CN116078364A - Mesoporous-load-target-protein-based biomembrane chromatographic stationary phase, and preparation method and application thereof - Google Patents
Mesoporous-load-target-protein-based biomembrane chromatographic stationary phase, and preparation method and application thereof Download PDFInfo
- Publication number
- CN116078364A CN116078364A CN202211712179.4A CN202211712179A CN116078364A CN 116078364 A CN116078364 A CN 116078364A CN 202211712179 A CN202211712179 A CN 202211712179A CN 116078364 A CN116078364 A CN 116078364A
- Authority
- CN
- China
- Prior art keywords
- stationary phase
- mesoporous
- mesoporous material
- biomembrane
- chromatographic stationary
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000005526 G1 to G0 transition Effects 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000013335 mesoporous material Substances 0.000 claims abstract description 80
- 102100033639 Acetylcholinesterase Human genes 0.000 claims abstract description 54
- 108010022752 Acetylcholinesterase Proteins 0.000 claims abstract description 54
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 238000012216 screening Methods 0.000 claims abstract description 18
- 241001673966 Magnolia officinalis Species 0.000 claims abstract description 17
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 15
- 239000002502 liposome Substances 0.000 claims abstract description 15
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims abstract description 13
- 229940022698 acetylcholinesterase Drugs 0.000 claims abstract description 13
- 229940083466 soybean lecithin Drugs 0.000 claims abstract description 13
- 241000037740 Coptis chinensis Species 0.000 claims abstract description 11
- 229930014626 natural product Natural products 0.000 claims abstract description 11
- 239000002904 solvent Substances 0.000 claims abstract description 11
- 239000000544 cholinesterase inhibitor Substances 0.000 claims abstract description 9
- 240000006464 Fibraurea tinctoria Species 0.000 claims abstract 2
- 239000012528 membrane Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 229940088598 enzyme Drugs 0.000 claims description 11
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 229920000428 triblock copolymer Polymers 0.000 claims description 8
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 7
- 239000011148 porous material Substances 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- 238000002425 crystallisation Methods 0.000 claims description 5
- 230000008025 crystallization Effects 0.000 claims description 5
- 238000002390 rotary evaporation Methods 0.000 claims description 5
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 229910052710 silicon Inorganic materials 0.000 claims description 4
- 239000010703 silicon Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000003837 high-temperature calcination Methods 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 abstract description 15
- 150000003904 phospholipids Chemical class 0.000 abstract description 10
- 210000000170 cell membrane Anatomy 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 6
- 238000007877 drug screening Methods 0.000 abstract description 5
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 230000014759 maintenance of location Effects 0.000 description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 239000003480 eluent Substances 0.000 description 12
- VVOAZFWZEDHOOU-UHFFFAOYSA-N magnolol Chemical compound OC1=CC=C(CC=C)C=C1C1=CC(CC=C)=CC=C1O VVOAZFWZEDHOOU-UHFFFAOYSA-N 0.000 description 12
- 241001111214 Fibraurea Species 0.000 description 11
- 239000003814 drug Substances 0.000 description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 10
- 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 description 9
- ZRJBHWIHUMBLCN-UHFFFAOYSA-N Shuangyiping Natural products N1C(=O)C=CC2=C1CC1C(=CC)C2(N)CC(C)=C1 ZRJBHWIHUMBLCN-UHFFFAOYSA-N 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- XWAIAVWHZJNZQQ-UHFFFAOYSA-N donepezil hydrochloride Chemical compound [H+].[Cl-].O=C1C=2C=C(OC)C(OC)=CC=2CC1CC(CC1)CCN1CC1=CC=CC=C1 XWAIAVWHZJNZQQ-UHFFFAOYSA-N 0.000 description 9
- 229960003135 donepezil hydrochloride Drugs 0.000 description 9
- 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 description 9
- 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 description 9
- 208000024827 Alzheimer disease Diseases 0.000 description 8
- 239000012488 sample solution Substances 0.000 description 8
- 229940079593 drug Drugs 0.000 description 6
- 238000005216 hydrothermal crystallization Methods 0.000 description 6
- 230000002035 prolonged effect Effects 0.000 description 6
- 229940100578 Acetylcholinesterase inhibitor Drugs 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241000218202 Coptis Species 0.000 description 4
- 235000002991 Coptis groenlandica Nutrition 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 description 4
- 229940093265 berberine Drugs 0.000 description 4
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 238000011140 membrane chromatography Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000011049 filling Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 229940124596 AChE inhibitor Drugs 0.000 description 2
- 241000123862 Fibraurea recisa Species 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229910002808 Si–O–Si Inorganic materials 0.000 description 2
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 2
- 229960004373 acetylcholine Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000001354 calcination Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- -1 polytetrafluoroethylene Polymers 0.000 description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000002210 silicon-based material Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 241001504070 Huperzia Species 0.000 description 1
- 241001090156 Huperzia serrata Species 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 229910018557 Si O Inorganic materials 0.000 description 1
- 229910008051 Si-OH Inorganic materials 0.000 description 1
- 229910006358 Si—OH Inorganic materials 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- LIVNPJMFVYWSIS-UHFFFAOYSA-N silicon monoxide Inorganic materials [Si-]#[O+] LIVNPJMFVYWSIS-UHFFFAOYSA-N 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of drug screening, and particularly relates to a mesoporous load target protein-based biomembrane chromatographic stationary phase, a preparation method thereof and application thereof in active molecular screening. The preparation method comprises the steps of placing a mesoporous material loaded with a receptor in a solution containing soybean lecithin and cholesterol, removing the solvent to obtain a mesoporous material coated with liposome, and removing free liposome to obtain a biomembrane chromatographic stationary phase; the receptor is acetylcholinesterase. The invention utilizes phospholipid and cholesterol to wrap mesoporous materials loaded with acetylcholinesterase as a chromatographic stationary phase, establishes a new platform for screening acetylcholinesterase inhibitors by combining HPLC, and screens active ingredients with inhibition effect on AChE from natural products of coptis chinensis, fibraurea stem and magnolia officinalis. The biomembrane chromatographic stationary phase has the advantages of small sample consumption, repeated use, high screening precision and the like, and compared with a cell membrane chromatographic column, the preparation method is simpler.
