CN107014847A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
- G01N24/088—Assessment or manipulation of a chemical or biochemical reaction, e.g. verification whether a chemical reaction occurred or whether a ligand binds to a receptor in drug screening or assessing reaction kinetics
Abstract
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes, belongs to tcm development applied technical field.The present invention dependent on set up based on prepares NiPt nano-probes be used for screen suppression anaerobic bacteria Chinese medicine preparation method.The characteristics of paramagnetic NiPt nano-probes prepared can be specifically bound to object bacteria is coated with using antibody, the influence using NiPt paramagnetic properties to the NMR relaxation time detects whether contain object bacteria in sample.Paramagnetic NiPt nano-probes, NMR relaxation time showing outlet sexual intercourse within the specific limits, i.e. Ni nanoparticle Pt contents are big, the nuclear magnetic resonance spin Lattice Relaxation Time and transverse relaxation time value of sample are smaller, detection object bacteria can be quantified in certain limit, so that the fungistatic effect of indirect assessment Chinese medicine preparation.This method can be used for the recipe determination for having the Chinese medicine of inhibitory action to anaerobic bacteria, accelerate the development rate of tcm product.
Description
Technical field
The present invention relates to a kind of screening technique of Chinese medicine preparation.
Background technology
Antibiotic simultaneously, will also result in human body infringement, influence liver, renal function, gastrointestinal reaction etc. in sterilization.Abuse is anti-
Raw element may cause the generation of some bacterial resistance phenomenons, and the treatment to infection can become very difficult.At present, almost without one
Plant antibacterials and resistance phenomenon is not present.Chinese medicine and formula of the exploitation with antibacterial action have positive effect.
Many Chinese medicines and magistral medicines have antibacterial and anti-inflammation functions, and some medium-height grass the effective elements of the medicines and molecular structure etc. are also
Through studying clear whole or in part.The main component berberine of such as coptis and golden cypress stop dysentery（Berberine）, radix scutellariae antibacterial
Main component baicalein etc..A host of facts prove, the medicine that ancient Chinese working people is accumulated by long-term practice
Legacy is extremely abundant, extremely valuable.We should value the great treasure-house of this motherland's medicine and pharmacology, make great efforts to excavate, be subject to
Improve, can therefrom develop the Chinese medicine with antibacterial and anti-inflammation functions, can replace or part substitution antibiotic.
Anaerobic bacteria be a class under anaerobic than the bacterium grown in aerobic environment, and can not be with the presence of air
In the case of the bacterium that grows.This kind of bacterium lacks complete metabolism enzyme system, and its energetic supersession is carried out in the way of anaerobic fermentation.
It can cause the infection of human body different parts, including appendicitis, cholecystitis, tympanitis, mouth infection, endocarditis, osteomyelitis,
A variety of diseases such as peritonitis.It is usually in sterilizing in Chinese medicine new formulation procedures of traditional exploitation for certain anaerobic bacteria
Traditional Chinese medicinal components are added after inoculation of medium anaerobic bacteria, are cultivated in special anaerobic culture box.Sampled again after a period of time into
Row line culture, calculates clump count, so that it is determined that whether certain Chinese medicine compositing formula has bacteriostasis.Line culture is also required to detesting
Cultivated in oxygen incubator, and cultivation cycle at least takes several days, therefore whole cycle is long, efficiency is low.This method is asked for above-mentioned
Inscribe a kind of rapid screening method of Chinese medicine preparation of invention.
The general principle of this method：Monoclonal antibody or antigen molecule are by Covalent bonding together, and this combination will not change
Monoclonal antibody, the immunological characteristic of antigen and bioactivity, specific monoclonal antibody only can be with specific antigen knots
Close.And NiPt materials have ferromagnetism, its outer layer has multiple unpaired electrons, small in particle diameter with high saturation and magnetic intensity
When to a certain extent, it may appear that superparamagnetic characteristic, NiPt nano materials can relax to nuclear magnetic resonance spin-lattice of surrounding water molecules
The influence of Henan time is very big, the effect with amplification nuclear magnetic resonance relaxation signals.Very micro NiPt nano materials will
The NMR relaxation time of water is caused to decline to a great extent.Therefore can be by building NiPt nano-probes, from the angle of magnetic resonance
To detect whether to have object bacteria, so that the bacteriostasis of indirect assessment traditional Chinese medicine ingredients.
