CN106729611A - A kind of bacteria inhibiting composition - Google Patents
A kind of bacteria inhibiting composition Download PDFInfo
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- CN106729611A CN106729611A CN201510817081.9A CN201510817081A CN106729611A CN 106729611 A CN106729611 A CN 106729611A CN 201510817081 A CN201510817081 A CN 201510817081A CN 106729611 A CN106729611 A CN 106729611A
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- silver
- inhibiting composition
- polymyxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/38—Silver; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
Abstract
The invention discloses a kind of bacteria inhibiting composition, Main Ingredients and Appearance is antibiotic and silver, wherein, the antibiotic is Polymyxin B sulfate, and the silver includes silver compound and silver nanoparticle.The bacteria inhibiting composition that silver compound of the invention or silver nanoparticle are made with antibiotic has bacteriostasis to Acinetobacter bauamnnii (Ab), and silver compound or silver nanoparticle have cooperative effect with antibiotic combination to Acinetobacter bauamnnii, the single pharmaceutical quantities of antibiotic, silver compound or silver nanoparticle to Acinetobacter bauamnnii can be reduced, so as to reduce toxic and side effect, reduce resistance and occur.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to a kind of bacteria inhibiting composition.
Background technology
Antibiotic has saved countless life from finding so far, highly important role to be play on human medical,
And for the health of the mankind is made that contribution.But, widely using and abusing due to current antibiotic, microorganism confrontation
The drug resistance of raw element is also increasingly stronger, many antibiotic is substantially reduced the anti-infection ability of microorganism, and for a long time
Also there is certain toxic and side effect to human body using antibiotic.Therefore new, more effective antiseptic is had to look for.
Silver is a kind of silvery white non-ferrous metal, and it has the antibacterial functions of wide spectrum to Gram-positive and negative bacteria, fungi,
And with preventing the ability of bacterial adhesion.German obstetrician Crede is molten in the silver nitrate concentration 1% in 1884
Drop prevents neonatal ophthalmia in entering neonate's eye, the blindness of baby is down to 0.2% from 10%.Until today
Untill, many countries are still using Crede preventive treatments.The sixties in 20th century, Moyer is molten by 0.5% silver nitrate
Liquid is used for the treatment burnt, and argentiferous medicine is applied again.The research of recent decades scientist both domestic and external shows
Certain density silver ion has extensive killing action to microorganisms such as various bacteriums, virus, fungies.
Another form --- the silver nanoparticle of silver, is domestic and international researcher one of focus of concern at present.It is that a class is new
Type antiseptic, with broad-spectrum antiseptic, have no drug resistance and be safe the characteristics of.The phase that domestic and international researcher develops
The silver nanoparticle product answered has been applied to each field.Increasingly serious with antibiotic resistance, silver nanoparticle is in disinfection
The research and application in field increasingly receive extensive concern, the generation antibacterials as great development prospect.
Research shows, it is that silver ion that Nano silver grain is discharged and nano effect are made jointly that silver nanoparticle has bactericidal action
：
1. silver ion upsets the metabolic activity inside bacterium, and the synthesis of disulfide bond in metabolic enzyme makes the metabolic enzyme of bacterium
Lose activity；Increase the generation of active oxygen, hinder the generation of Fe-S clusters in respiratory chain；
2. silver ion and peptide glycan reaction on bacteria cell wall, while silver nanoparticle is by nano effect interference cell wall
The bond energy of the chemical combination key of composition, makes cell membrane more easy fracture；
3. silver nanoparticle enters inside bacterium, may interfere with DNA synthesis, suppresses its transcription.
Although silver ion and silver nanoparticle bactericidal action are stronger, recent studies have shown that, their toxic and side effect is not allowed to neglect yet
Depending on.Silver ion due in ionic condition be easier pass in and out cell, and silver nanoparticle can also by the endocytosis of cell and to
Outer release action, through the physiologic barrier of some specific cells composition, tissue and cell to the position cause to damage.
Therefore, in order to solve the above technical problems, it is necessory to provide a kind of antibacterial scheme, not influenceing antibacterial effect
Reduce silver or silver nanoparticle concentration again simultaneously.
