CN105998065B - A kind of Nano carbon balls grain preparation and preparation method thereof and Helicobacter pylori Infection experimental method - Google Patents
A kind of Nano carbon balls grain preparation and preparation method thereof and Helicobacter pylori Infection experimental method Download PDFInfo
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Abstract
The invention discloses a kind of Nano carbon balls grain preparations and preparation method thereof and Helicobacter pylori Infection experimental method, Nano carbon balls grain preparation is made of Nano carbon balls grain and dispersion liquid mixing, the concentration of Nano carbon balls grain preparation is 0.1%~1.0%, wherein, dispersion liquid is deionized water, and the partial size of 95% or more Nano carbon balls grain is 5~30nm.It has the advantages that low in cost, preparation is easy, stability is good, highly-safe, Helicobacter pylori Infection effect is good.The preparation method of Nano carbon balls grain preparation has the characteristics that implement to be easy, is simple process, easy to operate.The experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection has the characteristics that easy to implement, scientific and reasonable.Nano carbon balls grain preparation of the invention can be used as the new drug development for the treatment of helicobacter pylori infections, opens new thinking to develop the drug of anti-helicobacter pylori, also provides new strategy for the prevention and treatment of Helicobacter pylori Infection.
Description
Technical field
The present invention relates to a kind of carbon nanomaterial, more particularly, to a kind of Nano carbon balls grain preparation and preparation method thereof, with
And the experimental method of the Nano carbon balls grain preparation Helicobacter pylori Infection.
Background technique
Helicobacter pylori (Helicobacter pylori, Hp) infectivity related disease, as gastritis, gastric ulcer, 12 refer to
Enterelcosis and gastric cancer seriously endanger the health of the mankind.According to the World Health Organization (WHO) statistics, there are about 50% in the whole world
Population infection helicobacter pylori, especially China the helicobacter pylori state of an illness it is more severe, about 60% people infects pylorus
Helicobacter pylori has been classified as a kind of carcinogen of gastric cancer by helicobacter, WHO.Currently, treatment helicobacter pylori infections are by adopting
With the conjoint therapy of antibiotic and acid inhibitor.But since the drug resistance of antibiotic is increasingly severe, dosage is caused to add
Greatly, the course for the treatment of extend, and along with adverse drug reaction aggravate, recurrence rate be still high the problem of.Therefore, clinic compels to be essential
Want the new treatment of non-antibiotic and the new strategy of Helicobacter pylori Infection, new material.
Carbon nanomaterial is a kind of novel nanotechnology material, there is multiple use.Carbon nanometer in carbon nanomaterial
Pipe has antibacterial action, and the Nano carbon balls grain (Carbon nanospheres, CNS) in carbon nanomaterial has transport drug
And the effects of fluorescent tracing.Since Nano carbon balls grain has unique structure and characteristic, potential biomedical applications need
Exploitation.Currently, the research of Nano carbon balls grain antibacterial action is reported not yet, also grinding without Nano carbon balls grain anti-helicobacter pylori
Study carefully report.On the one hand harm of the helicobacter pylori to body is the effect of helicobacter pylori itself, be on the other hand that its metabolism produces
The effect of object such as urease.Helicobacter pylori can generate a large amount of ureases, urease risen in helicobacter pylori infections field planting and
The effect of virulence is virulence factor, while urease is also the target spot for treating helicobacter pylori.Therefore, Nano carbon balls grain or its
If preparation can directly inhibit or kill helicobacter pylori thallus itself and or destroy the urea of helicobacter pylori
Enzyme, then the Nano carbon balls grain and its preparation just have huge anti-helicobacter pylori application prospect.
Although having the research that the nano-carbon powder in carbon nanomaterial is made to Eradication Therapy of Helicobacter pylori Infection agent, such as application No. is
2014104902353 and 2014104898659 patent application, but 95% or more the nano-carbon powder diameter of particle of both selection
Less than or equal to 5nm；Negatively charged, therapeutic agent is controlled in 7.8~9.1 using the dispersion preparation of human body isotonic solution, pH value.
It is primarily present the problem of following several respects: 1) since its 95% or more nano-carbon powder diameter of particle is less than or equal to 5nm, because of it
Partial size is too small to be penetrated readily through into human body cell, it is easier to be generated toxicity or be increased toxicity；2) because isotonic solution containing a large amount of sun from
Son, it is positively charged, certain influence can be generated on the long-time stability of the nano-carbon powder powder rich in negative electrical charge.
Summary of the invention
The object of the present invention is to provide a kind of Nano carbon balls grain preparation and preparation method thereof and Helicobacter pylori Infection are real
Proved recipe method, wherein Nano carbon balls grain preparation has the advantages that low in cost, preparation is easy, stability is good, highly-safe, preparation
Method has the characteristics that implement to be easy, is simple process, easy to operate, and experimental method has the characteristics that easy to implement, scientific and reasonable.
