CN108484693A - A kind of chitosan oligosaccharide-antibiotic conjugate and its preparation method and application - Google Patents
A kind of chitosan oligosaccharide-antibiotic conjugate and its preparation method and application Download PDFInfo
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- CN108484693A CN108484693A CN201810208430.0A CN201810208430A CN108484693A CN 108484693 A CN108484693 A CN 108484693A CN 201810208430 A CN201810208430 A CN 201810208430A CN 108484693 A CN108484693 A CN 108484693A
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- Prior art keywords
- chitosan oligosaccharide
- conjugate
- florfenicol
- antibiotic
- cos
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- 0 CS(c1ccc(C(C(CF)NC(C(Cl)Cl)=O)NC(CC(O)=O)=*)cc1)(=O)=O Chemical compound CS(c1ccc(C(C(CF)NC(C(Cl)Cl)=O)NC(CC(O)=O)=*)cc1)(=O)=O 0.000 description 2
- RINCXYDBBGOEEQ-UHFFFAOYSA-N O=C(CC1)OC1=O Chemical compound O=C(CC1)OC1=O RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
- C07H13/06—Fatty acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/722—Chitin, chitosan
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
- A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
Abstract
The invention discloses a kind of chitosan oligosaccharide antibiotic conjugates and its preparation method and application.The chitosan oligosaccharide antibiotic conjugate is the covalent complex of chitosan oligosaccharide and antibiotic, and the antibiotic is chloromycetin series antibiotics.The chloromycetin series antibiotics are Florfenicol, and the conjugate is chitosan oligosaccharide Florfenicol conjugate.The chitosan oligosaccharide Florfenicol conjugate of the present invention has good inhibition to pathogenetic bacteria, has stronger destruction particularly with the bacterium for having formed biofilm depths, and bactericidal range is wide, has good inhibiting effect to the formation of biofilm.Compared to Florfenicol, the bacteriostatic activity of chitosan oligosaccharide Florfenicol conjugate significantly improves.
Description
Technical field
The invention belongs to antiseptic technical fields, and in particular to a kind of chitosan oligosaccharide-antibiotic conjugate and preparation method thereof
And application.
Background technology
Excessive use due to the mankind to antibiotic results in drug resistance of the bacterium to existing antibiotic getting worse.This
Outside, the reason of clinical persistent infection and Nosocomial infection occur is mostly with pathogen in host or medical material surface
It is related to form biofilm (Biofilm).Biofilm is that have the bacterial community of special microenvironment and its extracellular matrix compound
Body.Bacterium or fungal colonization are attached to body surface or gas-liquid boundary, a large amount of exocellular polysaccharides, protein and DNA are secreted, to shape
At biofilm settlement.Biofilm state pathogen often improves the tolerance of antiseptic etc. hundreds of to thousands of than the state bacterium that swims
Times.Conventional medicament therefore clinically need to be for the treatment of pathogen for treating the less effective of biofilm infections relating
Exploitation simultaneously can be effectively for the drug of biofilm state pathogen, preferably to remove cause of disease on the basis of inhibiting pathogen
Bacterium.
Florfenicol is that veterinary clinic uses very extensive animal specific antibacterials, as Florfenicol faces in animal doctor
A large amount of uses on bed, the drug resistance problems of pathogenic bacteria are increasingly severe, are clinically badly in need of effectively solving the drug resistance of this drug
Problem.
Chitosan (chitosan) is that the chitin (chitin) being widely present by nature is obtained by deacetylation
, chitosan has many advantages, such as good biocompatibility, biodegradability, broad spectrum antibacterial, be widely used in medicine,
Food service industry.Chitosan oligosaccharide is that chitosan (is also had the report using chemical degradation, microwave degradation technology through special biological enzyme technology
Road) a kind of obtained degree of polymerization of degrading oligosaccharide product between 2~20, it has the unexistent higher solubility of chitosan, entirely
It is dissolved in water, is easy many unique functions such as to be absorbed and utilized by organism.
Invention content
It is an object of the present invention to provide a kind of chitosan oligosaccharide-antibiotic conjugates and its preparation method and application.Mainly solve
The increasingly severe problem of antibiotic resistance in the prior art.