Key step：
1）Prepared by NiPt nano-probes.Commercially paramagnetic NiPt nano materials, can also be prepared by other method
Nano level NiPt.Using silane coupler, its formula is:Y(CH2)nSiX3.Herein, n is 0-3；X is hydrolyzable group；Y
For organo-functional group.X is typically chloro, methoxyl group, ethyoxyl, acetoxyl group etc., generation silanol (Si when these groups are hydrolyzed
(OH)3), and combined with inorganic substances, form siloxanes.Y is vinyl, amino, epoxy radicals, methacryloxy, sulfydryl.
These reactive groups can react and combine with organic substance.Therefore, can be in inorganic substances and organic by using silane coupler
" molecular bridge " is erected between the interface of material, the material of two kinds of properties great disparity is linked together improve composite performance and
Increase the effect of adhesive strength.The NiPt for reacting the amination or carboxylated that can obtain surface modification by silane coupler receives
Rice material.Further by coupled antibody, surface-functionalized, formation specific immunity probe can be achieved, unnecessary activity is closed
Site, can separate excessive antibody after centrifugation, you can to obtain the NiPt nano-probes of resistance modification.
2）The sample cell made using polystyrene material carries out Chinese medicine screening.Polystyrene material and ELISA Plate class
Seemingly, detecting probe surface antibody can be combined on the surface of polystyrene material due to suction-operated, still, and the antibody of detecting probe surface is inhaled
The speed of polystyrene material surface is attached to compared with the speed of detecting probe surface antibody binding object bacteria, the former is slow-footed more,
Both are competitive relations.Therefore, the culture medium of the anaerobic bacteria of sterilizing, vaccination target anaerobic bacteria are added in polystyrene sample pipe
The Chinese medicine extract for needing to screen is added afterwards, after closing anaerobic culture for a period of time, adds step 1）Prepared NiPt nanometers
Probe, is sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, the antibody on NiPt nano-probes surface
It can be combined first with object bacteria, so that NiPt nano-probes are stayed in a liquid.If there is no object bacteria in sample cell, NiPt
The antibody on nano-probe surface can be adsorbed gradually in polystyrene sample pipe surface, so that not having NiPt nanometers of spies in liquid
Pin.Preceding to have addressed, the NMR relaxation time that micro NiPt nano materials will result in water declines to a great extent.Do not have with sterile
The medium liquid for having vaccination target bacterium is blank, and the relaxation time of the suspension measured then illustrates liquid compared to significantly reducing
Contain probe in body, so as to there is object bacteria in indirect proof sample, the amount of probe and the drop-out value in relaxation time are proportional, pass through
The quantitative probe of mark-on can go out the amount of object bacteria with indirect quantification.With the presence of object bacteria, then illustrate that Traditional Chinese medicine bacteriostatic effect is not obvious, instead
Then explanation have bacteriostasis.
The major advantage of this method is exactly quick, sensitivity height.Object bacteria can be realized and continuously existed to a certain extent
Line is monitored.Time of even several days 2-3 days relative to anaerobic bacteria culture, magnetic resonance detection only needs a few minutes.The method is not required to
Anaerobic culture box is wanted, can cultivate the sample seal of tube after need to being only inoculated with anaerobic, will be quantitative with syringe during detection
Probe injection can carry out follow-up magnetic resonance detection.Therefore, the screening of extensive Chinese medicine and formula can be made of this method.
The content of the invention
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes.This method is a kind of objective effective
The method for detecting object bacteria in sample, can do the quick screening of extensive Chinese medicine and formula, so as to a certain extent significantly
Reduction is directed to certain anaerobic bacteria effective tcm development time.
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes.Using NMR to paramagnet
The response sensibility of matter, proposes NMR relaxation Parameters variation and the correlation of NiPt nano-particle paramagnetic immunological probe contents
Index.This method is used to the detection of object bacteria in sample assess the suppression of Chinese medicine preparation dependent on paramagnetic nano NiPt nano-probes
Bacterium effect., can be to the specific anaerobism in sample using the paramagnetic NiPt nano-probes for being coupled monoclonal antibody specific
Bacterium is detected.Because NMR is very sensitive to NiPt nano-particles, there is micro paramagnetic NiPt nano-particles, then
The spin-lattice relaxation time of water and/spin-spin relaxation will be remarkably decreased.Quantitatively detected by blank control and mark-on
The immunological probe content gone out in sample, the anaerobic bacteria clump count gone out so as to indirect quantification in sample.The anaerobic bacteria detected
Content and probe content linear correlation, degree of fitting are preferable.Finally with the corresponding relation between paramagnetic NiPt nano-probes and anaerobic bacteria
For tie, the anaerobic bacteria clump count in sample is determined.So as to determine whether the bacteriostasis of Chinese medicine is effective indirectly.Relative to anaerobism
Bacterium cultivates the time of even several days 2-3 days, and magnetic resonance detection only needs a few minutes.Therefore, extensive Chinese medicine can be done with this method
And the screening of formula.