It is more next for traditional remedies antibiotic resistance existing for it for solving to treat helicobacter pylori infections in the prior art
It is more serious, cause the problem that dosage increases, the course for the treatment of extends, adverse drug reaction aggravates and infection and recurrence rate is high,
A kind of Nano carbon balls grain preparation provided by the invention is made of Nano carbon balls grain and dispersion liquid mixing, Nano carbon balls grain preparation
Concentration is 0.1%~1.0%, wherein dispersion liquid is deionized water, and the partial size of 95% or more Nano carbon balls grain is 5~30nm.
Further, a kind of Nano carbon balls grain preparation of the present invention, wherein the pH value of the Nano carbon balls grain preparation is 7.1
~7.6.
Further, a kind of Nano carbon balls grain preparation of the present invention, wherein the partial size frequency percentage of the Nano carbon balls grain
Are as follows: partial size is that the Nano carbon balls grain of 5~10nm accounts for 20~30%, and partial size is that the Nano carbon balls grain of 10~15nm accounts for 25~35%,
Partial size is that the Nano carbon balls grain of 15~20nm accounts for 15~25%, and partial size is that the Nano carbon balls grain of 20~25nm accounts for 6~10%, partial size
1~7% is accounted for for the Nano carbon balls grain of 25~30nm.
A kind of preparation method of Nano carbon balls grain preparation provided by the invention, comprising the following steps:
One, Nano carbon balls grain is prepared by original material processing of graphene；
Two, the satisfactory Nano carbon balls grain of partial size is screened using transmission electron microscope, wherein partial size is that the carbon of 5~30nm is received
Rice spherolite accounts for 95% or more；Nano carbon balls grain disperses in single spherical；
Three, it is required according to concentration, weigh the Nano carbon balls grain after step 2 is screened respectively and measures deionized water, it is standby
With；
Four, Nano carbon balls grain and deionized water the mixing dispersion obtained step 3；
Five, the mixed liquor being mixed to get through step 4 being stirred, wherein mixing time is 10~20 minutes,
Mixing speed is 60~80r/min；
Six, the mixed liquor after step 5 stir process is ultrasonically treated, wherein ultrasonic time is 15~20 points
Clock, ultrasound intensity are 80~100w；
Seven, pH value is adjusted to the mixed liquor after step 6 is ultrasonically treated using sodium bicarbonate-sodium carbonate buffer
Processing, is adjusted to 7.1~7.6 for the pH value of mixed liquor, obtains final Nano carbon balls grain preparation.
Further, a kind of preparation method of Nano carbon balls grain preparation of the present invention, wherein the partial size described in step 2 is
The Nano carbon balls grain of 5~30nm accounts for 95% or more, wherein the partial size frequency percentage of Nano carbon balls grain are as follows: partial size is 5~10nm
Nano carbon balls grain account for 20~30%, partial size is that the Nano carbon balls grain of 10~15nm accounts for 25~35%, and partial size is 15~20nm's
Nano carbon balls grain accounts for 15~25%, and partial size is that the Nano carbon balls grain of 20~25nm accounts for 6~10%, and partial size is that the carbon of 25~30nm is received
Rice spherolite accounts for 1~7%.
Further, a kind of preparation method of Nano carbon balls grain preparation of the present invention, wherein to through step described in step 6
Mixed liquor after rapid five stir process is ultrasonically treated, and is ultrasonically treated under low temperature water-bath.
Further, a kind of preparation method of Nano carbon balls grain preparation of the present invention, wherein described in step 6 when ultrasound
Between be 15~20 minutes, refer to that ultrasonic all times are 15~20 minutes, wherein single ultrasonic time is 3 seconds, and off time is
3 seconds.
A kind of experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection provided by the invention, including following step
It is rapid:
I, detection Nano carbon balls grain preparation is tested to the inhibiting rate of helicobacter Pylori urease by external fast urease；
II, it is acted on using coup injury of the transmission electron microscope observing Nano carbon balls grain preparation to helicobacter pylori；
III, the spy of Nano carbon balls grain preparation Helicobacter pylori Infection is determined according to the interpretation of result of step I and step II
Property.
