CN107271595A - A kind of cell membrane magnetic microsphere of quick screening natural product active ingredient is prepared and its applied - Google Patents
A kind of cell membrane magnetic microsphere of quick screening natural product active ingredient is prepared and its applied Download PDFInfo
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- CN107271595A CN107271595A CN201610220406.XA CN201610220406A CN107271595A CN 107271595 A CN107271595 A CN 107271595A CN 201610220406 A CN201610220406 A CN 201610220406A CN 107271595 A CN107271595 A CN 107271595A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
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- 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
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
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- 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
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2215/00—Separating processes involving the treatment of liquids with adsorbents
- B01D2215/02—Separating processes involving the treatment of liquids with adsorbents with moving adsorbents
- B01D2215/029—Centrifuge-like arrangements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
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Abstract
A kind of cell membrane magnetic microsphere of quick screening natural product active ingredient is prepared and its applied, and belongs to affinity chromatography screening natural product active ingredient field.The present invention is combined structure cell membrane magnetic microsphere using cell membrane with magnetic microsphere, and utilizes the active material in this affinitive material combination natural products (see Figure of abstract).The present invention obtains membrane structure from cell, and the structure is fixed on magnetic microsphere, the cell membrane magnetic microsphere for just having screening activity is made, is combined using the cell membrane magnetic material set up with hydrolysis and condensation and can quickly screen, identifies the active material contained in natural products.Whole separation qualification process only needs cell membrane microballoon affine combination and chromatographic mass spectrometry to identify two steps.Compared with conventional method, this law has separation short qualification cycle, advantage rapidly and efficiently, while chromatographic column need to be loaded by overcoming other membrane flexibilities, the shortcoming of separation qualification cycle length big to cell membrane consumption.
Description
Technical field
Patent of the present invention belongs to affinity chromatography screening natural product active ingredient field.It is related to a kind of preparation of the cell membrane affinity chromatography material of cell derived and using active component in material combination hydrolysis and condensation screening natural products.The technology can realize the purpose for the material with biological activity that the quick screening natural products of targeting contains.
Background technology
Natural products is the main source that reactive compound is found.For example, qinghaosu is separated from Chinese medicine sweet wormwood, found.The current compound and its derivative synthesis thing are widely used in clinic as effective antimalarial.However, drug candidate is screened from natural products often follows first Chemical Decomposition, post analysis identification, the method in conjunction with effect experiment to determine active ingredient.However, the separation of such chemical composition, Structural Identification and activity rating system, be confined to existing chromatographic technique isolates and purifies ability, can only isolated stabilization monomer organic chemical composition；Water soluble ingredient, micro constitutent and labile element are then relatively difficult to obtain.Therefore, how the reactive compound in quick discriminating natural products is still a urgent problem to be solved.
Because the main target of more than 50% marketed drug effect is exactly memebrane protein.Therefore Wainer professors and He Lang punching professors et al. develop a series of chromatographies based on cell membrane, these methods are respectively used to study in terms of the interaction of memebrane protein, the complex mixture for screening reactive compound.23 kinds of membrane flexibility models are had at present and have been used for effective component of chinese medicine screening, have received preferable effect.However, though membrane flexibility is feasible and had wide application prospects, because such method need to prepare stationary phase and chromatography column, also need to balance chromatographic in use.These operating process not only need the program of complexity, and need high-pressure pump to load chromatographic column.This process limits the further extensive use of membrane flexibility.
Therefore Zhu Quan professors et al. are improved to membrane flexibility, potential active component in Radix Angelicae Sinensis of directly being fished with erythrocyte cell film.With reference to high performance liquid chromatography, mass spectrum, nuclear magnetic resonance technique, they determine Ligustilide etc. in erythrocyte membrane dissociation solution has the compound of lateral reactivity.This prove using cell membrane " can fish " natural product active ingredient be can apply to predict composition Chinese medicine bioactivity.But this technology stills need repeatedly the cell membrane and extract solution that the separation of ultrafiltration centrifugation step is bonded with active component, and active component need to again be analyzed.Whole process is also very time-consuming.