The present invention is achieved in that step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic nano NiPt nano-probes.
2）The culture medium of the anaerobic bacteria of sterilizing, vaccination target anaerobic bacteria are added in polystyrene sample pipe.Adding needs
The Chinese medicine extract of screening, after culture a period of time, after culture a period of time, adds step 1）Prepared NiPt nanometers are visited
Pin, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, first can be anti-with NiPt nano-probes surface
Body is combined, so that NiPt nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, NiPt nano-probe tables
The antibody in face can gradually be adsorbed and separated in polystyrene sample pipe surface with liquid.Now, nuclear magnetic resonance is carried out to sample cell
The measure in relaxation time.Using the sterile medium liquid without vaccination target bacterium as blank, during the relaxation of the suspension measured
Between compared to significantly reducing, then explanation contains probe, so as to there is object bacteria in indirect proof sample, the amount of probe and relaxation time
Decline it is proportional, by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria.
The NiPt nano-probes are the nanoscale NiPt materials with paramagnetic properties, and nanometer particle size is less than 300 nanometers.
The change in final relaxation time of the detection evaluation method based on nuclear magnetic resonance technique of described object bacteria, during relaxation
Between include spin-lattice relaxation time and spin spin relaxation time.
Beneficial effects of the present invention：The present invention, which provides a kind of objectively quick screening, the Chinese medicine of inhibitory action to anaerobic bacteria
The method of formula, it is characterized in that using the corresponding relation between paramagnetic NiPt nano-probes and anaerobic bacteria as tie, determining in sample
Anaerobic bacteria clump count.So as to determine whether the bacteriostasis of Chinese medicine is effective indirectly.Relative to anaerobic bacteria culture 2-3 days even several days
Time, magnetic resonance detection only needs a few minutes.Therefore, the screening of extensive Chinese medicine and formula can be made of this method.
Embodiment
Example 1
Screening is to Propiobacterium（Anaerobic bacteria）There is the Chinese medicine preparation of suppression or therapeutic action.
1）It is prepared by Ni nanoparticle Pt immunological probes：
NiPt nano-particles can be commercially（Such as the Chao Wei nano materials company in Shanghai）, can also bibliography thing
Manage or be chemically synthesized.
Silicon dioxide coated Ni nanoparticle Pt：47.5g sodium metasilicate is taken, is dissolved in deionized water in beaker, is adjusted with hydrochloric acid
PH value is 12-13.5.0g Ni nanoparticles Pt is taken to be added in this beaker, mechanical agitation (using glass bar) 5min.By mixed liquor ultrasound
30min, in good time stirring.85 C are warming up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise.The deionization in Magneto separate
Water washing is precipitated, and is washed 3-4 times.Then, precipitation is scattered in 250mL methanol.Above procedure is in triplicate, it is ensured that silicon is depended on
On NiPt.
Amino containing silane NiPt nano materials：Obtained silicon dioxide coated Ni nanoparticle Pt is added to 25mL methanol
In, use 1mLH2O and methanol dilution are to 150mL.Then the mixing of 150mL glycerine is added.Ultrasonic 30min, has been transferred to agitating device
500mL three-necked flasks in.Add 10mL amino silicane coupling agents（AEAPS）, under 80-90 C after quick stirring 3h, transfer
Go out product.Product is washed with deionized 3 times, and methanol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.It should be noted
It is that suction filtration is due to there is glycerine, so slow, supernatant liquid can be sucked with suction pipe in good time by taking out after a period of time, suction filtration process is about
Need 6-8h.Obtained Aminosilylation NiPt nano materials are finally dried in vacuo 12h.
Antibody modification：A certain amount of amido modified NiPt nano-particles are weighed, the EDC and NHS of 2 times of quality is added, plus
Enter 0.01M each 1mL of PBS.It is placed on blending instrument, 15 turns of rotating speed, reacts 45min.）The sample that soak time is arrived is carried out
10000r/min is centrifuged, time 5min, and the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.By the sample centrifuged
Product respectively add excessive Propiobacterium antibody, and 1h is coupled on blending instrument.The 10%BSA confining liquids 45min added after the completion of coupling.