Further, a kind of experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection of the present invention, wherein in step
Detection Nano carbon balls grain preparation is tested to the inhibiting rate of helicobacter Pylori urease by external fast urease described in rapid I,
The following steps are included:
A, preparation and diluted sample: with physiological saline by Helicobacter pylori SS1 bacterium solution 109CFU/ml dilution
1000 times to 106CFU/ml, it is spare；Three kinds of Nano carbon balls grain preparations that concentration is respectively 1.0%, 0.5%, 0.4% are spent
Ionized water dilute 100 times, it is spare, the concentration of three kinds of Nano carbon balls grain preparations after dilution be respectively 100ug/ml, 50ug/ml,
40ug/ml；Urease inhibitor acetohydroxamic acid is first dissolved into 1% with deionized water, then again with deionized water dilution 100
Times, it is spare；Urease inhibitor acetohydroxamic acid concentration after dilution is 100ug/ml；Ionized water is removed, it is spare；Wherein, it urinates
Plain enzyme inhibitor acetohydroxamic acid is as positive control, and deionized water is as negative control；
B, the 10 of 10ul loading: is added in the hole of 96 orifice plates6CFU/ml bacterium solution, multiple three holes；After adding, according to grouping point
It is not rapidly added tri- kinds of Nano carbon balls grain preparations of 100ug/ml, 50ug/ml, 40ug/ml, deionized water, 100ug/ml acetyl oxygen
Each 90ul of oxime acid；
C, it is incubated for: 96 orifice plates being sealed using sealed membrane after loading, and are incubated for 24 hours in 4 DEG C of insulating box；
D, it develops the color: after being incubated for, the urea reagent of 50ul being added in corresponding hole, and is put in 37 DEG C of insulating box and incubates
Educate 30 points of kind colour developings；
E, it reads: after being incubated for 30 points of kinds, being read at 578nm wavelength, and record every group of OD value；
F, inhibiting rate: inhibiting rate=[1- (OD is calculatedMeasurement-ODBlank)/(ODControl-ODBlank)] × 100%.
Further, a kind of experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection of the present invention, wherein in step
It is acted on described in rapid II using coup injury of the transmission electron microscope observing Nano carbon balls grain preparation to helicobacter pylori, including following step
It is rapid:
G, prepared by bacterium solution: with physiological saline by helicobacter pylori NTCC11637 bacterium solution 109CFU/ml dilutes 10 times extremely
108CFU/ml, it is spare；
H, loading: taking 12 1ml test tubes, and the 10 of 360ul is added in every pipe8CFU/ml helicobacter pylori NTCC11637
Bacterium solution, multiple three holes；After adding, according to grouping be rapidly added respectively 0.4%, 0.1% Nano carbon balls grain preparation, deionized water,
Each 40ul of 0.1% clarithromycin is mixed；Wherein, deionized water is as negative control, and clarithromycin is as positive control；
I, it reacts: orifice plate being sealed using sealed membrane after loading, first the constant-temperature table at 37 DEG C shakes bacterium 10 points of kinds of culture, then
It takes out and is put into 30 points of kinds of culture in 37 DEG C of insulating box rapidly；
J, it dyes: 100ul reaction solution being taken to be placed on transmission electron microscope copper mesh, and with 1.0% phosphotungstic acid negative staining；
K, it observes: observing the Morphological Features of helicobacter pylori under transmission electron microscope, and judge Nano carbon balls grain preparation to deep and remote
Helicobacter pylori coup injury result.
A kind of Nano carbon balls grain preparation of the present invention and preparation method thereof and Helicobacter pylori Infection experimental method with it is existing
Technology is compared, have the advantage that (1) Nano carbon balls grain preparation provided by the invention use the Nano carbon balls grain of non-antibiotic for
Preparation raw material, and by 95% or more spherolite size controlling in 5~30nm, it avoids and toxicity is generated to body because partial size is too small
Or the problem of increasing toxicity, provide safety.(2) Nano carbon balls grain table is made full use of as dispersion liquid using deionized water
Face it is porous, enhance water imbibition, make the dispersion rate of Nano carbon balls grain in deionized water up to 99.5% or more, and because of deionization
Water is free of other metal cations, influences negatively charged Nano carbon balls grain by by cation, is in water in single spherical
Dispersion avoids the problem of spherolite is reunited, ensure that the high stability of Nano carbon balls grain preparation.(3) present invention is by by carbon
The pH value of nanometer spherolite preparation is controlled 7.1~7.6, is reduced or is eliminated in Nano carbon balls grain preparation PH (acid-resisting) to pylorus
The influence of Helicobacter growth can more protrude and embody Nano carbon balls grain itself to the antibacterial action of helicobacter pylori.(4) of the invention
Nano carbon balls grain preparation has the advantages that low in cost, preparation is easy, stability is good, highly-safe, and preparation method has real
Easy, simple process, easy to operate advantage are applied, experimental method has the advantages that easy to implement, scientific and reasonable.(5) table is tested
Bright, Nano carbon balls grain preparation of the invention has prominent and powerful anti-helicobacter pylori effect, to helicobacter Pylori urease
Maximum inhibition is high, and required dosage is small；Nano carbon balls grain has coup injury effect to helicobacter pylori, to helicobacter pylori flagellum
It is obvious with cell membrane cell wall damage.(6) Nano carbon balls grain preparation of the invention can be used as the new for the treatment of helicobacter pylori infections
Medicine exploitation opens new thinking to develop the drug of anti-helicobacter pylori, will also mention for the prevention and treatment of Helicobacter pylori Infection
For new strategy.
