CN106885822A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Pd Al O nano-probes - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Pd Al O nano-probes Download PDFInfo
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Abstract
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Pd Al O nano-probes, belongs to tcm development applied technical field.The present invention depend on set up based on prepares Co Pd Al O nano-probes for screen suppression anaerobic bacteria Chinese medicine preparation method.Whether the characteristics of paramagnetic Co Pd Al O nano-probes prepared using antibody coating can be specifically bound to object bacteria, the influence using the paramagnetic properties of Co Pd Al O to the NMR relaxation time is detected and contain object bacteria in sample.Paramagnetic Co Pd Al O nano-probes, NMR relaxation time showing outlet sexual intercourse within the specific limits, that is nano Co Pd Al O contents are 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 quaternary Co-Pd-Al-O materials have ferromagnetism, its Elements C o outer layers have multiple unpaired electrons, with saturation magnetic high
Change intensity, particle diameter it is small to a certain extent when, it may appear that paramagnetic and superparamagnetic characteristic Co-Pd-Al-O nano materials can be to surroundings
The influence of the nuclear magnetic resonance spin-Lattice Relaxation Time of hydrone is very big, with the effect for amplifying nuclear magnetic resonance relaxation signals.
The NMR relaxation time that very micro Co-Pd-Al-O nano materials will result in water declines to a great extent.Therefore can pass through
Co-Pd-Al-O nano-probes are built, detects whether there is object bacteria from the angle of magnetic resonance, so that indirect assessment Chinese medicine
The bacteriostasis of composition.
Key step：
1）Prepared by Co-Pd-Al-O nano-probes.Commercially paramagnetic Co-Pd-Al-O nano materials, it is also possible to pass through
Other method prepares nano level Co-Pd-Al-O.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., these groups
Silanol (Si (OH) is generated during hydrolysis3), 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, by using silane coupled
Agent, can erect " molecular bridge " between inorganic substances and the interface of organic substance, and the material of two kinds of property great disparities is connected to one
Play a part of to improve the performance of composite and increase adhesive strength.Being reacted by silane coupler can obtain surface modification
The Co-Pd-Al-O nano materials of amination or carboxylated.Further by coupled antibody, surface-functionalized, formation spy is capable of achieving
Specific immunological probe, closes unnecessary avtive spot, can be separated excessive antibody after centrifugation, you can to obtain resistance
The Co-Pd-Al-O nano-probes of 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 Co-Pd-Al-
O nano-probes, are sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, Co-Pd-Al-O nanometers of spy
The antibody of wire surface can be combined with object bacteria first, so that Co-Pd-Al-O nano-probes are stayed in a liquid.If sample cell
In there is no object bacteria, then the antibody on Co-Pd-Al-O nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface, from
And make there is no Co-Pd-Al-O nano-probes in liquid.Preceding to have addressed, micro Co-Pd-Al-O nano materials will result in water
The NMR relaxation time decline to a great extent.With the aseptic medium liquid without vaccination target bacterium as blank, what is measured is outstanding
The relaxation time of turbid liquid illustrates to contain probe in liquid compared to significantly reducing, then, so that have object bacteria in indirect proof sample,
The amount of probe is proportional with the drop-out value in relaxation time, can go out the amount of object bacteria with indirect quantification by the quantitative probe of mark-on.Have
Object bacteria is present, then illustrate that Traditional Chinese medicine bacteriostatic effect is not obvious, otherwise then explanation has 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 Co-Pd-Al-O nano-probes.The method is a kind of objective
The method of object bacteria, can do the quick screening of extensive Chinese medicine and formula, so as to a certain degree in effective detection sample
On significantly reduction be 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 Co-Pd-Al-O nano-probes.Using NMR pair
The response sensibility of paramagnetic meterial, proposes NMR relaxation Parameters variation and Co-Pd-Al-O nano-particle paramagnetic immunological probes
The correlation metric of content.The method depends on paramagnetic nano Co-Pd-Al-O nano-probes for the detection of object bacteria in sample
To assess the fungistatic effect of Chinese medicine preparation.Using the paramagnetic Co-Pd-Al-O nano-probes for being coupled monoclonal antibody specific,
Can to sample in specific anaerobic bacteria detect.Because NMR is very quick to Co-Pd-Al-O nano-particles
Sense, there is micro paramagnetic Co-Pd-Al-O nano-particles, then the spin-lattice relaxation time of water and/spin-spin relaxation just
Can be remarkably decreased.The immunological probe content gone out in sample by blank and mark-on quantitative determination, such that it is able to indirect quantification
The anaerobic bacteria clump count gone out in sample.The anaerobism bacterial content for detecting and probe content linear correlation, degree of fitting are preferable.It is final with
Corresponding relation between paramagnetic Co-Pd-Al-O nano-probes and anaerobic bacteria is tie, determines the anaerobic bacteria clump count in sample.From
And determine whether the bacteriostasis of Chinese medicine is effective indirectly.Relative to the anaerobic bacteria culture time of even several days 2-3 days, nuclear magnetic resonance
Detection only needs 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 Co-Pd-Al-O 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 Co-Pd-Al-O receives
Rice probe, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, first can be with Co-Pd-Al-O nanometers of spy
The antibody of wire surface is combined, so that Co-Pd-Al-O nano-probes are stayed in a liquid.If not there is object bacteria in sample cell,
Then the antibody on Co-Pd-Al-O nano-probes surface can gradually be adsorbed and separated with liquid in polystyrene sample pipe surface.This
When, the measure of NMR relaxation time is carried out to sample cell.With the aseptic medium liquid without vaccination target bacterium as empty
In vain, the relaxation time of the suspension for measuring, then explanation contained probe, so as to have mesh in indirect proof sample compared to significantly reducing
Mark bacterium, the amount of probe is proportional with the decline in relaxation time, and by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria.