Used technical solution is as follows to solve above-mentioned technical problem by the present invention：
A kind of chitosan oligosaccharide-antibiotic conjugate, the chitosan oligosaccharide-antibiotic conjugate are that chitosan oligosaccharide and the covalent of antibiotic are answered
Object is closed, the antibiotic is chloromycetin series antibiotics.
Preferably, the chloromycetin series antibiotics are Florfenicol, and the conjugate is chitosan oligosaccharide-Florfenicol coupling
Object.
Preferably, chitosan oligosaccharide (the wherein mole of Glucosamine) and fluorine in the chitosan oligosaccharide-Florfenicol conjugate
The molar ratio that benzene Buddhist nun examines is 1：1.
Preferably, the chitosan oligosaccharide is the oligomeric ammonia that molecular weight is more than or equal to 60% in 3000kDa or less and deacetylation
Base glucose mixture.
The present invention also provides a kind of preparation method of chitosan oligosaccharide-Florfenicol conjugate, which includes following step
Suddenly：
Step 1, Florfenicol and succinic anhydride are with molar ratio 1:1~1:1.5 are dissolved in organic solvent, and catalyst 4- is added
Dimethylamino naphthyridine reacts 2~5h, 20~30 DEG C of vacuum rotary steams, ethyl alcohol and water volume ratio 1 at 40~70 DEG C:Weight under the conditions of 1
Crystallize to obtain white solid；
Step 2, by product and the chitosan oligosaccharide obtained by step 1 in molar ratio 1:1 mixing, the amount of the chitosan oligosaccharide press comprising
(mole of Glucosamine and the molar ratio of step 1 products therefrom are i.e. in chitosan oligosaccharide for the mole calculating of Glucosamine
1:1)；Then mixture is dissolved in n,N-Dimethylformamide, and catalyst n-HOSu NHS, carbodiimides is added,
It is stirred overnight at 0~30 DEG C；Bag filter dialysis 1-5 d obtain dialyzate, are lyophilized, and the chitosan oligosaccharide-Florfenicol coupling is made
Object.
Preferably, organic solvent is acetone in the step 1, and the reaction time is 2~3h, and reaction temperature is 50~60
℃。
Preferably, in the step 1 catalyst 4-dimethylaminopyridine dosage be Florfenicol content 112mol%.
Preferably, catalyst n-HOSu NHS in the step 2, carbodiimides dosage be respectively step 1
The 112mol% of product amount.
Preferably, the reaction temperature of the step 2 is 20~30 DEG C, and dialysis time is 2~3d.
The present invention also provides application of the chitosan oligosaccharide-antibiotic conjugate in inhibiting microbial activity；Especially
It is the application inhibited in the microbial activity of biofilm depths.Preferably, the microorganism is gram-positive bacteria, preferably pig
Streptococcus.
The present invention also provides the chitosan oligosaccharide-antibiotic conjugates in preparing fungicide or preparing germ killing drugs
Using.
The present invention also provides a kind of pharmaceutical composition, which includes that chitosan oligosaccharide-Florfenicol conjugate is made
For active component.
As a kind of better choice of aforementioned pharmaceutical compositions, described pharmaceutical composition further includes pharmaceutically acceptable auxiliary
Material.The wherein described pharmaceutically acceptable auxiliary material is auxiliary material commonly used in the art.The excipient substance refers to production drug
With excipient and additives used when prescription being dispensed.In specific implementation process, the present invention can be by a variety of this fields
The method known practices.The present invention does not do particular determination to excipient and additives, and the two can use commonly used in the art
Type.For example, excipient can be syrup, sodium alginate, lactose etc., additives can be polysorbate, polyoxyethylene fat
Fat acid esters, chitin etc..
The present invention also provides application of any of the above-described composition in preparing antiseptic.The wherein described antiseptic can press down
The bacterium of free state processed and the bacterium of biofilm state.The bacterium is this field routine pathogenic bacteria.Preferably pig chain
Coccus.
Conjugate according to the present invention, wherein the composition further includes solvent, it is preferable that the solvent is N,
Dinethylformamide.The solvent of the application can be that common N,N-dimethylformamide can also be anhydrous N, N- dimethyl
Formamide, can be according to actually being selected.