With reference to the accompanying drawing shown specific embodiment to a kind of Nano carbon balls grain preparation of the present invention and preparation method thereof and
Helicobacter pylori Infection experimental method is described in further detail:
Detailed description of the invention
Fig. 1 is that Nano carbon balls grain preparation is observed by transmission electron microscope (TEM) in the experiment of the coup injury of helicobacter pylori,
The helicobacter pylori aspect graph of 400ug/ml high dose Nano carbon balls grain preparation group；
Fig. 2 is that Nano carbon balls grain preparation is observed by transmission electron microscope (TEM) in the experiment of the coup injury of helicobacter pylori,
The helicobacter pylori aspect graph of 100ug/ml low dosage Nano carbon balls grain preparation group；
Fig. 3 is that Nano carbon balls grain preparation is observed by transmission electron microscope (TEM) in the experiment of the coup injury of helicobacter pylori,
The helicobacter pylori aspect graph of 100ug/ml clarithromycin group；
Fig. 4 is that Nano carbon balls grain preparation is observed by transmission electron microscope (TEM) in the experiment of the coup injury of helicobacter pylori,
The helicobacter pylori aspect graph of deionized water group.
Specific embodiment
Firstly the need of explanation, this invention provides both a kind of Nano carbon balls grain preparations, and provide preparation method,
The experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection is additionally provided simultaneously.
As specific embodiment, Nano carbon balls grain preparation is made of Nano carbon balls grain and dispersion liquid mixing, and keeps its dense
Degree control is 0.1%~1.0%.Wherein, dispersion liquid is deionized water, the partial size of 95% or more Nano carbon balls grain is 5~
30nm.To reduce or eliminating influence of the PH (acid-resisting) to growth of H. pylori in Nano carbon balls grain preparation, can more protrude
Embody Nano carbon balls grain itself to the antibacterial action of helicobacter pylori, present embodiment allows the pH value of Nano carbon balls grain preparation
It is 7.1~7.6.
As prioritization scheme, present embodiment makes the partial size frequency percentage of Nano carbon balls grain using following values:
It allows partial size to be that the Nano carbon balls grain of 5~10nm accounts for 20~30%, partial size is allowed to be that the Nano carbon balls grain of 10~15nm accounts for 25~35%,
It allows partial size to be that the Nano carbon balls grain of 15~20nm accounts for 15~25%, partial size is allowed to be that the Nano carbon balls grain of 20~25nm accounts for 6~10%,
Partial size is allowed to be that the Nano carbon balls grain of 25~30nm accounts for 1~7%.
Nano carbon balls grain preparation provided by the invention uses the Nano carbon balls grain of non-antibiotic for preparation raw material, and will
95% or more spherolite size controlling is avoided to generate toxicity because partial size is too small to body or increase toxicity and be asked in 5~30nm
Topic, provides safety.And the porous enhancing on Nano carbon balls grain surface is made full use of as dispersion liquid by using deionized water
Water imbibition, makes the dispersion rate of Nano carbon balls grain in deionized water up to 99.5% or more, and because deionized water is without other gold
Belong to cation, influence negatively charged Nano carbon balls grain by by cation, disperses in water in single spherical, avoid ball
The problem of grain is reunited, it ensure that the high stability of Nano carbon balls grain preparation.Meanwhile Nano carbon balls grain preparation provided by the invention
Have the advantages that low in cost, preparation is easy, stability is good, highly-safe.Experiment shows Nano carbon balls grain provided by the invention
Preparation has prominent and powerful anti-helicobacter pylori effect, high, the required dosage to helicobacter Pylori urease maximum inhibition
It is small.Nano carbon balls grain has coup injury effect to helicobacter pylori, bright to helicobacter pylori flagellum and cell membrane cell wall damage
It is aobvious.Nano carbon balls grain preparation of the invention can be used as the new drug development for the treatment of helicobacter pylori infections, to develop anti-helicobacter pylorus
The drug of bacterium opens new thinking, also will provide new strategy for the prevention and treatment of Helicobacter pylori Infection.
