CN116640238B - Guanidyl pyridine chitosan onium salt and preparation method and application thereof - Google Patents
Guanidyl pyridine chitosan onium salt and preparation method and application thereof Download PDFInfo
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- CN116640238B CN116640238B CN202310618433.2A CN202310618433A CN116640238B CN 116640238 B CN116640238 B CN 116640238B CN 202310618433 A CN202310618433 A CN 202310618433A CN 116640238 B CN116640238 B CN 116640238B
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 115
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 title claims abstract description 108
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 title claims abstract description 87
- 150000003839 salts Chemical class 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- -1 pyridine compound Chemical class 0.000 claims abstract description 97
- 239000002904 solvent Substances 0.000 claims description 36
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 24
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 19
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 17
- 239000000126 substance Substances 0.000 claims description 16
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 15
- 239000013067 intermediate product Substances 0.000 claims description 15
- 238000007259 addition reaction Methods 0.000 claims description 14
- 238000005956 quaternization reaction Methods 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 9
- 238000007127 saponification reaction Methods 0.000 claims description 7
- 239000003513 alkali Substances 0.000 claims description 6
- DRTAALDHJVVZNT-UHFFFAOYSA-N guanidine;pyridine Chemical compound NC(N)=N.C1=CC=NC=C1 DRTAALDHJVVZNT-UHFFFAOYSA-N 0.000 claims description 6
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 claims description 5
- ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 claims description 4
- CUYKNJBYIJFRCU-UHFFFAOYSA-N 3-aminopyridine Chemical compound NC1=CC=CN=C1 CUYKNJBYIJFRCU-UHFFFAOYSA-N 0.000 claims description 4
- NUKYPUAOHBNCPY-UHFFFAOYSA-N 4-aminopyridine Chemical compound NC1=CC=NC=C1 NUKYPUAOHBNCPY-UHFFFAOYSA-N 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 229960004979 fampridine Drugs 0.000 claims description 4
- ALOCUZOKRULSAA-UHFFFAOYSA-N 1-methylpiperidin-4-amine Chemical compound CN1CCC(N)CC1 ALOCUZOKRULSAA-UHFFFAOYSA-N 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 23
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 12
- 238000004140 cleaning Methods 0.000 abstract description 6
- NVDXVBPBFJKQJG-UHFFFAOYSA-N 2-pyridin-2-ylguanidine Chemical compound NC(N)=NC1=CC=CC=N1 NVDXVBPBFJKQJG-UHFFFAOYSA-N 0.000 abstract description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 239000003242 anti bacterial agent Substances 0.000 abstract description 3
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 46
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 39
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 30
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 21
- 230000000052 comparative effect Effects 0.000 description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 18
- 238000001035 drying Methods 0.000 description 13
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 9
- 238000000108 ultra-filtration Methods 0.000 description 9
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 241000191967 Staphylococcus aureus Species 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 5
- 230000032770 biofilm formation Effects 0.000 description 5
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 5
- 238000010992 reflux Methods 0.000 description 5
- FKPUYTAEIPNGRM-UHFFFAOYSA-N 1-(diaminomethylidene)guanidine;hydron;chloride Chemical compound [Cl-].N\C([NH3+])=N/C(N)=N FKPUYTAEIPNGRM-UHFFFAOYSA-N 0.000 description 4
- 239000005711 Benzoic acid Substances 0.000 description 4
- 229940123208 Biguanide Drugs 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 235000010233 benzoic acid Nutrition 0.000 description 4
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- SATDLKYRVXFXRE-UHFFFAOYSA-N methyl 4-(chloromethyl)benzoate Chemical compound COC(=O)C1=CC=C(CCl)C=C1 SATDLKYRVXFXRE-UHFFFAOYSA-N 0.000 description 4
- 230000001376 precipitating effect Effects 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000012266 salt solution Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012065 filter cake Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 150000002357 guanidines Chemical class 0.000 description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 2
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- OITNBJHJJGMFBN-UHFFFAOYSA-N 4-(chloromethyl)benzoic acid Chemical compound OC(=O)C1=CC=C(CCl)C=C1 OITNBJHJJGMFBN-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 230000003214 anti-biofilm Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- JXHYCCGOZUGBFD-UHFFFAOYSA-N benzoic acid;hydrochloride Chemical compound Cl.OC(=O)C1=CC=CC=C1 JXHYCCGOZUGBFD-UHFFFAOYSA-N 0.000 description 1
- JGFASLCNXQGHPQ-UHFFFAOYSA-N butyl 4-(chloromethyl)benzoate Chemical compound CCCCOC(=O)C1=CC=C(CCl)C=C1 JGFASLCNXQGHPQ-UHFFFAOYSA-N 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- JTTXRFNOFFGPFI-UHFFFAOYSA-N ethyl 4-(chloromethyl)benzoate Chemical compound CCOC(=O)C1=CC=C(CCl)C=C1 JTTXRFNOFFGPFI-UHFFFAOYSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 231100000344 non-irritating Toxicity 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- UDAULNDFFKITRZ-UHFFFAOYSA-N tert-butyl 4-(chloromethyl)benzoate Chemical compound CC(C)(C)OC(=O)C1=CC=C(CCl)C=C1 UDAULNDFFKITRZ-UHFFFAOYSA-N 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Pest Control & Pesticides (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Polymers & Plastics (AREA)
- Agronomy & Crop Science (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Dentistry (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention provides a guanidino pyridine chitosan onium salt, a preparation method and application thereof, and belongs to the technical field of antibacterial agents. The invention firstly reacts the p-chloromethyl benzoate compound with the pyridine compound, then reacts the p-chloromethyl benzoate compound with the cyanamide compound to obtain the guanidyl pyridine quaternary ammonium salt, and then grafts the guanidyl pyridine quaternary ammonium salt onto a chitosan molecular chain, so that the obtained guanidyl pyridine chitosan onium salt has excellent water solubility, higher antibacterial and bacteriostatic activities, can inhibit the formation of a biological film, and has better cleaning effect on a mature bacterial biological film.
