CN106841271A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on ZnO nano probe - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on ZnO nano probe Download PDFInfo
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Abstract
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on ZnO nano probe, belongs to tcm development applied technical field.The present invention depend on set up based on prepare ZnO nano probe for screen suppression anaerobic bacteria Chinese medicine preparation method.Whether the characteristics of magnetic ZnO nano probe prepared using antibody coating can be specifically bound to object bacteria, the influence using the magnetic properties of ZnO to the NMR relaxation time is detected and contain object bacteria in sample.Magnetic ZnO nano probe, NMR relaxation time showing outlet sexual intercourse within the specific limits, that is nano-ZnO 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 ZnO material has magnetic, its element Zn outer layers have multiple unpaired electrons, with high saturation and magnetic intensity, in particle diameter
Small nano material to a certain extent can be very big to the influence of the nuclear magnetic resonance spin-Lattice Relaxation Time of surrounding water molecules,
With the effect for amplifying nuclear magnetic resonance relaxation signals.Very micro ZnO nano material will result in the NMR relaxation of water
Time declines to a great extent.Therefore can detect whether there is target from the angle of magnetic resonance by building ZnO nano probe
Bacterium, so that the bacteriostasis of indirect assessment traditional Chinese medicine ingredients.
Key step：
1）Prepared by ZnO nano probe.Commercially magnetic ZnO nano material, it is also possible to prepared by other method and received
The ZnO of meter level.Using silane coupler, its formula is:Y(CH2)nSiX3.Herein, n is 0-3；X is hydrolyzable group；Y is
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, 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.The ZnO 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 is capable of achieving, closes unnecessary activity
Site, can separate excessive antibody after centrifugation, you can to obtain the ZnO nano probe 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）Prepared ZnO nano is visited
Pin, is sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, the antibody of ZnO nano detecting probe surface is first
Can first be combined with object bacteria, so that ZnO nano probe is stayed in a liquid.If there is no object bacteria in sample cell, ZnO nano
The antibody of detecting probe surface can be adsorbed gradually in polystyrene sample pipe surface, so that not having ZnO nano probe in liquid.It is preceding
Address, the NMR relaxation time that micro ZnO nano material will result in water declines to a great extent.With aseptic without inoculation mesh
The medium liquid for marking bacterium is blank, and the relaxation time of the suspension for measuring illustrates to contain in liquid compared to significantly reducing, then
Probe, so as to there is object bacteria in indirect proof sample, the amount of probe is proportional with the drop-out value in relaxation time, quantitative by mark-on
Probe 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, otherwise then illustrate
There is bacteriostasis.
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 ZnO nano probe.The method is a kind of objective effective
The method of object bacteria, can do the quick screening of extensive Chinese medicine and formula, so as to a certain extent significantly in detection sample
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 ZnO nano probe.Using NMR to Magnetic Materials
The response sensibility of matter, proposes the correlation of NMR relaxation Parameters variation and ZnO nanoparticle magnetic immuno probe content
Index.The method depends on magnetic Nano ZnO nano probe for the detection of object bacteria in sample to assess the suppression of Chinese medicine preparation
Bacterium effect.Using the magnetic ZnO nano probe for being coupled monoclonal antibody specific, can to sample in specific anaerobic bacteria
Detected.Because NMR is very sensitive to ZnO nanoparticle, there is micro magnetic ZnO nanoparticle, then water
Spin-lattice relaxation time and/spin-spin relaxation will be remarkably decreased.Sample is gone out by blank and mark-on quantitative determination
Immunological probe content in product, such that it is able to the anaerobic bacteria clump count that indirect quantification goes out in sample.The anaerobism bacterial content for detecting
With probe content linear correlation, degree of fitting is preferable.Finally with the corresponding relation between magnetic ZnO nano probe and anaerobic bacteria as knob
Band, determines the anaerobic bacteria clump count in sample.So as to whether the bacteriostasis for determining Chinese medicine indirectly is effective.Trained relative to anaerobic bacteria
The time of even several days 2-3 days is supported, magnetic resonance detection only needs a few minutes.Therefore, extensive Chinese medicine can be done with the method and is matched somebody with somebody
The screening of side.
The present invention is achieved in that step is as follows：
1）Target bacteria antibody is coated with the preparation of magnetic Nano ZnO nano probe.
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 ZnO nano probe,
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 of ZnO nano detecting probe surface
Close, so that ZnO nano probe is stayed in a liquid.If not there is object bacteria in sample cell, ZnO nano detecting probe surface it is anti-
Cognition is gradually adsorbed and separated with liquid in polystyrene sample pipe surface.Now, when carrying out NMR relaxation to sample cell
Between measure.With the aseptic medium liquid without vaccination target bacterium as blank, the relaxation time of the suspension for measuring is compared
Significantly reduce, then explanation contains probe, so as to there is object bacteria in indirect proof sample, the amount of probe and the decline in relaxation time
Proportional, by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria.