After closing terminates, rotating speed 8000r/min centrifuges 5min, is centrifuged, is repeated 3 times with 0.01M PBS, then will be unnecessary
Antibody, BSA are washed away, and are eventually adding PBS and are resuspended.The NiPt nano-particles of the resistance modification of preparation are immunological probe, are protected
It is stand-by in the presence of 4 DEG C.
2）The culture medium sterilized in right amount is each added in a collection of polystyrene sample pipe, acne bar is inoculated with oxygen free condition
Bacterium, is separately added into after the Chinese medicine extract sealing for needing to screen, after culture a period of time, adds step 1）Prepared NiPt receives
Rice probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, antibody knot that first can be with NiPt nano-probes surface
Close, so that NiPt nano-probes are stayed in a liquid；If not there is object bacteria in sample cell, NiPt nano-probes surface
Antibody can be adsorbed gradually in polystyrene sample pipe surface.After reaction 1 hour, is carried out to sample cell the NMR relaxation time
Determine（Shanghai knob steps NMR20 NMRs）.Using the sterile medium liquid without vaccination target bacterium as blank, measure
The relaxation time spin-lattice relaxation time of suspension has been compared with spin spin relaxation time and significantly reduced, then explanation contains
Probe, illustrates there is object bacteria in sample cell, so that the bacteriostasis of side light Chinese medicine is not obvious, on the contrary then anti-.The amount of probe
Decline with spin-lattice relaxation time and spin spin relaxation time is proportional, the indirect quantification by mark-on quantitative probe
Go out the amount of object bacteria.Illustrate that the Chinese medicine added does not have inhibitory action with the presence of object bacteria.It is on the contrary then anti-.
Embodiment
Example 2
Screening is to clostridium（Anaerobic bacteria）There is the Chinese medicine preparation of suppression or therapeutic action.
1）It is prepared by Ni nanoparticle Pt immunological probes：
NiPt nano-particles can be commercially（Such as the Chao Wei nano materials company in Shanghai）, can also bibliography thing
Manage and be chemically synthesized.
Silicon dioxide coated Ni nanoparticle Pt：47.5g sodium metasilicate is taken, is dissolved in deionized water in beaker, is adjusted with hydrochloric acid
PH value is 12-13.5.0g Ni nanoparticles Pt is taken to be added in this beaker, mechanical agitation (using glass bar) 5min.By mixed liquor ultrasound
30min, in good time stirring.85 C are warming up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise.The deionization in Magneto separate
Water washing is precipitated, and is washed 3-4 times.Then, precipitation is scattered in 250mL methanol.Above procedure is in triplicate, it is ensured that silicon is depended on
On NiPt.
Amino containing silane NiPt nano materials：Obtained silicon dioxide coated Ni nanoparticle Pt is added to 25mL methanol
In, use 1mLH2O and methanol dilution are to 150mL.Then the mixing of 150mL glycerine is added.Ultrasonic 30min, has been transferred to agitating device
500mL three-necked flasks in.Add 10mL amino silicane coupling agents（AEAPS）, under 80-90 C after quick stirring 3h, transfer
Go out product.Product is washed with deionized 3 times, and methanol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.It should be noted
It is that suction filtration is due to there is glycerine, so slow, supernatant liquid can be sucked with suction pipe in good time by taking out after a period of time, suction filtration process is about
Need 6-8h.Obtained Aminosilylation NiPt nano materials are finally dried in vacuo 12h.
Antibody modification：A certain amount of amido modified NiPt nano-particles are weighed, the EDC and NHS of 2 times of quality is added, plus
Enter 0.01M each 1mL of PBS.It is placed on blending instrument, 15 turns of rotating speed, reacts 45min.）The sample that soak time is arrived is carried out
10000r/min is centrifuged, time 5min, and the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.By the sample centrifuged
Product respectively add excessive clostridium antibody, and 1h is coupled on blending instrument.The 10%BSA confining liquids added after the completion of coupling
45min.After closing terminates, rotating speed 8000r/min centrifuges 5min, is centrifuged, is repeated 3 times with 0.01M PBS, then will
Unnecessary antibody, BSA are washed away, and are eventually adding PBS and are resuspended.The NiPt nano-particles of the resistance modification of preparation are immune spy
Pin, be stored in 4 DEG C it is stand-by.
