CN106918612A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe nano-probes - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe 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 Fe nano-probes, belongs to tcm development applied technical field.The present invention depend on set up based on prepares Fe nano-probes for screen suppression anaerobic bacteria Chinese medicine preparation method.Whether the characteristics of paramagnetic Fe nano-probes prepared using antibody coating can be specifically bound to object bacteria, the influence using the paramagnetic properties of Fe to the NMR relaxation time is detected and contain object bacteria in sample.Paramagnetic Fe nano-probes, NMR relaxation time showing outlet sexual intercourse within the specific limits, that is nanometer Fe content is big, the nuclear magnetic resonance spin Lattice Relaxation Time and transverse relaxation time value of sample are smaller, certain limit can quantitative determination object bacteria so that the fungistatic effect of indirect assessment Chinese medicine preparation.The method can be used for the recipe determination of the Chinese medicine for having 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 will also result in human body infringement simultaneously sterilized, influence liver, renal function, gastrointestinal reaction etc..Abuse is anti-
Raw element may cause the generation of some bacterial resistance phenomenons, and the treatment to infecting can become very difficult.At present, almost without one
Plant antibacterials and do not exist resistance phenomenon.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）, root of large-flowered skullcap antibacterial
Main component baicalein etc..A host of facts prove that ancient Chinese working people passes through the medicine that long-term practice is accumulated
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, and 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 grow bacterium.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,
Various diseases such as peritonitis.In Chinese medicine new formulation procedures of traditional exploitation for certain anaerobic bacteria, usually in sterilizing
Traditional Chinese medicinal components are added after inoculation of medium anaerobic bacteria, is cultivated in special anaerobic culture box.A period of time after sample again 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 directed to above-mentioned asking
Inscribe a kind of rapid screening method of Chinese medicine preparation of invention.
The general principle of this method：, by Covalent bonding together, this combination will not change for monoclonal antibody or antigen molecule
Monoclonal antibody, the immunological characteristic of antigen and bioactivity, specific monoclonal antibody only can be with specific antigen knots
Close.And Fe materials have ferromagnetism, its outer layer has multiple unpaired electrons, with high saturation and magnetic intensity, arrives particle diameter is small
When to a certain degree, it may appear that superparamagnetic characteristic, Fe nano materials can to the nuclear magnetic resonance spin-lattice relaxation of surrounding water molecules when
Between influence it is very big, with amplify nuclear magnetic resonance relaxation signals effect.Very micro Fe nano materials will result in water
The NMR relaxation time decline to a great extent.Therefore can be detected from the angle of magnetic resonance by building Fe nano-probes
With the presence or absence of object bacteria, so that the bacteriostasis of indirect assessment traditional Chinese medicine ingredients.
Key step：
1）Prepared by Fe nano-probes.Commercially paramagnetic Fe nano materials, it is also possible to which nanometer is prepared by other method
The Fe of level.Using silane coupler, its formula is:Y(CH2)nSiX3.Herein, n is 0-3；X is hydrolyzable group；Y is organic
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, by using silane coupler, can be in inorganic substances and organic
" 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.Fe nanometers that the amination or carboxylated that can obtain surface modification is reacted by silane coupler
Material.Further by coupled antibody, surface-functionalized, formation specific immunity probe is capable of achieving, closes unnecessary active sites
Point, can separate excessive antibody after centrifugation, you can to obtain the Fe nano-probes of resistance modification.
2）The sample cell for being 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, but, 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 combining target bacterium, 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
Adding afterwards needs the Chinese medicine extract of screening, after closing anaerobic culture for a period of time, adds step 1）Fe nanometers of prepared spy
Pin, is sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, the antibody on Fe nano-probes surface is first
Can be combined with object bacteria, so that Fe nano-probes are stayed in a liquid.If there is no object bacteria in sample cell, Fe nano-probes
The antibody on surface can be adsorbed gradually in polystyrene sample pipe surface, so that not having Fe nano-probes in liquid.It is preceding to have addressed,
The NMR relaxation time that micro Fe nano materials will result in water declines to a great extent.With aseptic without vaccination target bacterium
Medium liquid is blank, and the relaxation time of the suspension for measuring illustrates to contain probe in liquid compared to significantly reducing, then, from
And having object bacteria in indirect proof sample, the amount of probe is proportional with the drop-out value in relaxation time, can by the quantitative probe of mark-on
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, on the contrary then explanation has antibacterial
Effect.