Description
Technical Field
The invention relates to the technical field of antibacterial agents, in particular to a guanidino pyridine chitosan onium salt, a preparation method and application thereof.
Background
Chitosan is a polysaccharide natural polymer obtained by deacetylation of chitin, has good biocompatibility and biodegradability, is nontoxic and nonirritating, and is widely applied to the fields of medical use, food, agriculture and the like. Chitosan has broad-spectrum antibacterial properties, but pure chitosan has insignificant antibacterial properties and limited solubility. Guanidine compounds refer to compounds and derivatives thereof containing-NH 2C(＝NH)NH2 groups in the structure. Guanidine is the organic base with the strongest electropositive bioactivity found in nature to date, and arginine is one of the guanidine salts most commonly found. Guanidine antibacterial agents are attracting attention because they are not likely to develop drug resistance due to their positive charges acting on bacterial cell membranes. The guanidine group grafted into the molecular structure of chitosan can not only promote the water solubility of chitosan, but also has positive effect on promoting the antibacterial activity of chitosan.
The most studied guanidine chitosan is mainly chitosan monoguanidine hydrochloride and chitosan biguanide hydrochloride. But the antibacterial performance is still more general and the cleaning effect on mature bacterial biofilms is poor. Therefore, how to further improve the antibacterial performance of the guanidine chitosan and the cleaning effect of the guanidine chitosan on mature bacterial biofilms becomes a difficult problem in the prior art.
Disclosure of Invention
The invention aims to provide a guanidino pyridine chitosan onium salt, a preparation method and application thereof. The guanidinopyridine chitosan onium salt prepared by the invention has higher antibacterial property and excellent cleaning effect on mature bacterial biofilm.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a preparation method of guanidinium pyridine chitosan onium salt, which comprises the following steps:
(1) Mixing a p-chloromethyl benzoate compound, a solvent and a pyridine compound, and then carrying out quaternization reaction to obtain pyridine quaternary ammonium salt;
(2) Mixing the pyridine quaternary ammonium salt obtained in the step (1) with a solvent, hydrochloric acid and a cyanamide compound, and then carrying out an addition reaction to obtain an intermediate product;
(3) Mixing the intermediate product obtained in the step (2) with a solvent and alkali for saponification, and adding acid to obtain guanidyl pyridine quaternary ammonium salt;
(4) Mixing the guanidyl pyridine quaternary ammonium salt obtained in the step (3) with a solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and chitosan solution, and performing grafting reaction to obtain the guanidyl pyridine chitosan onium salt.
Preferably, the ratio of the amounts of the substances of the p-chloromethylbenzoate compound and the pyridine compound in the step (1) is 1: (1-2).
Preferably, the temperature of the quaternization reaction in the step (1) is 50-140 ℃, and the quaternization reaction time is 4-48 h.
Preferably, the ratio of the amount of the pyridine quaternary ammonium salt to the amount of the cyanamide compound in the step (2) is 1: (1-3).
Preferably, the temperature of the addition reaction in the step (2) is 40-100 ℃, and the time of the addition reaction is 4-48 hours.
Preferably, the ratio of the guanidyl pyridine quaternary ammonium salt in the step (4) to the chitosan substance in the chitosan solution is (1-3): 1.
Preferably, the ratio of the amount of guanidyl pyridine quaternary ammonium salt to the amount of the substance of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in the step (4) is 1: (1-3).
Preferably, the grafting reaction temperature in the step (4) is 20-50 ℃, and the grafting reaction time is 12-48 h.
The invention provides the guanidyl pyridine chitosan onium salt prepared by the preparation method.
The invention also provides application of the guanidyl pyridine chitosan onium salt in bacteriostasis and inhibition of bacterial biofilm.
