CN107014851A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on FeZrB nano-probes - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on FeZrB nano-probes Download PDFInfo
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- G—PHYSICS
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- 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 FeZrB nano-probes, belongs to tcm development applied technical field.The present invention dependent on set up based on prepares FeZrB nano-probes be used for screen suppression anaerobic bacteria Chinese medicine preparation method.The characteristics of paramagnetic FeZrB nano-probes prepared can be specifically bound to object bacteria is coated with using antibody, the influence using FeZrB paramagnetic properties to the NMR relaxation time detects whether contain object bacteria in sample.Paramagnetic FeZrB nano-probes, NMR relaxation time showing outlet sexual intercourse within the specific limits, i.e. nanometer Fe ZrB 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 ternary FeZrB materials have ferromagnetism, its element of Fe outer layer has multiple unpaired electrons, strong with high saturation
Degree, particle diameter it is small to a certain extent when, it may appear that paramagnetic and superparamagnetic characteristic, FeZrB nano materials can be to surrounding water molecules
The influence of nuclear magnetic resonance spin-Lattice Relaxation Time is very big, the effect with amplification nuclear magnetic resonance relaxation signals.It is very micro
FeZrB nano materials will result in NMR relaxation time of water and decline to a great extent.Therefore can be by building FeZrB nanometers
Probe, detects whether there is object bacteria from the angle of magnetic resonance, so that the bacteriostasis of indirect assessment traditional Chinese medicine ingredients.
Key step：
1）Prepared by FeZrB nano-probes.Commercially paramagnetic FeZrB nano materials, can also be by other method system
Standby nano level FeZrB.Using silane coupler, its formula is:Y(CH2)nSiX3.Herein, n is 0-3；X is hydrolyzable base
Group；Y is organo-functional group.X is typically chloro, methoxyl group, ethyoxyl, acetoxyl group etc., and silicon is generated when these groups are hydrolyzed
Alcohol (Si (OH)3), and combined with inorganic substances, form siloxanes.Y be vinyl, amino, epoxy radicals, methacryloxy,
Sulfydryl.These reactive groups can react and combine with organic substance.Therefore, can be in inorganic substances by using silane coupler
" molecular bridge " is erected between the interface of organic substance, the material of two kinds of property great disparities is linked together and improves composite
The effect of performance and increase adhesive strength.The amination or carboxylated of surface modification can be obtained by being reacted by silane coupler
FeZrB nano materials.Further by coupled antibody, surface-functionalized, formation specific immunity probe can be achieved, it is unnecessary to close
Avtive spot, excessive antibody can be separated after centrifugation, you can to obtain the FeZrB 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 FeZrB nanometers
Probe, is sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, FeZrB nano-probes surface it is anti-
Body can be combined with object bacteria first, so that FeZrB nano-probes are stayed in a liquid.If there is no object bacteria in sample cell,
The antibody on FeZrB nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface, so that not having FeZrB to receive in liquid
Rice probe.Preceding to have addressed, the NMR relaxation time that micro FeZrB nano materials will result in water declines to a great extent.With nothing
The medium liquid without vaccination target bacterium of bacterium is blank, and the relaxation time of the suspension measured is compared and significantly reduced, then
Illustrate to contain probe in liquid, so as to there is object bacteria in indirect proof sample, the amount of probe and the drop-out value in relaxation time are in just
Than the amount of object bacteria can be gone out with indirect quantification by the quantitative probe of mark-on.With the presence of object bacteria, then illustrate Traditional Chinese medicine bacteriostatic effect not
Substantially, it is on the contrary 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 FeZrB nano-probes.This method is a kind of objective effective
Detection sample in object bacteria method, the quick screening of extensive Chinese medicine and formula can be done, so that big to a certain extent
Big 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 FeZrB nano-probes.Using NMR to paramagnetic
The response sensibility of material, proposes NMR relaxation Parameters variation and the phase of FeZrB nano-particle paramagnetic immunological probe contents
Closing property index.This method is matched somebody with somebody dependent on the detection that paramagnetic nano FeZrB nano-probes are used for object bacteria in sample to assess Chinese medicine
The fungistatic effect of side., can be to special in sample using the paramagnetic FeZrB nano-probes for being coupled monoclonal antibody specific
Property anaerobic bacteria is detected.Because NMR is very sensitive to FeZrB nano-particles, there is micro paramagnetic FeZrB and receive
Rice corpuscles, then the spin-lattice relaxation time of water and/spin-spin relaxation will be remarkably decreased.Pass through blank control and mark-on
The immunological probe content in sample is quantitatively detected, the anaerobic bacteria clump count gone out so as to indirect quantification in sample.Detect
Anaerobism bacterial content and probe content linear correlation, degree of fitting is preferable.Finally between paramagnetic FeZrB nano-probes and anaerobic bacteria
Corresponding relation is tie, determines the anaerobic bacteria clump count in sample.So as to determine whether the bacteriostasis of Chinese medicine is effective indirectly.Phase
For the anaerobic bacteria culture time of even several days 2-3 days, magnetic resonance detection only needs a few minutes.Therefore, it can be made greatly of this method
The screening of scale Chinese medicine and formula.