Compared with prior art, beneficial effects of the present invention are as follows：
Chitosan oligosaccharide-Florfenicol conjugate of the present invention has good inhibition to pathogenetic bacteria, particularly with
Bacterium through forming biofilm depths has stronger destruction, and bactericidal range is wide, has to the formation of biofilm good
Good inhibiting effect.Compared to Florfenicol, the bacteriostatic activity of chitosan oligosaccharide-Florfenicol conjugate significantly improves.
Description of the drawings
Fig. 1 is the mass spectrogram of the chitosan oligosaccharide-Florfenicol conjugate prepared in the embodiment of the present invention 1.
Fig. 2 is the bar shaped of Flo, COS, F-COS and Flo+COS to Streptococcus suis inhibition in the embodiment of the present invention 2
Datagram.
Fig. 3 is that Flo, COS, F-COS and Flo+COS destroy effect to streptococcus suis biofilm in the embodiment of the present invention 3
The bar shaped datagram of fruit.
Fig. 4 is the F-COS and Flo of various concentration in the embodiment of the present invention 4 to streptococcus suis biofilm execution
Data bar chart.
Fig. 5 is that Flo, COS, F-COS and Flo+COS inhibit to imitate to streptococcus suis biofilm in the embodiment of the present invention 5
The bar shaped datagram of fruit.
Fig. 6 is the F-COS and Flo of various concentration in the embodiment of the present invention 6 to streptococcus suis biofilm inhibition
Data bar chart.
Specific implementation mode
Technical scheme of the present invention is described in detail with reference to embodiment.Actual conditions are not specified in embodiment
Person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, being can be with
Conventional products that are commercially available.
The present invention takes following method to prepare the preparation method of chitosan oligosaccharide-Florfenicol chemical coupling thing：
(1) Florfenicol and succinic anhydride are with molar ratio 1：1~1：1.5 are dissolved in acetone and other organic solvent, and catalyst is added
4-dimethylaminopyridine reacts 2~5h, 20~30 DEG C of vacuum rotary steams, ethyl alcohol and water volume ratio 1 at 40~70 DEG C:Under the conditions of 1
Recrystallize to obtain white solid.
(2) by product and the chitosan oligosaccharide (being calculated by contained aminoglucose sugar monosaccharide mole) obtained by step (1) by mole
Than 1:1 mixing is dissolved in the organic solvents such as n,N-Dimethylformamide, and catalyst n-HOSu NHS, two Asia of carbonization is added
Amine is stirred overnight at 0~30 DEG C；Bag filter dialysis 1-5d obtains dialyzate, is lyophilized to get chitosan oligosaccharide-Florfenicol conjugate.
Wherein step (1) described catalyst is 4-dimethylaminopyridine, and dosage is the 112mol% of Florfenicol content,
The solvent is acetone and other organic solvent, and the reaction time is 2~3h, and reaction temperature is 50~60 DEG C.
Step (2) product and the molar ratio of chitosan oligosaccharide (being calculated by contained Glucosamine monosaccharide amount) are 1：1；It is described
Catalyst n-HOSu NHS, carbodiimides additive amount be the 112mol% of (1) product assay, it is described anti-
It is 20~30 DEG C to answer temperature, and the dialysis time is 2~3d.
Dialysis wherein described in step (2) is this field conventional dialysis procedures.The dialysis is a kind of selection through the membrane
Property diffusion process, can be used for detaching the different solute of molecular size range, the substance of threshold value molecular weight retained less than film can spread
Across film, the substance that threshold value molecular weight is retained higher than film is then retained in the other side of semi-permeable membrane.The wherein described bag filter is this
Field conventional dialysis bag.The molecular cut off of the bag filter is 500~1000Da, by dialysing to remove unreacted fluorobenzene
Buddhist nun examines and small molecule salt.The time of the wherein described dialysis is preferably 1~5d, is more preferably 2~3d, dialysis time is best
For 3d.
Drying wherein described in step (2) is this field conventional drying mode.It is dry that the drying is preferably vacuum refrigeration
Dry, vacuum drying, spray drying, drying or infra-red drying, are most preferably vacuum freeze drying.The ginseng of the vacuum freeze drying
Number is：Temperature -50~-80 DEG C, 20~30Pa of vacuum degree, 48~72 hours time.