Description
Technical Field
The invention belongs to the technical field of drug screening, and particularly relates to a mesoporous load target protein-based biomembrane chromatographic stationary phase, a preparation method thereof and application thereof in active molecular screening.
Background
Drug screening is a key step in the process of new drug development, which shortens the time, reduces the cost and reduces the risk. The most widely used natural product screening method is a traditional classical activity tracking method, and the method can effectively discover active ingredients, but has more defects. The development of cell and molecular level drug screening models has changed drug screening from traditional to automated. The screening method based on mesoporous load target protein biomembrane chromatography is one of cell and molecular level medicine screening models. Mesoporous materials have the advantages of ultrahigh specific surface area, high porosity, good adsorption performance, lower cytotoxicity and the like, so that the mesoporous materials are widely applied to the fields of adsorption and separation.
Alzheimer's Disease (AD), also known as senile dementia, is an irreversible neurodegenerative disease. The pathogenesis of AD is complex, and at present, the world has accepted the hypothesis of "central nervous system cholinergic injury", which suggests that the brain of AD patients lacks the neurotransmitter acetylcholine, and that the absence of acetylcholine is therefore considered a key cause of the disease. Currently, there are five main drugs for the treatment of AD, four of which are inhibitors of acetylcholinesterase AChE.
Common affinity chromatographic columns cannot simulate the transfer process of cell membranes to drugs, and in order to solve the problem, cell membrane chromatography was proposed at the end of the 20 th century. However, cell membrane chromatography has the disadvantages of short service life, high environmental requirements, high price, etc. In combination with the advantages and disadvantages of cell membrane chromatography, a biomembrane affinity chromatography method is provided, and the required receptor is selected and combined with an artificial biomembrane to compensate the disadvantages of the cell membrane chromatography to a certain extent. However, the problems of insufficient enzyme load, easy shedding and inactivation of the receptor under mobile phase washing and the like still exist.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a biomembrane chromatographic stationary phase based on mesoporous load target proteins, which combines affinity chromatography and biomembrane chromatography, selects a proper receptor, takes mesoporous materials as a main receptor carrier, adopts a technology of covalent bonding lipid biomembrane with a double-layer structure prepared by using materials such as soybean lecithin, cholesterol and the like on the surfaces of carriers such as silica gel and the like, can study the effects between medicines and the membranes and the receptor, can improve the problems of insufficient enzyme load, easy shedding and inactivation of the receptor under mobile phase flushing and the like, and has simple preparation process.
The technical scheme adopted for solving the technical problems is as follows:
the invention aims at providing a preparation method of a biological membrane chromatographic stationary phase based on mesoporous supported target proteins, which comprises the steps of placing mesoporous materials loaded with receptors in a solution containing soybean lecithin and cholesterol, removing a solvent to obtain mesoporous materials coated with liposome, and removing free liposome to obtain the biological membrane chromatographic stationary phase; the receptor is acetylcholinesterase.
Preferably, the preparation method comprises the following steps: the triblock copolymer P123 is used as a template, the tetraethyl silicate is used as a silicon source, and the mesoporous material is obtained by crystallizing in a reaction kettle, and removing the template agent through high-temperature calcination in a muffle furnace; after acetylcholinesterase AChE is loaded in a mesoporous material, the mesoporous material is placed in a solution containing soybean lecithin and cholesterol, the solvent is removed by rotary evaporation, free liposome is removed by washing, and the target product biomembrane chromatographic stationary phase is obtained by drying.
