CN108484693B - Chitosan oligosaccharide-antibiotic conjugate and preparation method and application thereof - Google Patents
Chitosan oligosaccharide-antibiotic conjugate and preparation method and application thereof Download PDFInfo
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- 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 chitosan oligosaccharide-antibiotic conjugate and a preparation method and application thereof. The chitosan oligosaccharide-antibiotic conjugate is a covalent complex of chitosan oligosaccharide and antibiotic, and the antibiotic is a chloramphenicol antibiotic. The chloramphenicol antibiotic is florfenicol, and the conjugate is a chitosan oligosaccharide-florfenicol conjugate. The chitosan oligosaccharide-florfenicol conjugate has a good inhibition effect on pathogenic bacteria, particularly has a strong destructive effect on bacteria in a deep part of a formed biofilm, is wide in sterilization range, and has a good inhibition effect on the formation of the biofilm. Compared with florfenicol, the chitosan oligosaccharide-florfenicol conjugate has obviously improved antibacterial activity.
Description
Technical Field
The invention belongs to the technical field of antibacterial agents, and particularly relates to a chitosan oligosaccharide-antibiotic conjugate as well as a preparation method and application thereof.
Background
The increasing use of antibiotics by humans has led to the development of increasingly severe bacterial resistance to existing antibiotics. In addition, the causes of persistent clinical infections and acquired nosocomial infections are often associated with the formation of biofilms (Biofilm) by pathogenic bacteria on the surfaces of host or medical materials. Biofilms are bacterial communities with special microenvironments and their extracellular matrix complexes. Bacterial or fungal communities attach to the surface of an object or air-liquid boundaries, secrete a large amount of extracellular polysaccharide, protein and DNA, and thus form biofilm colonies. The tolerance of biofilm pathogenic bacteria to antibacterial agents and the like is often increased by hundreds to thousands of times as compared with floating bacteria. The traditional medicine has poor effect on treating the infection related to the biofilm, so that the clinical treatment on the pathogenic bacteria needs to develop medicines capable of effectively aiming at the pathogenic bacteria in the biofilm state on the basis of inhibiting the pathogenic bacteria so as to better eliminate the pathogenic bacteria.
Florfenicol is an animal-specific antibacterial drug widely used in veterinary clinic, and with the large-scale use of florfenicol in veterinary clinic, the drug resistance problem of pathogenic bacteria is more and more serious, so that the drug resistance problem of the drug needs to be effectively solved in clinic urgently.
Chitosan (chitin) is obtained by deacetylation of chitin (chitin) widely existing in nature, has the advantages of good biocompatibility, biodegradability, broad-spectrum antibacterial property and the like, and is widely applied to the industries of medicine and food. Chitosan oligosaccharide is an oligosaccharide product with the polymerization degree of 2-20, which is obtained by degrading chitosan through a special biological enzyme technology (reports of using chemical degradation and microwave degradation technologies), has high solubility which chitosan does not have, is completely dissolved in water, is easily absorbed and utilized by organisms, and has a plurality of unique functions.
Disclosure of Invention
The invention aims to provide a chitosan oligosaccharide-antibiotic conjugate and a preparation method and application thereof. Mainly solves the problem that the antibiotic drug resistance in the prior art is more and more serious.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a chitosan oligosaccharide-antibiotic conjugate is a covalent complex of chitosan oligosaccharide and an antibiotic, wherein the antibiotic is a chloramphenicol antibiotic.
Preferably, the chloramphenicol antibiotic is florfenicol, and the conjugate is a chitosan oligosaccharide-florfenicol conjugate.
Preferably, the molar ratio of chitosan oligosaccharide (wherein the molar amount of glucosamine) to florfenicol in the chitosan oligosaccharide-florfenicol conjugate is 1:1.
preferably, the chitosan oligosaccharide is a mixture of oligoglucosamine having a molecular weight below 3000kDa and a degree of deacetylation of 60% or more.
The invention also provides a preparation method of the chitosan oligosaccharide-florfenicol conjugate, which comprises the following steps:
step 1, dissolving florfenicol and succinic anhydride in an organic solvent according to a molar ratio of 1: 1-1: 1.5, adding a catalyst 4-dimethylaminopyridine, reacting at 40-70 ℃ for 2-5 h, carrying out reduced pressure rotary evaporation at 20-30 ℃, and recrystallizing ethanol and water according to a volume ratio of 1:1 to obtain a white solid;
step 2, mixing the product obtained in the step 1 with chitosan oligosaccharide in a molar ratio of 1:1, wherein the amount of the chitosan oligosaccharide is calculated according to the molar amount of the glucosamine contained in the chitosan oligosaccharide (namely, the molar ratio of the molar amount of the glucosamine in the chitosan oligosaccharide to the product obtained in the step 1 is 1: 1); then dissolving the mixture in N, N-dimethylformamide, adding catalysts N-hydroxysuccinimide and carbodiimide, and stirring overnight at 0-30 ℃; dialyzing for 1-5 days by a dialysis bag to obtain dialysate, and freeze-drying to obtain the chitosan oligosaccharide-florfenicol conjugate.