As specific embodiment, the preparation method of Nano carbon balls grain preparation, comprising the following steps:
One, Nano carbon balls grain is prepared by original material processing of graphene；
Two, the satisfactory Nano carbon balls grain of partial size is screened using transmission electron microscope, wherein partial size is that the carbon of 5~30nm is received
Rice spherolite accounts for 95% or more；Nano carbon balls grain disperses in single spherical；
Three, it is required according to concentration, weigh the Nano carbon balls grain after step 2 is screened respectively and measures deionized water, it is standby
With；
Four, Nano carbon balls grain and deionized water the mixing dispersion obtained step 3；
Five, the mixed liquor being mixed to get through step 4 being stirred, wherein mixing time is 10~20 minutes,
Mixing speed is 60~80r/min；
Six, the mixed liquor after step 5 stir process is ultrasonically treated, wherein ultrasonic time is 15~20 points
Clock, ultrasound intensity are 80~100w；
Seven, the mixed liquor after step 6 is ultrasonically treated adjust at pH value using sodium bicarbonate-sodium carbonate buffer
Reason, is adjusted to 7.1~7.6 for the pH value of mixed liquor, obtains final Nano carbon balls grain preparation.
In above-mentioned steps two, the partial size is that the Nano carbon balls grain of 5~30nm accounts for 95% or more, wherein Nano carbon balls
The partial size frequency percentage of grain are as follows: partial size is that the Nano carbon balls grain of 5~10nm accounts for 20~30%, and partial size is that the carbon of 10~15nm is received
Rice spherolite accounts for 25~35%, and partial size is that the Nano carbon balls grain of 15~20nm accounts for 15~25%, and partial size is the carbon nanometer of 20~25nm
Spherolite accounts for 6~10%, and partial size is that the Nano carbon balls grain of 25~30nm accounts for 1~7%.
In above-mentioned steps six, it is in low temperature that the described pair of mixed liquor after step 5 stir process, which is ultrasonically treated,
It is ultrasonically treated under water-bath.
In above-mentioned steps six, the ultrasonic time is 15~20 minutes, refers to that ultrasonic all times are 15~20 minutes,
Wherein, single ultrasonic time is 3 seconds, and off time is 3 seconds.
Embodiment 1: Nano carbon balls grain and deionized water are prepared.
Nano carbon balls grain is prepared by original material processing of graphene, it is 5~30nm's that partial size is screened under transmission electron microscope
Nano carbon balls grain, it is spare.It should ensure that partial size is that the Nano carbon balls grain of 5~30nm accounts for 95% of total amount or more.
Deionized water is prepared, it is spare.
Embodiment 2: 1.0% Nano carbon balls grain preparation is prepared.
According to 1.0% concentration requirement, quantitative Nano carbon balls grain and deionized water mixing dispersion are taken, and stirs 20 points
Clock, wherein mixing speed is 80r/min.
Mixed liquor after stirring is ultrasonically treated, wherein ultrasonic all times are set as 20 minutes, single ultrasonic time
It is set as 3 seconds, off time is set as 3 seconds, and ultrasound intensity is set as 100w (ultrasonic under low temperature water-bath).
Mixed liquor after ultrasound is carried out to adjust pH value processing using sodium bicarbonate-sodium carbonate buffer, and mixing is mixed
Final pH value is adjusted to 7.6, to obtain satisfactory Nano carbon balls grain preparation.
Embodiment 3: 0.5% Nano carbon balls grain preparation is prepared.
According to 0.5% concentration requirement, quantitative Nano carbon balls grain and deionized water mixing dispersion are taken, and stirs 15 points
Clock, wherein mixing speed is 70r/min.
Mixed liquor after stirring is ultrasonically treated, wherein ultrasonic all times are set as 18 minutes, single ultrasonic time
It is set as 3 seconds, off time is set as 3 seconds, and ultrasound intensity is set as 100w (ultrasonic under low temperature water-bath).
Mixed liquor after ultrasound is carried out to adjust pH value processing using sodium bicarbonate-sodium carbonate buffer, and mixing is mixed
Final pH value is adjusted to 7.4, to obtain satisfactory Nano carbon balls grain preparation.
Embodiment 4: 0.2% Nano carbon balls grain preparation is prepared.
According to 0.2% concentration requirement, quantitative Nano carbon balls grain and deionized water mixing dispersion are taken, and stirs 10 points
Clock, wherein mixing speed is 60r/min.
Mixed liquor after stirring is ultrasonically treated, wherein ultrasonic all times are set as 15 minutes, single ultrasonic time
It is set as 3 seconds, off time is set as 3 seconds, and ultrasound intensity is set as 80w (ultrasonic under low temperature water-bath).
Mixed liquor after ultrasound is carried out to adjust pH value processing using sodium bicarbonate-sodium carbonate buffer, and mixing is mixed
Final pH value is adjusted to 7.2, to obtain satisfactory Nano carbon balls grain preparation.
Embodiment 5: 0.1% Nano carbon balls grain preparation is prepared.
According to 0.2% concentration requirement, quantitative Nano carbon balls grain and deionized water mixing dispersion are taken, and stirs 10 points
Clock, wherein mixing speed is 60r/min.