Preferably, the preparation method specifically comprises the following steps:
(1) The triblock copolymer P123 is used as a template, and tetraethyl silicate is used as a silicon source to prepare a mesoporous material;
(2) Dissolving acetylcholinesterase in phosphate buffer solution with pH value of 5.0-8.0, adding the mesoporous material prepared in the step (1), stirring and adsorbing for 1-5 h under ice bath condition, and centrifuging to obtain immobilized AChE mesoporous material;
(3) Dissolving soybean lecithin and cholesterol in a solvent to obtain a mixed solution, immersing the immobilized AChE mesoporous material prepared in the step (2) in the mixed solution, and removing the solvent by rotary evaporation to form a layer of phospholipid membrane on the surface of the AChE immobilized mesoporous material to obtain a mesoporous material coated with liposome;
(4) Washing the mesoporous material coated with the liposome in the step (3) with pure water to remove free liposome which does not participate in the coating, drying in a vacuum drying oven, volatilizing to remove the organic solvent and water, and obtaining the AChE biomembrane chromatographic stationary phase.
Preferably, the phosphate buffer solution in the step (2) is Na with the concentration of 0.1mol/L 2 HPO 3 ·H 2 O solution.
Preferably, the temperature of the ice bath conditions in step (2) is about 0 ℃.
Preferably, the solvent in the step (3) is chloroform.
Preferably, the mass ratio of the soybean lecithin to the cholesterol in the step (3) is 3:1. More preferably, the soybean lecithin: cholesterol: AChE loaded mesoporous material mass ratio = 0.21:0.07:2.
Preferably, the solvent in the step (3) is chloroform.
Preferably, the aperture of the biomembrane chromatographic stationary phase is 6.2 nm-12.8 nm, and the enzyme loading rate is 16-100.6U/g.
Preferably, the preparation method specifically comprises the following steps:
(1) dissolving a triblock copolymer P123 in a mixed solution of purified water and concentrated hydrochloric acid, and adding tetraethyl orthosilicate at 40 ℃ to react for 24 hours to obtain an emulsion;
further, the mass to volume ratio of the triblock copolymer P123 to the purified water and the concentrated hydrochloric acid was 2g:15mL:60mL, the concentration of the concentrated hydrochloric acid is 2mol/L;
further, the mass ratio of the triblock copolymer P123 to the tetraethyl orthosilicate is 2:4.25;
(2) transferring the emulsion obtained in the step (1) into a polytetrafluoroethylene reaction kettle for crystallization at 90-130 ℃ for 24 hours, washing with water and absolute ethyl alcohol in sequence, drying, and calcining in a muffle furnace for 6 hours to obtain a mesoporous material for later use; furthermore, mesoporous materials with different apertures can be prepared by adjusting crystallization temperature;
(3) dissolving acetylcholinesterase in phosphate buffer solution with pH value of 5.0-8.0, adding the mesoporous material obtained in the step (2), stirring and adsorbing for 1-5 h under ice bath condition, and centrifuging to obtain immobilized AChE mesoporous material for standby;
(4) dissolving soybean lecithin and cholesterol in chloroform to obtain a mixed solution;
(5) soaking the material obtained in the step (3) in the mixed solution obtained in the step (4), and slowly evaporating at normal temperature under reduced pressure to remove chloroform, thereby forming a layer of phospholipid membrane on the surface of the AChE immobilized mesoporous material;
(6) and (3) washing the material obtained in the step (5) with pure water, removing free liposome which does not participate in the coating, drying in a vacuum drying oven, volatilizing to remove the organic solvent and water, and obtaining the AChE biomembrane affinity chromatography stationary phase.
The invention also aims to provide an application of the mesoporous load target protein-based biomembrane chromatographic stationary phase prepared by the preparation method in the field of chromatographic columns: and loading the column by a wet method by taking pure water as homogenate and taking a biomembrane chromatographic stationary phase as a filler to obtain the biomembrane chromatographic column for mesoporous load target protein.
The invention also aims to provide an application of the mesoporous load target protein-based biomembrane chromatographic stationary phase prepared by the preparation method in the field of active compound screening: the biomembrane chromatographic stationary phase based on mesoporous load target protein or the corresponding liposome biomembrane chromatographic column is applied to screening of acetylcholinesterase inhibitors in natural products.
Still further, the natural product is at least one of coptis chinensis, fibraurea stem and magnolia officinalis.
Still further, the chromatographic conditions of the biofilm chromatography column are as follows: mobile phase Na 2 HPO 4 ·12H 2 O buffer (0.01M, pH 6.5), flow rate 0.2mL/min, column temperature 35 ℃, rhizoma Coptidis detection wavelength 345nm, cortex Magnoliae officinalis detection wavelength 294nm.
According to the invention, huperzine A and donepezil hydrochloride are selected as research objects, and screening performance of the two acetylcholinesterase inhibitors based on mesoporous load target protein biomembrane chromatographic stationary phase and corresponding chromatographic columns is examined. Huperzine A is a drug which is independently developed in China and is used for treating Alzheimer disease, is obtained by separating and extracting huperzia serrata of huperzia, and is a reversible acetylcholinesterase inhibitor. Donepezil hydrochloride is an FDA approved first-line drug for the treatment of light and medium alzheimer's disease, and can be highly efficiently and specifically combined with AChE. The difference of the retention behaviors of huperzine A and donepezil hydrochloride on the mesoporous load target protein-based biomembrane chromatographic column and the blank silica gel chromatographic column is compared. The experimental results show that: the retention time of the acetylcholinesterase inhibitor on the biological membrane chromatographic column based on the mesoporous load target protein and the blank chromatographic column are obviously different, which indicates that the biological membrane chromatographic column based on the mesoporous load target protein has selective action on the acetylcholinesterase inhibitor.