The invention provides a preparation method of guanidinium pyridine chitosan onium salt, which comprises the following steps: (1) Mixing a p-chloromethyl benzoate compound, a solvent and a pyridine compound, and then carrying out quaternization reaction to obtain pyridine quaternary ammonium salt; (2) Mixing the pyridine quaternary ammonium salt obtained in the step (1) with a solvent, hydrochloric acid and a cyanamide compound, and then carrying out an addition reaction to obtain an intermediate product; (3) Mixing the intermediate product obtained in the step (2) with a solvent and alkali for saponification, and adding acid to obtain guanidyl pyridine quaternary ammonium salt; (4) Mixing the guanidyl pyridine quaternary ammonium salt obtained in the step (3) with a solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and chitosan solution, and performing grafting reaction to obtain the guanidyl pyridine chitosan onium salt. According to the invention, the p-chloromethyl benzoate compound and the pyridine compound are reacted, then the p-chloromethyl benzoate compound and the pyridine compound are reacted to obtain the guanidyl pyridine quaternary ammonium salt, and then the guanidyl pyridine quaternary ammonium salt is grafted on a chitosan molecular chain to obtain the guanidyl pyridine chitosan onium salt, wherein the guanidyl and the pyridinium salt have good water solubility, and the guanidyl and the pyridinium salt have positive charges, and the charges of the guanidyl and the pyridinium salt are superposed, so that the antibacterial performance is improved, the antibacterial activity is higher, the formation of a biological film can be inhibited, and meanwhile, the mature bacterial biological film is better removed.
Drawings
FIG. 1 is a 1 H NMR chart of guanidinium chitosan salt prepared in example 1 of the present invention;
FIG. 2 is a 1 H NMR chart of the chitosan quaternary ammonium salt prepared in comparative example 1 of the present invention;
FIG. 3 is a 1 H NMR chart of chitosan biguanide hydrochloride prepared in comparative example 2 of the present invention.
Detailed Description
The invention provides a preparation method of guanidinium pyridine chitosan onium salt, which comprises the following steps:
(1) Mixing a p-chloromethyl benzoate compound, a solvent and a pyridine compound, and then carrying out quaternization reaction to obtain pyridine quaternary ammonium salt;
(2) Mixing the pyridine quaternary ammonium salt obtained in the step (1) with a solvent, hydrochloric acid and a cyanamide compound, and then carrying out an addition reaction to obtain an intermediate product;
(3) Mixing the intermediate product obtained in the step (2) with a solvent and alkali for saponification, and adding acid to obtain guanidyl pyridine quaternary ammonium salt;
(4) Mixing the guanidyl pyridine quaternary ammonium salt obtained in the step (3) with a solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and chitosan solution, and performing grafting reaction to obtain the guanidyl pyridine chitosan onium salt.
The source of each raw material is not particularly limited unless specifically stated, and commercially available products known to those skilled in the art may be used.
The invention mixes the p-chloromethyl benzoate compound with the solvent and the pyridine compound to carry out quaternization reaction, thus obtaining the pyridine quaternary ammonium salt.
In the present invention, the p-chloromethylbenzoate compound preferably includes methyl p-chloromethylbenzoate, ethyl p-chloromethylbenzoate, butyl p-chloromethylbenzoate or tert-butyl p-chloromethylbenzoate.
In the present invention, the solvent preferably includes one or more of ethyl acetate, tetrahydrofuran, acetone, petroleum ether, acetonitrile and toluene.
In the present invention, the mass ratio of the p-chloromethylbenzoate compound to the solvent is preferably (1-2): 20, more preferably (1.5 to 2): 20. the invention limits the mass ratio of the p-chloromethyl benzoate compound and the solvent in the above range, so that the raw materials can be dissolved more fully.
In the present invention, the pyridine compound preferably includes 2-aminopyridine, 3-aminopyridine, 4-aminopyridine or 4-amino-N-methylpiperidine.
In the present invention, the ratio of the amounts of the substances of the p-chloromethylbenzoate compound and the pyridine compound is preferably 1: (1-2), more preferably 1: (1.2-1.8). The present invention is capable of sufficiently reacting the p-chloromethylbenzoate compound and the pyridine compound by limiting the ratio of the amounts of the substances within the above-mentioned range.
In the present invention, the temperature of the quaternization reaction is preferably 50 to 140 ℃, more preferably 80 to 100 ℃; the quaternization reaction time is preferably 4 to 48 hours, more preferably 10 to 30 hours. The invention limits the temperature and time of the quaternization reaction within the above range, and can lead the p-chloromethyl benzoate compound and the pyridine compound to fully react.
After the quaternization reaction is finished, the product of the quaternization reaction is preferably filtered, washed and dried in sequence to obtain the pyridine quaternary ammonium salt.
The operation of the filtration, washing and drying is not particularly limited in the present invention, and the filtration, washing and drying techniques well known to those skilled in the art may be adopted.
After the pyridine quaternary ammonium salt is obtained, the pyridine quaternary ammonium salt is mixed with a solvent, hydrochloric acid and a cyanamide compound to carry out an addition reaction, so that an intermediate product is obtained.
