CN106814097A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on CdSe nano-probes - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on CdSe 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 CdSe nano-probes, belongs to tcm development applied technical field.The present invention depend on set up based on prepares CdSe nano-probes for screen suppression anaerobic bacteria Chinese medicine preparation method.Whether the characteristics of magnetic CdSe nano-probes prepared using antibody coating can be specifically bound to object bacteria, the influence using the magnetic properties of CdSe to the NMR relaxation time is detected and contain object bacteria in sample.Magnetic CdSe nano-probes, NMR relaxation time showing outlet sexual intercourse within the specific limits, that is nanocrystal CdSe 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 CdSe materials have magnetic, its Elements C d outer layers have multiple unpaired electrons, with high saturation and magnetic intensity, in grain
The small nano material to a certain extent in footpath can be to the influence of the nuclear magnetic resonance spin-Lattice Relaxation Time of surrounding water molecules very
Greatly, with the effect for amplifying nuclear magnetic resonance relaxation signals.Very micro CdSe nano materials will result in the nuclear magnetic resonance of water
Relaxation time declines to a great extent.Therefore can detect whether exist from the angle of magnetic resonance by building CdSe nano-probes
Object bacteria, so that the bacteriostasis of indirect assessment traditional Chinese medicine ingredients.
Key step：
1）Prepared by CdSe nano-probes.Commercially magnetic CdSe nano materials, it is also possible to prepared by other method
Nano level CdSe.Using silane coupler, its formula is:Y(CH2)nSiX3.Herein, n is 0-3；X is hydrolyzable group；Y
It 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 CdSe 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 CdSe 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 CdSe nanometers
Probe, is sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, the antibody on CdSe nano-probes surface
Can be combined with object bacteria first, so that CdSe nano-probes are stayed in a liquid.If there is no object bacteria in sample cell, CdSe
The antibody on nano-probe surface can be adsorbed gradually in polystyrene sample pipe surface, so that not having CdSe nanometers of spy in liquid
Pin.Preceding to have addressed, the NMR relaxation time that micro CdSe nano materials will result in water declines to a great extent.Do not have with aseptic
There is the medium liquid of vaccination target bacterium for blank, the relaxation time of the suspension for measuring then illustrates liquid compared to significantly reducing
Contain probe in body, 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, passes through
The quantitative probe of mark-on 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, instead
Then explanation have 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 CdSe nano-probes.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 CdSe nano-probes.Using NMR to Magnetic Materials
The response sensibility of matter, proposes the correlation of NMR relaxation Parameters variation and CdSe nano-particle magnetic immuno probe contents
Index.The method depends on magnetic Nano CdSe nano-probes for the detection of object bacteria in sample to assess the suppression of Chinese medicine preparation
Bacterium effect.Using the magnetic CdSe nano-probes for being coupled monoclonal antibody specific, can to sample in specific anaerobism
Bacterium is detected.Because NMR is very sensitive to CdSe nano-particles, there is micro magnetic CdSe nano-particles, then
The spin-lattice relaxation time of water and/spin-spin relaxation will be remarkably decreased.By blank and mark-on quantitative determination
The immunological probe content gone out in sample, such that it is able to the anaerobic bacteria clump count that indirect quantification goes out in sample.The anaerobic bacteria for detecting
Content and probe content linear correlation, degree of fitting are preferable.Finally with the corresponding relation between magnetic CdSe nano-probes and anaerobic bacteria
It is tie, determines the anaerobic bacteria clump count in sample.So as to whether the bacteriostasis for determining Chinese medicine indirectly is effective.Relative to anaerobism
Bacterium cultivates the time of even several days 2-3 days, and magnetic resonance detection only needs a few minutes.Therefore, extensive Chinese medicine can be done with the method
And the screening of formula.
The present invention is achieved in that step is as follows：
1）Target bacteria antibody is coated with the preparation of magnetic Nano CdSe 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）CdSe 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 anti-with CdSe nano-probes surface
Body is combined, so that CdSe nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, CdSe nano-probe tables
The antibody in face can gradually be adsorbed and separated with liquid in polystyrene sample pipe surface.Now, nuclear magnetic resonance is carried out to sample cell
The measure in relaxation time.With the aseptic medium liquid without vaccination target bacterium as blank, during the relaxation of the suspension for measuring
Between compared to significantly reducing, then explanation contains probe, so as to there is object bacteria in indirect proof sample, the amount of probe and relaxation time
Decline it is proportional, by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria.