The invention takes Chinese medicine coptis chinensis, fibraurea stem and magnolia officinalis as research objects, active ingredients in the Chinese medicine components are screened through the mesoporous load target protein-based biological membrane chromatographic column, and the active ingredients in natural products can be screened through the mesoporous load target protein-based biological membrane chromatographic column obtained through mass spectrometry.
Compared with the prior art, the invention has the beneficial effects that: the invention uses a mesoporous material coated by phospholipid and cholesterol and loaded with acetylcholinesterase as a chromatographic stationary phase, establishes a novel acetylcholinesterase inhibitor screening platform by combining HPLC, takes acetylcholinesterase inhibitor huperzine A and donepezil hydrochloride as objects, researches the effectiveness of the chromatographic model, and optimizes chromatographic screening conditions. And active ingredients with inhibition effect on AChE are screened from natural products of coptis chinensis, fibraurea stem and magnolia officinalis. The mesoporous load target protein-based biological membrane chromatographic column is applied to medicine screening, can be used for researching interaction between medicines and membranes and between the receptors, can also be used for screening effective components from complex compounds by utilizing the combination of the receptors and target components, has the advantages of small sample consumption, reusability, high screening precision and the like, and is simpler in preparation method compared with a cell membrane chromatographic column.
Drawings
FIG. 1 is a bar graph of pore size (A) of a synthesized mesoporous material at different hydrothermal crystallization temperatures, particle size (B) of the synthesized mesoporous material at different hydrothermal crystallization temperatures, and enzyme loading rate (C) of the synthesized mesoporous material at different hydrothermal crystallization temperatures in example 1 of the present invention.
Fig. 2 is an SEM image of the blank mesoporous material (a), the immobilized AChE mesoporous material (B), and the immobilized AChE biofilm mesoporous material (C) in example 1 of the present invention.
FIG. 3 is a FTIR image of the blank mesoporous material (A), the immobilized AChE mesoporous material (B) and the immobilized AChE biofilm mesoporous material (C) in example 1 of the present invention.
FIG. 4 is a graph showing the separation results of yellow even sample solutions in ABC and MBC in example 3 of the present invention.
FIG. 5 is a graph showing the separation results of the fibraurea stem sample solution in ABC and MBC in example 3 of the present invention.
FIG. 6 is a graph showing the separation results of sample solutions of Magnolia officinalis in example 3 of the present invention on ABC and MBC.
Detailed Description
The technical scheme of the invention is further specifically described below through specific embodiments and with reference to the accompanying drawings. The scope of the invention as claimed is not limited to the examples.
The acetylcholinesterase described in the present invention was purchased from Sigma-Aldrich Company, CAS number: 9000-81-1, MDL: MFCD00081268. Are used only as illustrations of the source of the product and do not constitute a limitation on the technical solution of the invention.
The coptis chinensis, fibraurea stem and magnolia officinalis are purchased from the genuine traditional Chinese medicine of the imperial mill. Are used only as illustrations of the source of the product and do not constitute a limitation on the technical solution of the invention.
Example 1
(1) 2.00g of triblock copolymer P123 is dissolved in a mixed solution of 15mL of purified water and 60mL (2 mol/L) of concentrated hydrochloric acid, 4.25g of tetraethyl orthosilicate is added at 40 ℃ and reacted for 24 hours to obtain emulsion;
(2) transferring the emulsion obtained in the step (1) into a polytetrafluoroethylene reaction kettle, crystallizing at 90 ℃,100 ℃,110 ℃,120 ℃ and 130 ℃ for 24 hours respectively, washing with water and absolute ethyl alcohol in sequence, drying, and calcining in a muffle furnace for 6 hours to obtain a mesoporous material for standby (blank mesoporous material); measuring the pore diameter, the particle diameter and the enzyme load rate of the mesoporous material;
(3) dissolving 5U of acetylcholinesterase in phosphate buffer solution (0.1M) with pH of 8.0, adding 50.0mg of mesoporous material obtained in the step (1), stirring and adsorbing for 4 hours under ice bath condition, and centrifuging to obtain immobilized AChE mesoporous material for standby (immobilized AChE mesoporous material);
(4) 0.21g of soybean lecithin and 0.07g of cholesterol are fully dissolved in 30mL of chloroform to obtain a mixed solution;
(5) 2.00g of the material obtained in the step (3) is immersed in the mixed solution obtained in the step (4), chloroform is slowly evaporated and removed by shaking for 15min under reduced pressure at the normal temperature of 25 ℃, and a layer of phospholipid membrane is formed on the surface of the AChE immobilized mesoporous material;
(6) washing the material obtained in the step (5) with pure water, removing liposome which does not participate in the coating, and drying in a vacuum drying oven to obtain AChE biomembrane affinity chromatography stationary phase (immobilized AChE biomembrane mesoporous material); the blank biomembrane stationary phase is coated by directly taking mesoporous material without enzyme, and the rest operation methods are consistent with the above.