In the present invention, the solvent preferably includes ethanol.
In the invention, the mass ratio of the pyridine quaternary ammonium salt to the solvent is preferably (2-3): 5, more preferably (2.5 to 3): 5. the invention limits the mass ratio of the pyridine quaternary ammonium salt to the solvent in the above range, so that the raw materials can be fully dissolved.
In the present invention, the concentration of the hydrochloric acid is preferably 10 to 15mol/L, more preferably 12mol/L; the ratio of the amounts of the substances of pyridine quaternary ammonium salt and hydrogen chloride in hydrochloric acid is preferably (0.5 to 1.5): 1, more preferably 1:1. In the present invention, the hydrochloric acid is used to stabilize the guanidino group formed.
In the present invention, the cyanamide compound preferably includes mono-cyanamide or dicyandiamide.
In the present invention, the ratio of the amounts of the substances of the pyridine quaternary ammonium salt and the cyanamide compound is preferably 1: (1 to 3), more preferably 1: (1.5-2.5). The present invention can sufficiently react the pyridine quaternary ammonium salt and the cyanamide compound by limiting the amount ratio of the substances to the above range.
In the present invention, the temperature of the addition reaction is preferably 40 to 100 ℃, more preferably 60 to 80 ℃; the time of the addition reaction is preferably 4 to 48 hours, more preferably 6 to 35 hours. The invention limits the temperature and time of the addition reaction within the above range, and can lead the pyridine quaternary ammonium salt and the cyanamide compound to fully react.
After the addition reaction is completed, the invention preferably sequentially concentrates and precipitates the products after the addition reaction to obtain intermediate products.
The concentration operation is not particularly limited in the present invention, and the concentration method known to those skilled in the art may be adopted.
In the present invention, the precipitation is preferably performed by adding acetone. The method is not particularly limited to the amount of the added acetone, and the product precipitation is ensured to be complete.
After the intermediate product is obtained, the intermediate product is mixed with a solvent and alkali for saponification reaction, and then acid is added to obtain the guanidyl pyridine quaternary ammonium salt.
In the present invention, the solvent preferably includes ethanol.
In the invention, the mass ratio of the pyridine quaternary ammonium salt to the solvent is preferably (2-3): 3, more preferably (2.5 to 3): 3. the invention limits the mass ratio of the pyridine quaternary ammonium salt to the solvent in the above range, and can fully dissolve the intermediate product.
In the present invention, the base preferably includes sodium hydroxide.
In the invention, the mass ratio of the pyridine quaternary ammonium salt to the alkali is preferably (2-3): 0.4, more preferably (2.5 to 3): 0.4. in the present invention, the base is used to saponify with the ester.
In the present invention, the temperature of the saponification reaction is preferably 50 to 70 ℃, more preferably 60 ℃; the saponification reaction time is preferably 8 to 15 hours, more preferably 10 to 12 hours.
In the present invention, the acid is preferably hydrochloric acid; the concentration of the hydrochloric acid is preferably 10 to 15mol/L, more preferably 12mol/L. In the present invention, the amount of the acid added is preferably such that the pH of the system is reduced to below 4. In the invention, the acid is added and then preferably reacted for 20-40 min, and then the filtration, concentration and precipitation are sequentially carried out. In the present invention, the acid is used to form carboxyl groups.
After the guanidyl pyridine quaternary ammonium salt is obtained, the guanidyl pyridine quaternary ammonium salt is mixed with a solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and chitosan solution and then subjected to grafting reaction, so that the guanidyl pyridine chitosan onium salt is obtained.
In the present invention, the solvent preferably includes a mixed solution of ethanol and water or ethanol. In the present invention, when the solvent is a mixed solution of ethanol and water, the volume content of ethanol is preferably 40 to 60%, more preferably 50%.
In the invention, the mass ratio of the guanidyl pyridine quaternary ammonium salt to the solvent is preferably (4-5): 50, more preferably (4.2 to 4.8): 50. the invention limits the mass ratio of the guanidyl pyridine quaternary ammonium salt to the solvent in the above range, so that the guanidyl pyridine quaternary ammonium salt can be fully dissolved.
In the present invention, the ratio of the amount of the guanidyl pyridine quaternary ammonium salt to the substance of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is preferably 1: (1 to 3), more preferably 1: (1.5-2.5).
In the present invention, the ratio of the amounts of the substances of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is preferably 1: (0.5 to 1.5), more preferably 1:1.
In the present invention, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are used to activate carboxyl groups to facilitate grafting into chitosan.
In the present invention, the solvent in the chitosan solution is preferably an aqueous acetic acid solution; the volume concentration of the aqueous acetic acid solution is preferably 1.5 to 2.5%, more preferably 2%.
In the present invention, the mass concentration of chitosan in the chitosan solution is preferably 0.1 to 5%, more preferably 1 to 4%, and most preferably 1 to 3%. The present invention limits the concentration of chitosan within the above range, and can sufficiently dissolve chitosan.