The major advantage of the method is exactly quick, sensitivity high.Object bacteria can be realized to a certain extent continuously existing
Line is monitored.Relative to the anaerobic bacteria culture time of even several days 2-3 days, magnetic resonance detection only needs a few minutes.The method is not required to
Anaerobic culture box is wanted, will be cultivated by 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 the method.
The content of the invention
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe nano-probes.The method is a kind of objective effective inspection
The method for going out object bacteria in sample, can do the quick screening of extensive Chinese medicine and formula, so as to contract significantly to a certain extent
Subtract for certain anaerobic bacteria effective tcm development time.
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe nano-probes.Using NMR to paramagnetic meterial
Response sensibility, propose NMR relaxation Parameters variation refer to the correlation of Fe nano-particle paramagnetic immunological probe contents
Mark.The method depends on paramagnetic nano Fe nano-probes for the detection of object bacteria in sample to assess the antibacterial effect of Chinese medicine preparation
Really.Using the paramagnetic Fe nano-probes for being coupled monoclonal antibody specific, can to sample in specific anaerobic bacteria carry out
Detection.Because NMR is very sensitive to Fe nano-particles, there is micro paramagnetic Fe nano-particles, then the spin of water-
Lattice Relaxation Time and/spin-spin relaxation will be remarkably decreased.Gone out in sample by blank and mark-on quantitative determination
Immunological probe content, such that it is able to the anaerobic bacteria clump count that indirect quantification goes out in sample.The anaerobism bacterial content and probe for detecting
Content linear correlation, degree of fitting is preferable.Finally with the corresponding relation between paramagnetic Fe nano-probes and anaerobic bacteria as tie, sample is determined
Anaerobic bacteria clump count in product.So as to whether the bacteriostasis for determining Chinese medicine indirectly is effective.Relative to anaerobic bacteria culture 2-3 days very
To the time of several days, magnetic resonance detection only needed a few minutes.Therefore, the screening of extensive Chinese medicine and formula can be made of the method.
The present invention is achieved in that step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic nano Fe 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 Fe nano-probes,
It is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, first can be with the antibody knot on Fe nano-probes surface
Close, so that Fe nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, the antibody on Fe nano-probes surface
Can gradually adsorb and be separated with liquid in polystyrene sample pipe surface.Now, is carried out to sample cell the NMR relaxation time
Measure.With the aseptic medium liquid without vaccination target bacterium as blank, the relaxation time of the suspension for measuring is compared and has
Significantly reduce, then explanation contains probe, so as to there is object bacteria in indirect proof sample, the amount of probe is in the decline in relaxation time
Direct ratio, by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria.
The Fe nano-probes are the nanoscale Fe materials with paramagnetic properties, and nanometer particle size is less than 300 nanometers.
The final detection evaluation method of described object bacteria is based on the change in the relaxation time of nuclear magnetic resonance technique, during relaxation
Between include spin-lattice relaxation time and spin spin relaxation time.
Beneficial effects of the present invention：The present invention 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 with the corresponding relation between paramagnetic Fe nano-probes and anaerobic bacteria as tie, determines detesting in sample
Oxygen bacterium clump count.So as to whether the bacteriostasis for determining Chinese medicine indirectly is effective.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 the method.
Specific 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 immunological probe：
Fe nano-particles can be commercially（Such as the Chao Wei nano materials company in Shanghai）, it is also possible to bibliography physics
Or be chemically synthesized.