Mixed liquor after stirring is ultrasonically treated, wherein ultrasonic all times are set as 15 minutes, single ultrasonic time
It is set as 3 seconds, off time is set as 3 seconds, and ultrasound intensity is set as 80w (ultrasonic under low temperature water-bath).
Mixed liquor after ultrasound is carried out to adjust pH value processing using sodium bicarbonate-sodium carbonate buffer, and mixing is mixed
Final pH value is adjusted to 7.1, to obtain satisfactory Nano carbon balls grain preparation.
The specific embodiment of experimental method as Nano carbon balls grain preparation Helicobacter pylori Infection, including following step
It is rapid:
I, detection Nano carbon balls grain preparation is tested to the inhibiting rate of helicobacter Pylori urease by external fast urease；
II, it is acted on using coup injury of the transmission electron microscope observing Nano carbon balls grain preparation to helicobacter pylori；
III, Nano carbon balls grain preparation Helicobacter pylori Infection is determined most according to the interpretation of result of step I and step II
Terminate fruit.
Embodiment 6: detection Nano carbon balls grain preparation is tested to helicobacter Pylori urease by external fast urease
Inhibiting rate.
Preparation and diluted sample: with physiological saline by Helicobacter pylori SS1 bacterium solution 109CFU/ml(OD578
=0.6) 1000 times to 10 are diluted6CFU/ml, it is spare；It is respectively 1.0%, 0.5%, 0.4% three kinds of Nano carbon balls by concentration
Grain preparation dilutes 100 times with deionized water, and spare, the concentration of three kinds of Nano carbon balls grain preparations after dilution is respectively 100ug/
Ml, 50ug/ml, 40ug/ml (with deionized water, sample is neutral in PH after 100 times of dilution)；By urease inhibitor second
Acyl hydroxamic acid is first dissolved into 1.0% with deionized water, spare then again with 100 times of deionized water dilution；Urease after dilution
Inhibitor acetohydroxamic acid concentration is 100ug/ml；Ionized water is removed, it is spare；Wherein, urease inhibitor acetohydroxamic acid is made
For positive control, deionized water is as negative control.
Loading: the 10 of 10ul is added in the hole of 96 orifice plates6CFU/ml bacterium solution, multiple three holes；After adding, distinguish according to grouping
It is rapidly added tri- kinds of Nano carbon balls grain preparations of 100ug/ml, 50ug/ml, 40ug/ml, deionized water, 100ug/ml acetyl oxygen oxime
Each 90ul of acid.
It is incubated for: 96 orifice plates being sealed using sealed membrane after loading, and are incubated for 24 hours in 4 DEG C of insulating box.
Colour developing: after being incubated for, the urea reagent of 50ul is added in corresponding hole, and is put in 37 DEG C of insulating box and is incubated for
30 points of kind colour developings.
Reading: it after being incubated for 30 points of kinds, is read at 578nm wavelength, and records every group of OD value.
Calculate inhibiting rate: inhibiting rate=[1- (ODMeasurement-ODBlank)/(ODControl-ODBlank)] × 100%.
OD value and inhibiting rate result such as table 1
Table 1: Nano carbon balls grain agent in vitro fast urease inhibitory enzyme activity experimental data table
By experimental result listed by table 1 it is found that the Nano carbon balls grain preparation of Helicobacter pylori Infection of the invention is to deep and remote
Helicobacter pylori urease has apparent inhibiting effect.
Embodiment 7: Nano carbon balls grain preparation is observed by transmission electron microscope (TEM), the coup injury of helicobacter pylori is made
With.
Bacterium solution preparation: with physiological saline by helicobacter pylori NTCC11637 bacterium solution 109CFU/ml (OD578=0.6) dilution
10 times to 108CFU/ml, it is spare.
Loading: taking 12 1ml test tubes, and 360ul 10 is added in every pipe8The helicobacter pylori NTCC11637 bacterium of CFU/ml
Liquid, multiple three holes；After adding, according to grouping be rapidly added respectively 0.4%, 0.1% Nano carbon balls grain preparation, deionized water,
Each 40ul of 0.1% clarithromycin, and mix；Wherein, deionized water is as negative control, and clarithromycin is as positive control.
Reaction: sealing orifice plate using sealed membrane after loading, and first the constant-temperature table at 37 DEG C shakes bacterium 10 points of kinds of culture, then takes
It is put into 30 points of kinds of culture in 37 DEG C of insulating box out and rapidly.
Dyeing: taking 100ul reaction solution to be placed on transmission electron microscope copper mesh, and with 1.0% phosphotungstic acid negative staining.
Observation: the Morphological Features of helicobacter pylori are observed at transmission electron microscope (TEM), and judge Nano carbon balls grain preparation pair
Helicobacter pylori coup injury result.