In the present invention, the ratio of the guanidyl pyridine quaternary ammonium salt to the amount of chitosan in the chitosan solution is preferably (1 to 3): 1, more preferably (1.5 to 2.5): 1. the invention limits the ratio of the guanidyl pyridine quaternary ammonium salt to the chitosan substance in the chitosan solution in the above range, can adjust the content of the guanidyl pyridine quaternary ammonium salt in the product, and further improves the antibacterial performance of the product.
In the present invention, the mixing of the guanidyl pyridine quaternary ammonium salt with the solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and chitosan solution is preferably: firstly, dissolving guanidyl pyridine quaternary ammonium salt in a solvent, then adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and finally adding chitosan solution.
In the present invention, the temperature of the grafting reaction is preferably 20 to 50 ℃, more preferably 20 to 30 ℃; the time of the grafting reaction is preferably 12 to 48 hours, more preferably 20 to 35 hours. The invention limits the temperature and time of the grafting reaction in the above range, and can lead the guanidyl pyridine quaternary ammonium salt and chitosan to fully react.
After the grafting reaction is completed, the grafted product is preferably subjected to ultrafiltration, concentration and drying in sequence to obtain the guanidyl pyridine chitosan onium salt.
The operations of ultrafiltration, concentration and drying are not particularly limited in the present invention, and the technical schemes of ultrafiltration, concentration and drying, which are well known to those skilled in the art, may be adopted.
The invention provides the guanidyl pyridine chitosan onium salt prepared by the preparation method.
The guanidino pyridine chitosan onium salt provided by the invention has excellent water solubility, higher antibacterial and bacteriostatic activity, can inhibit the formation of a biological film, and has a better cleaning effect on a mature bacterial biological film.
The invention also provides application of the guanidyl pyridine chitosan onium salt in bacteriostasis and inhibition of bacterial biofilm.
The operation of the application of the guanidyl pyridine chitosan onium salt in bacteriostasis and inhibition of bacterial biofilm is not particularly limited, and the application of the guanidyl pyridine chitosan onium salt in bacteriostasis and inhibition of bacterial biofilm is realized by adopting a technical scheme which is well known to a person skilled in the art.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) 1.85Kg of methyl p-chloromethylbenzoate is weighed and dissolved in 20kg of ethyl acetate (the mass ratio of the methyl p-chloromethylbenzoate to the ethyl acetate is 1.85:20), and is put into a reaction kettle, 1.13kg of 4-aminopyridine (the mass ratio of the methyl p-chloromethylbenzoate to the 4-aminopyridine is 1:1.2) is added under stirring, the temperature is increased to 85 ℃, the mixture is stirred under reflux for 10 hours, a large amount of white solid is separated out during the reaction, suction filtration is carried out after the reaction is finished, a filter cake is washed for a plurality of times by ethyl acetate, and 4-amino-1- (4-methoxycarbonyl) benzyl pyridine chloride is obtained after drying;
(2) Weighing 2.78kg of 4-amino-1- (4-methoxycarbonyl) benzyl pyridine chloride, dissolving in 5kg of ethanol (the mass ratio of the 4-amino-1- (4-methoxycarbonyl) benzyl pyridine chloride to the ethanol is 2.78:5), adding hydrochloric acid solution (the concentration is 12mol/L, the ratio of the 4-amino-1- (4-methoxycarbonyl) benzyl pyridine chloride to the hydrogen chloride in hydrochloric acid is 1:1), adding 0.84kg of dicyandiamide (the ratio of the 4-amino-1- (4-methoxycarbonyl) benzyl pyridine chloride to the dicyandiamide is 1:1 after complete dissolution, reflux stirring at 60 ℃ for 6 hours, concentrating, precipitating with acetone to obtain an intermediate product, dissolving the intermediate product in 3kg of ethanol (the mass ratio of 4-amino-1- (4-methoxycarbonyl) benzyl pyridine chloride to ethanol is 2.78:3), adding 0.4kg of sodium hydroxide (the mass ratio of 4-amino-1- (4-methoxycarbonyl) benzyl pyridine chloride to sodium hydroxide is 2.78:0.4), refluxing at 60 ℃ overnight, cooling, regulating the pH value of the system to be below 4 by using 12mol/L hydrochloric acid, stirring for 30 minutes, filtering, concentrating the filtrate, and precipitating with acetone to obtain the biguanidino-1- (4-carboxybenzyl) pyridine hydrochloride;
(3) 1.61kg of chitosan is weighed and dissolved in 100kg of acetic acid water with the volume fraction of 2% to obtain chitosan acetic acid solution, and the mass percentage concentration of the chitosan is 1.6%;