The preparation of 1 chitosan oligosaccharide of embodiment-Florfenicol chemical coupling thing
Chitosan oligosaccharide and the reaction equation of Florfenicol chemical coupling are as shown in Scheme1.Compound 1 is Florfenicol,
Compound 2 is obtained after being reacted with succinic anhydride, being obtained by the reaction of amino of the carboxyl in compound 2 and compound 3 (chitosan oligosaccharide)
Close object 4, i.e. chitosan oligosaccharide-Florfenicol conjugate.
The specific implementation step of chemical coupling reaction is as follows：
(1) Florfenicol and succinic anhydride are with molar ratio 1:1~1:1.5 are dissolved in acetone and other organic solvent, and catalyst is added
4-dimethylaminopyridine reacts 2~5h, 20~30 DEG C of vacuum rotary steams, ethyl alcohol and water volume 1 at 40~70 DEG C:Weight under the conditions of 1
Crystallize to obtain white solid.
(2) by product and the chitosan oligosaccharide obtained by step (1) in molar ratio 1:1 mixing is dissolved in N, N- dimethylformamides etc.
In organic solvent, catalyst n-HOSu NHS is added, carbodiimides is stirred overnight at 0~30 DEG C；Bag filter is dialysed
1-5d obtains dialyzate, is lyophilized to get chitosan oligosaccharide-Florfenicol conjugate.
For the chitosan oligosaccharide being prepared-Florfenicol conjugate through mass spectral analysis, spectrogram is as shown in Figure 1.To the mass spectrogram of Fig. 1
Interpretation of result is as follows：
The corresponding compound structure of 1218.01 spectral peaks is in Fig. 1：
The corresponding compound structure of 1379.96 spectral peaks is in Fig. 1：
The corresponding compound structure of 1541.22 spectral peaks is in Fig. 1：
The corresponding compound structure of 1701.59 spectral peaks is in Fig. 1：
The corresponding compound structure of 1862.01 spectral peaks is in Fig. 1：
The corresponding compound structure of 2184.57 spectral peaks is in Fig. 1：
2 chitosan oligosaccharides of embodiment-Florfenicol conjugate inhibits bacterial growth
In the present embodiment, to the chitosan oligosaccharide of preparation-Florfenicol conjugate inhibit Streptococcus suis growth effect into
Research is gone.Streptococcus suis used comes from China General Microbiological culture presevation administrative center.Streptococcus suis is trained in MRS liquid
After supporting 37 DEG C of shake overnight incubations of base, it is diluted to 4mL about 1 × 105CFU bacterium solutions are separately added into Florfenicol (abbreviation Flo), shell
Oligosaccharides (COS), chitosan oligosaccharide-Florfenicol conjugate (abbreviation F-COS) and Florfenicol and chitosan oligosaccharide mixture (abbreviation Flo+
COS) (mixing quality ratio is 1:1), respectively at 37 DEG C of dynamic cultivations, the bacterial population under different time is counted.
It is the bar shaped datagram of Flo, COS, F-COS and Flo+COS to Streptococcus suis inhibition referring to Fig. 2.Experiment
The result shows that：Flo groups and Flo+COS groups are substantially consistent the inhibition that Streptococcus suis grows, therefore may determine that
The simple fungistatic effect mixed Florfenicol with chitosan oligosaccharide and do not reinforce Flo.And relative to Flo groups and Flo+COS
Group, then highly significant improves the inhibiting effect (p grown to Streptococcus suis to F-COS groups<, therefore chitosan oligosaccharide-fluorobenzene 0.01)
Buddhist nun, which examines conjugate, has apparent inhibition bacterial growth effect.
3 chitosan oligosaccharides of embodiment-Florfenicol conjugate is to streptococcus suis biofilm execution
In the present embodiment, the COS chemical modifications F-COS of Flo is destroyed the effect of streptococcus suis biofilm into
Research is gone.Streptococcus suis used comes from China General Microbiological culture presevation administrative center.Streptococcus suis is trained in MRS liquid
After supporting 37 DEG C of shake overnight incubations of base, 100 L~2 × 10 μ are taken7CFU bacterium solutions are added to 37 DEG C of 96 orifice plate static culture 24 hours
Form ripe biofilm.