Drug combination is clinically most simple and easy to do at present to overcome one of strategy of bacterial drug resistance, the reason of drug combination
By advantage：Different pharmaceutical acts on bacterial metabolism different phase；Act on different molecular target position；So that antibacterial effect is big
Big is higher than single medicine, such that it is able to reduce single pharmaceutical quantities, and reduces toxic and side effect, reduces resistance generation.For example, Chinese
In patent CN101394741B, it is related to the combination of a kind of imazalil and silver compound, it provides the biocidal for improving and makees
With for protecting all lived or abiotic material.But it is not that any two antibacterial material combines all
There can be more preferable effect, the index for being usually used in drug combination evaluation is index of cooperation, and only index of cooperation is in certain limit
When interior, the possibility of drug combination is just considered to have.And silver element can be used with which Antibiotic combination, combined
The dosage range that uses and the problems such as there is relatively more excellent insect killing effect for which microorganism, so far also
Without very clear and definite result of study.
The content of the invention
Inventor is combined from various different antibiotic respectively by by silver compound and silver nanoparticle, investigate its for
The antibacterial activity of different microorganisms, as a result finds, when silver compound or silver nanoparticle are combined with Polymyxin B sulfate, for
Anti- Acinetobacter bauamnnii (Ab) has more obvious synergistic function, thus can significantly reduce silver and polymyxin
The consumption of B so that silver can be used to use in vivo, and the exploitation for new antimicrobial agent is significant.
Therefore, first purpose of the application is to provide a kind of bacteria inhibiting composition, it is possible to reduce the list of antibiotic and silver
Pharmaceutical quantities, so as to reduce toxic and side effect, reduce resistance generation etc..
A kind of bacteria inhibiting composition, its Main Ingredients and Appearance be antibiotic and silver, wherein, the antibiotic be Polymyxin B sulfate,
The silver includes silver compound and silver nanoparticle.
According to the present invention, the silver nanoparticle is 40 with the weight ratio of Polymyxin B sulfate:1~5:4.
Preferably, the silver nanoparticle and the weight ratio of Polymyxin B sulfate are 20:1~5:2.
According to the present invention, the silver compound is silver nitrate.
Further, the silver nitrate and the weight ratio of Polymyxin B sulfate are 80:1~5:4.
Preferably, the silver nitrate and the weight ratio of Polymyxin B sulfate are 40:1~5:2.
Second object of the present invention, is to provide the bacteria inhibiting composition to cause for preparing treatment Acinetobacter bauamnnii
Infection medicine application.
According to a preferred embodiment of the invention, the formulation of the medicine is preferably injection.
According to the present invention, the injection also includes water for injection and auxiliary material, and described is amino acid, sweet dew including auxiliary material
Alcohol, PVP.
The present invention prepares silver nanoparticle Ag-cit with water phase trisodium citrate-sodium borohydride reduction, with PVP stabilization second
Alcohol reduction method for preparing silver nanometer Ag-PVP, silver compound of the invention selects silver nitrate.The present invention uses micro-dilution method
Drug susceptibility is determined, the anti-Acinetobacter bauamnnii of bacteria inhibiting composition of silver compound or silver nanoparticle and Polymyxin B sulfate is evaluated
Bacteriostatic activity.Compare the anti-Acinetobacter bauamnnii of independent silver compound, silver nanoparticle or Polymyxin B sulfate, present invention tool
Have the following advantages：
1st, silver compound or silver nanoparticle and Polymyxin B sulfate combination have cooperative effect to Acinetobacter bauamnnii, it is possible to reduce
Polymyxin B sulfate, silver compound or silver nanoparticle, so as to reduce toxic and side effect, subtract to single pharmaceutical quantities of Acinetobacter bauamnnii
Few resistance occurs.
2nd, silver compound or silver nanoparticle and Polymyxin B sulfate combination enhance bactericidal effect in vivo, meanwhile, can subtract significantly
Few internal viable bacteria amount.
3rd, the present invention silver compound or silver nanoparticle and Polymyxin B sulfate are made antibacterial injection, with good antimicrobial effect,
Many advantages, such as cost is relatively low, it has compared with high inhibition effect to Acinetobacter bauamnnii.
4th, silver compound or silver nanoparticle concentration are reduced, silver compound or silver nanoparticle is further improved as antibacterial note
Penetrate the security of agent.
Brief description of the drawings
Fig. 1 schemes for the TEM of Ag-PVP；
Fig. 2 is Ag-PVP grain size distributions；
Fig. 3 schemes for the TEM of Ag-cit；
Fig. 4 is Ag-cit grain size distributions；
Fig. 5 is the fungistatic effect figure that silver nanoparticle, silver nitrate and PMB combination are carried disease germs to mouse kidney；
Fig. 6 is the fungistatic effect figure that silver nanoparticle, silver nitrate and PMB combination are carried disease germs to mouse lung；
Fig. 7 is the fungistatic effect figure that silver nanoparticle, silver nitrate and PMB combination are carried disease germs to mouse blood；
Fig. 8 is the fungistatic effect figure that silver nanoparticle, silver nitrate and PMB combination are carried disease germs to mouse ascites.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following examples are merely to illustrate
The present invention is not for limitation the scope of the present invention.