(4) Weighing 4.21kg of biguanide-1- (4-carboxybenzyl) pyridine hydrochloride, dissolving the biguanide-1- (4-carboxybenzyl) pyridine hydrochloride in 50kg of ethanol water with the volume fraction of 50% (the mass ratio of the biguanide-1- (4-carboxybenzyl) pyridine hydrochloride to the ethanol water is 4.21:50), adding 1.92kg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (the mass ratio of the biguanide-1- (4-carboxybenzyl) pyridine hydrochloride to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1, and 1.15kg of N-hydroxysuccinimide (the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to the N-hydroxysuccinimide) is 1:1), and obtaining a guanylpyridine quaternary ammonium salt solution after complete dissolution, wherein the mass percentage concentration of the biguanide-1- (4-carboxybenzyl) pyridine hydrochloride in the solution is 8.4%;
(5) Slowly dripping the guanidyl pyridine quaternary ammonium salt solution into chitosan acetic acid aqueous solution in a reaction kettle, and reacting for 24 hours at room temperature after the addition is finished, wherein the ratio of the guanidyl-1- (4-carboxybenzyl) pyridine hydrochloride to the chitosan substance in the chitosan acetic acid aqueous solution is 1:1;
(6) After the reaction at room temperature for 24 hours is finished, transferring the product into an ultrafiltration device, adding water for ultrafiltration purification, concentrating and drying to obtain guanidyl pyridine chitosan onium salt;
The reaction procedure of this example is as follows:
comparative example 1
1, Weighing 1.70kg of p-chloromethyl benzoic acid, dissolving in 20kg of ethyl acetate, adding into a reaction kettle, adding 0.95kg of pyridine under stirring, raising the temperature to 85 ℃, refluxing and stirring for 10 hours, precipitating a large amount of white solid in the reaction process, filtering after the reaction is finished, washing a filter cake with ethyl acetate for multiple times, and drying to obtain N-pyridine- (4-carboxybenzyl) onium salt;
Step 2, weighing 1.61kg of chitosan, and dissolving the chitosan in 50kg of acetic acid water with the volume fraction of 2% to obtain chitosan acetic acid solution, wherein the mass percentage concentration of the chitosan is 3.2%;
Step 3, weighing 2.49kg of N-pyridine- (4-carboxybenzyl) onium salt to dissolve in 50kg of water, adding 1.92kg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1.15kg of N-hydroxysuccinimide, and obtaining a small molecule solution after complete dissolution, wherein the mass percentage concentration of the N-pyridine- (4-carboxybenzyl) onium salt in the solution is 3.84%, and the mole percentage of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:1;
Step 4, slowly dropwise adding the pyridine quaternary ammonium salt solution into the chitosan acetic acid aqueous solution in the reaction kettle, and reacting for 24 hours at 30 ℃ after the addition is finished, wherein the molar ratio of the N-pyridine- (4-carboxybenzyl) onium salt to the chitosan added in the step 2 is 1:1;
and 5, after the reaction at room temperature is finished for 24 hours, transferring the product into an ultrafiltration device, adding water for ultrafiltration purification, concentrating and drying to obtain chitosan quaternary ammonium salt.
Comparative example 2
1, Weighing 1.37kg of p-aminobenzoic acid, dissolving in 10kg of ethanol, adding hydrochloric acid solution, adding 0.84kg of dicyandiamide, refluxing and stirring at 60 ℃ for 6 hours, concentrating, precipitating acetone to obtain a crude product, and recrystallizing acetone and water to obtain p-biguanidino benzoic acid hydrochloride;
Step 2, weighing 1.61kg of chitosan acetic acid solution, and dissolving the chitosan acetic acid solution in 100kg of acetic acid water with the volume fraction of 2%, wherein the mass percentage concentration of the chitosan is 1.6%;
step 3, weighing 2.94kg of p-biguanide benzoic acid, dissolving in 50kg of ethanol water with the volume fraction of 50%, adding 1.92kg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1.15kg of N-hydroxysuccinimide, and obtaining a small molecule solution after complete dissolution, wherein the mass percentage concentration of the p-biguanide benzoic acid in the solution is 5.9%, and the mole percentage of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:1;
step 4, slowly dropwise adding the p-biguanide benzoic acid solution into the chitosan acetic acid aqueous solution in the reaction kettle, and reacting for 24 hours at room temperature after the addition is finished, wherein the molar ratio of the p-biguanide benzoic acid to the chitosan added in the step 2 is 1:1 in the embodiment;
And 5, after the reaction at room temperature is finished for 24 hours, transferring the product into an ultrafiltration device, adding water for ultrafiltration purification, concentrating and drying to obtain chitosan biguanide hydrochloride.
The figure shows 1 H NMR of guanidinium chitosan salt prepared in example 1, chitosan quaternary ammonium salt prepared in comparative example 1, and chitosan biguanide hydrochloride prepared in comparative example 2, respectively. The structure of the resulting product is demonstrated from figures 1 to 3.
Antibacterial property test
The antibacterial properties of the chitosan derivatives of example 1 and comparative examples 1 to 2 were tested by the plate count method, and the antibacterial rate was calculated by formula 1, and the results are shown in tables 1 and 2.
