CN107014931B - A kind of film modified nano carbon microsphere material of cell and its preparation method and application - Google Patents
A kind of film modified nano carbon microsphere material of cell and its preparation method and application Download PDFInfo
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- CN107014931B CN107014931B CN201710132304.7A CN201710132304A CN107014931B CN 107014931 B CN107014931 B CN 107014931B CN 201710132304 A CN201710132304 A CN 201710132304A CN 107014931 B CN107014931 B CN 107014931B
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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
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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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
Abstract
The present invention relates to a kind of drug screening material more particularly to a kind of nano carbon microsphere materials film modified by cell, and its for the application study of effective component of chinese medicine screening.A kind of film modified nano carbon microsphere material of cell, the film modified nano carbon microsphere material of the cell are made by the film modified nano carbon microsphere of cell.The material has preferable adsorption capacity, can either play the selective retention effect of membrane flexibility, and the adsorbance to active constituent can be greatly improved, and provides experiment basis for the verifying of further pharmacological activity.
Description
Technical field
The present invention relates to a kind of drug screening material more particularly to a kind of nano carbon microsphere materials film modified by cell, and
Its application study for being used for effective component of chinese medicine screening.
Background technique
The screening of effective component of chinese medicine is the source sex work of new medicinal products research.Due to Chinese medicine effect globality,
The research inhibition of the complexity of chemical composition of Chinese materia medica and mechanism of action, bioactive components is made slow progress, and is become in restriction
One of the bottleneck of medicine modernization.For a long time, natural product active ingredient has been used for reference in the screening of effective component of chinese medicine substantially
The thinking of research separates chemical composition of Chinese materia medica, is identified, pharmacological model is recycled to carry out Analysis on Biological Activity, or
Active tracking, separation and Structural Identification are carried out to Chinese medicine using pharmacological model.These traditional effective component of chinese medicine screen thinking
The characteristics of being unable to actual response Chinese medicine multicomponent, multiple target point and integration.Therefore, modernization of cmm needs to find
Recent studies on strategy suitable for traditional Chinese medicine complex system.
Cell separation membrane is a kind of biological chromatography separation material that cell membrane is bonded to fixed phase surface, is had thin
The bioactivity of after birth, so that traditional Chinese medicine complex system does not need traditional extraction step, it can be real on cell separation membrane
Existing activity identification process, has the more target affinities of multicomponent-.Currently, only membrane flexibility technology uses cell membrane point
From material, carrier of the macro porous silica gel as cell membrane, i.e. cell membrane-silica gel material are all used.Membrane flexibility technology is Chinese medicine
Active ingredient screening provide a kind of new thinking and means, but due to following disadvantage: (1) traditional Chinese medicine ingredients concentration difference compared with
Greatly, but cell membrane-silica gel material adsorption capacity is very low, is easily saturated absorption high concentration ingredient, and is difficult to most of low
Concentration effective component；(2) complicated for operation.Cell membrane stationary phase is served only in chromatographic column at present, needs to customize the color of special requirement
Column blank pipe is composed, complicated chromatograph packing material filling process is carried out, chromatogram research could be used for.The equipment that the process needs is more, hand
Continue many and diverse, is unfavorable for the universal of cell separation membrane.
Summary of the invention
In order to solve the above technical problems, the first purpose of the invention is to provide a kind of film modified nano carbon microspheres of cell
Material, the material have preferable adsorption capacity, can either play the selective retention effect of membrane flexibility, and can be very big
The adsorbance to active constituent is improved, provides experiment basis for the verifying of further pharmacological activity.Second object of the present invention is
The preparation method of the film modified nano carbon microsphere material of above-mentioned cell is provided.Third object of the present invention is to provide above-mentioned cells
The application of film modified nano carbon microsphere material.
In order to realize first above-mentioned purpose, present invention employs technical solutions below:
A kind of film modified nano carbon microsphere material of cell, the film modified nano carbon microsphere material of the cell is by the film modified nano-sized carbon of cell
Ball is made.
Preferably, cell membrane suspension, which is added, by nano carbon microsphere in the film modified nano carbon microsphere material of the cell carries out reaction system
, the mass ratio of the nano carbon microsphere and cell membrane suspension is 0.005 ~ 0.2:1 ~ 100.
Preferably, the nano carbon microsphere selects fowler polysaccharide microsphere.
Preferably, the cell membrane selects biological tissue cell film or biological cells film.