Silicon dioxide coated nanometer Fe：47.5g sodium metasilicate is taken, is dissolved in beaker with deionized water, use salt acid for adjusting pH
It is 12-13 to be worth.Take 5.0g nanometer Fes to be added in this beaker, mechanical agitation (using glass bar) 5min.By mixed liquor ultrasound
30min, in good time stirring.85 C are warmed up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise over.The deionization in Magneto separate
Water washing is precipitated, and is washed 3-4 times.Then, precipitation is scattered in 250mL methyl alcohol.Above procedure is in triplicate, it is ensured that silicon is depended on
On Fe.
Amino containing silane Fe nano materials：Obtained silicon dioxide coated nanometer Fe is added in 25mL methyl alcohol, is used
1mLH2O and methanol dilution are to 150mL.It is subsequently adding the mixing of 150mL glycerine.Ultrasonic 30min, is transferred to agitating device
In 500mL there-necked flasks.Add 10mL amino silicane coupling agents（AEAPS）, after quick stirring 3h under 80-90 C, it is transferred out of
Product.Product is washed with deionized 3 times, and methyl alcohol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.It should be noted that
Suction filtration so slow, supernatant liquid can be sucked after taking out a period of time with suction pipe in good time due to there is glycerine, and suction filtration process about needs 6-
8h.The Aminosilylation Fe nano materials vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified Fe nano-particles are weighed, 2 times of EDC and NHS of quality are added, added
Each 1mL of PBS of 0.01M.It is placed on blending instrument, 15 turns of rotating speed, reacts 45min.）By soak time to sample carry out
10000r/min is centrifuged, and time 5min, the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.The sample that will be 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 centrifugation 5min are centrifuged with the PBS of 0.01M, are repeated 3 times, then will be unnecessary
Antibody, BSA are washed away, be eventually adding PBS carry out it is resuspended.The Fe nano-particles of the resistance modification of preparation are immunological probe, preserve
It is stand-by at 4 DEG C.
2）The culture medium of appropriate sterilizing is each added in a collection of polystyrene sample pipe, acne bar is inoculated with oxygen free condition
Bacterium, after being separately added into the Chinese medicine extract sealing for needing screening, after culture a period of time, adds step 1）Prepared Fe nanometers
Probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, can be combined with the antibody on Fe nano-probes surface first,
So that Fe nano-probes are stayed in a liquid；If not there is object bacteria in sample cell, the antibody meeting on Fe nano-probes surface
Gradually adsorb in polystyrene sample pipe surface.After reaction 1 hour, the measure of NMR relaxation time is carried out to sample cell
（Shanghai knob steps NMR20 NMRs）.With the aseptic medium liquid without vaccination target bacterium as blank, what is measured is suspended
The relaxation time spin-lattice relaxation time of liquid 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 it is 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.The Chinese medicine added with the presence of object bacteria explanation does not have inhibitory action.Otherwise it is then anti-.
Specific 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 immunological probe：
Fe nano-particles can be commercially（Such as the Chao Wei nano materials company in Shanghai）, it is also possible to bibliography physics
And be chemically synthesized.
Silicon dioxide coated nanometer Fe：47.5g sodium metasilicate is taken, is dissolved in beaker with deionized water, use salt acid for adjusting pH
It is 12-13 to be worth.Take 5.0g nanometer Fes to be added in this beaker, mechanical agitation (using glass bar) 5min.By mixed liquor ultrasound
30min, in good time stirring.85 C are warmed up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise over.The deionization in Magneto separate
Water washing is precipitated, and is washed 3-4 times.Then, precipitation is scattered in 250mL methyl alcohol.Above procedure is in triplicate, it is ensured that silicon is depended on
On Fe.