Preferably, the organic solvent in the step 1 is acetone, the reaction time is 2-3 h, and the reaction temperature is 50-60 ℃.
Preferably, the catalyst 4-dimethylaminopyridine in the step 1 is used in an amount of 112 mol% of the florfenicol content.
Preferably, the N-hydroxysuccinimide and the carbodiimide used as catalysts in the step 2 are respectively 112 mol% of the product in the step 1.
Preferably, the reaction temperature of the step 2 is 20-30 ℃, and the dialysis time is 2-3 d.
The invention also provides the application of the chitosan oligosaccharide-antibiotic conjugate in inhibiting the activity of microorganisms; especially inhibiting the activity of microorganisms deep in the biofilm. Preferably, the microorganism is a gram-positive bacterium, preferably streptococcus suis.
The invention also provides application of the chitosan oligosaccharide-antibiotic conjugate in preparation of bactericides or bactericidal drugs.
The invention also provides a pharmaceutical composition which comprises the chitosan oligosaccharide-florfenicol conjugate as an active component.
As a better choice of the pharmaceutical composition, the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials. Wherein the pharmaceutically acceptable excipients are excipients conventionally used in the art. The pharmaceutical adjuvant refers to excipient and additive used in medicine production and prescription preparation. In particular embodiments, the present invention may be practiced using a variety of methods known in the art. The excipient and the additive of the present invention are not particularly limited, and those commonly used in the art may be used for both of them. For example, the excipient may be syrup, sodium alginate, lactose, etc., and the additive may be polysorbate, polyoxyethylene fatty acid ester, chitin, etc.
The invention also provides application of any one of the compositions in preparation of an antibacterial agent. Wherein the antimicrobial agent is capable of inhibiting bacteria in a free state and bacteria in a biofilm state. The bacteria are conventional pathogens in the art. Preferably streptococcus suis.
The conjugate according to the present invention, wherein the composition further comprises a solvent, preferably the solvent is N, N-dimethylformamide. The solvent can be ordinary N, N-dimethylformamide or anhydrous N, N-dimethylformamide and can be selected according to actual conditions.
Compared with the prior art, the invention has the following beneficial effects:
the chitosan oligosaccharide-florfenicol conjugate has a good inhibition effect on pathogenic bacteria, particularly has a strong destructive effect on bacteria in a deep part of a formed biofilm, is wide in sterilization range, and has a good inhibition effect on the formation of the biofilm. Compared with florfenicol, the chitosan oligosaccharide-florfenicol conjugate has obviously improved antibacterial activity.
Drawings
FIG. 1 is a mass spectrum of a chitosan oligosaccharide-florfenicol conjugate prepared in example 1 of the present invention.
FIG. 2 is a bar graph of the inhibitory effect of Flo, COS, F-COS, and Flo + COS on Streptococcus suis in example 2 of the present invention.
FIG. 3 is a bar graph of the effect of Flo, COS, F-COS, and Flo + COS on the biofilm disruption of Streptococcus suis in example 3 of the present invention.
FIG. 4 is a bar graph of data for the biofilm disruption effect of Streptococcus suis at various concentrations of F-COS and Flo in example 4 of the present invention.
FIG. 5 is a bar graph of the inhibitory effect of Flo, COS, F-COS, and Flo + COS on Streptococcus suis biofilm in example 5 of the present invention.
FIG. 6 is a bar graph of data for the inhibitory effect of F-COS and Flo at different concentrations on Streptococcus suis biofilm in example 6 of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail with reference to examples. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The invention adopts the following method to prepare the chitosan oligosaccharide-florfenicol chemical conjugate:
(1) florfenicol and succinic anhydride are mixed in a molar ratio of 1: 1-1: 1.5 dissolving in organic solvents such as acetone and the like, adding 4-dimethylaminopyridine as a catalyst, reacting for 2-5 h at 40-70 ℃, carrying out reduced pressure rotary evaporation at 20-30 ℃, and recrystallizing ethanol and water at a volume ratio of 1:1 to obtain a white solid.
