CN104307368B - Method for screening active ingredients in plant extracting liquid and special electrophoretic separation device - Google Patents
Method for screening active ingredients in plant extracting liquid and special electrophoretic separation device Download PDFInfo
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- CN104307368B CN104307368B CN201410611855.8A CN201410611855A CN104307368B CN 104307368 B CN104307368 B CN 104307368B CN 201410611855 A CN201410611855 A CN 201410611855A CN 104307368 B CN104307368 B CN 104307368B
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Abstract
The invention discloses a method for screening active ingredients in plant extracting liquid. The method comprises the following steps: (1) incubating protein and the plant extracting liquid in a buffer solution for 30 minutes at 37 DEG C, so that the active ingredients in the plant extracting liquid are specifically bound with the protein; (2) adding the mixed solution in the step (1) into a sample chamber of a special electrophoretic separation device, adding the buffer solution into other chambers and putting platinum gauze electrodes into electrode chambers respectively; (3) opening a buffer solution circulation system and condensing system, and opening a power supply system to form an electric field in a separation passage; (4) transferring the protein-active ingredient compound into a receiving chamber from the sample chamber under the effect of the electric field and leaving other ingredients except for the active ingredients in the plant extracting liquid in the sample chamber; (5) further analyzing the ingredients in both the sample chamber and the receiving chamber to obtain the active ingredients in the plant extracting liquid so as to achieve the purpose of screening the active ingredients in the plant extracting liquid. The invention further discloses the special electrophoretic separation device used in the method.
Description
Technical field
The present invention relates to active component in a kind of utilization electrophoretic techniques and target molecule affine technolog screening plant extraction liquid
Method and special purpose device, specifically refer to the screening technique of active component and special electrophoresis separator in a plant extract liquor.
Background technology
At present, the important channel that bioactive ingredients have become new drug development is screened from natural plants, how from again
Active component is fast and efficiently filtered out in miscellaneous plant substrates, is one of key areas of modern medicines research.Traditional sieve
Selecting technology needs separation and the purification step for repeating, and time-consuming, laborious, efficiency is low.In recent years, target molecule affine technolog is fast because of its
Speed, efficiently, high specificity the features such as be rapidly developed in drug screening field.The drug screening for growing up on this basis
Technology has affinity chromatography technology, hyperfiltration technique, magnetic bead triage techniques, doughnut triage techniques etc., but these technologies are present
The shortcomings of enzyme immobilization reduces activity, the enzyme-active component compound separation process complex effects the selection result of enzyme.More than being based on
Limitation, needs exploitation a kind of without the need for making enzyme under physiological environment in enzyme immobilizatio, screening process, simple to operate and can be quick
Separate the technology of the other compositions in enzyme-active component compound and plant extraction liquid in addition to the active ingredient (s.
The content of the invention
The invention aims to overcome enzyme immobilization in existing drug screening technology to reduce activity, the enzyme-activity of enzyme
The deficiencies such as composition compound separation process complex effects the selection result, and provide the sieve of active component in a plant extract liquor
Choosing method and special electrophoresis separator.The present invention without the need for enzyme immobilizatio, under close physiological environment, using electric field by albumen
Other compositions quick separating in the compound and plant extraction liquid of matter-active component in addition to the active ingredient (s.
To solve above-mentioned technical problem, technical scheme is as follows：
The screening technique of active component, comprises the following steps in one plant extract liquor：
1. protein and plant extraction liquid are hatched 30 minutes in cushioning liquid at 37 DEG C, in making plant extraction liquid
Active component specifically binds with protein, forms protein-reactive composition compound；
2. the mixed solution after protein and plant extraction liquid hatching is added in the sample room of special electrophoresis separator,
Cushioning liquid is added in cathode chamber, positive receiving chamber, negative receiving chamber, the anode chamber of electrophoretic separation device, and cathode chamber and anode chamber are divided
It is not placed with the platinum plate electrode of connection power supply；
3. open electrophoretic separation device the cushioning liquid circulatory system, condenser system, turn on the power supply system make its
Electric field is formed in split tunnel；
4. protein-reactive composition compound is moved in receiving chamber under electric field action by sample room, plant extraction liquid
In other compositions in addition to the active ingredient (s rest in sample room；
5. the component in sample room and receiving chamber is further analyzed, obtains the active component in plant extraction liquid.
The pH value of described cushioning liquid is 6-8.