Amino containing silane Fe nano materials：Obtained silicon dioxide coated nanometer Fe is added in 25mL methyl alcohol, is used
1mLH2O and methanol dilution are to 150mL.It is subsequently adding the mixing of 150mL glycerine.Ultrasonic 30min, is transferred to agitating device
In 500mL there-necked flasks.Add 10mL amino silicane coupling agents（AEAPS）, after quick stirring 3h under 80-90 C, it is transferred out of
Product.Product is washed with deionized 3 times, and methyl alcohol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.It should be noted that
Suction filtration so slow, supernatant liquid can be sucked after taking out a period of time with suction pipe in good time due to there is glycerine, and suction filtration process about needs 6-
8h.The Aminosilylation Fe nano materials vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified Fe nano-particles are weighed, 2 times of EDC and NHS of quality are added, added
Each 1mL of PBS of 0.01M.It is placed on blending instrument, 15 turns of rotating speed, reacts 45min.）By soak time to sample carry out
10000r/min is centrifuged, and time 5min, the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.The sample that will be 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 centrifugation 5min are centrifuged with the PBS of 0.01M, are repeated 3 times, then will
Unnecessary antibody, BSA are washed away, be eventually adding PBS carry out it is resuspended.The Fe 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 of appropriate sterilizing is each added in a collection of polystyrene sample pipe, fusiform bud is inoculated with oxygen free condition
Spore bacillus, after being separately added into the Chinese medicine extract sealing for needing screening, after culture a period of time, adds step 1）Prepared Fe
Nano-probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with the antibody knot on Fe nano-probes surface
Close, so that Fe nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, the antibody on Fe nano-probes surface
Can gradually adsorb in polystyrene sample pipe surface.After reaction 1 hour, the measure of NMR relaxation time is carried out to sample cell
（Shanghai knob steps NMR20 NMRs）.With the aseptic medium liquid without vaccination target bacterium as blank, what is measured is suspended
The spin-lattice relaxation time of liquid has been compared with spin spin relaxation time and significantly reduced, then explanation contains probe, illustrates sample
There is object bacteria in QC, so that the bacteriostasis of side light Chinese medicine is not obvious, on the contrary it is then anti-.The amount and SPIN LATTICE of probe
The decline of relaxation time and spin spin relaxation time is proportional, and by mark-on quantitative probe, indirect quantification goes out object bacteria
Amount.The Chinese medicine added with the presence of object bacteria explanation does not have inhibitory action.Otherwise it is then anti-.
Claims (3)
1. a kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe nano-probes, its characterization step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic Fe 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 for a period of time, step 1 is quantitatively adding with syringe）Prepared Fe nano-probes,
It is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, the antibody on Fe nano-probes surface first can be with mesh
Bacterium combines mark, so that Fe nano-probes are stayed in a liquid；If not there is object bacteria in sample cell, Fe nano-probes surface
Antibody can gradually adsorb and leave nutrient solution in polystyrene sample pipe surface；Now, nuclear magnetic resonance relaxation is carried out to sample cell
The measure of Henan time；With the aseptic medium liquid without vaccination target bacterium as blank, the relaxation time of the suspension for measuring
Compared to significantly reducing, then explanation contains probe, so that have object bacteria in indirect proof sample, amount and the relaxation time of probe
Decline is proportional, and by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria；There is no object bacteria to illustrate to add in sample cell
Traditional Chinese medicine ingredients there is fungistatic effect, otherwise it is then anti-.
2. it is according to claim 1 based on Fe nano-probes screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, it is characterized in that institute
It is the nano-Fe materials with paramagnetic properties to state nanometer Fe nano-probe, and nanometer particle size is less than 300 nanometers.
3. it is according to claim 1 based on Fe nano-probes screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, it is characterized in that institute
The final detection evaluation method of the object bacteria stated is based on the change in the relaxation time of nuclear magnetic resonance technique.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0280894B1 (en) * | 1987-02-03 | 1991-06-26 | Mitsubishi Kasei Corporation | Novel antibiotic vermisporin, process for the production thereof and pharmaceutical composition comprising it as an active antibacterial agent |
| JPH0468319B2 (en) * | 1985-05-22 | 1992-11-02 | Kitasato Inst | |
| 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 CN201710100646.0A patent/CN106918612A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0468319B2 (en) * | 1985-05-22 | 1992-11-02 | Kitasato Inst | |
| EP0280894B1 (en) * | 1987-02-03 | 1991-06-26 | 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 (3)
| Title |
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| 孙建宁: "《中药药理学 供中药学 中医学 护理学等专业》", 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: 20170704 |