(7) And (3) taking pure water as homogenate, taking AChE mesoporous material which is wrapped by a phospholipid membrane as a filler, and carrying out wet column packing to obtain the target product mesoporous loaded target protein biological membrane chromatographic column.
The pore diameter, particle diameter and enzyme load rate of the synthesized mesoporous material at different hydrothermal crystallization temperatures are shown in figure 1; it can be seen from fig. 1 a that as the crystallization temperature increases, the pore size of the mesoporous material increases, and as the hydrothermal crystallization temperature increases from 90 ℃ to 130 ℃, the pore size of the mesoporous material increases from 6.2nm to 12.8nm; it can be seen from FIG. 1B that as the crystallization temperature increases, the particle size of the mesoporous material increases, and as the hydrothermal crystallization temperature increases from 90℃to 130℃the particle size of the mesoporous material increases from 3.0 μm to 5. Mu.m; as can be seen from fig. 1C, the enzyme activity of the blank group is almost 0, which indicates that the blank mesoporous material itself does not interfere with the AChE enzyme activity assay; five experimental groups all have certain enzyme activities, so that the prepared mesoporous materials with five apertures can load AChE, and the fourth group has the highest enzyme activity, which indicates that the mesoporous materials synthesized at 120 ℃ are most suitable for the load of AChE.
SEM images of the blank mesoporous material (a), the immobilized AChE mesoporous material (B) and the immobilized AChE biofilm mesoporous material (C) are shown in fig. 2; from fig. 2 a, it can be found that the synthesized mesoporous material is aggregated into a long stripe shape, and has a plurality of pores, and the porous structure is favorable for loading AChE, so as to meet the requirement of the preset target. From fig. 2C, it can be seen that the volume of the coated mesoporous material is increased and the lines are blurred, and it can be presumed that the morphology is changed due to the coating of the phospholipid membrane.
The FTIR diagrams of the blank mesoporous material (A), the immobilized AChE mesoporous material (B) and the immobilized AChE biomembrane mesoporous material (C) are shown in FIG. 3, 463.49cm -1 The nearby absorption peak is the bending vibration peak of skeleton Si-O-Si bond, 803.01cm -1 The nearby peak is the absorption peak of Si-O symmetrical telescopic vibration, 1081.20cm -1 Is an antisymmetric telescopic vibration peak of Si-O-Si bond, 1632.11cm -1 The nearby peak is C=O stretching vibration in peptide bond, at 3442.94cm -1 The absorption peak is probably generated by Si-OH stretching vibration of the surface; at 2930.84cm -1 The immobilized AChE biomembrane mesoporous material has an obvious absorption peak at the left and right positions, but the absorption peak corresponding to the characteristic peak of C-H stretching vibration of phospholipid is not found in the blank mesoporous material, and the fact that the phospholipid membrane is coated on the mesoporous silicon material is proved.
The surface energy spectrum analysis of the blank mesoporous material, the immobilized AChE mesoporous material and the immobilized AChE biomembrane mesoporous material is shown in the following table, and the immobilized AChE biomembrane mesoporous material contains the P element, so that the phospholipid membrane is further proved to be coated on the mesoporous silicon material.
TABLE 1 surface energy Spectrometry of blank mesoporous Material, AChE mesoporous Material and AChE biofilm mesoporous Material
Example 2
Taking the blank biomembrane stationary phase and the AChE biomembrane affinity chromatographic stationary phase prepared in the embodiment 1 respectively, taking pure water as homogenate, and respectively filling the prepared chromatographic packing into a stainless steel column by using a column filling machine until the pressure of the column filling machine is not changed; a blank Mesoporous Biofilm Chromatography (MBC) column and an AChE Biofilm Chromatography (ABC) column were prepared. Wherein the specification of the chromatographic column is 100 x 4.6mm.
Placing 10mg of huperzine A and 10mg of donepezil hydrochloride standard substance into 5ml volumetric flasks respectively, adding a proper amount of aqueous hydrochloric acid solution respectively for dissolution and volume fixing to obtain 2mg/ml of huperzine A solution and 2mg/ml of donepezil hydrochloride Ji Rongye, taking 20ul of huperzine A solution and donepezil hydrochloride solution respectively, and carrying out sample injection analysis on ABC and MBC, wherein chromatographic conditions are as follows: mobile phase 0.01M (mol/L) pH6.5 Na 2 HPO 4 ·H 2 O, the column temperature is 35 ℃, the flow rate is 0.2mL/min, the detection wavelength of huperzine A is 271nm, and the detection wavelength of donepezil hydrochloride is 312nm;
the retention time of huperzine A in MBC is 6.14min, and the retention time in ABC is 10.83min. The retention time of donepezil hydrochloride on MBC was 6.27min and on ABC was 12.15min. The retention time of the component with the inhibiting effect on the acetylcholinesterase on the ABC chromatographic column is prolonged, and the component with the inhibiting effect on the acetylcholinesterase can be effectively retained.