(2) Mixing the product obtained in the step (1) and chitosan oligosaccharide (calculated according to the molar weight of glucosamine monosaccharide contained) in a molar ratio of 1:1, dissolving the mixture in an organic solvent such as N, N-dimethylformamide and the like, adding catalysts N-hydroxysuccinimide and carbodiimide, and stirring the mixture at 0-30 ℃ for overnight; dialyzing for 1-5 days by a dialysis bag to obtain dialysate, and freeze-drying to obtain the chitosan oligosaccharide-florfenicol conjugate.
The catalyst in the step (1) is 4-dimethylaminopyridine, the dosage of the catalyst is 112 mol% of the content of florfenicol, the solvent is an organic solvent such as acetone, the reaction time is 2-3 h, and the reaction temperature is 50-60 ℃.
The molar ratio of the product obtained in the step (2) to the chitosan oligosaccharide (calculated according to the glucosamine monosaccharide content) is 1: 1; the addition amount of the catalysts N-hydroxysuccinimide and carbodiimide is 112 mol% of the product content in the step (1), the reaction temperature is 20-30 ℃, and the dialysis time is 2-3 d.
Wherein the dialysis in step (2) is a conventional dialysis method in the art. Dialysis is a selective diffusion process across a membrane that separates solutes of different molecular weights, species below the cut-off threshold molecular weight of the membrane can diffuse across the membrane, and species above the cut-off threshold molecular weight of the membrane are retained on the other side of a semi-permeable membrane. Wherein the dialysis bag is a dialysis bag conventional in the art. The cut-off molecular weight of the dialysis bag is 500-1000 Da, and unreacted florfenicol and small molecular salt are removed through dialysis. Wherein the dialysis time is preferably 1-5 days, more preferably 2-3 days, and the dialysis time is preferably 3 days.
Wherein the drying in the step (2) is a conventional drying mode in the field. The drying is preferably vacuum freeze drying, vacuum drying, spray drying, oven drying or infrared drying, most preferably vacuum freeze drying. The parameters of the vacuum freeze drying are as follows: the temperature is minus 50 ℃ to minus 80 ℃, the vacuum degree is 20 Pa to 30Pa, and the time is 48 hours to 72 hours.
Example 1 preparation of Chitosan oligosaccharide-florfenicol chemical conjugate
The reaction equation for chemical coupling of chitosan oligosaccharide and florfenicol is shown in Scheme 1. The compound 1 is florfenicol, and reacts with succinic anhydride to obtain a compound 2, and carboxyl on the compound 2 reacts with amino of a compound 3 (chitosan oligosaccharide) to obtain a compound 4, namely the chitosan oligosaccharide-florfenicol conjugate.
The specific implementation steps of the chemical coupling reaction are as follows:
(1) the florfenicol and succinic anhydride are dissolved in an organic solvent such as acetone according to a molar ratio of 1: 1-1: 1.5, a catalyst 4-dimethylaminopyridine is added to react for 2-5 hours at the temperature of 40-70 ℃, reduced pressure rotary evaporation is carried out at the temperature of 20-30 ℃, and recrystallization is carried out on ethanol and water under the condition that the volume is 1:1 to obtain a white solid.
(2) Mixing the product obtained in the step (1) and chitosan oligosaccharide according to a molar ratio of 1:1, dissolving the mixture in an organic solvent such as N, N-dimethylformamide and the like, adding catalysts N-hydroxysuccinimide and carbodiimide, and stirring the mixture overnight at 0-30 ℃; dialyzing for 1-5 days by a dialysis bag to obtain dialysate, and freeze-drying to obtain the chitosan oligosaccharide-florfenicol conjugate.
The prepared chitosan oligosaccharide-florfenicol conjugate is analyzed by mass spectrometry, and the spectrogram is shown in figure 1. The mass spectrum results of figure 1 were analyzed as follows:
the structure of the compound corresponding to the 1218.01 spectrum peak in FIG. 1 is:
the structure of the compound corresponding to the 1379.96 spectrum peak in FIG. 1 is:
the structure of the compound corresponding to the 1541.22 spectrum peak in FIG. 1 is:
the structure of the compound corresponding to the 1701.59 spectrum peak in FIG. 1 is:
the structure of the compound corresponding to the 1862.01 spectrum peak in FIG. 1 is:
the structure of the compound corresponding to the 2184.57 spectrum peak in FIG. 1 is:
example 2 Chitosan oligosaccharide-florfenicol conjugate inhibits bacterial growth
In bookIn the examples, the effect of the prepared chitosan oligosaccharide-florfenicol conjugate on the inhibition of growth of Streptococcus suis was studied, the Streptococcus suis used was from China general microbiological culture Collection center, and after culturing the Streptococcus suis overnight at 37 ℃ in MRS liquid medium with shaking, it was diluted to 4mL of about 1 × 105And respectively adding florfenicol (Flo for short), Chitosan Oligosaccharide (COS), a chitosan oligosaccharide-florfenicol conjugate (F-COS) and a mixture of the florfenicol and the chitosan oligosaccharide (Flo + COS for short) (the mixing mass ratio is 1:1) into the CFU bacterial liquid, dynamically culturing at 37 ℃, and counting the bacterial number at different times.