Under transmission electron microscope (TEM), each group experimental result is as shown in Figures 1 to 4:
Shown in 400ug/ml high dose Nano carbon balls grain preparation group Fig. 1: helicobacter pylori has no that flagellum, cell wall after birth are thinning
Imperfect, bacterium soft edge, structure is unclear.
Shown in 100ug/ml low dosage Nano carbon balls grain preparation group Fig. 2: helicobacter pylori flagellum is reduced, and cell wall after birth is endless
It is whole.
Shown in 100ug/ml clarithromycin group Fig. 3: helicobacter pylori cell wall after birth is thinning imperfect, and flagellum reduces or disappears
It loses.
Shown in deionized water group Fig. 4: helicobacter pylori flagellum is abundant, and cell wall after birth is complete, and bacterium is clearly outlined, and thallus is just
Often.
It follows that Nano carbon balls grain formulation C NS has coup injury effect to helicobacter pylori.
Above embodiments are only the descriptions carried out to the preferred embodiment for the present invention, and range not is claimed to the present invention
The restriction of progress, without departing from the spirit of the design of the present invention, the technology of this field engineers and technicians according to the present invention
The various forms of deformations that scheme is made, should fall within the scope of protection determined by the claims of the present invention.
Claims (9)
1. a kind of Nano carbon balls grain preparation, which is characterized in that the Nano carbon balls grain preparation is mixed by Nano carbon balls grain and dispersion liquid
It closes and constitutes, the concentration of Nano carbon balls grain preparation is 0.1%~1.0%, wherein dispersion liquid is deionized water, 95% or more carbon
The partial size of nanometer spherolite is greater than 5nm and is less than 30nm, and the pH value of Nano carbon balls grain preparation is 7.1~7.6.
2. a kind of Nano carbon balls grain preparation described in accordance with the claim 1, which is characterized in that the partial size frequency of the Nano carbon balls grain
Number percentage are as follows: partial size is that the Nano carbon balls grain of 5~10nm accounts for 20~30%, and partial size is that the Nano carbon balls grain of 10~15nm accounts for 25
~35%, partial size is that the Nano carbon balls grain of 15~20nm accounts for 15~25%, partial size be the Nano carbon balls grain of 20~25nm account for 6~
10%, partial size is that the Nano carbon balls grain of 25~30nm accounts for 1~7%.
3. the preparation method of Nano carbon balls grain preparation described in a kind of claim 1, which comprises the following steps:
One, Nano carbon balls grain is prepared by original material processing of graphene；
Two, the satisfactory Nano carbon balls grain of partial size is screened using transmission electron microscope, wherein partial size is the Nano carbon balls of 5~30nm
Grain accounts for 95% or more；Nano carbon balls grain disperses in single spherical；
Three, it is required according to concentration, weigh the Nano carbon balls grain after step 2 is screened respectively and measures deionized water, it is spare；
Four, Nano carbon balls grain and deionized water the mixing dispersion obtained step 3；
Five, the mixed liquor being mixed to get through step 4 is stirred, wherein mixing time is 10~20 minutes, stirring
Speed is 60~80r/min；
Six, the mixed liquor after step 5 stir process is ultrasonically treated, wherein ultrasonic time is 15~20 minutes, is surpassed
Sound intensity degree is 80~100w；
Seven, the mixed liquor after step 6 is ultrasonically treated is adjusted at pH value using sodium bicarbonate-sodium carbonate buffer
Reason, is adjusted to 7.1~7.6 for the pH value of mixed liquor, obtains final Nano carbon balls grain preparation.
4. a kind of preparation method of Nano carbon balls grain preparation described in accordance with the claim 3, which is characterized in that the institute in step 2
It states the Nano carbon balls grain that partial size is 5~30nm and accounts for 95% or more, wherein the partial size frequency percentage of Nano carbon balls grain are as follows: partial size
20~30% are accounted for for the Nano carbon balls grain of 5~10nm, partial size is that the Nano carbon balls grain of 10~15nm accounts for 25~35%, partial size 15
The Nano carbon balls grain of~20nm accounts for 15~25%, and partial size is that the Nano carbon balls grain of 20~25nm accounts for 6~10%, and partial size is 25~
The Nano carbon balls grain of 30nm accounts for 1~7%.
5. a kind of preparation method of Nano carbon balls grain preparation described in accordance with the claim 3, which is characterized in that the institute in step 6
It states and the mixed liquor after step 5 stir process is ultrasonically treated, is ultrasonically treated under low temperature water-bath.
6. a kind of preparation method of Nano carbon balls grain preparation described in accordance with the claim 3, which is characterized in that the institute in step 6
Stating ultrasonic time is 15~20 minutes, refers to that ultrasonic all times are 15~20 minutes, wherein single ultrasonic time is 3 seconds,
The gap time is 3 seconds.