TABLE 1 antibacterial effect of chitosan derivatives at different concentrations on Staphylococcus aureus in example 1 and comparative examples 1-2
| Concentration of | 2.5mg/mL | 1.0mg/mL | 0.5mg/mL |
| Example 1 | 100 | 100 | 100 |
| Comparative example 1 | 100 | 100 | 100 |
| Comparative example 2 | 97.92 | 91.90 | 88.16 |
TABLE 2 antibacterial effect of chitosan derivatives at different concentrations on E.coli in example 1 and comparative examples 1-2
| Concentration of | 2.5mg/mL | 1.0mg/mL | 0.5mg/mL |
| Example 1 | 73.22 | 58.48 | 43.25 |
| Comparative example 1 | 68.24 | 50.00 | 34.12 |
| Comparative example 2 | 48.73 | 38.24 | 26.06 |
The Minimum Inhibitory Concentration (MIC) and minimum biofilm inhibition concentration (MBC) of the chitosan derivatives of example 1 and comparative examples 1 to 2 were measured by a nephelometry, and the Minimum Bactericidal Concentration (MBC) and minimum biofilm killing concentration (MBBC) of the chitosan derivatives of example 1 and comparative examples 1 to 2 were measured in combination with a plate count method, and the results are shown in tables 3 and 4, and the specific methods are as follows:
100 mu L of diluted bacterial solution and 100 mu L of chitosan solution after being subjected to sesquidilution are added into a 96-well plate, a control group which is only added with chitosan derivative solution and no bacterial solution and a control group which is only added with bacterial solution and no chitosan derivative solution are respectively prepared, 3 groups of chitosan derivatives with each concentration are parallel, the culture is carried out in a culture box at 37 ℃ for 18 hours, the observation result is carried out, the lowest concentration of the chitosan derivatives corresponding to the clarified holes is taken as MIC, 100 mu L of mixed solution of bacterial solution and sample solution in the clarified holes is taken for plating, and the lowest concentration corresponding to the colony number of each dish is less than 5, and the MBC is marked.
Adding diluted bacterial liquid into a 96-well plate, placing the bacterial liquid into a 37 ℃ incubator for culturing for 36-48 hours, enabling the bacterial liquid to form a biological film on the well plate, then discarding planktonic bacteria, rinsing with sterile physiological saline or PBS, drying and fixing, adding 100 mu L of diluted chitosan derivative sample solution and 100 mu L of culture medium into the 96-well plate, taking sterile water as a control group, placing the culture medium into the 37 ℃ incubator for culturing for 18 hours, observing results, taking the lowest concentration of the chitosan derivative corresponding to the clarified holes as MBIC, taking 100 mu L of mixed liquid of the bacterial liquid and the sample solution in the clarified holes for plating, and recording the concentration of the chitosan derivative corresponding to the lowest concentration of less than 5 bacterial colonies per dish as MBBC.
TABLE 3 variation of resistance before and after the formation of Staphylococcus aureus BF
TABLE 4 variation of drug resistance before and after the formation of E.coli BF
The crystal violet staining method can be used for rapidly and simply measuring the intervention effect of the medicine on the biological envelope. The inhibition strength of chitosan derivatives on biofilm formation can be reflected by the OD values under the action of different drugs, and the specific method is as follows:
100 mu L of diluted bacterial solution and 100 mu L of chitosan derivative solution (4 MIC, 2MIC and MIC) diluted to a certain concentration are added into a 96-well plate, water is used as a control group, and the final concentration of the chitosan derivative is 2MIC, MIC and 1/2MIC. 3 groups of medicines with each concentration are prepared in parallel, cultured for 36-48 hours in a constant temperature box at 37 ℃, then plankton and chitosan derivative solution are removed, the solution is rinsed with sterile physiological saline, dried and fixed, 0.1% crystal violet solution is added for dyeing for 15min, the crystal violet solution is removed, the solution is rinsed for 3 times with sterile physiological saline, the solution is dried by sterile air, 95% ethanol solution is added for 5min, absorbance is measured at 595nm by an enzyme-labeled instrument, and the biofilm formation inhibition rate is calculated by a formula 2, and the results are shown in tables 5 and 6.
TABLE 5 inhibition of Staphylococcus aureus biofilm formation by chitosan derivatives
| Concentration of | 1/2MIC | MIC | 2MIC |
| Example 1 | 69.06％ | 79.77％ | 90.62％ |
| Comparative example 1 | 60.72％ | 71.2％ | 83.42％ |
| Comparative example 2 | 62.99％ | 74.96％ | 79.98％ |
TABLE 6 inhibition effect of chitosan derivatives on E.coli biofilm formation
200 Mu L of diluted bacterial liquid is added into a 96-well plate, then the mixture is placed into a constant temperature incubator at 37 ℃ for culturing for 36-48 hours, bacterial liquid is removed, sterile physiological saline is used for rinsing, drying and fixing are carried out, 200 mu L of chitosan derivative solution is added, sterile water is used as a control group, the temperature is kept constant for 1 hour in the incubator at 37 ℃, the chitosan derivative solution is removed, sterile physiological saline is used for rinsing, crystal violet is dyed, drying is carried out, 95% ethanol solution is added, absorbance at 595nm is measured by an enzyme-labeled instrument after 5 minutes, and the clearance of a biological envelope is calculated by a formula 3, and the results are shown in tables 7 and 8.