Embodiment 2 and embodiment 9 are using the Acinetobacter bauamnnii (Huashan hospital with Carbapenem-resistant class antibiotic
Aba1604) as test bacterium；The silver nanoparticle of embodiment 2~9 is the silver nanoparticle synthesized using the method for embodiment 1.
The synthesis of the silver nanoparticle of embodiment 1
Ag-PVP synthesizes：0.78g PVP and the 49mL absolute ethyl alcohol that will be weighed are added in three neck round bottom, ultrasound
Fully mix, be placed in 70 DEG C of oil baths, meanwhile, magnetic stirrer solution is slowly added to 1mL after 15min
1%AgNO3Solution, keeps 70 DEG C of heating 120min.Question response liquid is cooled to room temperature, and now the color of colloidal sol is in
It is orange-yellow.TEM detects that Fig. 1 is the TEM figures of silver nanoparticle prepared by the method, and Fig. 2 is silver nanoparticle particle diameter distribution,
Average grain diameter is 13nm.
Ag-cit synthesizes：During 1% citric acid three sodium solution of 20mL and 75mL water added into three neck round bottom,
It is placed in 70 DEG C of oil baths, meanwhile, magnetic stirrer solution adds the 1%AgNO of 1.7mL after 15min3
Solution, the 0.1%NaBH that then rapid addition 2mL is newly prepared4Solution.Mixing speed 400r/min, reaction 1
Terminate reaction after hour.Question response liquid is cooled to room temperature, is diluted with water to 100mL, and now the color of colloidal sol is in pale yellow
Color.TEM detects that Fig. 3 is the TEM figures of silver nanoparticle prepared by the method, and Fig. 4 is silver nanoparticle particle diameter distribution, averagely
Particle diameter is 8nm.
The bacteriostatic experiment of the silver medal of embodiment 2 and Polymyxin B sulfate
1st, method
(1) silver nitrate or silver nanoparticle and determination of antibiotic susceptibility：First by silver nitrate or silver nanoparticle and Polymyxin B sulfate
Contrast is diluted to a series of concentration on 96 empty flat boards of sterilizing respectively, silver nitrate concentration is respectively 50,25,12.5,
6.3rd, 3.1,1.6ppm, silver nanoparticle concentration is respectively 25,12.5,6.3,3.1,1.6ppm, Polymyxin B sulfate.
The fresh colony of LB flat boards is grown on oese picking to be placed in LB culture mediums, 37 DEG C incubate 18h, LB cultures
Base is diluted to 0.5 Maxwell turbidity, then dilutes 10,000 times, makes final bacterial concentration be 5 × 105CFU/mL.Will be dilute
The bacterium solution released is moved in 96 orifice plates respectively, is added by different proportion and is diluted to a series of concentration each sample.37 DEG C incubate
18h is educated, result is observed, inhibiting rate and MIC value is calculated.
(1) chessboard method determines Compound Resisting microbial inoculum effect joint susceptibility：Silver nitrate or silver nanoparticle press micro LB with antibiotic
Broth dilution method is diluted in 96 orifice plate files and row respectively, is added in 90 μ LLB culture mediums are contained in every hole
Silver nitrate or the μ L of silver nanoparticle antibiotic compound antiseptic 10, dilution factor is according to bacterial strain is to silver nitrate or silver nanoparticle and resists
The MIC value result of raw element determines, and boxed out with plate and do the MIC value of the micro meat soup of single medicine and determine.By inoculation to examination
Guan Zhong, 37 DEG C of incubation 18h, LB culture mediums are diluted to 0.5 Maxwell turbidity, then dilute 10,000 times, make final
Bacterial concentration is 5 × 105CFU/mL.37 DEG C of incubation 18h, observe result, calculate inhibiting rate, determine MIC value
With collaboration inhibition index (FIC).
2nd, result
Silver compound or silver nanoparticle and Polymyxin B sulfate are matched somebody with somebody to the MIC value area Results and difference of Acinetobacter bauamnnii
Combination effect than under.
The MIC (ppm) of the silver medal of table 1. and Polymyxin B sulfate to Acinetobacter bauamnnii
| AgNO3 | Silver nanoparticle | Polymyxin B sulfate | |
| MIC(ppm) | 2.5 | 1.25 | 0.25 |
Conclusion：Silver nanoparticle is 1.25ppm to the MIC of Ab, and silver nitrate is 2.5ppm to the MIC of Ab, many
Colistin B is 0.25ppm to the MIC of Ab.