7. a kind of experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection, which comprises the following steps:
I, detection Nano carbon balls grain preparation is tested to the inhibiting rate of helicobacter Pylori urease by external fast urease；
II, it is acted on using coup injury of the transmission electron microscope observing Nano carbon balls grain preparation to helicobacter pylori；
III, the characteristic of Nano carbon balls grain preparation Helicobacter pylori Infection is determined according to the interpretation of result of step I and step II.
8. a kind of experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection according to claim 7, feature
It is, detection Nano carbon balls grain preparation is tested to helicobacter Pylori urease by external fast urease described in step I
Inhibiting rate, comprising the following steps:
A, preparation and diluted sample: with physiological saline by Helicobacter pylori SS1 bacterium solution 109CFU/ml dilution 1000
Again to 106CFU/ml, it is spare；It is respectively 1.0%, 0.5%, 0.4% three kinds of Nano carbon balls grain preparation deionizations by concentration
Water dilutes 100 times, and spare, the concentration of three kinds of Nano carbon balls grain preparations after dilution is respectively 100 μ g/ml, 50 μ g/ml, 40 μ g/
ml；Urease inhibitor acetohydroxamic acid is first dissolved into 1% with deionized water, it is standby then again with 100 times of deionized water dilution
With；Urease inhibitor acetohydroxamic acid concentration after dilution is 100 μ g/ml；Ionized water is removed, it is spare；Wherein, urease presses down
Preparation acetohydroxamic acid is as positive control, and deionized water is as negative control；
B, the 10 of 10 μ l loading: are added in the hole of 96 orifice plates6CFU/ml bacterium solution, multiple three holes；It is fast respectively according to grouping after adding
100 μ g/ml, 50 μ g/ml, 40 μ g/ml, tri- kinds of Nano carbon balls grain preparations, deionized water, 100 μ g/ml acetohydroxamic acids are added in speed
Each 90 μ l；
C, it is incubated for: 96 orifice plates being sealed using sealed membrane after loading, and are incubated for 24 hours in 4 DEG C of insulating box；
D, it develops the color: after being incubated for, the urea reagent of 50 μ l being added in corresponding hole, and is put in 37 DEG C of insulating box and is incubated for 30
Divide kind of a colour developing；
E, it reads: after being incubated for 30 points of kinds, being read at 578nm wavelength, and record every group of OD value；
F, inhibiting rate: inhibiting rate=[1- (OD is calculatedMeasurement-ODBlank)/(ODControl-ODBlank)] × 100%.
9. a kind of experimental method of Nano carbon balls grain preparation Helicobacter pylori Infection according to claim 7, feature
It is, is acted on described in step II using coup injury of the transmission electron microscope observing Nano carbon balls grain preparation to helicobacter pylori,
The following steps are included:
G, prepared by bacterium solution: with physiological saline by helicobacter pylori NTCC11637 bacterium solution 109CFU/ml dilutes 10 times to 108CFU/
Ml, it is spare；
H, loading: taking 12 1ml test tubes, and the 10 of 360 μ l are added in every pipe8CFU/ml helicobacter pylori NTCC11637 bacterium solution,
Multiple three holes；After adding, it is rapidly added 0.4%, 0.1% Nano carbon balls grain preparation, deionized water, 0.1% respectively according to grouping
Each 40 μ l of clarithromycin is mixed；Wherein, deionized water is as negative control, and clarithromycin is as positive control；
I, it reacts: orifice plate being sealed using sealed membrane after loading, first the constant-temperature table at 37 DEG C shakes bacterium 10 points of kinds of culture, then takes out
And it is put into 30 points of kinds of culture in 37 DEG C of insulating box rapidly；
J, it dyes: 100 μ l reaction solutions being taken to be placed on transmission electron microscope copper mesh, and with 1.0% phosphotungstic acid negative staining；
K, it observes: observing the Morphological Features of helicobacter pylori under transmission electron microscope, and judge Nano carbon balls grain preparation to pylorus spiral shell
Bacillus coup injury result.
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| CN103316044A (en) * | 2013-06-07 | 2013-09-25 | 逢甲大学 | Composition structure used for inhibiting gastrointestinal bacterial infections |
| CN103316043A (en) * | 2013-06-07 | 2013-09-25 | 逢甲大学 | Medical application of composition comprising carbon material and active particles |
| CN104224838A (en) * | 2014-09-23 | 2014-12-24 | 贵州特力达纳米碳素科技有限公司 | Nanometer carbon powder and application thereof |
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| CN103316043A (en) * | 2013-06-07 | 2013-09-25 | 逢甲大学 | Medical application of composition comprising carbon material and active particles |
| CN104224838A (en) * | 2014-09-23 | 2014-12-24 | 贵州特力达纳米碳素科技有限公司 | Nanometer carbon powder and application thereof |
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