In order to realize second above-mentioned purpose, present invention employs technical solutions below:
A method of the film modified nano carbon microsphere material of above-mentioned cell being prepared, this method, which includes the following steps: to take, to be received
The mass ratio of rice carbon ball material, addition cell membrane suspension, the nano carbon microsphere material and cell membrane suspension is 0.005 ~ 0.2:1
~100；Vortex oscillation 5-100min stands 10-30h；5000-10000 rpm is centrifuged 10-30min, and precipitating is slow with 0.5-50mL
Salting liquid or brine are rushed, the film modified nano carbon microsphere material of cell is prepared into.
Preferably, the preparation method of the nano carbon microsphere includes the following steps: to weigh 1-10g glucose, dissolution
In 20-1000ml distilled water, stirs 5-30 minutes, glucose solution is transferred in the water heating kettle of inner liner polytetrafluoroethylene, it will
Water heating kettle is put into electric heating constant-temperature blowing drying box, the hydro-thermal reaction 2-20h under 100-300 DEG C of constant temperature；Then natural
It is cooled to room temperature, obtains brown suspension, the carbon ball of generation is collected by centrifugation, washs 2 ~ 3 with deionized water and dehydrated alcohol respectively
Time, it finally dries to get nano carbon microsphere is arrived.
Preferably, the preparation method of the cell membrane suspension includes the following steps: to take 0.2-10g animal tissue,
Residual blood is cleaned, is blotted with filter paper, is ground, with buffer salt solution or physiological saline the preparation homogenate of 5-30 times of volume, 2 layers of yarn
Cloth filtering, filtrate 5000-20000rpm are centrifuged 10-60 min；Precipitating is homogenized 2 times repeatedly, the supernatant after collecting 3 centrifugations,
5000-20000rpm is centrifuged 10-60 min, abandons supernatant, and precipitating uses buffer salt solution or normal saline flushing twice, every time
5000-20000rpm is centrifuged 10-60 min, must finally precipitate；10-100mg precipitating 1-10mL physiological saline is suspended, both
Cell membrane suspension.
Preferably, the preparation method of the cell membrane suspension includes the following steps: to take 0.1-0.5% trypsin
The cell that density is 104-108 is digested, cell suspension is centrifuged 10-30 min in 500-10000rpm, 1-20mL is added in precipitating
Physiological saline, ultrasonic 100-500W handles 10-60 min, and taking part supernatant observe under inverted microscope has confirmed it
Rupture of membranes, 5000-20000rpm are centrifuged 10-60 min, abandon supernatant, must finally precipitate.By 10-100mg precipitating 1-10mL physiology
Salt water is suspended, and both obtains cell membrane suspension.
In order to realize above-mentioned third purpose, present invention employs technical solutions below:
The film modified nano carbon microsphere material of above-mentioned cell is as dispersive solid-phase extraction material.
A kind of screening technique of effective component of chinese medicine, this method include the following steps:
1) it adsorbs
Buffer salt solution or normal saline suspension 0.5-20mL containing 5-200mg cell membrane nano carbon microsphere are taken, with
0.001-10 mL Chinese medicine extract vortex mixed 1-10min；Mixture 10000-20000 rpm is centrifuged 10-30min, collects
Supernatant；
2) it analyzes
By supernatant after 0.22 μm of membrane filtration in injection=analysis instrument, the separation condition of optimization is selected, carries out chemistry
Analysis；The change of the chemical component and content of comparison absorption front and back Chinese medical extract sample, screening obtain effectively
Components group.
The present invention due to the adoption of the above technical solution, using the fowler poly being carbonized under glucose water heat condition
For sugared microballoon as carrier, outer layer contains a large amount of hydrophilic radicals, can adherent cell film well, it is film modified to be prepared as cell
Nano carbon microsphere material.The nano carbon microsphere carrier has preferable adsorption capacity, and the selectivity that can either play membrane flexibility is protected
Use is given over to, and the adsorbance to active constituent can be greatly improved, provides experiment basis for the verifying of further pharmacological activity.This is thin
After birth modifies nano carbon microsphere material as dispersive solid-phase extraction material, and for the screening of effective component of chinese medicine, other use cell
There are notable differences with the present invention in contexture, working principle for the separation material of film.
The present invention is due to having the characteristics that following using above-mentioned experimental provision and applying step:
(1) material adsorption capacity is big
The adsorption capacity of current cell membrane silica gel material is very low, it is difficult to capture low concentration effective component.The present invention adopts
Use nano carbon microsphere as carrier, cell membrane is prepared as the drug screening material of high-adsorption-capacity as functional coating.Chinese medicine has
It imitates ingredient and enters nano carbon microsphere after cell membrane selectively penetrates, nano carbon microsphere will adsorb mass efficient ingredient, and be extracted by comparison
The content and type for taking front and back can filter out effective component.