TABLE 7 Effect of chitosan derivatives on the removal of biofilm from Staphylococcus aureus
| Concentration of | 2.5mg/mL | 5.0mg/mL |
| Example 1 | 90.06％ | 94.01％ |
| Comparative example 1 | 83.81％ | 89.04％ |
| Comparative example 2 | 88.46％ | 91.83％ |
TABLE 8 Effect of chitosan derivatives on E.coli biofilm removal
| Concentration of | 2.5mg/mL | 5.0mg/mL |
| Examples | 72.87％ | 77.98％ |
| Comparative example 1 | 46.18％ | 50.22％ |
| Comparative example 2 | 65.61％ | 76.79％ |
The above results show that the guanidinium pyridine chitosan onium salt prepared by the preparation method provided by the invention has effects on staphylococcus aureus and escherichia coli, can inhibit biofilm formation of staphylococcus aureus and escherichia coli, has a certain cleaning effect on mature biofilm, and can be found by comparison example that the antibacterial activity and the anti-biofilm activity of the prepared guanidinium pyridine chitosan onium salt are superior to those of corresponding guanidinium chitosan and corresponding chitosan quaternary ammonium salt.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for preparing guanidinium pyridine chitosan onium salt, comprising the following steps:
(1) Mixing a p-chloromethyl benzoate compound, a solvent and a pyridine compound, and then carrying out quaternization reaction to obtain pyridine quaternary ammonium salt; the pyridine compound comprises 2-aminopyridine, 3-aminopyridine, 4-aminopyridine or 4-amino-N-methylpiperidine;
(2) Mixing the pyridine quaternary ammonium salt obtained in the step (1) with a solvent, hydrochloric acid and a cyanamide compound, and then carrying out an addition reaction to obtain an intermediate product;
(3) Mixing the intermediate product obtained in the step (2) with a solvent and alkali for saponification, and adding acid to obtain guanidyl pyridine quaternary ammonium salt;
(4) Mixing the guanidyl pyridine quaternary ammonium salt obtained in the step (3) with a solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and chitosan solution, and performing grafting reaction to obtain the guanidyl pyridine chitosan onium salt.
2. The method according to claim 1, wherein the ratio of the amounts of the substances of the p-chloromethylbenzoate compound and the pyridine compound in the step (1) is 1: (1-2).
3. The method according to claim 1, wherein the temperature of the quaternization reaction in the step (1) is 50 to 140 ℃ and the time of the quaternization reaction is 4 to 48 hours.
4. The method according to claim 1, wherein the ratio of the amounts of the substances of the pyridine quaternary ammonium salt and the cyanamide compound in the step (2) is 1: (1-3).
5. The method according to claim 1, wherein the temperature of the addition reaction in the step (2) is 40 to 100℃and the time of the addition reaction is 4 to 48 hours.
6. The method according to claim 1, wherein the ratio of the guanidyl pyridine quaternary ammonium salt to the chitosan substance in the chitosan solution in the step (4) is (1-3): 1.
7. The method according to claim 1 or 6, wherein the ratio of the amount of guanidyl pyridine quaternary ammonium salt to the amount of the substance of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in the step (4) is 1: (1-3).
8. The method according to claim 1, wherein the grafting reaction in the step (4) is carried out at a temperature of 20 to 50℃for a period of 12 to 48 hours.
9. A guanidinium chitosan salt prepared by the process of any one of claims 1 to 8.
10. Use of a guanidinium chitosan onium salt according to claim 9 for the preparation of a medicament for inhibiting bacteria and biofilm.
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| CN1662501A (en) * | 2002-05-17 | 2005-08-31 | 利奥制药有限公司 | Cyanoguanidine prodrugs |
| CN104672346A (en) * | 2014-12-15 | 2015-06-03 | 四川吉百瑞生物医疗科技有限公司 | Preparation method of guanidyl chitosan quaternary ammonium salt |
| CN111057164A (en) * | 2019-12-30 | 2020-04-24 | 华侨大学 | Preparation method of guanidino chitosan quaternary ammonium salt antibacterial agent |
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| CN1662501A (en) * | 2002-05-17 | 2005-08-31 | 利奥制药有限公司 | Cyanoguanidine prodrugs |
| CN104672346A (en) * | 2014-12-15 | 2015-06-03 | 四川吉百瑞生物医疗科技有限公司 | Preparation method of guanidyl chitosan quaternary ammonium salt |
| CN111057164A (en) * | 2019-12-30 | 2020-04-24 | 华侨大学 | Preparation method of guanidino chitosan quaternary ammonium salt antibacterial agent |
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