Hereinafter suppress Acinetobacter bauamnnii for Polymyxin B sulfate is closed with Unionpay, Polymyxin B sulfate shows and silver nanoparticle or nitre
Sour silver collaboration suppresses the effect of Acinetobacter bauamnnii.
Wherein, FIC≤0.5 cooperative effect；1>FIC>0.5 part cooperative effect；FIC=1 is summation action；
2>FIC>1 unrelated effect；FIC >=2 antagonistic effect.
The silver nanoparticle of table 2. is combined optimum ratio (mass ratio) research with Polymyxin B sulfate
Conclusion：When silver nanoparticle and PMB are combined, different ratio is different to the activity of Acinetobacter bauamnnii.40:1~
5:Within the scope of 4, acted synergistically with collaboration or part, and 20:1~5:Within the scope of 2, with stronger
Synergy.
Table 3.AgNO3Optimum ratio (mass ratio) is combined with Polymyxin B sulfate to study
Conclusion：When silver nitrate and PMB are combined, different proportion is different to the activity of Acinetobacter bauamnnii.80:1~
5:Within the scope of 4, acted synergistically with collaboration or part, and 40:1~5:Within the scope of 2, with stronger
Synergy.
It is prepared by the parenteral solution of embodiment 3
Formula：
Silver nitrate 52g, PB 12.5g, glycine 1.5g, mannitol 3.5g, PVP 1.5g,
1000 are prepared altogether.
By in silver nitrate, PB, glycine, mannitol, PVP addition 8000ml waters for injection, note is added
Penetrate with water to 10000ml, plus activated carbon stirring, filtering, sterilize, it is filling.
It is prepared by the parenteral solution of embodiment 4
Formula：Silver nitrate 62.5g, PB 12.5g, glycine 1.7g, mannitol 3.6g, PVP
1.8g, prepares 1000 altogether.
By in silver nitrate, PB, glycine, mannitol, PVP addition 8000ml waters for injection, note is added
Penetrate with water to 10000ml, plus activated carbon stirring, filtering, sterilize, it is filling.
It is prepared by the parenteral solution of embodiment 5
Formula：
Silver nitrate 105g, PB 12.5g, glycine 1.8g, mannitol 3.8g, PVP 1.7g,
1000 are prepared altogether.
By in silver nitrate, PB, glycine, mannitol, PVP addition 8000ml waters for injection, note is added
Penetrate with water to 10000ml, plus activated carbon stirring, filtering, sterilize, it is filling.
It is prepared by the parenteral solution of embodiment 6
Formula：
Silver nanoparticle 16g, PB 12.5g, glycine 1.5g, mannitol 3.5g, PVP 1.5g,
1000 are prepared altogether.
By in silver nanoparticle, PB, glycine, mannitol, PVP and addition 8000ml waters for injection, add
Water for injection is to 10000ml, plus activated carbon stirring, and filtering, sterilizing is filling.
It is prepared by the parenteral solution of embodiment 7
Formula：
Silver nanoparticle 24g, PB 12.5g, glycine 1.6g, mannitol 3.7g, PVP 1.6g,
1000 are prepared altogether.
By in silver nanoparticle, PB, glycine, mannitol, PVP and addition 8000ml waters for injection, add
Water for injection is to 10000ml, plus activated carbon stirring, and filtering, sterilizing is filling.
It is prepared by the parenteral solution of embodiment 8
Formula：
Silver nanoparticle 31g, PB 12.5g, glycine 1.6g, mannitol 3.8g, PVP 1.8g,
1000 are prepared altogether.
By in silver nanoparticle, PB, glycine, mannitol, PVP addition 8000ml waters for injection, note is added
Penetrate with water to 10000ml, plus activated carbon stirring, filtering, sterilize, it is filling.
Fungistatic effect evaluation in the body of embodiment 9
1st, Acinetobacter bauamnnii Murine Model of Intraperitoneal Infection model is set up
1.1 Acinetobacter bauamnnii mouse severe infection models are set up：Acinetobacter bauamnnii is placed in nutrient solution containing LB
Test tube in, 37 DEG C, 220rpm culture 16h.Then 100 times are diluted in fresh LB nutrient solutions, 37 DEG C, 220rpm
Zengjing Granule 3h, normal saline dilution, about 5 × 1011CFU Acinetobacter bauamnniis are expelled to mouse peritoneal, mouse 24h
It is dead.Average mice body weight is 20g.
