere as claimed in claim 1, which is used as a carrier for preparing immobilized acetylcholinesterase, and is characterized in that: the pH value of the phosphate buffer solution is 5-11.
5. The dopamine-coated echinoid manganese dioxide hollow microsphere as claimed in claim 4 used as a carrier for preparing immobilized acetylcholinesterase, wherein: the mass percentage of the glutaraldehyde solution is 5-25%; the concentration of the acetylcholinesterase solution is 0.2-1 mg/mL.
6. The dopamine-coated echinoid manganese dioxide hollow microsphere prepared by the method of claim 4 is used as a carrier for preparing immobilized acetylcholinesterase, and is characterized in that: the mass ratio of h-MnO2@ PDA to glutaraldehyde is 1: 940-1: 1900; the mass ratio of h-MnO2@ PDA to acetylcholinesterase is 1: 0.04-1: 0.09.
7. The use of the immobilized acetylcholinesterase of claim 4 in screening acetylcholinesterase inhibitors in Chinese medicinal materials.
8. The use of the immobilized acetylcholinesterase of claim 7 in screening acetylcholinesterase inhibitors in Chinese medicinal materials, wherein: the method comprises the following steps:
(1) preparing a traditional Chinese medicine extracting solution: air-drying the natural medicine by using a pulverizer, pulverizing, ultrasonically extracting for 1-3 h by using ultrapure water, and centrifuging; mixing the supernatants, evaporating to dryness with rotary evaporator, and dissolving with phosphate buffer (30 mM, pH 9.0) to obtain 250mg/mL Chinese medicinal extractive solution;
(2) adding immobilized acetylcholinesterase and a color-developing agent 5, 5-dithiobis- (2-nitrobenzoic acid) into an enzyme catalysis substrate acetylthiocholine chloride for incubation, collecting supernatant after incubation is finished, and determining the absorbance of a reaction product 5-thio-2-nitrobenzoic acid of a blank group by capillary electrophoresis;
(3) adding a natural medicine extracting solution into immobilized acetylcholinesterase for pre-incubation, adding an enzyme reaction substrate, namely acetylthiocholine chloride and a color-developing agent DTNB into the pre-incubation solution for incubation, collecting supernatant after the incubation is finished, and determining the absorbance of a reaction product, namely 5-thio-2-nitrobenzoic acid, through capillary electrophoresis;
(4) the calculation formula of the inhibition rate of the acetylcholine esterase is as follows: inhibition (%) = (1-Ab/Aa) × 100, wherein Aa represents absorbance with or without addition of an enzyme; ab represents the absorbance of the added enzyme and the extract solution of the Chinese medicinal materials.
9. The use of the immobilized acetylcholinesterase of claim 8 in screening acetylcholinesterase inhibitors in Chinese medicinal materials, wherein: in the steps (2) and (3), the dosage of the immobilized acetylcholinesterase is 50-200 mu L, the dosage of the enzyme catalysis substrate AThCH is 50-200 mu L, the dosage of the color development agent DTNB is 25-100 mu L, and the dosage of the huperzine A is 25-100 mu L; the incubation conditions were: 35 to 37oC, incubating for 15-35 min; the pre-incubation conditions in step (3) are as follows: 35 to 37oAnd C, incubating for 10-20 min.
10. The use of the immobilized acetylcholinesterase of claim 8 in screening acetylcholinesterase inhibitors in Chinese medicinal materials, wherein: the natural medicines are Inula salix leaf, herba Inulae, semen Ziziphi Spinosae, rhizoma corydalis, Atractylodis rhizoma, fructus Zanthoxyli and fructus Schisandrae.
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| CN114917863A (en) * | 2022-04-24 | 2022-08-19 | 西安工业大学 | Dopamine-coated nano calcium hydroxide and preparation method thereof |
| CN114804209A (en) * | 2022-04-28 | 2022-07-29 | 中山大学 | Burr microspheric MnO 2 Preparation method of material and application of material in sterilization and disinfection |
| CN114804209B (en) * | 2022-04-28 | 2023-11-28 | 中山大学 | Burr microsphere type MnO 2 Preparation method of material and sterilization and disinfection application |
| CN114717602A (en) * | 2022-05-13 | 2022-07-08 | 中山大学 | Proton transfersome modified nano array electrode and preparation method and application thereof |
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| CN116987467A (en) * | 2023-09-26 | 2023-11-03 | 太仓斯迪克新材料科技有限公司 | Compressed low-rebound high-level difference filling pressure-sensitive adhesive, pressure-sensitive adhesive tape and preparation method |
| CN116987467B (en) * | 2023-09-26 | 2023-12-15 | 太仓斯迪克新材料科技有限公司 | Compressed low-rebound high-level difference filling pressure-sensitive adhesive, pressure-sensitive adhesive tape and preparation method |
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