See FIG. 2, bar graphs for the inhibitory effects of Flo, COS, F-COS, and Flo + COS on Streptococcus suis. The experimental results show that: the inhibition effect of the Flo group and the Flo + COS group on the growth of the streptococcus suis is basically consistent, so that the florfenicol and the chitosan oligosaccharide are simply mixed without enhancing the inhibition effect of the Flo. Compared with the Flo group and the Flo + COS group, the F-COS group has remarkably improved inhibition effect (p is less than 0.01) on the growth of the streptococcus suis, so the chitosan oligosaccharide-florfenicol conjugate has obvious bacterial growth inhibition effect.
Example 3 Chitosan oligosaccharide-florfenicol conjugate on disruption of Streptococcus suis biofilm
In this example, the effect of disrupting a biofilm of Streptococcus suis, which is a chemical modification product of COS of Flo, F-COS, was investigated, Streptococcus suis was used from China general microbiological culture Collection center, and after culturing Streptococcus suis overnight at 37 ℃ in MRS liquid medium with shaking 100. mu.L-2 × 107And adding the CFU bacterial liquid into a 96-well plate, and statically culturing at 37 ℃ for 24 hours to form a mature biofilm.
Samples of Flo, COS, F-COS, and a mixture of Flo and COS Flo + COS (mixed mass ratio 1:1) were prepared for this study, and after the biofilm was matured, the supernatant was removed and 100. mu.L of MRS liquid medium containing 1mg/mL of the above samples was added. The untreated group was added 100 μ LMRS liquid medium. After 24 hours at 37 ℃, the effect of biofilm disruption was tested by MTT staining.
See FIG. 3, bar graphs for Flo, COS, F-COS, and Flo + COS on Streptococcus suis biofilm disruption effects. The experimental results show that: the destruction effect of the Flo group and the Flo + COS group on the streptococcus suis biofilm is not obvious, and the F-COS group has a remarkable biofilm removal effect; compared with the Flo group, the F-COS group has obviously improved biofilm clearing effect (p < 0.01).
Example 4 Effect of different concentrations of Chitosan oligosaccharide-florfenicol conjugate on biofilm disruption in Streptococcus suis
In this example, the effect of F-COS at various concentrations on the destruction of Streptococcus suis in biofilm state was studied, specifically, Streptococcus suis was obtained from China general microbiological culture Collection center, and 100. mu.L-2 × 10 of Streptococcus suis was cultured overnight at 37 ℃ in MRS liquid medium under shaking7Adding the CFU bacterial liquid into a 96-well plate, performing static culture at 37 ℃ for 24 hours to form a mature biofilm, removing supernatant liquid after the biofilm is mature, and respectively adding 100 mu L of MRS culture medium containing F-COS with different concentrations (1, 50, 150, 250, 500, 750 and 1000 mu g/mL). The untreated group was added 100 μ LMRS liquid medium with unmodified Flo as control. After 24 hours at 37 ℃, the effect of biofilm disruption was tested by MTT staining.
See fig. 4, bar graph of data for the effect of different concentrations of F-COS and Flo on streptococcus suis biofilm disruption. The experimental results show that: F-COS can obviously destroy the mature biofilm of streptococcus suis at 500 ug/ml; compared with Flo, the F-COS has the advantages that the destroy effect on the streptococcus suis biofilm is remarkably enhanced, and the use concentration of the antibiotic can be obviously reduced by coupling the chitosan oligosaccharide and the florfenicol.
Example 5 Chitosan oligosaccharide-florfenicol conjugate against Streptococcus suis biofilm inhibition Effect
In this example, the effect of F-COS, a chemically modified product of COS of Flo, in inhibiting biofilm formation by Streptococcus suis was investigated. The specific implementation is as follows: the streptococcus suis used is from China general microbiological culture collection center. After Streptococcus suis was cultured overnight at 37 ℃ in MRS liquid medium with shaking, the supernatant was removed, Flo, COS, F-COS containing 1mg/mL, and a mixture of Flo and COS were addedAdding Flo + COS (mixed mass ratio of 1:1) into MRS liquid culture medium until the bacterial liquid concentration is 2 × 107CFU, 100. mu.L of the above-mentioned bacterial liquid was added to a 96-well plate and statically cultured at 37 ℃ for 24 hours to form a biofilm. The untreated group was added with 100. mu.L of MRS liquid medium. The MTT staining method was used to detect biofilm inhibition.