The CdSe nano-probes are the nanoscale CdSe materials 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 CdSe 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 nanocrystal CdSe immunological probe：
CdSe nano-particles can be commercially, it is also possible to which bibliography is with either physically or chemically synthesizing.
Silicon dioxide coated nanocrystal CdSe：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 nanocrystal CdSes 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 CdSe.
Amino containing silane CdSe nano materials：Obtained silicon dioxide coated nanocrystal CdSe 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 CdSe nano materials vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified CdSe 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 CdSe 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 CdSe receives
Rice probe, is sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with the antibody knot on CdSe nano-probes surface
Close, so that CdSe nano-probes are stayed in a liquid；If not there is object bacteria in sample cell, CdSe nano-probes surface
Antibody can be adsorbed gradually in polystyrene sample pipe surface.After reaction 1 hour, is carried out to sample cell the NMR relaxation time
Determine（Shanghai knob steps NMR20 NMRs）.With the aseptic medium liquid without vaccination target bacterium as blank, measure
The relaxation time spin-lattice relaxation time of suspension has been compared with spin spin relaxation time and significantly reduced, then explanation contains
Probe, 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
Decline with spin-lattice relaxation time and spin spin relaxation time is proportional, the indirect quantification by mark-on quantitative probe
Go 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 nanocrystal CdSe immunological probe：
CdSe nano-particles can be commercially, it is also possible to bibliography physics and is chemically synthesized.
Silicon dioxide coated nanocrystal CdSe：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 nanocrystal CdSes 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 CdSe.
Amino containing silane CdSe nano materials：Obtained silicon dioxide coated nanocrystal CdSe 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 CdSe nano materials vacuum drying 12h that will finally obtain.
Antibody modification：A certain amount of amido modified CdSe 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 CdSe 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
CdSe nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with CdSe nano-probes surface
Antibody is combined, so that CdSe nano-probes are stayed in a liquid.If not there is object bacteria in sample cell, CdSe 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 spin-lattice relaxation time of the suspension for measuring 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-.
Claims (3)
1. a kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on CdSe nano-probes, its characterization step is as follows：
1）Target bacteria antibody is coated with the preparation of magnetic CdSe 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）CdSe 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 CdSe nano-probes surface first can
Combined with object bacteria, so that CdSe nano-probes are stayed in a liquid；If there is no object bacteria in sample cell, CdSe nanometers of spy
The antibody of wire surface can gradually be adsorbed and leave nutrient solution 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 time of the suspension for measuring is notable compared to having
Reduce, then explanation contains probe, so as to there is object bacteria in indirect proof sample, the amount of probe is with the decline in relaxation time in just
Than by mark-on quantitative probe, indirect quantification goes out the amount of object bacteria；Do not have in sample cell object bacteria illustrate add Chinese medicine into
Dividing has fungistatic effect, otherwise then anti-.
2. it is according to claim 1 based on CdSe nano-probes screen anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs method, it is characterized in that
The nanocrystal CdSe nano-probe is the nanocrystal CdSe material with magnetic properties, and nanometer particle size is less than 300 nanometers.
3. it is according to claim 1 based on CdSe 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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Citations (3)
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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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| CN104371733A (en) * | 2014-12-04 | 2015-02-25 | 东华大学 | Micro-fluid synthesizing method for CdSe/ZnS core-shell-structured quantum dots with controllable light emitting performance |
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2017
- 2017-03-22 CN CN201710174344.8A patent/CN106814097A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN103446595A (en) * | 2013-08-01 | 2013-12-18 | 华中科技大学 | Multifunctional probe for CT (Computed Tomography) and dual-mode fluorescent imaging based on nanometer bismuth sulfide |
| CN104371733A (en) * | 2014-12-04 | 2015-02-25 | 东华大学 | Micro-fluid synthesizing method for CdSe/ZnS core-shell-structured quantum dots with controllable light emitting performance |
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| Title |
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Application publication date: 20170609 |