(2) manufacturing process is easy to be controllable
Cell membrane stationary phase is served only in chromatographic column at present, needs to customize the chromatographic column blank pipe of special requirement, is carried out complicated
Chromatograph packing material load process, chromatogram research could be used for.The equipment that the process needs is more, and formality is many and diverse, is unfavorable for cell
Separation membrane is popularized.The present invention uses easy to operate, each factor in energy strict control manufacturing process, adsorbent material
Dosage can be precisely controlled, as a result favorable reproducibility, can be screened for the effective component of traditional Chinese medicine complex system and be provided reliable drug
Screening plant.
(3) methodological science is reasonable
Current membrane flexibility column has the problems such as hypertonia, complicated for operation, cost is excessively high, limits cell membrane color
The application of spectrum.The present invention is to disperse the film modified nano carbon microsphere of cell in Chinese medicine extract to operate, used
Swirling device and centrifuge are the common laboratory apparatus in all kinds of laboratories, with cheap, materials are convenient, easy to operate excellent
Point, it is easy to operate, it can be used for the effective elements of the medicine screening study in the overwhelming majority, and can be repeated as many times and use, Neng Gouwei
Effective component screening time is reduced, experimental expenses is reduced, plays positive effect.
(4) it is good to can be changed application prospect for cell membrane
The present invention obtains the cell membrane nano carbon microsphere material of different selection performances by changing cell membrane type, for screening
Effective component of chinese medicine with different drug effects.Nano carbon microsphere is prepared as a raw material with glucose, has good biofacies
Capacitive can effectively be combined with most cells film, form a variety of drug screening solid phase extraction materials, and there is selectivity flexibly may be used
Become, production is simple, reproducible advantage.
Detailed description of the invention
Scanning electron microscope (SEM) photograph (the 1- nano carbon microsphere, 2- cell membrane nanometer of Fig. 1 nano carbon microsphere material and cell membrane nano carbon microsphere
Carbon ball).
Chromatography figure (A: Chinese medicine extract, the B: the traditional Chinese medicine extraction after nano carbon microsphere absorption of Fig. 2 extraction front and back sample
Liquid, C: the Chinese medicine extract 1 after the absorption of cell membrane nano carbon microsphere: magnoflorine, 2: epiberberine, 3: Palmatine, 4: fixed towards the leaves of pulse plants
A, 5: Epimedin B, 6- epimedin C, 7- icariin).
Specific embodiment
1) preparation of carbon ball
10g glucose is weighed, is dissolved in 200ml distilled water, stirs 10 minutes, glucose solution is transferred to liner and is gathered
In the water heating kettle of tetrafluoroethene (compactedness about 80%), water heating kettle is put into electric heating constant-temperature blowing drying box, in 200 DEG C of perseverance
Hydro-thermal reaction 10h under the conditions of temperature.Then cooled to room temperature obtains brown suspension, and the carbon ball of generation is collected by centrifugation, respectively
It is washed 3 times with deionized water and dehydrated alcohol, finally in 80 DEG C of constant temperature 6h drying to get template carbon ball is arrived, grinding is standby
With.
2) preparation of cell membrane suspension
When preparing cell membrane suspension using osteoblast, taking 0.1-0.5% trypsin digestion density first is 108At
Cell suspension is centrifuged 10-30 min in 5000rpm by osteocyte, and the physiological saline of 10mL, ultrasonic 300W processing 10 is added in precipitating
Min, taking part supernatant to observe under inverted microscope confirms that its rupture of membranes, 10000rpm are centrifuged 20 min, abandons supernatant, most
It must precipitate afterwards.30mg precipitating 10mL physiological saline is suspended, osteoblast film suspension is both obtained.
3) preparation of cell membrane nano carbon microsphere
Cell membrane nano carbon microsphere is fixed on carbon ball carrier by cell membrane and is prepared.Carbon ball material 50mg is taken, cell is added
Film suspension 10mL, vortex oscillation 10min stand 20h.5000 rpm are centrifuged 10min, and precipitating is prepared with 10mL brine
Into cell membrane nano carbon microsphere.
4) it adsorbs
Buffer salt solution or normal saline suspension 1mL containing 50mg cell membrane nano carbon microsphere are taken, is mentioned with 20 μ L Chinese medicines
Take liquid vortex mixed 10min.20000 rpm of mixture is centrifuged 10min, collects supernatant.
5) it analyzes
20 μ L supernatants are injected in high performance liquid chromatograph after 0.22 μm of membrane filtration, carry out chemical component and content
Analysis.The change of the chemical component of comparison absorption front and back Chinese medical extract sample, screening obtain effective component group.