Flo, COS, F-COS and Flo and COS mixtures Flo+COS (mixing quality ratios 1 are prepared respectively:1) sample is used
Supernatant fluid is removed after biofilm maturation in this research, is separately added into the MRS liquid that 100 μ L contain the above-mentioned samples of 1mg/mL
Body culture medium.100 μ LMRS fluid nutrient mediums are added in untreated fish group.Using the detection life of MTT decoration methods after being acted on 24 hours at 37 DEG C
Object envelope execution.
It is the figurate number of Flo, COS, F-COS and Flo+COS to streptococcus suis biofilm execution referring to Fig. 3
According to figure.The experimental results showed that：Flo groups and Flo+COS groups are not obvious the execution of streptococcus suis biofilm, and F-
COS groups then have significant biofilm elimination effect；Flo groups are compared, F-COS groups significantly improve the removing effect of biofilm
Fruit (p<0.01).
4 various concentration chitosan oligosaccharide of embodiment-Florfenicol conjugate is to streptococcus suis biofilm execution
In the present embodiment, to various concentration F-COS to the destruction effect of Streptococcus suis under biofilm state into
Research is gone.Specific implementation is as follows：Streptococcus suis used comes from China General Microbiological culture presevation administrative center.Pig hammer
Bacterium takes 100 L~2 × 10 μ after 37 DEG C of shake overnight incubations of MRS fluid nutrient mediums7It is quiet that CFU bacterium solutions are added to 37 DEG C of 96 orifice plate
State culture forms ripe biofilm for 24 hours and removes supernatant fluid after biofilm maturation, is separately added into 100 μ L and contains
There are the MRS culture mediums of various concentration (1,50,150,250,500,750,1000 μ g/mL) F-COS.100 μ L are added in untreated fish group
MRS fluid nutrient mediums, while unmodified Flo is as a contrast.Using the detection life of MTT decoration methods after being acted on 24 hours at 37 DEG C
Object envelope execution.
It is the data bar chart of F-COS and Flo to streptococcus suis biofilm execution of various concentration referring to Fig. 4.
The experimental results showed that：F-COS can significantly destroy Streptococcus suis maturation biofilm in 500ug/ml；Compared to Flo, F-
The destruction of COS highly significants enhanced to streptococcus suis biofilm, by the way that chitosan oligosaccharide and Florfenicol are coupled energy
It is substantially reduced the use concentration of antibiotic.
5 chitosan oligosaccharides of embodiment-Florfenicol conjugate is to streptococcus suis biofilm inhibition
In the present embodiment, the effect that streptococcus suis biofilm is formed is inhibited to the COS chemical modifications F-COS of Flo
Fruit is studied.Specific implementation is as follows：Streptococcus suis used comes from China General Microbiological culture presevation administrative center.Pig
Streptococcus removes supernatant fluid after 37 DEG C of MRS fluid nutrient mediums shake overnight incubations, be added Flo, COS containing 1mg/mL,
F-COS and Flo and COS mixtures (Flo+COS) (mixing quality ratio 1:1) it is in MRS fluid nutrient mediums to bacterial concentration
2×107CFU takes the 100 above-mentioned bacterium solutions of μ L to be added to 37 DEG C of 96 orifice plate static culture 24 hours and forms biofilm.Untreated fish group
100 μ L MRS fluid nutrient mediums are added.MTT decoration methods detect biofilm inhibition.
It is the figurate number of Flo, COS, F-COS and Flo+COS to streptococcus suis biofilm inhibition referring to Fig. 5
According to figure.The experimental results showed that：Compared with Flo, F-COS has good biofilm inhibition, significantly suppresses biological quilt
Film is grown.
6 various concentration chitosan oligosaccharide of embodiment-Florfenicol conjugate is to streptococcus suis biofilm inhibition
In the present embodiment, the various concentration F-COS function and effect for inhibiting streptococcus suis biofilm to be formed are carried out
Research.Specific implementation is as follows：Streptococcus suis used comes from China General Microbiological culture presevation administrative center.Streptococcus suis exists
After 37 DEG C of MRS fluid nutrient mediums shake overnight incubation, 100 L~2 × 10 μ are taken7CFU bacterium solutions, wherein containing various concentration (1,50,
150,250,500,750,1000 μ g/mL) F-COS, 96 orifice plates are added to, 100 L~2 × 10 μ are added in untreated fish group7CFU bacterium
Liquid, while unmodified Flo is as a contrast.Effect is destroyed using MTT decoration methods detection biofilm after being acted on 24 hours at 37 DEG C
Fruit.