Part method of fishing is initially to be fixed on miniature or nanosphere and then screening reactive compound from the extractions target biological molecules of complicated cellular matrix albumen and enzyme, applied magnetic microballoon is fished, the magnetic force for relying primarily on its exterior is separated, factor in system can more be avoided (for example, pH value, ionic strength, or surface charge) to analyze work influence.Moaddel in 2007 et al. first time applied magnetic microballoons in part fishes experiment.At work, human serum albumins is fixed on silica gel magnetic microsphere surface by researcher, and mixture of fishing contains known part and non-part.Hereafter, increasing protein is fixed to magnetic microsphere Surface testing reactive compound.
However, at present both at home and abroad, having no that cell membrane is fixed in the report of this such magnetic microsphere, the report using the active component in cell membrane magnetic microsphere screening natural products is also had no.Cell membrane is fixed on magnetic microsphere surface by this patent based on membrane flexibility technology, sets up cell membrane magnetic microsphere, and screen the active component in natural products using the microballoon combination mass-spectrometric technique.
The content of the invention
For these reasons, the active component in natural products is screened it is an object of the invention to provide a kind of cell membrane magnetic microsphere preparation method of new quick screening natural product active ingredient and using it.Comprise the following steps：
(1) cell membrane suspension is added after protease inhibitors, and cell, sonicated cells is resuspended with pH7-8 hypotonic medium.Under the conditions of 4-10 DEG C, 5-10min is centrifuged using 200-1000 × g, supernatant is taken, then 15-30min is centrifuged using 10000-20000 × g, supernatant is abandoned, takes precipitation, precipitated 3~5 times using 5mM phosphate buffers (PBS) cleaning, obtain cell membrane.4 DEG C storage, it is standby.
(2) magnetic microsphere is suctioned out from protection liquid using magnetic force or the method for centrifugation.
(3) under oscillating condition, the cell membrane that step 1 is obtained is adsorbed in by magnetic microsphere surface using decompression method, cell membrane magnetic microsphere is prepared, the microballoon prepared is placed in 4-10 DEG C, constant temperature oscillation 2-8h so that cell membrane is fully merged is produced.
(4) active ingredients of medicinal materials is extracted 1-3 times using water or alcohol heat reflux, each 30min-120min merges extract solution, let cool, after filtration, 4 DEG C of preservations are standby.
(5) under the conditions of 37 DEG C, cell membrane magnetic microsphere and natural product extraction liquid are incubated 10-60min altogether.The cell membrane magnetic microsphere for having adsorbed active component is separated from extract solution using magnetic force, centrifugal force etc., uncombined compound is washed away using phosphate buffer again, the dissociation solution constituted using 10%-30% acetic acid aqueous solutions dissociates the target spot on reactive compound and cell membrane magnetic microsphere surface, using the method for combined gas chromatography mass spectrometry determine compound group in dissociation solution into.
Advantages of the present invention compared with prior art：
1st, the affine adsorption technology of analog cell film that this patent is built using magnetic microsphere is core technology.By setting up cell membrane magnetic microsphere, and the screening by the technology applied to the active component of natural products.Its advantage protrude be embodied in it is following some.(1) using magnetic microsphere as carrier, the magnetic force for relying primarily on its exterior is separated, so as to avoid influence of the factor (for example, pH value, ionic strength, or surface charge) to analysis work in system.(2) the necessary steps of conventional cell membrane chromatography such as post, balance chromatographic column need not be filled, so as to simplify Active Components process, analytical cycle are shortened.(3) compared with traditional membrane flexibility post, cell membrane consumption is significantly reduced, (4) due to post and balance chromatographic column need not be filled, therefore The method reduces the consumption of buffer solution.