As a result, it has been found that nano carbon microsphere has stronger absorption after cell is film modified, to part chemical component in Chinese medicine extract.
The absorption property and nano carbon microsphere of cell membrane nano carbon microsphere have significant difference.Using osteoblast model, Herba Epimedii portion is carried out
Divide the drug effect verifying of chemical component, what the result and present invention screening of cell Proliferation and cytoactive detection (MTT experiment) obtained
As a result it coincide, it includes magnoflorine, epiberberine, icariin, Epimedin A, Epimedin B and towards the leaves of pulse plants that screening, which obtains effective component,
Determine C.The results are shown in Table 1.
The selection result of the invention of table 1 and MTT experiment verification result (average value, n=6)
Claims (7)
1. a kind of method of the film modified nano carbon microsphere material of cell, it is characterised in that: this method includes the following steps: to take nanometer
Carbon ball material, is added cell membrane suspension, the mass ratio of the nano carbon microsphere material and cell membrane suspension be 0.005 ~ 0.2:1 ~
100；Vortex oscillation 5-100min stands 10-30h；5000-10000 rpm is centrifuged 10-30min, and precipitating is buffered with 0.5-50mL
Salting liquid or brine are prepared into the film modified nano carbon microsphere material of cell；
Wherein, the preparation method of cell membrane suspension includes the following steps: to take 0.2-10g animal tissue, cleans residual blood, uses
Filter paper blots, and grinds, with buffer salt solution or physiological saline the preparation homogenate of 5-30 times of volume, 2 layers of filtered through gauze, filtrate
5000-20000rpm is centrifuged 10-60 min；Precipitating is homogenized 2 times repeatedly, the supernatant after collecting 3 centrifugations, 5000-
20000rpm is centrifuged 10-60 min, abandons supernatant, and precipitating uses buffer salt solution or normal saline flushing twice, each equal 5000-
20000rpm is centrifuged 10-60 min, must finally precipitate；10-100mg precipitating 1-10mL physiological saline is suspended, cell is both obtained
Film suspension；, wherein the preparation method of the nano carbon microsphere includes the following steps: to weigh 1-10g glucose, is dissolved in 20-
In 1000ml distilled water, stirs 5-30 minutes, glucose solution is transferred in the water heating kettle of inner liner polytetrafluoroethylene, by water heating kettle
It is put into electric heating constant-temperature blowing drying box, the hydro-thermal reaction 2-20h under 100-300 DEG C of constant temperature；Then it naturally cools to
Room temperature obtains brown suspension, and the carbon ball of generation is collected by centrifugation, and washs 2 ~ 3 times with deionized water and dehydrated alcohol respectively, most
After dry to get to nano carbon microsphere.
2. according to the method described in claim 1, it is characterized by: the preparation method of cell membrane suspension includes the following steps:
The cell that 0.1-0.5% trypsin digestion density is 104-108 is taken, cell suspension is centrifuged 10-30 in 500-10000rpm
The physiological saline of 1-20mL is added in min, precipitating, and ultrasonic 100-500W handles 10-60 min, takes part supernatant micro- in inversion
It confirms that its rupture of membranes, 5000-20000rpm are centrifuged 10-60 min under the microscope, abandons supernatant, must finally precipitate；By 10-
100mg precipitating is suspended with 1-10mL physiological saline, both obtains cell membrane suspension.
3. the film modified nano carbon microsphere material of the cell that method according to claim 1 or 2 prepares.
4. a kind of application of film modified nano carbon microsphere material of cell as claimed in claim 3 as dispersive solid-phase extraction material.
5. application according to claim 4, it is characterised in that: nano carbon microsphere selects fowler polysaccharide microsphere.
6. application according to claim 4, it is characterised in that: cell membrane selects biological tissue cell film or biological cell
Cell membrane.
7. a kind of film modified nano carbon microsphere material of cell as claimed in claim 3 is in epimedium active constituent screening technique
Using this method includes the following steps: that 1) absorption takes the buffer salt solution containing 5-200mg cell membrane nano carbon microsphere or life
Salt aqueous suspension 0.5-20mL is managed, with 0.001-10 mL extraction ofHerba Epimedii vortex mixed 1-10min；Mixture 10000-
20000 rpm are centrifuged 10-30min, collect supernatant；2) supernatant is injected analyzer after 0.22 μm of membrane filtration by analysis
In device, the separation condition of optimization is selected, carries out chemical component and content analysis；Comparison absorption front and back Shorthorned Epimedium P.E sample
The change of chemical component and content, screening obtain effective component group.
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| CN102951638B (en) * | 2012-11-30 | 2014-12-31 | 上海大学 | Hydrothermal synthesis method for hollow carbon nanomaterials |
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