Example 3
(1) Respectively taking 2.0g of coptis chinensis, fibraurea stem and magnolia officinalis, adding 30mL of 60% ethanol water solution, refluxing for 2 hours at 70 ℃, filtering, and removing ethanol and water from the filtrate by rotary evaporation at 65 ℃ to obtain a natural product extract.
(2) Taking 0.10g of each of coptis chinensis, fibraurea stem and magnolia bark extract, respectively using a mobile phase to fix the volume to 2mL, and passing through a membrane for standby.
(3) Taking 10ul of coptis chinensis, fibraurea stem and magnolia officinalis sample solutions respectively, and separating on ABC and MBC respectively, wherein chromatographic conditions are as follows: mobile phase Na 2 HPO 4 ·12H 2 O buffer (0.01M, pH 6.5), flow rate 0.2mL/min, column temperature 35 ℃, rhizoma Coptidis detection wavelength 345nm, cortex Magnoliae officinalis detection wavelength 294nm.
(4) Taking collected rhizoma Coptidis, caulis Fibraureae, cortex Magnoliae officinalis eluent, berberine, fibrauretine, and magnolol standard as samples, and performing reverse phase separation under the following chromatographic conditions: XDB-C18 (4.6X1250 mm) reversed phase chromatographic column with flow rate of 0.8mL/min, column temperature of 25deg.C, detection wavelength of Coptidis rhizoma, berberine, caulis Fibraureae, fibrauretine 345nm, detection wavelength of cortex Magnolia officinalis, and detection wavelength of magnolol 294nm.
(5) The active eluent composition was then subjected to quaternary rod-time of flight tandem mass spectrometry (Q-TOF/MS) analysis.
The coptis extract has only three peaks on MBC, but has four peaks on ABC, the peak time of the fourth peak is 12.1min, the peak time of the last peak on MBC is 8.7min, and the retention time is prolonged; the peak time of the last peak of the fibraurea stem extract on MBC is 15.2min, and the peak time of the peak on ABC is prolonged to 20.5min; the peak time of the magnolia bark extract at the last peak of MBC is 11min, and the peak time of the peak at ABC is prolonged to 14.3min. Since the AChE inhibitor has longer retention time in ABC than MBC, it is primarily inferred that the active ingredient in the extractive solution of Coptidis rhizoma, caulis Fibraureae and cortex Magnolia officinalis with prolonged retention time in ABC is AChE inhibitor. The separation results of the coptis chinensis, fibraurea stem and magnolia bark sample solutions on ABC and MBC are shown in figures 4 to 6.
The collected eluent (peak with prolonged retention time) of rhizoma Coptidis, caulis Fibraureae and cortex Magnoliae officinalis is subjected to reverse chromatography. Fig. 4 is a coptis sample solution, fig. 5 is a fibraurea stem sample solution, and fig. 6 is a magnolia bark sample solution. Q-TOF/MS analysis is carried out on the coptis active ingredient eluent with t=9.77 min, and mass spectrum results prove that the active ingredient in the coptis active ingredient eluent is mainly berberine; the retention time of the berberine standard substance is consistent with the components in the eluent of the active components of the coptis chinensis by HPLC determination. Q-TOF/MS analysis is carried out on the fibraurea recisa pierre active ingredient eluent with t=9.80 min, and the mass spectrum result proves that the active ingredient in the fibraurea recisa pierre active ingredient eluent is mainly fibrauretine; the retention time of the fibrauretine standard substance is consistent with the components in the fibrauretine active component eluent by HPLC determination. Q-TOF/MS analysis is carried out on the magnolia officinalis active ingredient eluent with t=20.86 min, and mass spectrum results prove that the active ingredient in the magnolia officinalis active ingredient eluent is mainly magnolol; the magnolol standard substance is measured by HPLC, and the retention time of the magnolol standard substance is consistent with the components in the magnolol active component eluent. The invention is based on mesoporous load target protein biomembrane chromatographic stationary phase and corresponding chromatographic column, can be successfully used for screening natural product active components, and can effectively screen and separate natural product active components by combining with corresponding HPLC conditions.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the invention in any way, but other variations and modifications are possible without exceeding the technical solutions described in the claims.
Claims (10)
1. A preparation method of a biological membrane chromatographic stationary phase based on mesoporous load target protein is characterized in that the preparation method comprises the steps of placing a mesoporous material loaded with a receptor into a solution containing soybean lecithin and cholesterol, removing the solvent to obtain a mesoporous material coated with liposome, and removing free liposome to obtain the biological membrane chromatographic stationary phase; the receptor is acetylcholinesterase.
2. The method for preparing the mesoporous load target protein based biomembrane chromatographic stationary phase according to claim 1, wherein triblock copolymer P123 is used as a template, tetraethyl silicate is used as a silicon source for crystallization in a reaction kettle, and a muffle furnace is used for high-temperature calcination to obtain a mesoporous material; and loading acetylcholinesterase in the mesoporous material, placing the mesoporous material in a solution containing soybean lecithin and cholesterol, removing the solvent by rotary evaporation, and washing and drying to obtain the biomembrane chromatographic stationary phase.