2）The culture medium sterilized in right amount is each added in a collection of polystyrene sample pipe, fusiform bud is inoculated with oxygen free condition
Spore bacillus, is separately added into after the Chinese medicine extract sealing for needing to screen, after culture a period of time, adds step 1）Prepared
NiPt nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with NiPt nano-probes surface
Antibody binding, so that NiPt nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, NiPt nano-probes
The antibody on surface can be adsorbed gradually in polystyrene sample pipe surface.After reaction 1 hour, NMR relaxation is carried out to sample cell
The measure of time（Shanghai knob steps NMR20 NMRs）.Using the sterile medium liquid without vaccination target bacterium as blank,
The spin-lattice relaxation time of the suspension measured has been compared with spin spin relaxation time and significantly reduced, then explanation contains spy
Pin, illustrates there is object bacteria in sample cell, so that the bacteriostasis of side light Chinese medicine is not obvious, on the contrary then anti-.The amount of probe with
The decline of spin-lattice relaxation time and spin spin relaxation time is proportional, and by mark-on quantitative probe, indirect quantification goes out
The amount of object bacteria.Illustrate that the Chinese medicine added does not have inhibitory action with the presence of object bacteria.It is on the contrary then anti-.
Claims (3)
1. a kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes, its characterization step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic NiPt nano-probes；
2）The culture medium of the anaerobic bacteria of sterilizing is added in polystyrene sample pipe, vaccination target anaerobic bacteria, addition needs screening
Chinese medicine preparation extract solution, after closed culture a period of time, step 1 is quantitatively adding with syringe）Prepared NiPt nanometers are visited
Pin, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, and the antibody on NiPt nano-probes surface first can
Combined with object bacteria, so that NiPt nano-probes are stayed in a liquid；If not there is object bacteria in sample cell, NiPt nanometers
The antibody of detecting probe surface can gradually adsorb and leave nutrient solution in polystyrene sample pipe surface；Now, core is carried out to sample cell
The measure in magnetic resonance relaxation time；Using the sterile medium liquid without vaccination target bacterium as blank, the suspension measured
Relaxation time, then explanation contained probe compared to significantly reducing, so that have object bacteria in indirect proof sample, the amount and relaxation of probe
The decline of Henan time is proportional, and by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria；There is no object bacteria in sample cell
Illustrate that the traditional Chinese medicine ingredients added have a fungistatic effect, it is on the contrary then anti-.
2. the method according to claim 1 that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes, it is characterized in that
The Ni nanoparticle Pt nano-probes are the Ni nanoparticle Pt alloy materials with paramagnetic properties, and nanometer particle size is less than 300 nanometers.
3. the method according to claim 1 that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on NiPt nano-probes, it is characterized in that
The change in final relaxation time of the detection evaluation method based on nuclear magnetic resonance technique of described object bacteria.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61268187A (en) * | 1985-05-22 | 1986-11-27 | Kitasato Inst:The | Novel antibiotic substance sk-1071 and production thereof |
| EP0280894A1 (en) * | 1987-02-03 | 1988-09-07 | Mitsubishi Kasei Corporation | Novel antibiotic vermisporin, process for the production thereof and pharmaceutical composition comprising it as an active antibacterial agent |
| CN1808106A (en) * | 2005-01-21 | 2006-07-26 | 中国科学院上海药物研究所 | Method for screening acetylcholine esterase inhibitor by nuclear magnetic resonance |
-
2017
- 2017-02-23 CN CN201710100983.XA patent/CN107014847A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61268187A (en) * | 1985-05-22 | 1986-11-27 | Kitasato Inst:The | Novel antibiotic substance sk-1071 and production thereof |
| EP0280894A1 (en) * | 1987-02-03 | 1988-09-07 | Mitsubishi Kasei Corporation | Novel antibiotic vermisporin, process for the production thereof and pharmaceutical composition comprising it as an active antibacterial agent |
| CN1808106A (en) * | 2005-01-21 | 2006-07-26 | 中国科学院上海药物研究所 | Method for screening acetylcholine esterase inhibitor by nuclear magnetic resonance |
Non-Patent Citations (1)
| Title |
|---|
| 申静 等: "基于NMR纳米探针的生物传感器用于快速检测沙门氏菌", 《食品工业科技》 * |
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Application publication date: 20170804 |