The Co-Pd-Al-O nano-probes are the nanoscale Co-Pd-Al-O materials with paramagnetic properties, and nanometer particle size is small
In 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 Co-Pd-Al-O nano-probes and anaerobic bacteria as tie, determining sample
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.
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 Co-Pd-Al-O immunological probes：
Co-Pd-Al-O nano-particles can be commercially, it is also possible to which bibliography is with either physically or chemically synthesizing.
Silicon dioxide coated nano Co-Pd-Al-O：47.5g sodium metasilicate is taken, is dissolved in beaker with deionized water, use salt
Acid for adjusting pH value is 12-13.Take 5.0g nano Cos-Pd-Al-O to be added in this beaker, mechanical agitation (using glass bar) 5min.
By mixed liquor ultrasound 30min, stirring in good time.85 C are warmed up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise over.Side magnetic point
Precipitation is washed with deionized from side, washs 3-4 times.Then, precipitation is scattered in 250mL methyl alcohol.Above procedure repeats three
It is secondary, it is ensured that silicon is attached on Co-Pd-Al-O.
Amino containing silane Co-Pd-Al-O nano materials：Obtained silicon dioxide coated nano Co-Pd-Al-O is added
To in 25mL methyl alcohol, 1mLH is used2O and methanol dilution are to 150mL.It is subsequently adding the mixing of 150mL glycerine.Ultrasonic 30min, is transferred to
In having a 500mL there-necked flasks of agitating device.Add 10mL amino silicane coupling agents（AEAPS）, quickly stirred under 80-90 C
After mixing 3h, product is transferred out of.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 is due to there is glycerine, thus it is slow, supernatant liquid can be sucked with suction pipe in good time after taking out a period of time,
Suction filtration process about needs 6-8h.The Aminosilylation Co-Pd-Al-O nano materials vacuum drying 12h that will finally obtain.
Antibody modification：Weigh a certain amount of amido modified Co-Pd-Al-O nano-particles, add 2 times of EDC of quality and
NHS, adds 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 enter
Row 10000r/min is centrifuged, and time 5min, the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.By what is be centrifuged
Sample respectively adds excessive Propiobacterium 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 Co-Pd-Al-O nano-particles of the resistance modification of preparation are
Immunological probe, 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, 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 Co-Pd-
Al-O nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with Co-Pd-Al-O nano-probes
The antibody on surface is combined, so that Co-Pd-Al-O nano-probes are stayed in a liquid；If not there is object bacteria in sample cell,
The antibody on Co-Pd-Al-O nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface.After reaction 1 hour, to sample
Pipe carries out the measure of NMR relaxation time（Shanghai knob steps NMR20 NMRs）.There is no vaccination target bacterium with aseptic
Medium liquid be blank, the relaxation time spin-lattice relaxation time and spin spin relaxation time of the suspension for measuring
Compared to significantly reducing, then explanation contains probe, illustrates there is object bacteria in sample cell, so that the bacteriostasis of side light Chinese medicine
Not substantially, it is otherwise then anti-.The amount of probe is proportional with the decline of spin-lattice relaxation time and spin spin relaxation time, leads to
Cross the quantitative probe of mark-on and 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 Co-Pd-Al-O immunological probes：
Co-Pd-Al-O nano-particles can be commercially, it is also possible to bibliography physics and is chemically synthesized.