The ZnO nano probe is the nanoscale ZnO material with magnetic 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 magnetic ZnO nano probe 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 nano-ZnO immunological probe：
ZnO nanoparticle can be commercially, it is also possible to which bibliography is with either physically or chemically synthesizing.
Silicon dioxide coated nano-ZnO：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 nano-ZnOs 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 ZnO.
Amino containing silane ZnO nano material：Obtained silicon dioxide coated nano-ZnO is added in 25mL methyl alcohol,
Use 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 ZnO nano material vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified ZnO nanoparticle is 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 ZnO nanoparticle of the resistance modification of preparation is immunological probe, preserves
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 ZnO receives
Rice probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with the antibody knot of ZnO nano detecting probe surface
Close, so that ZnO nano probe is stayed in a liquid；If not there is object bacteria in sample cell, ZnO nano detecting probe surface it is anti-
Cognition is gradually adsorbed in polystyrene sample pipe surface.After reaction 1 hour, the survey of NMR relaxation time is carried out to sample cell
It is fixed（Shanghai knob steps NMR20 NMRs）.With the aseptic medium liquid without vaccination target bacterium as blank, what is measured is outstanding
The relaxation time spin-lattice relaxation time of turbid 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 nano-ZnO immunological probe：
ZnO nanoparticle can be commercially, it is also possible to bibliography physics and is chemically synthesized.
Silicon dioxide coated nano-ZnO：47.5g sodium metasilicate is taken, is dissolved in beaker with deionized water, be with salt acid for adjusting pH value
12-13.Take 5.0g nano-ZnOs to be added in this beaker, mechanical agitation (using glass bar) 5min.By mixed liquor ultrasound 30min, fit
When stir.85 C are warmed up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise over.It is heavy to be washed with deionized in Magneto separate
Form sediment, wash 3-4 times.Then, precipitation is scattered in 250mL methyl alcohol.Above procedure is in triplicate, it is ensured that silicon is attached on ZnO.
Amino containing silane ZnO nano material：Obtained silicon dioxide coated nano-ZnO is added in 25mL methyl alcohol,
Use 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 ZnO nano material vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified ZnO nanoparticle is 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 ZnO nanoparticle of the resistance modification of preparation is 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
ZnO nano probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be anti-with ZnO nano detecting probe surface
Body is combined, so that ZnO nano probe is stayed in a liquid.If not there is object bacteria in sample cell, ZnO nano detecting probe surface
Antibody can gradually adsorb in polystyrene sample pipe surface.After reaction 1 hour, is carried out to sample cell the NMR relaxation time
Measure（Shanghai knob steps NMR20 NMRs）.With the aseptic medium liquid without vaccination target bacterium as blank, measure
The spin-lattice relaxation time of suspension compared with spin spin relaxation time and significantly reduced, then explanation contains probe,
Illustrate 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 from
The decline of rotation-Lattice Relaxation Time and spin spin relaxation time is proportional, and by mark-on quantitative probe, indirect quantification goes out mesh
Mark the amount of bacterium.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 ZnO nano probe, its characterization step is as follows：
1）Target bacteria antibody is coated with the preparation of magnetic ZnO nano probe；
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 ZnO nano is visited
Pin, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, the antibody of ZnO nano detecting probe surface first can
Combined with object bacteria, so that ZnO nano probe is stayed in a liquid；If there is no object bacteria in sample cell, ZnO nano probe
The antibody on surface can gradually be adsorbed and leave nutrient solution in polystyrene sample pipe surface；Now, nuclear-magnetism is carried out to sample cell to be total to
Shake the measure in relaxation time；With the aseptic medium liquid without vaccination target bacterium as blank, the relaxation of the suspension for measuring
Time, then explanation contained probe compared to significantly reducing, so that have object bacteria in indirect proof sample, when the amount of probe is with relaxation
Between decline it is proportional, by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria；There is no object bacteria to illustrate in sample cell
The traditional Chinese medicine ingredients of addition have fungistatic effect, otherwise then anti-.
2. it is according to claim 1 based on ZnO nano probe screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, it is characterized in that institute
It is the nano ZnO material with magnetic properties to state nano-ZnO nano-probe, and nanometer particle size is less than 300 nanometers.
3. it is according to claim 1 based on ZnO nano probe 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109932399A (en) * | 2017-12-15 | 2019-06-25 | Tcl集团股份有限公司 | Nanocomposite and its preparation method and application |
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Application publication date: 20170613 |