3. The method for preparing the mesoporous supported target protein based biomembrane chromatographic stationary phase according to claim 1, which is characterized by comprising the following steps:
(1) The triblock copolymer P123 is used as a template, and tetraethyl silicate is used as a silicon source to prepare a mesoporous material;
(2) Dissolving acetylcholinesterase in phosphate buffer solution with pH value of 5.0-8.0, adding mesoporous material, stirring and adsorbing for 1-5 hr in ice bath condition, and centrifuging to obtain immobilized AChE mesoporous material;
(3) Dissolving soybean lecithin and cholesterol in a solvent to obtain a mixed solution, immersing an immobilized AChE mesoporous material in the mixed solution, and removing the solvent by rotary evaporation to obtain a liposome-coated mesoporous material;
(4) Washing the mesoporous material covered with the liposome with pure water, and drying in a vacuum drying oven to obtain the biomembrane chromatographic stationary phase.
4. The method for preparing a mesoporous supported target protein based biomembrane chromatographic stationary phase according to claim 3, wherein the phosphate buffer solution in the step (2) is Na with a concentration of 0.1mol/L 2 HPO 3 ·H 2 O solution.
5. The method for preparing a mesoporous supported target protein based biomembrane chromatographic stationary phase according to claim 3, wherein the mass ratio of soybean lecithin to cholesterol in the step (3) is 3:1.
6. The method for preparing the biological membrane chromatographic stationary phase based on mesoporous supported target protein according to claim 1, wherein the pore diameter of the biological membrane chromatographic stationary phase is 6.2-12.8 nm, and the enzyme loading rate is 16-100.6U/g.
7. A biofilm chromatography stationary phase prepared according to the preparation method of any one of claims 1-6.
8. Use of a biofilm chromatography stationary phase as claimed in claim 7 in the field of active compound screening.
9. The use according to claim 8, wherein the biofilm chromatography is immobilized for screening of acetylcholinesterase inhibitors in a natural product.
10. The use according to claim 9, wherein the natural product is at least one of coptis chinensis, fibraurea stem and magnolia officinalis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211712179.4A CN116078364A (en) | 2022-12-29 | 2022-12-29 | Mesoporous-load-target-protein-based biomembrane chromatographic stationary phase, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211712179.4A CN116078364A (en) | 2022-12-29 | 2022-12-29 | Mesoporous-load-target-protein-based biomembrane chromatographic stationary phase, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116078364A true CN116078364A (en) | 2023-05-09 |
Family
ID=86207591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211712179.4A Pending CN116078364A (en) | 2022-12-29 | 2022-12-29 | Mesoporous-load-target-protein-based biomembrane chromatographic stationary phase, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116078364A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1369707A (en) * | 2001-02-16 | 2002-09-18 | 中国科学院大连化学物理研究所 | Biological membrane as chromatographic medium and its preparing process |
| CN1865929A (en) * | 2006-06-13 | 2006-11-22 | 中国科学院上海有机化学研究所 | Acetylcholine esterase inhibitor screening method and application therefor |
| CN101339167A (en) * | 2008-08-27 | 2009-01-07 | 中国药科大学 | Active ingredient high throughput screen method based on target protein and selection |
| WO2014109704A1 (en) * | 2013-01-14 | 2014-07-17 | Transientic Interactions Ab | New material for use in high performance liquid chromatography |
| CN106770564A (en) * | 2016-11-10 | 2017-05-31 | 河南工业大学 | Acetylcholinesterasebiosensor biosensor for detection of organic phosphorus pesticide and preparation method thereof |
| CN108931604A (en) * | 2018-05-03 | 2018-12-04 | 暨南大学 | A kind of online Screening Platform of reactive compound based on enzyme reactor and application |
| CN111077251A (en) * | 2019-12-30 | 2020-04-28 | 浙江工业大学 | Mesoporous biomembrane chromatographic column based on target protein and application of mesoporous biomembrane chromatographic column in screening active components in natural products |
| CN111122759A (en) * | 2019-12-30 | 2020-05-08 | 浙江工业大学 | Target protein-based liposome biological membrane chromatographic column and application thereof in screening active components in natural products |
| CN111721870A (en) * | 2020-06-24 | 2020-09-29 | 兰州大学 | Method for high-throughput screening of acetylcholinesterase inhibitor |
| CN112816592A (en) * | 2020-12-31 | 2021-05-18 | 兰州大学 | Preparation of immobilized acetylcholinesterase and application thereof in screening and identifying enzyme inhibitor |
-
2022
- 2022-12-29 CN CN202211712179.4A patent/CN116078364A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1369707A (en) * | 2001-02-16 | 2002-09-18 | 中国科学院大连化学物理研究所 | Biological membrane as chromatographic medium and its preparing process |