2nd, this patent can be that raw material prepares the active component that cell membrane magnetic microsphere " is fished " in natural products using various kinds of cell cell membrane, and method is simple, has wide practical use in terms of the active ingredient screening of natural products.
Embodiment
Following examples are used merely to explain the present invention, and protection scope of the present invention is not intended to be limited to following examples.Person of an ordinary skill in the technical field takes scope according to present invention below disclosure and each parameter, can be achieved.
Example (1) erythrocyte membrane magnetic microsphere quickly screens Radix Angelicae Sinensis containing active component
New fresh rabbit blood is placed in Alsever's Solution, 4 DEG C of preservations.1mL is taken to centrifuge 5min in 3000rpm before use, precipitation is red blood cell.The hypotonic medium containing protease inhibitors is added into precipitation, using sonicated cells.Under the conditions of 4 DEG C, 5min is centrifuged using 200 × g, supernatant is taken and 20min is centrifuged using 15000 × g, abandon supernatant, take precipitation, precipitated 3 times using the cleaning of 5mM PBS solutions, obtain cell membrane.With pH7.1, cell membrane is resuspended in 5mM PBS, and adjustment epicyte protein concentration is 1.2mg/mL, is placed on 4 DEG C of storages of centrifuge tube, standby.Magnetic microsphere is suctioned out from protection liquid using magnetic force.Under oscillating condition, the cell membrane of acquisition is adsorbed in by magnetic microsphere surface using decompression method, cell membrane magnetic microsphere is prepared, the microballoon prepared is placed in 4 DEG C, constant temperature oscillation 6h so that cell membrane is fully merged is produced.
Cell membrane magnetic microsphere is marked using fluorescein isothiocynate (FITC)：10 μ L 1mg/mL FITC solution are taken to add 1mL cell membrane suspensions, 8h is kept in dark place at 4 DEG C in magnetic microsphere, cell membrane magnetic microsphere.Unnecessary FITC is gone using 5mM PBS rinsings.Observed using confocal microscope, as a result show that cell membrane has attached to magnetic microsphere surface (accompanying drawing 1).
Radix Angelicae Sinensis medicinal powder 10g is taken to use 80mL hydro-thermal refluxing extractions 1h.Extract solution is placed in -20 DEG C of pre-freezes, is subsequently placed in vacuum freeze drier and freezes (24h), is redissolved using 5mM PBS, 0.22 μm of membrane filtration, 4 DEG C of preservations, standby.Under the conditions of 37 DEG C, cell membrane magnetic microsphere and natural product extraction liquid are incubated 30min altogether.The cell membrane magnetic microsphere for having adsorbed active component is separated from extract solution using magnetic force, uncombined compound is washed away using 5mM PBS again, the target spot for making reactive compound and cell membrane magnetic microsphere using 20% acetic acid aqueous strip solution is dissociated, using GC/MS method determine compound group in dissociation solution into.Gas Chromatographic Determination condition：Aglient 7890A-5973C types gas-mass spectrometer is determined.Chromatographic column is Aglient HP-5ms capillary gas chromatographic columns, and carrier gas is He gas, flow velocity 1.0mL/min.280 DEG C of injector temperature, 300 DEG C of ion source temperature, 50 DEG C of initial temperature, with 10 DEG C/min temperature programmings to 100 DEG C, then with 5 DEG C/min temperature programmings to 280 DEG C, scanning of the mass spectrum scope 50-550amu (chromatogram is shown in accompanying drawing 2).Compared by using NIST databases, determine the composition contained in dissociation solution.