It is described component in sample room and receiving chamber to be done into liquid chromatogram-tandem mass spectrum and NMR spectrum enters one
Step analysis, obtains the active component in plant extraction liquid.
The presentation mode of the electric field is dc source.
A kind of special electrophoresis separator, by the cushioning liquid circulatory system, piece-rate system, cooling separation condensed system
System and power system are constituted, described piece-rate system successively by cathode chamber, positive receiving chamber, sample room, negative receiving chamber and
Five room compositions of anode chamber, form split tunnel, fine by acetic acid respectively between sample room therein and positive receiving chamber, negative receiving chamber
The plain film of dimension separates, and is separated by milipore filter respectively between cathode chamber and positive receiving chamber, between anode chamber and negative receiving chamber, cathode chamber and
Anode chamber has the buffer solution inlet and buffering taphole, cathode chamber and negative pole being connected with the cushioning liquid circulatory system
Room is respectively put into the platinum plate electrode of connection power system.
Described buffer solution inlet is located at a side-lower of cathode chamber and anode chamber, and described cushioning liquid outlet is located at
The cathode chamber opposite side top corresponding with anode chamber.
The mechanism of active component in present invention screening plant extraction liquid：In hatching process in protein and plant extraction liquid
Protein-reactive composition compound, i.e. target molecule affine technolog formed and specific binding in active component there is.Protein has
Different from carrying electric charge in the cushioning liquid of the pH value of its isoelectric point, therefore protein-reactive composition compound carries identical electricity
Lotus.After applied voltage, the protein-reactive composition compound with electric charge can occur migration i.e. electrophoretic techniques.Protein-reactive
Composition compound is migrated through CAM from sample room to receiving chamber, and due to the crown_interception of milipore filter, albumen
Matter-active component compound will not move to electrode chamber and rest in receiving chamber, in plant extraction liquid in addition to the active ingredient (s
Other compositions are not rested in sample room by electric field action, and then have separated protein-reactive composition compound and plant extraction
Other compositions in addition to the active ingredient (s in liquid are taken, the purpose for filtering out active component in plant extraction liquid is reached.
Description of the drawings
Fig. 1 is the schematic diagram of special electrophoresis separator of the invention.
Fig. 2 is the piece-rate system structural representation in special electrophoresis separator of the invention.
Specific embodiment
In fig. 1 and 2, the 1. cushioning liquid circulatory system, 2. pump, 3. piece-rate system, 4. condenser system, 5. power supply supply
System, 6. lucite clamping plate, 7. cathode chamber, 8. positive receiving chamber, 9. sample room, 10. negative receiving chamber, 11. anode chambers, 12. vinegar
Acid cellulose film, 13. milipore filters, 14a. platinized platinum positive electrodes, 14b. platinized platinum negative electrodes, 15. buffer solution inlets, 16. bufferings are molten
Liquid is exported, 17. screw rods, 18. nuts.
With reference to Fig. 1 and Fig. 2, a kind of multicell is separated by electrophoresis device, by the cushioning liquid circulatory system 1, piece-rate system 3, cooling
The condenser system 4 and power system 5 of piece-rate system is constituted.Its piece-rate system 3 is successively by cathode chamber 7, positive receiving chamber 8, sample
Product room 9, negative receiving chamber 10,11 5 rooms compositions of anode chamber, between sample room 9 and positive receiving chamber 8, sample room 9 and negative receiving chamber
Separated by CAM 12 respectively between 10, between cathode chamber 7 and positive receiving chamber 8, anode chamber 11 and negative receiving chamber 10 it
Between separated by milipore filter 13 respectively, a side-lower of cathode chamber 7 and anode chamber 11 is respectively provided with buffer solution inlet 15, corresponding
Opposite side top is respectively provided with cushioning liquid outlet 16, for the circulation of cushioning liquid.Cathode chamber 7 and anode chamber 11 have been respectively put into
The platinized platinum positive electrode 14a and platinized platinum negative electrode 14b of connection power system.Cathode chamber 7 and the outside of anode chamber 11 respectively have one piece
Lucite clamping plate 6, and two pieces of both sides of lucite clamping plate 6 respectively have screw rod 17 to connect, and be used to fix by nut 18.Buffering is molten
Fluid circulation 1 is made up of the pump 2 arranged on the container equipped with cushioning liquid, circulating line, pipeline, slow with piece-rate system 3
Rush solution inlet 15, cushioning liquid outlet 16 to be connected to form loop.