Silicon dioxide coated nano Co-Pd-Al-O：47.5g sodium metasilicate is taken, is dissolved in beaker with deionized water, use salt
Acid for adjusting pH value is 12-13.Take 5.0g nano Cos-Pd-Al-O to be added in this beaker, mechanical agitation (using glass bar) 5min.
By mixed liquor ultrasound 30min, stirring in good time.85 C are warmed up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise over.Side magnetic point
Precipitation is washed with deionized from side, washs 3-4 times.Then, precipitation is scattered in 250mL methyl alcohol.Above procedure repeats three
It is secondary, it is ensured that silicon is attached on Co-Pd-Al-O.
Amino containing silane Co-Pd-Al-O nano materials：Obtained silicon dioxide coated nano Co-Pd-Al-O is added
To in 25mL methyl alcohol, 1mLH is used2O and methanol dilution are to 150mL.It is subsequently adding the mixing of 150mL glycerine.Ultrasonic 30min, is transferred to
In having a 500mL there-necked flasks of agitating device.Add 10mL amino silicane coupling agents（AEAPS）, quickly stirred under 80-90 C
After mixing 3h, product is transferred out of.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 is due to there is glycerine, thus it is slow, supernatant liquid can be sucked with suction pipe in good time after taking out a period of time,
Suction filtration process about needs 6-8h.The Aminosilylation Co-Pd-Al-O nano materials vacuum drying 12h that will finally obtain.
Antibody modification：Weigh a certain amount of amido modified Co-Pd-Al-O nano-particles, add 2 times of EDC of quality and
NHS, adds 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 enter
Row 10000r/min is centrifuged, and time 5min, the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.By what is be centrifuged
Sample respectively adds 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 Co-Pd-Al-O nano-particles of the resistance modification of preparation are
Immunological probe, 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
Co-Pd-Al-O nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, can be received with Co-Pd-Al-O first
The antibody of rice detecting probe surface is combined, so that Co-Pd-Al-O nano-probes are stayed in a liquid.If not there is mesh in sample cell
Bacterium is marked, then the antibody on Co-Pd-Al-O nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface.Reaction 1 hour
Afterwards, the measure of NMR relaxation time is carried out to sample cell（Shanghai knob steps NMR20 NMRs）.Do not connect with aseptic
The medium liquid for planting object bacteria is blank, the spin-lattice relaxation time and spin spin relaxation time of the suspension for measuring
Compared to significantly reducing, then explanation contains probe, illustrates there is object bacteria in sample cell, so that the bacteriostasis of side light Chinese medicine
Not substantially, it is otherwise then anti-.The amount of probe is proportional with the decline of spin-lattice relaxation time and spin spin relaxation time, leads to
Cross the quantitative probe of mark-on and 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-.
Claims (3)
1. a kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co-Pd-Al-O nano-probes, its characterization step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic Co-Pd-Al-O 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 Co-Pd-Al-O
Nano-probe, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, Co-Pd-Al-O nano-probes surface
Antibody can be combined with object bacteria first so that Co-Pd-Al-O nano-probes are stayed in a liquid；If not having in sample cell
Object bacteria, then the antibody on Co-Pd-Al-O nano-probes surface can gradually adsorb and leave culture in polystyrene sample pipe surface
Liquid；Now, the measure of NMR relaxation time is carried out to sample cell；With the aseptic medium liquid without vaccination target bacterium
It is blank, the relaxation time of the suspension for measuring, then explanation contained probe, so that in indirect proof sample compared to significantly reducing
There is object bacteria, the amount of probe is proportional with the decline in relaxation time, indirect quantification goes out object bacteria by mark-on quantitative probe
Amount；There is no object bacteria to illustrate that the traditional Chinese medicine ingredients for adding have fungistatic effect in sample cell, on the contrary it is then anti-.
2. it is according to claim 1 based on Co-Pd-Al-O nano-probes screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, its
It is characterized in that the nano Co-Pd-Al-O nano-probes are the nano Co-Pd-Al-O materials with paramagnetic properties, nanometer particle size is small
In 300 nanometers.
3. it is according to claim 1 based on Co-Pd-Al-O nano-probes screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, its
It is characterized in the change in the relaxation time of the final detection evaluation method based on nuclear magnetic resonance technique of described object bacteria.
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| 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 |
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| 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 |
| CN103207202A (en) * | 2013-03-26 | 2013-07-17 | 南昌大学 | Paramagnetic nano Fe-Ni alloy probe based quick detecting method for NMR (nuclear magnetic resonance) food-borne pathogenic bacteria |
| CN103217449A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment |
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Application publication date: 20170623 |
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