Brief description of the drawings
Fig. 1 is the confocal microscope figure (cell membrane (A1) of the red blood cell under visible ray of cell membrane magnetic microsphere, the erythrocyte cell film (B1) of FITC- marks, the magnetic microsphere (C1) of FITC- marks, the cell membrane magnetic microsphere (D1) of FITC- marks, the cell membrane (A2) of red blood cell under fluorescence, the erythrocyte cell film (B2) of FITC- marks, the magnetic microsphere (C2) of FITC- marks, the cell membrane magnetic microsphere (D2) of FITC- marks)
Fig. 2 is that (A is the GC/MS figures of angelica extract to erythrocyte cell film magnetic microsphere analysis Radix Angelicae Sinensis active component chromatogram, B is after being incubated altogether with angelica extract, the GC/MS figures of dissociation solution, C is the GC/MS figures (blank control) of dissociation solution after cell membrane magnetic microsphere is incubated altogether with water).
Claims (4)
1. a kind of cell membrane magnetic microsphere of quick screening natural product active ingredient is prepared and its applied, it is characterised in that including following step
Suddenly：
(1) cell membrane suspension is added after protease inhibitors, and cell is resuspended with pH7-8 hypotonic medium.Sonicated cells.In 4-10
Under the conditions of DEG C, 5-10min is centrifuged using 200-1000 × g, supernatant is taken, then 15-30min is centrifuged using 10000-20000 × g,
Supernatant is abandoned, precipitation is taken, is precipitated 3~5 times using 5mM phosphate buffers (PBS) cleaning, obtains cell membrane.4 DEG C storage,
It is standby.
(2) magnetic microsphere is suctioned out from protection liquid using magnetic force or the method for centrifugation.
(3) under oscillating condition, the cell membrane that step 1 is obtained is adsorbed in by magnetic microsphere surface using decompression method, cell membrane magnetic is prepared
Property microballoon, is placed in 4-10 DEG C, constant temperature oscillation 2-8h by the microballoon prepared so that cell membrane is fully merged, produces.
(4) active ingredients of medicinal materials is extracted 1-3 times using water or alcohol heat reflux, each 30min-120min merges extract solution, let cool,
After filtration, 4 DEG C of preservations are standby.
(5) under the conditions of 37 DEG C, cell membrane magnetic microsphere and natural product extraction liquid are incubated 10-60min altogether.Using magnetic force, from
Mental and physical efforts etc. separate the cell membrane magnetic microsphere for having adsorbed active component from extract solution, then use phosphate buffer is washed away and do not tied
The compound of conjunction, the dissociation solution constituted using 10%-30% acetic acid aqueous solutions makes reactive compound and cell membrane magnetic microsphere surface
Target spot dissociate, using combined gas chromatography mass spectrometry method determine dissociation solution in compound group into.
2. a kind of cell membrane magnetic microsphere of quick screening natural product active ingredient according to claim 1 is prepared and its applied,
It is characterized in that the cell membrane of live body or the cell of culture is fixed on into magnetic microsphere, cell membrane magnetic microsphere is prepared.
3. a kind of cell membrane magnetic microsphere of quick screening natural product active ingredient according to claim 1 is prepared and its applied,
It is characterized in that being incubated altogether with natural products using cell membrane magnetic microsphere, so that the active component with reference to contained by natural products.
4. a kind of cell membrane magnetic microsphere of quick screening natural product active ingredient according to claim 1 is prepared and its applied,
It is characterized in that analyzing active component in cell membrane magnetic microsphere dissociation solution using hydrolysis and condensation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108896647A (en) * | 2018-07-20 | 2018-11-27 | 遵义医学院 | A kind of method of high-flux fast screening ERp57 inhibitor |
| CN109856278A (en) * | 2019-02-01 | 2019-06-07 | 广东药科大学 | A method of the screening active constituent based on three-phase laminar flow micro-fluidic chip |
-
2016
- 2016-04-06 CN CN201610220406.XA patent/CN107271595A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108896647A (en) * | 2018-07-20 | 2018-11-27 | 遵义医学院 | A kind of method of high-flux fast screening ERp57 inhibitor |
| CN109856278A (en) * | 2019-02-01 | 2019-06-07 | 广东药科大学 | A method of the screening active constituent based on three-phase laminar flow micro-fluidic chip |
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