A kind of disclosed method for screening active component in plant extraction liquid using above-mentioned special electrophoresis separator, should
Method will remove active component using electric field under close physiological environment in the compound of protein-reactive composition and plant extraction liquid
Outer other compositions are separated, explanation as a example by negatively charged, selected plant is as the red sage root in PBS with protein.Tool
Body experimentation is as follows：1. dry red sage root meal 1g is taken, with 10ml, 50% methyl alcohol ultrasonic extraction twice, combining extraction liquid,
Rotary evaporation, by the residual solution for obtaining 20ml PBSs are dissolved in, and obtain red sage root extract；Protein and the red sage root are extracted
Take liquid to hatch 30 minutes at 37 DEG C in cushioning liquid, the pH value of cushioning liquid is 6-8, makes the activity in plant extraction liquid
Composition specifically binds with protein, forms protein-reactive composition compound；2. by the mixed solution after hatching by micro-
Amount syringe is introduced in sample room 9, and cushioning liquid is introduced in other rooms, and platinized platinum positive electrode 14a, platinized platinum negative electrode 14b are put respectively
In entering cathode chamber 7 and anode chamber 11；3. open pump 2 and open the cushioning liquid circulatory system 1, open condenser system 4, turn on the power confession
Answer system 5 that electric field is formed in split tunnel, the presentation mode of the electric field is dc source；4. protein-reactive composition is answered
Compound is moved in positive receiving chamber 8 by sample room 9 under electric field action through CAM 12, except work in red sage root extract
Other compositions outside property composition are rested in sample room 9；5. by the component liquid chromatogram in sample room 9 and positive receiving chamber 8-
Tandem mass spectrum and NMR spectrum are further analyzed, and obtain the active component in red sage root extract.
Claims (4)
1. in a plant extract liquor active component screening technique, comprise the following steps：
1. protein and plant extraction liquid are hatched 30 minutes in cushioning liquid at 37 DEG C, makes the activity in plant extraction liquid
Composition specifically binds with protein, forms protein-reactive composition compound；
2. the mixed solution after protein and plant extraction liquid hatching is added in the sample room of electrophoretic separation device, cushioning liquid
In adding cathode chamber, positive receiving chamber, negative receiving chamber, the anode chamber of electrophoretic separation device, cathode chamber and anode chamber have been respectively put into
The platinum plate electrode of connection power supply；
3. the cushioning liquid circulatory system, the condenser system of electrophoretic separation device are opened, turning on the power supply system is separating it
Electric field is formed in passage；
4. protein-reactive composition compound is moved in receiving chamber under electric field action by sample room, is removed in plant extraction liquid
Other compositions outside active component are rested in sample room；
5. the component in sample room and receiving chamber is further analyzed, obtains the active component in plant extraction liquid.
2. in a plant extract liquor according to claim 1 active component screening technique, it is characterised in that it is described
Cushioning liquid pH value is 6-8.
3. in a plant extract liquor according to claim 1 active component screening technique, it is characterised in that it is described
Component in sample room and receiving chamber is done into liquid chromatogram-tandem mass spectrum and NMR spectrum is further analyzed, obtain plant
Active component in extract.
4. in a plant extract liquor according to claim 1 active component screening technique, it is characterised in that the electricity
The presentation mode of field is dc source.
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| CN107246985A (en) * | 2017-06-10 | 2017-10-13 | 延边大学 | A kind of method that utilization electrophoresis screens target protein natural products multicomponent complex |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1127157A (en) * | 1995-01-20 | 1996-07-24 | 清华大学 | Preparation type isoelectric point electrophoresis separating method and equipment |
| CN101716465A (en) * | 2009-12-02 | 2010-06-02 | 中国科学院过程工程研究所 | Method and device for separating electric field-separation membrane coupling |
| US8366897B2 (en) * | 2006-10-04 | 2013-02-05 | National Institute Of Standards And Technology | Gradient elution electrophoresis and detectorless electrophoresis apparatus |
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2014
- 2014-11-05 CN CN201410611855.8A patent/CN104307368B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1127157A (en) * | 1995-01-20 | 1996-07-24 | 清华大学 | Preparation type isoelectric point electrophoresis separating method and equipment |
| US8366897B2 (en) * | 2006-10-04 | 2013-02-05 | National Institute Of Standards And Technology | Gradient elution electrophoresis and detectorless electrophoresis apparatus |
| CN101716465A (en) * | 2009-12-02 | 2010-06-02 | 中国科学院过程工程研究所 | Method and device for separating electric field-separation membrane coupling |
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