See FIG. 5, bar graphs for Flo, COS, F-COS, and Flo + COS on Streptococcus suis biofilm inhibition. The experimental results show that: compared with Flo, F-COS has good biofilm inhibiting effect, and can significantly inhibit biofilm growth.
Example 6 Effect of different concentrations of Chitosan oligosaccharide-florfenicol conjugate on inhibition of Streptococcus suis biofilm
In this example, the effect of inhibiting the formation of a biofilm of Streptococcus suis by F-COS was studied, and the specific implementation was that Streptococcus suis was collected from China general microbiological culture Collection center, and after culturing Streptococcus suis overnight at 37 ℃ in MRS liquid medium under shaking, 100. mu.L-2 × 10 was collected7CFU bacterial liquid containing F-COS with different concentrations (1, 50, 150, 250, 500, 750, 1000 mu g/mL) is added into a 96-well plate, and 100 mu L-2 × 10 is added into an untreated group7CFU broth, with unmodified Flo as control. After 24 hours at 37 ℃, the effect of biofilm disruption was tested by MTT staining.
See fig. 6, bar graph of data for the effect of different concentrations of F-COS and Flo on inhibition of streptococcus suis biofilm. The experimental results show that: F-COS can obviously inhibit the formation of a mature biofilm of streptococcus suis at 500 ug/ml; compared with Flo (>1000ug/ml), F-COS can obviously reduce the use concentration of antibiotics.
The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.
Claims (7)
1. A chitosan oligosaccharide-antibiotic conjugate, characterized in that: the chitosan oligosaccharide-antibiotic conjugate is a covalent complex of chitosan oligosaccharide and antibiotic, and the antibiotic is a chloramphenicol antibiotic; the chloramphenicol antibiotic is florfenicol; the chitosan oligosaccharide is an oligomeric glucosamine mixture with the molecular weight below 3000kDa and the deacetylation degree of more than or equal to 60 percent.
2. A preparation method of a chitosan oligosaccharide-florfenicol conjugate is characterized by comprising the following steps:
step 1, dissolving florfenicol and succinic anhydride in an organic solvent according to a molar ratio of 1: 1-1: 1.5, adding a catalyst 4-dimethylaminopyridine, reacting at 40-70 ℃ for 2-5 h, carrying out reduced pressure rotary evaporation at 20-30 ℃, and recrystallizing ethanol and water according to a volume ratio of 1:1 to obtain a white solid;
step 2, mixing the product obtained in the step 1 with chitosan oligosaccharide in a molar ratio of 1:1, wherein the amount of the chitosan oligosaccharide is calculated according to the molar amount of the glucosamine contained in the mixture; then dissolving the mixture in N, N-dimethylformamide, adding catalysts N-hydroxysuccinimide and carbodiimide, and stirring the mixture at 0-30 ℃ overnight; dialyzing for 1-5 days by a dialysis bag to obtain dialysate, and freeze-drying to obtain the chitosan oligosaccharide-florfenicol conjugate.
3. The method for preparing a chitosan oligosaccharide-florfenicol conjugate of claim 2, wherein the chitosan oligosaccharide-florfenicol conjugate comprises the following steps: the organic solvent in the step 1 is acetone, the reaction time is 2-3 h, and the reaction temperature is 50-60 ℃; the using amount of the catalyst 4-dimethylaminopyridine is 112mol percent of the content of the florfenicol.
4. The method for preparing a chitosan oligosaccharide-florfenicol conjugate of claim 2, wherein the chitosan oligosaccharide-florfenicol conjugate comprises the following steps: the dosage of the N-hydroxysuccinimide and the carbodiimide used as the catalysts in the step 2 is respectively 112 mol% of the product in the step 1.
5. Use of the chitosan oligosaccharide-antibiotic conjugate of claim 1 for non-therapeutic purposes in inhibiting microbial activity or inhibiting microbial activity deep in a biofilm; the microorganism is streptococcus suis.
6. Use of the chitosan oligosaccharide-antibiotic conjugate of claim 1 in the preparation of a bactericide or in the preparation of a bactericidal medicament.
7. A pharmaceutical composition characterized by: the pharmaceutical composition comprises a chitosan oligosaccharide-florfenicol conjugate as an active component.
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