| CN1865929A (en) * | 2006-06-13 | 2006-11-22 | 中国科学院上海有机化学研究所 | Acetylcholine esterase inhibitor screening method and application therefor |
| CN101339167A (en) * | 2008-08-27 | 2009-01-07 | 中国药科大学 | Active ingredient high throughput screen method based on target protein and selection |
| WO2014109704A1 (en) * | 2013-01-14 | 2014-07-17 | Transientic Interactions Ab | New material for use in high performance liquid chromatography |
| CN106770564A (en) * | 2016-11-10 | 2017-05-31 | 河南工业大学 | Acetylcholinesterasebiosensor biosensor for detection of organic phosphorus pesticide and preparation method thereof |
| CN108931604A (en) * | 2018-05-03 | 2018-12-04 | 暨南大学 | A kind of online Screening Platform of reactive compound based on enzyme reactor and application |
| CN111077251A (en) * | 2019-12-30 | 2020-04-28 | 浙江工业大学 | Mesoporous biomembrane chromatographic column based on target protein and application of mesoporous biomembrane chromatographic column in screening active components in natural products |
| CN111122759A (en) * | 2019-12-30 | 2020-05-08 | 浙江工业大学 | Target protein-based liposome biological membrane chromatographic column and application thereof in screening active components in natural products |
| CN111721870A (en) * | 2020-06-24 | 2020-09-29 | 兰州大学 | Method for high-throughput screening of acetylcholinesterase inhibitor |
| CN112816592A (en) * | 2020-12-31 | 2021-05-18 | 兰州大学 | Preparation of immobilized acetylcholinesterase and application thereof in screening and identifying enzyme inhibitor |
Non-Patent Citations (1)
| Title |
|---|
| 毛希琴, 邹汉法, 罗权舟, 孔亮, 厉欣, 孙乃昌: "卵磷脂涂敷生物膜色谱固定相的制备及其稳定性的考察", 色谱, no. 05, 30 September 2001 (2001-09-30) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hou et al. | Using self-polymerization synthesis of boronate-affinity hollow stannic oxide based fragment template molecularly imprinted polymers for the selective recognition of polyphenols | |
| CN100482657C (en) | Process for purifying (-)-delta 9-trans-tetrahydrocannabinol | |
| Zheng et al. | Identification of acetylcholinesterase inhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautography | |
| CN108445114B (en) | Method for screening trace neuraminidase inhibitor in honeysuckle | |
| Bai et al. | Highly selective separation and purification of chicoric acid from Echinacea purpurea by quality control methods in macroporous adsorption resin column chromatography | |
| CN101002905A (en) | Medicine for treating infantile anorexia, its preparing method and quality control method | |
| Yu et al. | Application of choline chloride deep eutectic solvents and high‐speed counter‐current chromatography to the extraction and purification of flavonoids from the thorns of Gleditsia sinensis Lam | |
| Sheng et al. | Separation of compounds interacting with liposome membrane in combined prescription of traditional Chinese medicines with immobilized liposome chromatography | |
| CN110652975B (en) | (S) -BINOL derivative CSP filler and preparation method and application thereof | |
| EP3954371A1 (en) | Anti-acetylcholinesterase active composition in caulis mahoniae and screening method therefor and application thereof | |
| Ma et al. | One-step fabrication of hydrophobic metal-organic framework@ covalent organic framework hybrid as sorbent for high-performance solid-phase extraction of flavonoids | |
| CN116078364A (en) | Mesoporous-load-target-protein-based biomembrane chromatographic stationary phase, and preparation method and application thereof | |
| Li et al. | Screening and isolation of cyclooxygenase‐2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry, and complex chromatography | |
| Rosa et al. | Biological sample preparation by using restricted-access nanoparticles prepared from bovine serum albumin: application to liquid chromatographic determination of β-blockers | |
| Kim et al. | UPLC-MS/MS-based profiling of 31 neurochemicals in the mouse brain after treatment with the antidepressant nefazodone | |
| Pande et al. | Preparation and evaluation of phytosomes of pomegrane peels | |
| Li et al. | Separation of flavonoid and alkaloid using collagen fiber adsorbent | |
| CN1660380A (en) | Method for preparing liangfuwan and method for controlling quality | |
| Bosch et al. | Analytical methodologies for the determination of sertraline | |
| Liu et al. | Biological evaluation, molecular modeling and dynamics simulation of phenanthrenes isolated from Bletilla striata as butyrylcholinesterase inhibitors | |
| CN111077251B (en) | Mesoporous biomembrane chromatographic column based on target protein and application thereof in screening active components in natural products | |
| FR2915900A1 (en) | NEW PROCESS FOR THE PREPARATION OF PURIFIED EXTRACTS OF HARPAGOPHYTUM PROCUMBENS. | |
| CN106333994A (en) | Perilla seed extract and pharmacological effect thereof | |
| WO2013044570A1 (en) | Detection method of medicine for curing mastitis and hyperplasia of mammary glands | |
| Xue et al. | In vitro metabolic study of Rhizoma coptidis extract using liver microsomes immobilized on magnetic nanoparticles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |