CN106885820A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Cr nano-probes - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Cr 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 Cr nano-probes, belongs to tcm development applied technical field.The present invention depend on set up based on prepares Co Cr nano-probes for screen suppression anaerobic bacteria Chinese medicine preparation method.Whether the characteristics of paramagnetic Co Cr nano-probes prepared using antibody coating can be specifically bound to object bacteria, the influence using the paramagnetic properties of Co Cr to the NMR relaxation time is detected and contain object bacteria in sample.Paramagnetic Co Cr nano-probes, NMR relaxation time showing outlet sexual intercourse within the specific limits, i.e. nano Co Cr 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 ternary Co-Cr materials have ferromagnetism, its Elements C o outer layers have 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, Co-Cr nano materials can be to surrounding water molecules
The influence of nuclear magnetic resonance spin-Lattice Relaxation Time is very big, with the effect for amplifying nuclear magnetic resonance relaxation signals.It is very micro
Co-Cr nano materials will result in NMR relaxation time of water and decline to a great extent.Therefore can be by building Co-Cr 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 Co-Cr nano-probes.Commercially paramagnetic Co-Cr nano materials, it is also possible to by other method system
Standby nano level Co-Cr.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, by using silane coupler, can be in inorganic substances
" molecular bridge " is erected and the interface of organic substance between, the material of two kinds of property great disparities is linked together and is improved composite
The effect of performance and increase adhesive strength.Being reacted by silane coupler can obtain the amination or carboxylated of surface modification
Co-Cr nano materials.Further by coupled antibody, surface-functionalized, formation specific immunity probe is capable of achieving, it is unnecessary to close
Avtive spot, excessive antibody can be separated after centrifugation, you can to obtain the Co-Cr 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）Prepared Co-Cr nanometers
Probe, is sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, Co-Cr nano-probes surface it is anti-
Body can be combined with object bacteria first, so that Co-Cr nano-probes are stayed in a liquid.If there is no object bacteria in sample cell,
The antibody on Co-Cr nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface, so that not having Co-Cr to receive in liquid
Rice probe.Preceding to have addressed, the NMR relaxation time that micro Co-Cr 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 for measuring is compared and significantly reduces, then
Illustrate to contain probe in liquid, so as to there is object bacteria in indirect proof sample, the amount of probe is with the drop-out value in relaxation time 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 Traditional Chinese medicine bacteriostatic effect is illustrated not
Substantially, 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 Co-Cr nano-probes.The 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 Co-Cr nano-probes.Using NMR to paramagnetic
The response sensibility of material, proposes the phase of NMR relaxation Parameters variation and Co-Cr nano-particle paramagnetic immunological probe contents
Closing property index.The method depends on paramagnetic nano Co-Cr nano-probes for the detection of object bacteria in sample and matches somebody with somebody assessing Chinese medicine
The fungistatic effect of side.Using the paramagnetic Co-Cr nano-probes for being coupled monoclonal antibody specific, can to sample in it is special
Anaerobic bacteria is detected property.Because NMR is very sensitive to Co-Cr nano-particles, there is micro paramagnetic Co-Cr and receive
Rice corpuscles, then the spin-lattice relaxation time of water and/spin-spin relaxation will be remarkably decreased.By blank and mark-on
The immunological probe content that quantitative determination goes out in sample, such that it is able to the anaerobic bacteria clump count that indirect quantification goes out in sample.Detect
Anaerobism bacterial content and probe content linear correlation, degree of fitting is preferable.Finally between paramagnetic Co-Cr nano-probes and anaerobic bacteria
Corresponding relation is tie, determines the anaerobic bacteria clump count in sample.So as to whether the bacteriostasis for determining Chinese medicine indirectly is effective.Phase
For the anaerobic bacteria culture time of even several days 2-3 days, magnetic resonance detection only needs a few minutes.Therefore, can be made greatly of the 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 Co-Cr 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）Co-Cr nanometers of prepared spy
Pin, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, first can be with Co-Cr nano-probes surface
Antibody is combined, so that Co-Cr nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, Co-Cr nanometers of spy
The antibody of wire surface can gradually be adsorbed and separated with liquid in polystyrene sample pipe surface.Now, nuclear-magnetism is carried out to sample cell
The measure in resonance relaxation time.With the aseptic medium liquid without vaccination target bacterium as blank, the relaxation of the suspension for measuring
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 Co-Cr nano-probes are the nanoscale Co-Cr 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 Co-Cr nano-probes and anaerobic bacteria as tie, in determining sample
Anaerobic bacteria 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 Co-Cr immunological probes：
Co-Cr nano-particles can be commercially, it is also possible to which bibliography is with either physically or chemically synthesizing.
Silicon dioxide coated nano Co-Cr：47.5g sodium metasilicate is taken, is dissolved in beaker with deionized water, adjusted with hydrochloric acid
PH value is 12-13.Take 5.0g nano Cos-Cr 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 Co-Cr.
Amino containing silane Co-Cr nano materials：Obtained silicon dioxide coated nano Co-Cr is added to 25mL methyl alcohol
In, use 1mLH2O and methanol dilution are to 150mL.It is subsequently adding the mixing of 150mL glycerine.Ultrasonic 30min, has been transferred to agitating device
500mL there-necked flasks in.Add 10mL amino silicane coupling agents（AEAPS）, after quick stirring 3h under 80-90 C, transfer
Go out product.Product is washed with deionized 3 times, and methyl alcohol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.Should be noted
It is 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 is about
Need 6-8h.The Aminosilylation Co-Cr nano materials vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified Co-Cr nano-particles are weighed, 2 times of EDC and NHS of quality are added, plus
Enter 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 Co-Cr nano-particles of the resistance modification of preparation are immunological probe, protect
It is stand-by in the presence of 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 Co-Cr
Nano-probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be anti-with Co-Cr nano-probes surface
Body is combined, so that Co-Cr nano-probes are stayed in a liquid；If not there is object bacteria in sample cell, Co-Cr 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）.With the aseptic medium liquid without vaccination target bacterium as blank,
The relaxation time spin-lattice relaxation time of the suspension for measuring has been compared with spin spin relaxation time and significantly reduced, then said
It is bright to illustrate there is object bacteria in sample cell containing probe, so that the bacteriostasis of side light Chinese medicine is not obvious, on the contrary it is then anti-.Visit
The amount of pin is proportional with the decline of spin-lattice relaxation time and spin spin relaxation time, by the quantitative probe of mark-on
Connect the amount for quantifying 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-Cr immunological probes：
Co-Cr nano-particles can be commercially, it is also possible to bibliography physics and is chemically synthesized.
Silicon dioxide coated nano Co-Cr：47.5g sodium metasilicate is taken, is dissolved in beaker with deionized water, adjusted with hydrochloric acid
PH value is 12-13.Take 5.0g nano Cos-Cr 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 Co-Cr.
Amino containing silane Co-Cr nano materials：Obtained silicon dioxide coated nano Co-Cr is added to 25mL methyl alcohol
In, use 1mLH2O and methanol dilution are to 150mL.It is subsequently adding the mixing of 150mL glycerine.Ultrasonic 30min, has been transferred to agitating device
500mL there-necked flasks in.Add 10mL amino silicane coupling agents（AEAPS）, after quick stirring 3h under 80-90 C, transfer
Go out product.Product is washed with deionized 3 times, and methyl alcohol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.Should be noted
It is 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 is about
Need 6-8h.The Aminosilylation Co-Cr nano materials vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified Co-Cr nano-particles are weighed, 2 times of EDC and NHS of quality are added, plus
Enter 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 Co-Cr 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 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-Cr nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with Co-Cr nano-probes surface
Antibody combine so that Co-Cr nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, Co-Cr 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）.It is with the aseptic medium liquid without vaccination target bacterium
Blank, the spin-lattice relaxation time of the suspension for measuring has been compared with spin spin relaxation time and significantly reduced, then illustrated
Containing 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-.Probe
Amount it is proportional with the decline of spin-lattice relaxation time and spin spin relaxation time, it is indirect by the quantitative probe of mark-on
Quantify 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-Cr nano-probes, its characterization step is as follows：
1）Target bacteria antibody is coated with the preparation of paramagnetic Co-Cr 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）Co-Cr nanometers of prepared spy
Pin, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, the antibody on Co-Cr nano-probes surface is first
Can be combined with object bacteria, so that Co-Cr nano-probes are stayed in a liquid；If not having object bacteria in sample cell, Co-Cr
The antibody on nano-probe surface can gradually be adsorbed and leave nutrient solution in polystyrene sample pipe surface；Now, sample cell is entered
The measure of row NMR relaxation time；With the aseptic medium liquid without vaccination target bacterium as blank, what is measured is suspended
The relaxation time of liquid, then explanation contained probe compared to significantly reducing, so that have object bacteria in indirect proof sample, the amount of probe
Decline with the relaxation time is proportional, and by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria；There is no mesh in sample cell
The traditional Chinese medicine ingredients that mark bacterium explanation is added have fungistatic effect, otherwise then anti-.
2. it is according to claim 1 based on Co-Cr nano-probes screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, it is characterized in that
Nano Co-Cr the nano-probes are the nano Co-Cr materials with paramagnetic properties, and nanometer particle size is less than 300 nanometers.
3. it is according to claim 1 based on Co-Cr nano-probes screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, it is characterized in that
The final detection evaluation method of described object bacteria is based on the change in the relaxation time of nuclear magnetic resonance technique.
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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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| 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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- 2017-03-30 CN CN201710200595.9A patent/CN106885820A/en active Pending
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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 |
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Application publication date: 20170623 |