It is the data bar chart of F-COS and Flo to streptococcus suis biofilm inhibition of various concentration referring to Fig. 6.
The experimental results showed that：F-COS can significantly inhibit Streptococcus suis maturation biofilm in 500ug/ml and be formed；Compared to Flo
(>1000ug/ml), F-COS can be substantially reduced the use concentration of antibiotic.
The part preferred embodiment of the present invention is above are only, the present invention is not limited in the content of embodiment.For ability
For technical staff in domain, can there is various change and change in the conception range of technical solution of the present invention, made by appoint
What changes and change, within the scope of the present invention.
Claims (10)
1. a kind of chitosan oligosaccharide-antibiotic conjugate, it is characterised in that：The chitosan oligosaccharide-antibiotic conjugate is chitosan oligosaccharide and resists
The covalent complex of raw element, the antibiotic are chloromycetin series antibiotics.
2. chitosan oligosaccharide as described in claim 1-antibiotic conjugate, it is characterised in that：The chloromycetin series antibiotics are fluorine
Benzene Buddhist nun examines.
3. chitosan oligosaccharide as described in claim 1-antibiotic conjugate, it is characterised in that：The chitosan oligosaccharide is that molecular weight exists
3000kDa or less and deacetylation are more than or equal to 60% oligo-glucosamine mixture.
4. a kind of preparation method of chitosan oligosaccharide-Florfenicol conjugate, which is characterized in that the preparation method includes the following steps：
Step 1, Florfenicol and succinic anhydride are with molar ratio 1:1~1:1.5 are dissolved in organic solvent, and catalyst 4- diformazans are added
Aminopyridine reacts 2~5h, 20~30 DEG C of vacuum rotary steams, ethyl alcohol and water volume ratio 1 at 40~70 DEG C:It is recrystallized under the conditions of 1
Obtain white solid；
Step 2, by product and the chitosan oligosaccharide obtained by step 1 in molar ratio 1:1 mixing, the amount of the chitosan oligosaccharide press comprising amino
The mole of glucose calculates；It is then dissolved in n,N-Dimethylformamide, catalyst n-HOSu NHS, carbonization is added
Diimine is stirred overnight at 0~30 DEG C；Bag filter dialysis 1-5d obtains dialyzate, is lyophilized, the chitosan oligosaccharide-fluorobenzene Buddhist nun is made
Examine conjugate.
5. a kind of preparation method of chitosan oligosaccharide-Florfenicol conjugate as claimed in claim 4, it is characterised in that：The step
Organic solvent is acetone in rapid 1, and the reaction time is 2~3h, and reaction temperature is 50~60 DEG C；The catalyst 4- diformazan ammonia
Yl pyridines dosage is the 112mol% of Florfenicol content.
6. a kind of preparation method of chitosan oligosaccharide-Florfenicol conjugate as claimed in claim 4, it is characterised in that：The step
Catalyst n-HOSu NHS in rapid 2, carbodiimides dosage be respectively the 112mol% of step 1 product amount.
7. chitosan oligosaccharide-antibiotic conjugate described in claim 1-3 is inhibiting microbial activity or is inhibiting biofilm depths
Application in microbial activity.
8. purposes of the chitosan oligosaccharide as claimed in claim 7-antibiotic conjugate in inhibiting microbial activity, feature exist
In：The microorganism is gram-positive bacteria, preferably Streptococcus suis.
9. application of the chitosan oligosaccharide-antibiotic conjugate in preparing fungicide or preparing germ killing drugs described in claim 1-3.
10. a kind of pharmaceutical composition, it is characterised in that：The pharmaceutical composition includes chitosan oligosaccharide-Florfenicol conjugate conduct
Active component.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810208430.0A CN108484693B (en) | 2018-03-14 | 2018-03-14 | Chitosan oligosaccharide-antibiotic conjugate and preparation method and application thereof |
Applications Claiming Priority (1)
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