The present invention is achieved in that step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic nano FeZrB 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 FeZrB 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 with FeZrB nano-probes surface
Antibody binding, so that FeZrB nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, FeZrB nanometers of spies
The antibody of wire surface can gradually be adsorbed and separated in polystyrene sample pipe surface with liquid.Now, nuclear-magnetism is carried out to sample cell
The measure in resonance relaxation time.Using the sterile medium liquid without vaccination target bacterium as blank, the relaxation of the suspension measured
The Henan 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 time is proportional, and by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria.
The FeZrB nano-probes are the nanoscale FeZrB 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 FeZrB 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 nanometer Fe ZrB immunological probes：
FeZrB nano-particles can commercially, can also bibliography with either physically or chemically synthesizing.
Silicon dioxide coated nanometer Fe ZrB：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 nanometer Fes ZrB 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 FeZrB.
Amino containing silane FeZrB nano materials：Obtained silicon dioxide coated nanometer Fe ZrB 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 FeZrB nano materials are finally dried in vacuo 12h.
Antibody modification：A certain amount of amido modified FeZrB 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 FeZrB 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 FeZrB
Nano-probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be anti-with FeZrB nano-probes surface
Body is combined, so that FeZrB nano-probes are stayed in a liquid；If not there is object bacteria in sample cell, FeZrB 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 relaxation time spin-lattice relaxation time of the suspension measured has been compared with spin spin relaxation time and significantly reduced, then is said
It is bright to illustrate there is object bacteria in sample cell containing probe, it is on the contrary then anti-so that the bacteriostasis of side light Chinese medicine is not obvious.Visit
The amount of pin and the decline of spin-lattice relaxation time and spin spin relaxation time are proportional, by the quantitative probe of mark-on
Connect the amount for quantifying 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 nanometer Fe ZrB immunological probes：
FeZrB nano-particles bibliography physics and can also commercially can be chemically synthesized.
Silicon dioxide coated nanometer Fe ZrB：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 nanometer Fes ZrB 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 FeZrB.
Amino containing silane FeZrB nano materials：Obtained silicon dioxide coated nanometer Fe ZrB 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 FeZrB nano materials are finally dried in vacuo 12h.
Antibody modification：A certain amount of amido modified FeZrB 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 FeZrB nano-particles of the resistance modification of preparation are immune
Probe, 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
FeZrB nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with FeZrB nano-probes surface
Antibody binding so that FeZrB nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, FeZrB nanometers
The antibody of detecting probe surface can be adsorbed gradually in polystyrene sample pipe surface.After reaction 1 hour, nuclear magnetic resonance is carried out to sample cell
The measure in relaxation 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 is illustrated
Containing probe, illustrate there is object bacteria in sample cell, it is on the contrary then anti-so that the bacteriostasis of side light Chinese medicine is not obvious.Probe
Amount and the decline of spin-lattice relaxation time and spin spin relaxation time it is proportional, it is indirect by the quantitative probe of mark-on
Quantify 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 FeZrB nano-probes, its characterization step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic FeZrB 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 FeZrB 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 FeZrB nano-probes surface is first
It can be combined with object bacteria, so that FeZrB nano-probes are stayed in a liquid；If not having object bacteria in sample cell, FeZrB receives
The antibody of rice detecting probe surface can be adsorbed gradually leaves nutrient solution in polystyrene sample pipe surface；Now, sample cell is carried out
The measure of NMR relaxation time；Using the sterile medium liquid without vaccination target bacterium as blank, the suspension measured
Relaxation time compared to significantly reducing, then explanation contains probe, so as to there is object bacteria in indirect proof sample, the amount of probe with
The decline in relaxation time is proportional, and by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria；There is no target in sample cell
Bacterium illustrates that the traditional Chinese medicine ingredients added have a fungistatic effect, 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 FeZrB nano-probes, it is characterized in that
The nanometer Fe ZrB nano-probes are the nanometer Fe ZrB 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 FeZrB 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
- 2017-03-31 CN CN201710205328.0A patent/CN107014851A/en not_active Withdrawn
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 (2)
| Title |
|---|
| 孙建宁: "《中药药理学》", 31 January 2014 * |
| 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 |