1.2 models are set up：The μ L 1 × 10 of intraperitoneal injection 50011Acinetobacter bauamnnii solution (8% mucin of CFU/mL
Normal saline solution)
1.3 administration process：After model is set up and finishes 1h, difference the μ L silver nano solutions of intraperitoneal injection 100 and nitric acid
Silver-colored solution, observation.
2nd, silver nanoparticle, the LD50 of silver nitrate are determined
The μ L silver nano solutions of healthy mice intraperitoneal injection 100 and silver nitrate solution, determine Ag-PVP, the LD50 of silver nitrate.
The LD50 of silver nanoparticle is determined：Silver nano solution concentration is that 22.431mM, i.e. silver content are 0.2423mg, LD50
It is 12.12mg/kg；Silver nitrate LD50 is determined：Silver nitrate solution concentration is for 43.285mM, i.e. silver content
0.4675mg, the LD50 of silver nitrate is 36.79mg/kg.
3rd, silver nanoparticle, silver nitrate treatment Acinetobacter bauamnnii mouse model dosage determine
Silver nanoparticle, silver nitrate respectively select three therapeutic doses, (1/10LD50) respectively high, in (1/20LD50),
Low (1/30LD50).Mouse Weight is based on average every 20g.
Mouse survival ratio under the different silver dosage of table 4.
Group observes mouse survival rate and is 0% with silver nanoparticle group in, low two dosage, after 24h.Follow-up zoopery
Middle silver nitrate dosage and silver nanoparticle dosage choice are between (1/20~1/10) LD50.
4th, silver nanoparticle (16.2 μ g), silver nitrate (28.9 μ g) and PMB combination treatment Acinetobacter bauamnnii infecting mouses
Model, each group mouse organs compare with bacterium number after 16 hours.
The fungistatic effect that 4.1 silver nanoparticles, silver nitrate and PMB combination are carried disease germs to mouse kidney, is shown in Fig. 5.
The fungistatic effect that 4.2 silver nanoparticles, silver nitrate and PMB combination are carried disease germs to mouse lung, is shown in Fig. 6.
The fungistatic effect that 4.3 silver nanoparticles, silver nitrate and PMB combination are carried disease germs to mouse blood, is shown in Fig. 7.
The fungistatic effect that 4.4 silver nanoparticles, silver nitrate and PMB combination are carried disease germs to mouse ascites, is shown in Fig. 8.
Conclusion is carried disease germs analysis from each organ of mouse, and silver greatly reduces viable bacteria amount in Mice Body with Polymyxin B sulfate combination.
Silver and the respective consumption of Polymyxin B sulfate can effectively be reduced.
Claims (9)
1. a kind of bacteria inhibiting composition, it is characterised in that the Main Ingredients and Appearance of the bacteria inhibiting composition is antibiotic and silver,
Wherein, the antibiotic is Polymyxin B sulfate, and the silver includes silver compound and silver nanoparticle.
2. bacteria inhibiting composition as claimed in claim 1, it is characterised in that the silver nanoparticle and Polymyxin B sulfate
Weight ratio is 40:1~5:4.
3. described bacteria inhibiting composition according to claim 2, it is characterised in that the silver nanoparticle is sticked more with
The weight ratio of rhzomorph B is 20:1~5:2.
4. described bacteria inhibiting composition according to claim 1, it is characterised in that the silver compound is nitre
Sour silver.
5. bacteria inhibiting composition as claimed in claim 4, it is characterised in that the silver nitrate and Polymyxin B sulfate
Weight ratio is 80:1~5:4.
6. bacteria inhibiting composition according to claim 5, it is characterised in that the silver nitrate and Polymyxin B sulfate
Weight ratio be 40:1~5:2.
7. the bacteria inhibiting composition as any one of claim 1~6 causes for preparing treatment Acinetobacter bauamnnii
Infection medicine application.
8. application as claimed in claim 7, it is characterised in that the formulation of the medicine is injection.
9. injection as claimed in claim 8, it is characterised in that the injection also includes water for injection and auxiliary
Material, described is amino acid, mannitol, PVP including auxiliary material.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021036923A1 (en) * | 2019-08-23 | 2021-03-04 | The University Of Hong Kong | Silver-/gold-compounds and methods thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021036923A1 (en) * | 2019-08-23 | 2021-03-04 | The University Of Hong Kong | Silver-/gold-compounds and methods thereof |
| CN114258308A (en) * | 2019-08-23 | 2022-03-29 | 香港大学 | Silver/gold compounds and methods thereof |
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Application publication date: 20170531 |